Microarray

Abstract

The present invention relates to microarrays. More particularly, the present invention relates to low-density microarrays, e.g., low-density protein microarrays, in which the surface density of analytes within individual, predetermined regions (e.g., microarray spots) of the surface is low. Additionally, the present invention relates to microarrays, e.g., protein microarrays, in which the surface coverage of analytes within individual, predetermined regions (e.g., microarray spots) of the surface is low. The present invention also relates to methods for producing such microarrays.

Description

[Technical Field]

[0001] The present invention relates to microarrays. More particularly, the present invention relates to low-density microarrays, e.g., low-density protein microarrays, in which the surface density of analytes within individual, predetermined regions (e.g., microarray spots) of the surface is low. Additionally, the present invention relates to microarrays, e.g., protein microarrays, in which the surface coverage of analytes within individual, predetermined regions (e.g., microarray spots) of the surface is low. The present invention also relates to methods for producing such microarrays. [Background technology]

[0002] Microarray technology allows for the high-throughput, parallel analysis of several different molecular interactions under uniform assay conditions, making it a useful tool in many areas of biological science research, such as the identification of novel disease-specific serological markers, the evaluation of the activity of lead compounds in the drug discovery process against potential therapeutic targets, and the functional analysis of unidentified proteins.

[0003] A microarray is typically an ordered spatial arrangement of purified analytes, such as recombinant or native proteins. Multiple different analytes can be immobilized in nanoliter quantities at spatially defined locations on a solid support, allowing high-throughput microanalytical (e.g., antibody binding) and functional (e.g., protein-protein interaction, protein-DNA interaction, or protein-small molecule interaction) assays to be performed in parallel on each immobilized analyte. Commonly used microarray technologies include both solid (planar or 2D) microarrays and bead-based suspension microarrays (Dunbar, S., et al. (2018) Solid and Suspension Microarrays for Detection and Identification of Infectious Diseases. In: Tang, YW., Stratton, C. (eds) Advanced Techniques in Diagnostic Microbiology. Springer, Cham, https: / / doi.org / 10.1007 / 978-3-319-33900-9_20). An ideal microarray would generate highly sensitive and specific quantitative data that accurately reflects the thermodynamics of true, physiologically relevant biomolecular interactions.

[0004] With respect to protein microarrays, to measure true physiologically relevant biomolecular interactions in highly multiplexed protein array-type assays, each protein immobilized on the protein array must retain its physiologically relevant folded structure and be presented on the surface in a manner such that true specific biomolecular recognition events can occur. Thus, an ideal surface for protein microarray fabrication is one that captures and retains only folded proteins in a controlled orientation with minimal nonspecific interactions in array-type assays.

[0005] Although several common methods are used to fabricate protein arrays, all of them suffer from problems that reduce the effectiveness of the final protein array. Nonspecific covalent attachment of proteins to surfaces can result in proteins immobilized in a random orientation, obscuring functionally important regions of the protein that are accessed in assays. Nonspecific, noncovalent physical adsorption of proteins to surfaces also results in proteins immobilized in a random orientation and typically results in protein unfolding and loss of activity on the surface. Encapsulation within hydrogels can preserve protein structure and function, but limits access to macromolecular interactors, and affinity capture to surfaces does not control protein orientation.

[0006] In addition, all of these methods of protein array fabrication typically result in a high density of immobilized proteins at each location on the resulting protein array, which can drive nonspecific interactions, including physiologically irrelevant nonspecific aggregation-driven interactions. Furthermore, the immobilized protein molecules are typically bound in a fixed orientation on the surface, potentially occluding true binding sites.

[0007] In contrast, in the natural cellular environment, the low concentrations of most soluble proteins and the low densities of most membrane-bound proteins favor true specific biomolecular interactions and strongly thermodynamically discourage nonspecific interactions. Furthermore, for true physiologically relevant biomolecular interactions to occur, the specific binding sites of interacting proteins must be physically accessible and must be able to find each other in 3D diffusion space, typically with rotational and conformational freedom preserved, especially for membrane-associated proteins.

[0008] Typically, protein arrays known in the art are blocked using proteinaceous reagents, most commonly bovine serum albumin, casein, or powdered milk, to reduce nonspecific binding of macromolecules to the surface, however, these blocking agents themselves exhibit significant nonspecific binding to other macromolecules.

[0009] Therefore, protein arrays known in the art are not always well suited to measuring true physiologically relevant biomolecular interactions in highly multiplexed assays, because it is typically difficult to distinguish between true specific binding on the array surface and background nonspecific interactions. They suffer from high nonspecific background binding and poor accessibility to specific binding sites of proteins immobilized in a fixed orientation. They also do not control the density of immobilization and do not allow for local rotational or conformational freedom on the surface.

[0010] Thus, there is a need in the art for improved protein microarrays that are capable of performing multiplexed, quantitative, and physiologically relevant measurements of biomolecular interactions across multiple different proteins in parallel. Summary of the Invention

[0011] According to a first aspect, the present invention provides a microarray comprising a surface having a plurality of reactive groups attached thereto, wherein an analyte is immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface density of the analyte within at least one discrete, predetermined region of the surface is less than about 20%. As part of this first aspect, the present invention also provides a microarray comprising a surface having a plurality of reactive groups attached thereto, wherein the analyte is immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface coverage of the analyte within at least one discrete, predetermined region of the surface is less than about 20%.

[0012] According to a second aspect, the present invention provides a method for manufacturing a microarray, the method comprising: The method includes the steps of: (i) providing a surface having a plurality of reactive groups attached thereto, wherein no more than about 20% of the reactive groups are capable of reacting at any one time; (ii) optionally contacting the reactive groups with a linking moiety under conditions whereby the linking moiety reacts with the one or more reactive groups capable of reacting, thereby binding the linking moiety to the surface; and (iii) depositing a sample of analyte on the surface within at least one discrete predetermined region of the surface such that the analyte is immobilized on the surface via direct binding to the one or more reactive groups capable of reacting or via indirect binding to the reactive groups via the linking moiety, wherein accordingly, the surface coverage of bound analyte within at least one discrete predetermined region of the surface is less than about 20%.

[0013] According to a third aspect, the present invention provides a method for reducing the density of analyte binding to the surface of a microarray, the method comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached; (ii) inactivating or rendering inaccessible a portion of the reactive groups such that no more than about 20% of the reactive groups are available to react at any one time; (iii) optionally contacting the reactive groups with a linking moiety under conditions that allow one or more reactive groups that retain reactivity to react with the linking moiety, thereby binding the linking moiety to the surface; and (iv) depositing a sample of analyte on the surface in at least one discrete predetermined area of ​​the surface such that the analyte is immobilized on the surface via direct binding to one or more reactive groups or via indirect binding to one or more reactive groups via the linking moiety, wherein accordingly, the surface coverage of bound analyte in at least one discrete predetermined area of ​​the surface is less than about 20%.

[0014] According to a fourth aspect, the present invention provides a method comprising: i) increasing the analyte signal to background noise ratio of the microarray; ii) increasing the rotational and conformational freedom of analytes immobilized on the microarray; and / or iii) increasing the rate of physiologically relevant interactions between analytes immobilized on a microarray and test molecules applied to the microarray, wherein the method comprises the steps of providing a microarray comprising a surface having a plurality of reactive groups attached thereto, wherein the analytes are immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface coverage of the immobilized analytes within at least one individual predetermined region of the surface is less than about 20%.

[0015] According to a fifth aspect, the present invention provides the use of a surface of a microarray as a low-density surface having a surface coverage (or surface density) of less than about 20% of analytes immobilized within at least one individual predetermined area of ​​said surface, wherein the analytes are optionally immobilized to said microarray via a linking moiety.

[0016] According to a sixth aspect, the present invention provides the use of a surface for the manufacture of a microarray comprising immobilised analytes, wherein the surface coverage (or surface density) of the immobilised analytes within at least one individual predetermined area of ​​said surface is less than about 20%, and the analytes are optionally immobilised to said microarray via a linking moiety.

[0017] According to a seventh aspect, the present invention provides the use of reactive groups on a surface suitable for forming a microarray to reduce the density of analytes immobilized on said surface, wherein some of the reactive groups are incapable of reacting with said analytes, and wherein said analytes are optionally immobilized on said surface via a linking moiety, such that the surface coverage (or surface density) of analytes immobilized on at least one individual predetermined area of ​​said surface is less than about 20%.

[0018] According to an eighth aspect, the present invention provides the use of reactive groups on a surface suitable for forming a microarray for the manufacture of a low-density protein microarray, wherein some of the reactive groups on the surface are incapable of reacting with an analyte, and the analyte is optionally immobilized on the surface via a linking moiety, such that the surface coverage (or surface density) of the analyte immobilized on at least one individual predetermined area of ​​the surface is less than about 20%.

[0019] In a ninth aspect, as disclosed herein, the present invention relates to the use of a microarray as defined in the first aspect for: i) identifying interactions between the analyte and a test molecule applied to the analyte; ii) determining the antibody profile of the subject; iii) identification of biomolecules that specifically bind to the immobilized analyte; iv) identification of an antibody that specifically binds to the immobilized analyte and is suitable for diagnosing or treating disease; or v) Identification of biomolecules that specifically bind to the immobilized analyte and are capable of treating a disease mediated by the immobilized analyte.

[0020] Further, in a tenth aspect of the present invention disclosed herein, there is provided a method for manufacturing a microarray, the method comprising the steps of: (i) preparing a surface to which a plurality of reactive groups are attached; (ii) contacting the reactive groups with a linking moiety comprising a biotin-binding molecule under conditions in which the linking moiety reacts with one or more of the reactive groups to which it is capable of reacting, thereby binding the linking moiety to the surface; (iii) depositing a sample of a biotinylated analyte such that the analyte is immobilized on the surface via indirect binding to the reactive groups via the linking moiety; and thereafter, (iv) applying a solution of biotin to the surface of the microarray.

[0021] In an eleventh aspect of the present invention, there is provided a method for increasing the analyte signal-to-background noise ratio of a microarray, the method comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached, wherein no more than about 20% of the reactive groups are reactive at any one time; (ii) contacting the reactive groups with a linking moiety that is a biotin-binding moiety under conditions in which the linking moiety reacts with one or more of the reactive groups to thereby bind the linking moiety to the surface; (iii) depositing a sample of biotinylated analytes onto the surface in at least one discrete predetermined area of ​​the surface such that the analytes are immobilized on the surface via indirect binding to the reactive groups via the linking moiety, wherein accordingly, the surface coverage (surface density) of bound analytes within the predetermined area of ​​the surface is less than about 20%; and thereafter (iv) applying a solution of biotin to the surface of the microarray.

[0022] In a twelfth aspect of the present invention, there is provided a microarray comprising a surface having a plurality of reactive groups attached thereto, wherein an analyte is immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the number density of the analyte (i.e., the number of immobilized analyte molecules) within at least one individual defined area of ​​the surface is less than about 300 per square micrometer.

[0023] In a thirteenth aspect of the present invention, there is provided a method of manufacturing a microarray, the method comprising: The method includes the steps of: (i) providing a surface to which a plurality of reactive groups are attached; and (ii) contacting the reactive groups with a solution of linking moieties, wherein the concentration of linking moieties in the solution is less than 1 mg / ml, and wherein the linking moieties react with one or more of the reactive groups capable of reacting, thereby attaching the linking moieties to the surface.

[0024] In a fourteenth aspect of the invention, the invention provides a microarray obtainable by either the method of the tenth or thirteenth aspects of the invention.

[0025] It will be understood that features described in relation to one aspect of the invention may be incorporated in other aspects of the invention, for example, the methods and uses of the invention may incorporate any of the features described in relation to the microarrays of the invention, and vice versa. [Brief explanation of the drawings]

[0026] [Figure 1] Figures A-B show quality control checks to determine the reproducibility of fabricated slides. Both figures show Cy5 biotin-labeled bovine serum albumin (Cy5 BBSA) fluorescence to demonstrate slide coating uniformity (A) and spot uniformity (B). [Figure 2] 1 shows confirmation of His-tagged CYP450 binding using an anti-6xHis probe. [Figure 3] 1 is a flow chart showing the experimental plan for comparing biotin and BSA treatment of replica protein arrays after array fabrication. [Figure 4A] Microarray layout of BCCP-tagged full-length and truncated SARS-CoV-2 nucleocapsid protein antigens is shown. [Figure 4B] Array images of a biotin block array scanned at 532 and 635 nm are shown. [Figure 4C] Array images of a BSA block array scanned at 532 and 635 nm are shown. [Figure 5] AB shows a comparison of the foreground signal intensity (635 nm) of spots printed on a biotin block array (A) and a BSA block array (B). [Figure 6A] A comparison of the signal intensity (635 nm) of the surrounding background area adjacent to the printed spots of a biotin block array is shown. [Figure 6B]A comparison of the signal intensity (635 nm) of the surrounding background area adjacent to the printed spots of a BSA block array is shown. [Figure 7] Shown is a scanning atomic force microscopy (AFM) topography image of a 1 μm×1 μm area within one CYP450 spot on a protein array. [Figure 8] Figures A-B show schematic diagrams of an embodiment of the present invention in which a test molecule (5) interacts with an analyte (4) immobilized on a surface (1), e.g., a microarray. In Figure A, the analyte (4) is bound to a reactive group (2) on the surface via a linking moiety (3). In Figure B, the analyte (4) is directly bound to a reactive group (2) on the surface. The analyte (4) can be, e.g., a recombinant or native protein, a BCCP-tagged protein, a biotinylated protein, a nucleic acid, a sugar, a bacterium, or another type of molecule. The linking moiety can be, e.g., streptavidin. The reactive group (2) can be, e.g., an NHS-activated PEG polymer. In both cases, the reactive group (2) is attached to a surface (1), which can be, e.g., a glass slide, a plate well, a bead surface, or other surface. Solid lines represent the connections between the individual elements: surface (1), reactive group (2), linking moiety (3), if present, and analyte (4). Such a linkage can be any suitable linkage, e.g., a covalent or non-covalent interaction, e.g., the interaction between streptavidin and biotin. The dashed line represents an interaction between the analyte and the test molecule (5), which can be, for example, a protein-ligand interaction or an antibody-antigen interaction. [Figure 9A] Fluorescence images of Nexterion H slides coated with streptavidin solutions at concentrations ranging from 0.05 to 2 mg / ml and incubated with Cy3-biotin-BSA are shown. [Figure 9B] Fluorescence images of Nexterion H slides coated with streptavidin solutions at concentrations ranging from 0.05 to 2 mg / ml and incubated with Cy3-biotin-BSA in the presence of competitor 50 mM glycine are shown. [Figure 9C]Fluorescence images of time-lapsed Nexterion H slides derivatized with streptavidin solutions at concentrations ranging from 0.03 to 2 mg / ml and incubated with Cy3-biotin-BSA are shown. [Figure 9D] Fluorescence images of Nexterion H slides are shown that were derivatized with streptavidin solution at a concentration of 1 mg / ml, pH 9, or pH 4.5, and incubated with Cy3-biotin-BSA, as well as negative controls (no streptavidin), such as slide coating buffer, slide coating buffer with 50 mM glycine, or empty gasket wells (which were also incubated with Cy3-biotin-BSA). [Figure 9E] Binding curves for Nexterion H-slides derivatized with streptavidin alone, in the presence of 50 mM glycine, or on aged H-slides, followed by incubation with Cy3-biotin-BSA are shown. [Figure 9F] Shown is a bar graph of the mean fluorescence intensity of Nexterion H slides derivatized with streptavidin and incubated with Cy3-biotin-BSA at pH 8.5, 9, or 4.5. [Figure 9G] Shown is a bar graph of the mean fluorescence intensity of negative control Nexterion H slides coated with coating buffer only (no glycine), slide coating buffer containing 50 mM glycine (Glycine), or empty wells followed by incubation with Cy3-biotin-BSA. [Figure 10] Streptavidin binding on Nexterion H slides derivatized with streptavidin solutions of streptavidin concentrations ranging from 0.125 to 8 mg / ml and incubated with Cy3-biotin-BSA, with or without a subsequent blocking step with 50 mM biotin, is shown. [Figure 11A] Shown is an SEM image of a Nexterion H slide derivatized with 8 mg / ml streptavidin and incubated with Cy3-biotin-BSA. [Figure 11B] Shown is an SEM image of a Nexterion H slide derivatized with 0.25 mg / ml streptavidin and incubated with Cy3-biotin-BSA. [Figure 11C] Shown are SEM images of Nexterion H slides incubated with slide coating buffer only (negative control) and incubated with Cy3-biotin-BSA. [Figure 12] Shown is a microarray layout of SARS-CoV-2 proteins, including the full-length S ectodomain trimer and the C-terminal domain of the N protein, printed in triplicate on derivatized Nexterion H slides under various conditions. [Figure 13] A shows an anti-c-Myc assay for the detection of SARS-CoV-2 S and CTD proteins detected on Nexterion H slides derivatized with 0.03-2 mg / ml streptavidin. B shows an anti-c-Myc assay for the detection of SARS-CoV-2 S and CTD proteins detected on Nexterion H slides derivatized with 0.03-2 mg / ml streptavidin in the presence of 50 mM glycine. C shows an anti-c-Myc assay for the detection of SARS-CoV-2 S and CTD proteins detected on timed Nexterion H slides derivatized with 0.03-2 mg / ml streptavidin. D shows anti-c-Myc assay for detection of SARS-CoV-2 S and CTD proteins at pH 9 and pH 4.5 on streptavidin-derivatized H slides, as well as negative controls, e.g., slide coating buffer (SCB) alone, slide coating buffer containing 50 mM glycine (SCB + 50 mM glycine), or empty wells. [Figure 14]A shows a microarray image of IgG detection against SARS-CoV-2 S protein printed on Nexterion H slides derivatized with 0.03-2 mg / ml streptavidin. B shows a microarray image of IgG detection against SARS-CoV-2 S protein printed on Nexterion H slides derivatized with 0.03-2 mg / ml streptavidin in the presence of 50 mM glycine. C shows a microarray image of IgG detection against SARS-CoV-2 S protein printed on aged Nexterion H slides derivatized with 0.03-2 mg / ml streptavidin. D shows microarray images of IgG detection against SARS-CoV-2 S protein printed on streptavidin-derivatized Nexterion H slides at pH 9 and pH 4.5, as well as negative controls, e.g., slide coating buffer (SCB) only, slide coating buffer containing 50 mM glycine (SCB + 50 mM glycine), or empty wells. [Figure 15A] Shown is an IgG assay for SARS-CoV-2 S protein detected on Nexterion H slides derivatized with 0.03–2 mg / ml streptavidin. [Figure 15B] IgG assay of SARS-CoV-2 S protein detected on Nexterion H slides derivatized with 0.03–2 mg / ml streptavidin in the presence of 50 mM glycine. [Figure 15C] IgG assay of SARS-CoV-2 S protein detected on time-aged Nexterion H slides derivatized with 0.03–2 mg / ml streptavidin. [Figure 15D] IgG assay of SARS-CoV-2 S protein detected on streptavidin-derivatized Nexter ion H slides at pH 9 and pH 4.5. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to improved microarrays, particularly microarrays in which analytes are immobilized at low density on the surface of the microarray. For a full understanding of the terms used in the context of the present invention (e.g., "analyte," "linking moiety," "reactive group," "surface," and "test molecule"), reference is made to Figures 8A and 8B and their accompanying descriptions. As can be seen from these figures, an "analyte" in the context of the present invention is a moiety that is immobilized on the microarray and whose properties it is desired to analyze. Such analysis may take the form, for example, of determining the interaction of the analyte with a "test molecule" that is applied to the immobilized analyte. In certain embodiments of the present invention, the analyte is immobilized on the surface of the microarray via direct binding to a "reactive group" on the surface. In other embodiments, the analyte is immobilized on the surface of the microarray via indirect binding to a reactive group on the surface via a "linking moiety."

[0028] More specifically, the present invention is based on the surprising observation that immobilizing analytes at low density on the surface of a microarray results in improved properties, e.g., when immobilized analytes are sufficiently separated from one another on the surface of the array, the analytes are less likely to aggregate and maintain local rotational and structural freedom. This allows the immobilized analytes to behave more like they do in a cellular environment, resulting in significantly improved specificity (increased physiological relevance) of biomolecular interactions, lower detection limits, and significantly reduced nonspecific binding in array-based assays because the inventive configurations make such nonspecific binding thermodynamically less likely. In particular, the inventive low-density arrays reduce steric hindrance and / or blockage of relevant binding sites, making the immobilized analytes more physically accessible. Therefore, the risk of steric hindrance between analytes within a spot, which could prevent proper interaction between the analyte and its binding partner, is reduced. Therefore, such immobilized analytes are better able to find their (physiologically relevant) interacting molecules (e.g., binding partners) in 3D diffusion space. Such interactions include, for example, protein-protein interactions, protein-ligand interactions, protein-nucleic acid interactions, and protein-small molecule interactions.

[0029] Furthermore, when the analytes of the present invention are proteins or polypeptides, the surfaces of the microarrays of the present invention favor the capture of properly folded proteins / polypeptides in a controlled orientation, thus favoring physiologically relevant interactions (with applied test molecules) while again minimizing nonspecific interactions, such as nonspecific aggregation-driven interactions. Thus, the microarrays of the present invention allow the immobilized analytes to behave more like they would in a natural cellular environment, where, for example, most soluble proteins are present at low concentrations and most membrane-bound proteins are present at low densities. Immobilization in a controlled (constant) orientation is achieved across all arrays, rather than just one, thereby improving the reproducibility of interactions with a given analyte.

[0030] In protein arrays known in the art, such arrays are typically blocked using proteinaceous reagents (most commonly bovine serum albumin, casein, or powdered milk) to reduce nonspecific binding of macromolecules to the surface. However, such blocking agents themselves exhibit significant nonspecific binding to other macromolecules, thus resulting in physiologically irrelevant interactions. The present invention eliminates the need for blocking with proteinaceous reagents and thus avoids the resulting disadvantages.

[0031] A further benefit associated with the present invention (when the binding analyte is a protein or polypeptide) is the use of protein folding markers to report the folded state of individual recombinant proteins. Thus, using such folding markers, only folded and biologically functional proteins can be immobilized (at low density) on the surface of the array. One of the main advantages of this is that the interaction / binding partners indeed require 3D structural moieties generated by folding for specific analyte recognition and binding, allowing for in vivo-like interaction specificity, which would be lost if the protein is not properly folded.

[0032] Due to the above advantages, the interaction of analytes immobilized on the array of the present invention exhibits high specificity, low background, and little non-specific interaction. Therefore, the array of the present invention produces relevant analyte interactions with the applied test molecules with a high signal-to-noise ratio. This provides accurate quantitative measurements with a low detection limit.

[0033] Furthermore, due to the particular techniques used in the context of the arrays of the present invention for immobilizing analytes on the surface of the array, the present invention allows for the combination of immobilization and purification of desired analytes in a single step, as described in more detail elsewhere herein.

[0034] Finally, the methods for fabricating microarrays as described herein allow for control of the density at which proteins are immobilized on the surface of the microarray.

[0035] In a first aspect, the present invention relates to a microarray comprising a surface to which a plurality of reactive groups are attached, wherein analytes are immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface density of the analytes within at least one individual predetermined region of the surface is less than about 20%, e.g., the surface density of the analytes within an analyte spot of the microarray is less than about 20%.

[0036] As part of this first aspect, the present invention also relates to a microarray comprising a surface having a plurality of reactive groups attached thereto, wherein an analyte is immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface coverage of the analyte within at least one individual predetermined area of ​​the surface is less than about 20% of the predetermined area. Microarrays are sometimes referred to as low-density microarrays or microarrays with low surface coverage of immobilized analytes, e.g., the surface coverage of the analyte within at least one analyte spot of the microarray is less than about 20% of the area of ​​the analyte spot.

[0037] In one embodiment of this first aspect, the invention relates to a low-density microarray comprising a surface having a plurality of reactive groups attached thereto, wherein an analyte is immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface coverage of the analyte in at least one individual defined area of ​​the surface is less than about 20% of the defined area.

[0038] In one embodiment of this first aspect, the surface density of the analyte within at least one discrete predetermined area of ​​the surface is less than about 18%, e.g., less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4%, or less than about 2%.

[0039] In one embodiment of this first aspect, the surface coverage of or by the analyte within at least one individual defined area of ​​the surface is less than about 18%, e.g., less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4%, or less than about 2% of the defined area.

[0040] In one embodiment of this first aspect, the surface density of the analytes within at least one discrete predetermined area of ​​the surface is at least about 0.05%, preferably at least about 0.5%, and more preferably at least about 1%. For example, in one embodiment of this first aspect, the surface density of the analytes within at least one discrete predetermined area of ​​the surface is in the range of about 0.05% to about 20%, about 0.5% to about 20%, about 1% to about 20%, about 0.05% to about 15%, about 0.05% to about 12%, about 0.05% to about 10%, about 0.5% to about 10%, or about 1% to about 10%.

[0041] In one embodiment of this first aspect, the surface coverage of or by the analyte within at least one distinct predefined area of ​​the surface is at least about 0.05%, preferably at least about 0.5%, and more preferably at least about 1% of the predefined area. For example, in one embodiment of this first aspect, the surface coverage of the analyte within at least one distinct predefined area of ​​the surface is in the range of about 0.05% to about 20%, about 0.5% to about 20%, about 1% to about 20%, about 0.05% to about 15%, about 0.05% to about 12%, about 0.05% to about 10%, about 0.5% to about 10%, or about 1% to about 10% of the predefined area.

[0042] In the context of the present invention, "surface coverage" of an analyte within an individual predetermined region refers to the percentage of a particular individual region on a microarray that is covered by bound analyte molecules (e.g., the percentage of the area of ​​an individual analyte "spot" on the surface of the microarray). Surface coverage can be measured, for example, by microscopy, for example, by atomic force microscopy (AFM) or, for example, by scanning electron microscopy (SEM). When the surface coverage of an analyte within a predetermined region is referred to as less than 10%, this means that the analyte molecules cover less than 10% of the predetermined region (e.g., spot) of the array.

[0043] In one embodiment of this first aspect, the surface coverage of the analyte within substantially all or all individual predetermined regions on the surface (i.e., all individual analyte "spots" on the surface of the microarray) is less than about 20%, e.g., less than about 18%, e.g., less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4%, or less than about 2%. For example, in one embodiment of this first aspect, the surface coverage of the analyte within substantially all or all individual predetermined regions on the surface (i.e., individual analyte "spots" on the surface of the microarray) is in the range of 0.05% to 20%, 0.5% to 20%, 1% to 20%, 0.05% to 15%, 0.05% to 12%, 0.05% to 10%, 0.5% to 10%, or 1% to 10% of the predetermined region.

[0044] As will be apparent, in one embodiment, the "individual predetermined areas" referred to herein are analyte "spots" on the surface of the microarray, i.e., areas where the analyte is printed on the microarray. Outside of the individual predetermined areas, the analyte may not be immobilized on the surface of the microarray.

[0045] Alternatively, it is contemplated that reference to the surface coverage of an analyte within a given region (as referred to herein) refers to the percentage of reactive groups within the given region of the surface of the microarray that actually bind to the analyte (either directly or indirectly via a linking moiety). To provide a simple example for illustrative purposes only, if a given region of the surface of the microarray is characterized as containing a 10x10 grid of reactive groups (i.e., a total of 100 reactive groups within the given region), a surface coverage of less than 10% correlates with fewer than 10 of the 100 reactive groups binding to the analyte (either directly or indirectly via a linking moiety). It should be noted that in the context of the present invention, reference to low surface coverage does not refer to the density (number) of spots on the array (or how close such spots are to one another), but rather refers to the percentage of the area of ​​each individual microarray spot on which analyte molecules are immobilized. In this context, reference to a "high density array" may be a term in the art, but it should be noted that this term typically refers to an array in which a high density (large number) of spots are printed in the array, i.e., a large number of spots are printed in close proximity to each other in the array. This is therefore a different concept from the concepts in which the terms surface density or surface coverage are used in the context of the present invention.

[0046] Analyte surface density in the context of the present invention can alternatively be considered as the number of analyte molecules immobilized on the microarray per unit area. Thus, an alternative definition of low surface density can be defined as less than a predetermined number of analyte molecules immobilized per unit area, optionally when analyzed by atomic force microscopy (AFM), or optionally when analyzed by scanning electron microscopy (SEM).

[0047] In one embodiment of this first aspect, the number of immobilized analyte molecules, optionally when analyzed by AFM, or optionally when analyzed by SEM, is less than about 300 per square micrometer, e.g., less than about 275 per square micrometer, less than about 250 per square micrometer, less than about 225 per square micrometer, less than about 200 per square micrometer, less than about 175 per square micrometer, less than about 150 per square micrometer, less than about 125 per square micrometer, less than about 100 per square micrometer, less than about 75 per square micrometer, less than about 50 per square micrometer, less than about 25 per square micrometer, less than about 20 per square micrometer, less than about 15 per square micrometer, less than about 10 per square micrometer, or less than about 5 per square micrometer.

[0048] Alternatively, the surface density or surface coverage of analytes in the context of the present invention can be considered as the number of reactive groups, e.g., NHS-activated PEG polymers, attached to linking moieties, e.g., streptavidin tetramers, per unit area. Tagged (e.g., biotinylated) analytes specifically bind only to linking moieties (e.g., streptavidin). Therefore, the distribution of linking moieties (e.g., streptavidin) attached to reactive groups (e.g., NHS-activated PEG polymers) across the entire surface of the slide ultimately controls the density or coverage of analytes ultimately bound to the slide. When the reactive groups are PEG polymers (e.g., NHS-activated PEG polymers), the percentage of PEG polymers that are NHS-activated is very high, essentially 100%, but the inventors have concluded that the percentage of NHS-activated polymers that are available and can be bound to linking moieties such as streptavidin can be surprisingly low. This means that the surface density or surface coverage of the bound linking moieties (e.g., streptavidin) can be low, resulting in a low surface density or low surface coverage of the bound analyte. Thus, alternatively, low surface density can be defined as less than a predetermined number of bound linking moieties per unit area of ​​the surface of the microarray when analyzed, for example, by AFM or, for example, by SEM.In one embodiment of the first aspect of the invention, the number of linking moieties (e.g., streptavidin) attached to the surface of the microarray, optionally when analyzed by AFM, or optionally when analyzed by SEM, is less than about 300 per square micrometer, e.g., less than about 275 per square micrometer, less than about 250 per square micrometer, less than about 225 per square micrometer, less than about 200 per square micrometer, less than about 175 per square micrometer, less than about 150 per square micrometer, less than about 125 per square micrometer, less than about 100 per square micrometer, less than about 75 per square micrometer, less than about 50 per square micrometer, less than about 25 per square micrometer, less than about 20 per square micrometer, less than about 15 per square micrometer, less than about 10 per square micrometer, or less than about 5 per square micrometer. Similarly, low surface coverage can be defined as less than a certain percentage of a given area of ​​a surface being covered by linking moieties, as analyzed, for example, by AFM or, for example, by SEM. In one embodiment of this first aspect of the invention, the surface coverage of or by linking moieties within a given area of ​​the surface is, optionally, less than 20%, e.g., less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, or less than 2%, as analyzed by AFM. In one embodiment of this first aspect, the surface coverage of or by linking moieties within a given area of ​​the surface is at least 0.05%, preferably at least 0.5%, and more preferably at least 1%. For example, in one embodiment of this first aspect, the surface coverage of or by the linking moieties within a given area of ​​the surface ranges from 0.05% to 20%, 0.5% to 20%, 1% to 20%, 0.05% to 15%, 0.05% to 12%, 0.05% to 10%, 0.5% to 10%, or 1% to 10%.

[0049] For the avoidance of doubt, references herein to "predefined regions" refer to subregions (e.g., "spots") of the surface of the microarray, and not the entire surface area of ​​the microarray. An individual "predefined region" (e.g., an analyte "spot" or "dot") has a diameter of approximately 200 μm (if circular), and therefore an area of ​​approximately 0.03 mm 2 In one embodiment of this first aspect, the "predetermined area" referred to herein has a diameter in the range of 1 μm to 500 μm (if circular). In one embodiment of this first aspect, the "predetermined area" referred to herein has a surface area of ​​0.80 μm 2 ~0.2mm 2 As will be apparent, microarrays typically have a plurality of "predetermined regions" (e.g., a plurality of "spots") on a surface. Such "predetermined regions" may be printed on the surface at high density (i.e., close together), but within those predetermined regions, the analytes are immobilized at low density.

[0050] Additionally, it should be noted that in all aspects and embodiments of the present invention, within a given region (e.g., a microarray "spot"), the analyte molecules (and linking moieties, if present) are advantageously uniformly distributed across the surface of the given region. In other words, the arrays of the present invention and methods for preparing them do not result in given regions containing subregions where analyte molecules (and linking moieties, if present) are immobilized ("aggregated") at high densities, interspersed with subregions that are substantially devoid of surface-bound analyte, resulting in an overall average low surface density.

[0051] Thus, a microarray of the present invention may contain one or, more typically, multiple spots, each of which constitutes a "predetermined region" as defined herein. Surrounding or between one or more spots, the microarray may contain one or more interspot regions (which may be contiguous) that are free or substantially free of analyte. Those skilled in the art will appreciate that the interspot regions may contain reactive groups and, optionally, linking moieties. After depositing analyte molecules on the microarray as one or more spots, any unbound reactive groups and / or linking moieties on the microarray may be blocked as disclosed herein.

[0052] In all contexts of the aspects of the invention, reference to the "surface" of a microarray should be interpreted to mean not only a flat slide (i.e., a solid, planar or 2D microarray, e.g., made of glass), but also other suitable surfaces such as beads (as found in suspension or 3D microarrays), or the wells of a plate (e.g., a 96-well plate). Thus, the term "microarray" should be interpreted to include both planar, solid microarrays and suspension microarrays. In one embodiment of each of the aspects of the invention, the microarray is a planar microarray (also known as a solid or 2D microarray). In another embodiment of each of the aspects of the invention, the microarray is a suspension microarray, e.g., a bead-based suspension microarray (also known as a 3D microarray). Suspension arrays of such beads (sometimes called microparticles) are well known in the art (Nolan, JP and Sklar, LA, (2002) Trends in Biotechnology, vol. 20(1), p. 9-12, https: / / doi.org / 10.1016 / S0167-7799(01)01844-3).

[0053] In the context of all aspects and embodiments of the present invention, references to surface density may alternatively be read as references to surface coverage, and vice versa.

[0054] In one embodiment of this first aspect, the analyte is immobilized on the surface via indirect binding to one or more of the reactive groups via a linking moiety.

[0055] In one embodiment of this first aspect, the analyte comprises a tag that allows for immobilization of said analyte to said surface.

[0056] In one embodiment of this first aspect, the analyte comprises one or more of a polypeptide, a nucleic acid, a lipid, and a carbohydrate.

[0057] For purposes of the present invention, a protein is considered to be a polypeptide chain of 50 or more amino acids, a peptide is considered to be a polypeptide chain of 50 or fewer amino acids and more than 20 amino acids, and an oligopeptide is considered to be a polypeptide chain of at least 2 and not more than 20 amino acids. Thus, as defined herein, oligopeptides, peptides, and proteins are collectively referred to as polypeptides.

[0058] In one embodiment of this first aspect, the analyte is a polypeptide (ie, an oligopeptide, peptide, or protein) or a nucleic acid.

[0059] In one embodiment of this first aspect, the analyte is a polypeptide (ie, an oligopeptide, peptide, or protein), for example, a glycoprotein.

[0060] In one embodiment of this first aspect, the analyte is biotinylated, for example a biotinylated polypeptide.

[0061] 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid, also known as biotin, is a small chemical that is widely distributed in living organisms and is involved in several metabolic processes. The protein streptavidin has an extremely high affinity for biotin. Approximately 10-14 Dissociation constant of M (K d ), and the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. For the purposes of the present invention, the term biotin refers to biotin itself and its derivatives that retain the functionality of biotin, i.e., -9 Less than M, e.g., 10 -12 M or less.

[0062] In one embodiment of this first aspect, the biotinylated analyte, e.g., biotinylated polypeptide, is a chemically biotinylated analyte or an enzymatically biotinylated analyte. In one embodiment of this first aspect, the biotinylated analyte, e.g., biotinylated polypeptide, is an enzymatically biotinylated polypeptide.

[0063] In one embodiment of this first aspect, the analyte is a polypeptide, preferably a biotinylated polypeptide, and said tag allowing immobilization of said polypeptide to said surface is fused to the N-terminus or C-terminus of the polypeptide.

[0064] In one embodiment of this first aspect, the polypeptide is properly folded. For purposes of the present invention, a "properly folded" polypeptide means that the polypeptide chain itself is organized into the proper secondary or tertiary structure and is functionally active.

[0065] The polypeptides or other analytes contemplated herein can be biotinylated at available primary amine groups. In polypeptides, such primary amine groups can be present on lysine side chains or N- or C-terminal residues. In these cases, the biotin molecule begins to be covalently bound to the polypeptide after a dehydration reaction between the carboxyl group of biotin and the amine group of the polypeptide.

[0066] Biotinylation can be performed chemically or enzymatically. When performed enzymatically, the reaction is catalyzed by biotin ligase, which recognizes the biotinylation motif methionine-lysine-methionine within the polypeptide of interest. Such a biotinylation motif can be present, for example, within a biotin carboxyl carrier protein (BCCP) domain. Biotin ligase preferentially biotinylates the lysine within this motif. The polypeptide of interest can be expressed, for example, in insect cells (such as Spodoptera frugiperda cells) or E. coli with the BCCP domain fused to it (e.g., at the N- or C-terminus). Thus, using such methods of biotinylation, the site of biotinylation on the protein can be controlled. In vitro biotinylation methods are also available using E. coli biotin ligase (BirA), as described in Schatz, PJ (1993), Bio / Technology (Nature Publishing Company), 11(10), 1138-1143, https: / / doi.org / 10.1038 / nb t1093-1138. Such methods are useful for certain BCCP-tagged proteins (e.g., secreted, membrane-bound) that cannot be biotinylated in insect cells due to maturation within the endoplasmic reticulum (ER), which does not contain the biotinylation machinery present in the cytosol.

[0067] Chemical methods offer greater flexibility in the type of biotinylation required than enzymatic approaches and can be performed both in vitro and in vivo. Furthermore, chemical methods do not require the coexpression of bacterial biotin ligase or the modification of the polypeptide of interest to carry a biotin acceptor peptide. Chemical biotinylation reagents possess a reactive moiety that can crosslink biotin to reactive moieties (e.g., primary amines, sulfhydryls, carboxyls, or carbonyls) within the polypeptide of interest. Nonselective biotinylation reagents are also available that can be used to label macromolecules that do not have available primary amines, sulfhydryls, carboxyls, or carbonyls. A so-called spacer arm can be present between the reactive moiety and the biotin molecule itself. Amines are the most common target functional group for biotinylation due to the abundance of lysine side chain ε-amines and N-terminal α-amines. N-hydroxysuccinimide (NHS) esters readily form stable bonds with primary amines, and reactive groups are easily incorporated and stabilized into a variety of useful, ready-to-use biotinylation reagents. NHS esters can be modified to be water-soluble by sulfonating the N-hydroxysuccinimide ring to form sulfo-NHS esters. Tetrafluorophenyl (TFP) esters contain a commonly used amine-reactive group that has similar reactivity with primary amines but is more hydrophobic than NHS. Sulfhydryl groups found on exposed cysteine ​​residues are the second most common target for biotinylation. Examples of reactive sites on sulfhydryl-reactive biotinylation reagents include maleimide, iodoacetyl, and pyridyl disulfide groups. Carboxyl groups are found at the carboxy-terminus of proteins and on aspartic acid and glutamic acid side chains. Biotinylation reagents targeting carboxyl groups require a zero-length crosslinker, such as EDC (carbodiimide), to conjugate to the primary amines of the biotinylation reagent. Therefore, the amines of carboxyl-reactive biotinylation reagents are not reactive themselves; they are the site of conjugation to the target protein. In addition to amines, biotinylation reagents containing hydrazide moieties can also be used with EDC to react with carboxyl groups.Although carbonyls are not readily present in proteins, carbohydrate residues on glycoproteins can be modified to aldehydes and labeled with hydrazide or alkoxyamine derivative biotinylation reagents. These aldehydes on glycoproteins are generated by oxidation of carbohydrate sialic acids using sodium periodate. The aldehydes are then specifically reacted with hydrazides or alkoxyamines at pH 4-6 to form stable linkages. Nonselective, photocleavable biotinylation reagents are available for labeling target proteins without available amines, sulfhydryls, carboxyls, or carbohydrates. Most photoreactive biotinylation reagents are based on aryl azides, which become activated by UV light (>350 nm) and initiate addition reactions that insert into CH and NH sites. Subsequent ring expansion drives the reaction toward conjugation to nucleophiles such as primary amines. Photoactivatable reagents are usually chosen when primary amines and other functional groups are scarce or when initiation of conjugation must occur at a specific time during the incubation period (i.e., by exposure to UV light).

[0068] In one embodiment of this first aspect, the tag that enables immobilization of an analyte to the surface of a microarray comprises a biotin carboxyl carrier protein (BCCP) motif, Avi tag (SEQ ID NO: 1), SNAP tag®, SpyTag tag, or SpyCatcher protein. Avi tag is a 15-residue peptide (sequence GLNDIFEAQKIEWHE, defined herein as SEQ ID NO: 1) that mimics the biotin acceptor function of the very large BCCP domain that is normally recognized by biotin ligase. The advantage of this is that Avi tag is much smaller than BCCP domains and can therefore be used as a biotinylation site on recombinant proteins where potential steric conflicts need to be minimized. SNAP tag® is a self-labeling protein tag and is commercially available in various expression vectors from New England Biolabs, Inc. SNAP-tag® is a 182-residue polypeptide (19.4 kDa) that can be fused to an analyte of interest, e.g., a polypeptide, and then specifically and covalently tagged with a suitable ligand, e.g., biotin. Thus, an analyte of the present invention can be fused to SNAP-tag® and then labeled with biotin. The peptide SpyTag (13 amino acids) naturally reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair (PNAS, 109(12)E690-E697, https: / / doi.org / 10.1073 / pnas.1115485109). The SpyTag or SpyCatcher protein can bind to the analyte of interest; for example, a DNA sequence encoding SpyTag or SpyCatcher can be recombinantly introduced into a DNA sequence encoding the polypeptide analyte of interest, thereby forming a fusion protein with its cognate pair that is bound to the surface of a microarray. The analyte fusion protein can then be covalently attached to the microarray via a reaction mediated by the SpyTag / SpyCatcher system.

[0069] In one embodiment of this first aspect, the tag that enables immobilization of an analyte to the surface of the microarray comprises a BCCP motif, or an Avi tag (SEQ ID NO: 1). In one embodiment of this first aspect, the tag that enables immobilization of an analyte to the surface of the microarray comprises a biotin carboxyl carrier protein (BCCP) motif.

[0070] In one embodiment of this first aspect, the BCCP motif has at least 80% sequence identity, preferably at least 90% sequence identity, and more preferably 100% sequence identity to SEQ ID NO: 2. In one embodiment of this first aspect, the BCCP motif is preferably derived from E. coli and corresponds to residues 74-156 of the AccB protein, i.e., AAAEISGHIV RSPMVGTFYR TPSPDAKAFI EVGQKVNVGD TLCIVEAMKM MNQIEADKSG TVKAILVESG QPVEFDEPLV VIE (defined herein as SEQ ID NO: 2). This BCCP motif is cross-recognized by eukaryotic biotin ligases, allowing for efficient biotinylation in yeast, insect, and mammalian cells without the need to co-express E. coli biotin ligase. Advantageously, the N- and C-termini of the BCCP domain are physically separated from the site of biotinylation by 50 Å, which is ideal for displaying recombinant proteins away from the surface, thus minimizing any deleterious effects due to immobilization. In one embodiment of this first aspect, insect cells are used to express recombinant BCCP-tagged proteins. In one embodiment of this first aspect, the insect cells are Spodoptera frugiperda cells. Insect cell expression systems allow for complex eukaryotic post-translational modifications (maintaining protein function and epitope detection) while being compatible with mild lysis conditions. Mammalian systems are also compatible with the expression and biotinylation of BCCP-tagged recombinant proteins.

[0071] In one embodiment of this first aspect, the BCCP motif is properly folded. For purposes of the present invention, a "properly folded" BCCP motif refers to a motif that is a suitable substrate for a biotin ligase enzyme, such as E. coli biotin ligase. Advantageously, it has been demonstrated that biotinylation of the BCCP motif in a fusion protein is a reliable marker of the folded state of the fusion partner, and that only properly folded recombinant fusion proteins are biotinylated by the biotin ligase enzyme.

[0072] In one embodiment of this first aspect, biotin is attached to (i) the biotin-binding domain within said BCCP motif, or (ii) said Avi tag.

[0073] In one embodiment of this first aspect, biotin is attached to the biotin-binding domain within the BCCP motif or to the Avi tag via enzymatic biotinylation, preferably via a biotin ligase, such as E. coli biotin ligase.

[0074] In one embodiment of this first aspect, the linking moiety is a biotin-binding molecule and the analyte is biotinylated.

[0075] In one embodiment of this first aspect, the linking moiety that is a biotin-binding molecule is a protein.

[0076] In one embodiment of this first aspect, the linking moiety that is a biotin-binding molecule is selected from the group consisting of: (i) an anti-biotin antibody, (ii) avidin, (iii) neutravidin, (iv) streptavidin, or (iii) a fragment, variant, or analog of streptavidin, avidin, neutravidin, or the anti-biotin antibody described above that retains biotin-binding ability. In the context of this embodiment of this first aspect of the invention, the streptavidin, avidin, neutravidin, or fragment, variant, or analog of the anti-biotin antibody described above that retains biotin-binding ability has a binding affinity for biotin of less than 10 M, e.g., less than 10 M, e.g., less than 10 M.

[0077] Avidin is a tetrameric biotin-binding protein found naturally in egg white. Each monomeric unit of the protein can bind to biotin, and this binding interaction between avidin and biotin is one of the strongest non-covalent interactions known, with a dissociation constant of approximately 10-15 M.

[0078] NeutrAvidin is a deglycosylated homolog of avidin, which reduces nonspecific binding of lectins and lowers the pI of the molecule to near neutral, thus reducing nonspecific binding of charged molecules such as nucleic acids.

[0079] Streptavidin is another tetrameric biotin-binding protein originally isolated from the bacterium Streptomyces avidinii. Although streptavidin has relatively low primary sequence identity with avidin, the two molecules share nearly identical secondary and tertiary structures. Consequently, streptavidin also has a high binding interaction with biotin, similar to avidin. However, streptavidin has a better binding affinity for biotin-conjugated molecules than avidin, and, like neutral avidin, has a neutral charge.

[0080] In one embodiment of this first aspect, the biotin-binding molecule comprises a sequence having at least 80% sequence identity, at least 90% sequence identity, or 100% sequence identity to SEQ ID NO: 3, the amino acid sequence of the core streptavidin molecule (residues 37-159) of Streptomyces avidinii, i.e., AEAG ITGTWYNQLG STFIVTAGAD GALTGT YESA VGNAESRYVL TGRYDSAPAT DGSGTALGWT VAWK NNYRNA HSATTWSGQY VGGAEARINT QWLLTSGTTE ANAWKSTLVG HDTFTKVKP. In one embodiment of this first aspect, the biotin-binding molecule comprises a sequence having at least 80% sequence identity, at least 90% sequence identity, or 100% sequence identity to SEQ ID NO:4, the amino acid sequence of the full-length streptavidin molecule (residues 1-183) of Streptomyces avidinii, i.e., MRKIVVAAIAVSLTTVSITASASADPSKDSKAQVSAAEAGITGTWYN QLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPA TDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQ WLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDA. In one embodiment of this first aspect, the biotin-binding molecule, e.g., streptavidin, is a tetramer (i.e., in tetrameric form). In one embodiment of this first aspect, the biotin-binding molecule, e.g., streptavidin, is a tetramer (i.e., in tetrameric form) and does not aggregate. In one embodiment of this first aspect, the biotin-binding molecule, e.g., streptavidin, is a tetramer (i.e., in tetrameric form), does not aggregate, and binds to one, two, three, or four biotinylated analyte molecules. When a streptavidin tetramer binds to three or fewer biotinylated analyte molecules, the remaining biotin-binding sites in the tetramer can bind to free biotin.

[0081] In one embodiment of this first aspect, the microarray comprises a plurality of binding sites for biotin (i.e., via biotin-binding molecules such as streptavidin), where at least a portion of the biotin-binding sites do not bind to biotinylated analytes, and those that do not bind to biotinylated analytes bind to free biotin. This can be achieved by treating the surface of the array with a solution containing free biotin after immobilization of the biotinylated analytes. This significantly reduces (approximately 60-fold) background, nonspecific binding of test molecules applied to the surface (the applied test molecules are molecules applied to the array to determine whether they can bind to the immobilized analytes). This is unexpected and highly useful because it significantly increases signal-to-noise. In one embodiment of this first aspect, substantially all of the biotin-binding sites on the microarray that do not bind to biotinylated analytes bind to free biotin, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the biotin-binding sites that do not bind to biotinylated analytes bind to free biotin. Free biotin refers to biotin that does not bind to an analyte, e.g., a naked biotin molecule. In one embodiment of this first aspect, the biotin-binding molecule is streptavidin. Streptavidin is a tetramer and therefore contains four binding sites for biotin. Thus, in one embodiment of this first aspect (when the biotin-binding molecule is streptavidin), in substantially all of the streptavidin tetramers present on the array surface, all four biotin-binding sites in the streptavidin tetramer bind to either biotinylated analytes or free biotin. In this context, "substantially all" means at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the streptavidin tetramers present on the surface of the array, and all four biotin binding sites in the streptavidin molecules bind either biotinylated analyte or free biotin.For example, one binding site may bind to a biotinylated analyte and the remaining three binding sites may bind to free biotin. In another example, two binding sites may bind to a biotinylated analyte and the remaining two binding sites may bind to free biotin.

[0082] The very high affinity of the streptavidin-biotin interaction (Kd approx. 10-14 to 10 -15 The high M and specificity of the biotinylated recombinant proteins eliminate the need for laborious pre-purification of each expressed biotinylated recombinant protein prior to array fabrication. Therefore, array fabrication is simplified because crude lysates containing expressed recombinantly biotinylated proteins are printed onto streptavidin-coated slides, which have low nonspecific protein binding capacity. The printed microarrays can then be washed to remove non-biotinylated proteins from the array surface.

[0083] The high affinity of the streptavidin-biotin interaction allows rapid saturation of available biotin-binding sites on the surface, meaning that rough normalization of protein loading can be achieved without prior adjustment of crude lysate concentration to compensate for differences in the individual expression levels of different recombinant proteins.

[0084] In one embodiment of this first aspect, the surface density or surface coverage is calculated using microscopy.

[0085] In one embodiment of this first aspect, the surface density or surface coverage is calculated using a microscopy technique selected from the group consisting of atomic force microscopy (AFM), electron microscopy (EM), e.g., scanning electron microscopy (SEM), and super-resolution microscopy. In one embodiment of this first aspect, the surface density or surface coverage is calculated using AFM. In one embodiment of this first aspect, the surface density or surface coverage is calculated using AFM when contact mode is used with a 2 μm thick AFM pin. In one embodiment of this first aspect, the surface density or surface coverage is calculated using AFM when contact mode is used with a 2 μm thick AFM pin, and the tip scan is 50 μm. 2 or 1 μm 2 In one embodiment of this first aspect, the surface density or surface coverage is calculated using SEM. In one embodiment of this first aspect, the surface density or surface coverage is calculated using SEM, where the sample to be analyzed is coated with a layer of carbon (e.g., 5 nm thick, e.g., using a Quorum Q150VEplus combined carbon / sputter coater) and then scanned using, e.g., a Tescan MIRA3 electron microscope at an appropriate magnification (e.g., 100,000x) and an appropriate voltage (e.g., 10 kV).

[0086] Electron microscopy refers to microscopy techniques that use electrons as radiation. Transmission electron microscopy (TEM) is very similar in configuration to traditional light microscopy, using lenses to focus a radiation beam onto a slide prepared with a slice of the sample; however, in this case, the radiation used is electrons rather than visible light. TEM has an advantage over traditional light microscopy because the wavelength of electrons is several orders of magnitude smaller than that of visible light, resulting in a much higher degree of resolution, typically less than one nanometer compared to the theoretical limit of 200 nm imposed by light microscopy. One major difference in sample preparation for TEM is the need to stain the sample with heavy metals to create contrast within the image. Scanning electron microscopy (SEM) is also related, but instead of passing an electron beam through a slice of the sample, the beam is reflected off the sample's surface and can be used to create a three-dimensional image of the sample. The resolution achievable with SEM is typically around 50 nm. A major consideration with both EM techniques is that the system requires imaging to be performed in a vacuum to avoid obstructing the electron path.

[0087] Atomic force microscopy (AFM) is a type of scanning microscopy. See Microsc.Res.Tech.2017;80:75-84 (DOI 10.1002 / jemt.22776). In the most typical configuration, a laser is shone onto the cantilever while it is scanned across the surface of a sample. As the cantilever moves across the sample's surface, its small tip contacts the surface, causing the cantilever to bend in response to its interaction with the sample. Changes in the laser light reflected from the cantilever and measured can be used to construct an image of the sample. A distinct advantage of this method over other high-resolution microscopy techniques is that AFM does not require the use of lenses, vacuums, or sample staining methods to be effective at typical image resolutions of less than one nanometer.

[0088] Super-resolution microscopy refers to optical microscopy techniques such as structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM), and photoactivated localization microscopy (PALM), which allow for the recording of images at resolutions higher than the diffraction limit of conventional optical microscopes. Super-resolution microscopy typically has a resolution of 10-100 nm. The advantage of this method is that it is compatible with the use of multiple fluorescent labeling reagents, such as antibodies, allowing for the simultaneous identification of specific protein components in a sample (e.g., within a cell or on a surface).

[0089] In one embodiment of this first aspect, the reactive groups on the surface of the microarray are selected from the group consisting of carboxylic acid groups, activated carboxylic acid groups, amine groups, imidoester groups, maleimide groups, haloacetyl groups, pyridyldithiol groups, azide groups, hydrazide groups, alkoxyamine groups, thiol groups, arylazide groups, and diazirine groups.

[0090] In one embodiment of this first aspect, the reactive groups on the surface are selected from the group consisting of carboxylic acid groups, activated carboxylic acid groups, amine groups, and maleimide groups.

[0091] In one embodiment of this first aspect, the reactive groups on the surface are activated carboxylic acid groups, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-activated carboxylic acid groups, or N-hydroxysuccinimide (NHS)-activated carboxylic acid groups.

[0092] In one embodiment of this first aspect, a portion of the reactive groups on the surface are non-reactive, e.g., about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, or about 90% of the reactive groups are non-reactive. Non-reactive reactive groups on the surface may be physically inaccessible or may become inactivated over time (e.g., reactive groups such as NHS esters may spontaneously hydrolyze). Alternatively, reactive groups on the surface may be actively inactivated, e.g., via specific chemical treatments. In one embodiment of this first aspect, the microarray is a 2D solid slide (e.g., Nexterion H slide) containing reactive groups that has passed the manufacturer's recommended use date. In one embodiment of this first aspect, the microarray is a 2D solid slide (e.g., Nexterion H slide) containing reactive groups that has been stored, e.g., at -20°C, for at least 3 months, at least 6 months, at least 1 year, at least 18 months, or at least 2 years before use. Such slides have a low proportion of reactive groups (eg, NHS esters) that cannot react due to spontaneous hydrolysis of the reactive groups over time.

[0093] In one embodiment of this first aspect, the reactive groups are attached to the surface of the microarray via a hydrophilic organic polymer.

[0094] In one embodiment of this first aspect, the hydrophilic organic polymer is selected from the group consisting of polyacrylamide, polyurethane, polyethyleneimine, and polyethylene glycol, preferably polyethylene glycol.

[0095] In one embodiment of this first aspect, the hydrophilic organic polymer is polyethylene glycol having an average molecular weight ranging from 500 to 20,000, e.g., 500 to 15,000, 500 to 10,000, 500 to 7,500, 500 to 5,000, or 1,000 to 5,000. Specific PEGs include PEG1000, PEG2000, PEG3000, PEG3500, and PEG5000. The PEG itself blocks nonspecific adsorption sites on the glass slide, thereby eliminating the need for additional proteinaceous surface blocking agents that may themselves alter the behavior of surface-bound analytes or cause nonspecific binding.

[0096] In one embodiment of this first aspect, the immobilized analytes or linking moieties on the surface are spaced apart by at least 10, at least 20, at least 50, at least 75, at least 100, at least 180, at least 200, at least 250, or at least 500 nm, e.g., as measured by AFM or SEM. In one embodiment of this first aspect, the immobilized analytes or linking moieties on the surface are spaced apart by at least 50, or at least 100, or at least 180 nm, e.g., as measured by AFM or SEM. In one embodiment of this first aspect, the immobilized analytes or linking moieties on the surface are spaced apart by at least 50, at least 100, at least 150, or at least 200 nm, e.g., 50-500, 50-250, 50-200, 50-150, or 50-100 nm, e.g., When performing AFM measurements according to this embodiment, optimal empirically determined recommendations and parameters should be followed for factors such as calibration, vibration amplitude, cantilever / probe tip properties, cantilever stability, and image processing pipeline.

[0097] In a second aspect, the present invention relates to a method for producing a microarray, the method comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached, wherein no more than about 20% of the reactive groups are capable of reacting at any one time; (ii) optionally contacting the reactive groups with a linking moiety under conditions under which the linking moiety reacts with the reactive group or groups to which it is capable of reacting, thereby binding the linking moiety to the surface; and (iii) depositing a sample of analyte on the surface in at least one individual predetermined region of the surface such that the analyte is immobilized on the surface via direct binding to the reactive group or groups or via indirect binding to the reactive groups via the linking moiety, wherein accordingly, the surface density of bound analyte in the at least one individual predetermined region of the surface is less than about 20%.

[0098] Also in this second aspect, the present invention relates to a method for producing a microarray, the method comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached, wherein no more than about 20% of the reactive groups are reactive at any one time; (ii) optionally contacting the reactive groups with a linking moiety under conditions under which the linking moiety reacts with the reactive group or groups to which it is capable of reacting, thereby binding the linking moiety to the surface; and (iii) depositing a sample of analyte on the surface in at least one discrete predetermined region of the surface such that the analyte is immobilized on the surface via direct binding to the reactive group or groups or via indirect binding to the reactive groups via the linking moiety, wherein accordingly, the surface coverage of bound analyte in the at least one predetermined region of the surface is less than about 20%.

[0099] In one embodiment of this second aspect of the invention, the microarray is a low density microarray.

[0100] In all embodiments of the present invention, e.g., in the context of this second aspect of the present invention, conditions that affect the ability of a linking moiety to react with one or more reactive groups, or conditions that affect the ability of an analyte to react directly with one or more reactive groups, can be varied to vary the surface density / surface coverage of the linking moiety and / or analyte on the surface of the microarray, thereby achieving the low surface density / coverage of the present invention. Such conditions include: i) the concentration of the solution of the linking moiety (e.g., streptavidin) or the concentration of the analyte applied to the reactive group; ii) the length of time that the reactive group is incubated with the analyte or with a solution of the linking moiety (e.g., streptavidin); iii) the temperature at which the reactive group is incubated with the analyte or with a solution of the linking moiety (e.g., streptavidin); iv) the pH at which the reactive group is incubated with the analyte or with a solution of the linking moiety (e.g., streptavidin); v) the immobilization of the analyte or the linking moiety (e.g., streptavidin); These include: conditions under which the microarray slide was stored / equilibrated prior to exposure to streptavidin (which may result in passive hydrolysis of some of the reactive groups (e.g., NHS esters) over time); vi) co-incubation of the linking moiety (e.g., streptavidin) or analyte with a defined molar ratio of a competitor molecule that can compete for reaction with the reactive groups (but the reactive groups themselves are not linking moieties), such as bovine serum albumin, powdered milk, casein hydrolysate, ethanolamine, glycine, chemically reactive nucleic acid polymers, or nucleic acid sugars. The appropriate combination of conditions can be selected to allow binding of a particular analyte or linking moiety (e.g., streptavidin) to the reactive groups at the appropriate surface density / coverage.Typical conditions include: i) a concentration of a linking moiety (e.g., streptavidin) of 0.01 to 0.9 mg / ml, or 0.1 to 10 mg / ml, e.g., 0.05 to 0.5 mg / ml, 0.05 to 0.3 mg / ml, 0.05 to 0.2 mg / ml, 0.01 to 0.2 mg / ml, about 0.08 mg / ml, about 0.1 mg / ml, or about 0.25 mg / ml, 0.2 to 5 mg / ml, 0.5 to 2 mg / ml, 0.8 to 1.2 mg / ml, or about 1 mg / ml; ii) an incubation time of 1 minute to 24 hours, e.g., 10 minutes to 12 hours, 15 minutes to 5 hours, 20 minutes to 2 hours, 30 minutes to 1.5 hours, or about 10 minutes, 20 minutes, 30 minutes, or 1 hour. These may include: iii) incubation time, 4°C to 50°C, e.g., 4°C to 15°C, 10°C to 40°C, 15°C to 30°C, or about 4°C or about 20°C (room temperature); iv) incubation pH, e.g., 6.5 to 9.5, 7 to 9, 8 to 9, or about 8.5; and iv) using the microarray slide according to the manufacturer's instructions and observing the recommended use date, or past the recommended use date, or storing / equilibrating the microarray slide, e.g., at -20°C, for at least 3 months, at least 6 months, at least 1 year, at least 18 months, or 2 years before use. Furthermore, prior to analyte binding, an activation chemical step may also be performed to hydrolyze or protect reactive groups (e.g., NHS esters). Such activation chemical steps are well known to those skilled in the art.

[0101] In one embodiment of this second aspect of the invention, step (ii) is not optional. In one embodiment of this second aspect of the invention, step (ii) is not optional, the linking moiety is a biotin-binding protein as described elsewhere herein, and the analyte is biotinylated. In one embodiment of this second aspect of the invention, step (ii) is not optional, the linking moiety is a biotin-binding protein as described elsewhere herein, the analyte is biotinylated, and the method comprises a further step (iv) following step (iii), in which a solution of biotin (i.e., free biotin that is not bound to the analyte) is applied to the surface of the microarray, such that biotin-binding sites on the linking moiety that are not bound to the biotinylated analyte are occupied by biotin.

[0102] Thus, in an embodiment of this second aspect of the present invention, there is provided a method of manufacturing a microarray, the method comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached, wherein no more than about 20% of the reactive groups are reactive at any one time; (ii) contacting the reactive groups with a linking moiety under conditions whereby the linking moiety reacts with one or more of the reactive groups to which it is reactive, thereby binding the linking moiety to the surface; (iii) depositing a sample of biotinylated analytes onto the surface in at least one discrete predetermined region of the surface such that the analytes are immobilized on the surface via indirect binding to the reactive groups via the linking moiety, wherein accordingly, the surface density of bound analytes in the at least one predetermined region of the surface is less than about 20%; and thereafter (iv) applying a solution of biotin to the surface of the microarray.

[0103] In a further embodiment of this second aspect of the invention, there is provided a method of manufacturing a microarray, comprising the steps of: (i) providing a surface having attached thereto a plurality of reactive groups, wherein no more than about 20% of the reactive groups are capable of reacting at any one time; (ii) contacting the reactive groups with a linking moiety that is a biotin-binding molecule selected from the group consisting of: (a) an anti-biotin antibody, (b) avidin, (c) neutravidin, (d) streptavidin, and (e) a fragment, variant, or analog of streptavidin, avidin, neutravidin, or the anti-biotin antibody that retains biotin-binding ability, wherein the contacting is performed under conditions in which the linking moiety reacts with the reactive group or groups, thereby binding the linking moiety to the surface; (iii) contacting the microarray with a sample of biotinylated polypeptides, wherein biotin is bound to the polypeptides via a biotin carboxyl carrier protein (BCCP) motif or an Avi tag (SEQ ID NO: 1), such that the polypeptides are immobilized on the surface via indirect binding to a reactive group via the linking moiety, and accordingly, the surface density of bound polypeptides within a given area of ​​the surface is less than 20%; and thereafter, (iv) Applying a solution of biotin to the surface of the microarray.

[0104] In one embodiment of this second aspect of the invention, there is provided a method of manufacturing a microarray, the method comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached, wherein only about 20% or less of the reactive groups are reactive at any one time; (ii) contacting the reactive groups with a linking moiety under conditions under which the linking moiety reacts with one or more of the reactive groups to which it is reactive, thereby binding the linking moiety to the surface; (iii) depositing a sample of biotinylated analyte on the surface in at least one discrete predetermined region of the surface such that the analyte is immobilized on the surface via indirect binding to the reactive groups via the linking moiety, wherein accordingly, surface coverage of bound analyte in at least one discrete predetermined region of the surface is less than 20%; and thereafter (iv) applying a solution of biotin to the surface of the microarray.

[0105] In a further embodiment of this second aspect of the invention, there is provided a method of manufacturing a microarray, comprising the steps of: (i) providing a surface having attached thereto a plurality of reactive groups, wherein no more than about 20% of the reactive groups are capable of reacting at any one time; (ii) contacting the reactive groups with a linking moiety that is a biotin-binding molecule selected from the group consisting of: (a) an anti-biotin antibody, (b) avidin, (c) neutravidin, (d) streptavidin, and (e) a fragment, variant, or analog of streptavidin, avidin, neutravidin, or the anti-biotin antibody that retains biotin-binding ability, wherein the contacting is performed under conditions in which the linking moiety reacts with the reactive group or groups, thereby binding the linking moiety to the surface; (iii) depositing a sample of biotinylated polypeptides onto the surface within at least one respective predetermined region of the surface, wherein biotin is bound to the polypeptide via a biotin carboxyl carrier protein (BCCP) motif or an Avi tag (SEQ ID NO: 1), such that the polypeptide is immobilized on the surface via indirect binding to a reactive group via the linking moiety, and accordingly, the surface coverage of bound polypeptides within the at least one predetermined region of the surface is less than about 20%; and thereafter (iv) Applying a solution of biotin to the surface of the microarray.

[0106] In additional embodiments of this second aspect of the invention, the reactive groups are attached to the surface via a hydrophilic organic polymer, e.g., polyethylene glycol, which may be selected from the group consisting of polyacrylamide, polyurethane, polyethyleneimine, and polyethylene glycol.

[0107] In the preceding embodiments referring to a solution of biotin being applied to the surface of the microarray, the solution of biotin is applied to the surface at a concentration of 10-100 μM, for example, 25-75 μM, for example, 25-75 μM, for example, about 50 μM.

[0108] In a third aspect, the present invention relates to a method for reducing the density of analytes bound to the surface of a microarray to form a low-density (or low surface coverage) microarray, the method comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached; (ii) inactivating or rendering inaccessible a portion of the reactive groups such that no more than about 20% of the reactive groups are available to react at any one time; (iii) optionally contacting the reactive groups with a linking moiety under conditions that allow one or more reactive groups that retain their reactivity to react with the linking moiety, thereby binding the linking moiety to the surface; and (iv) contacting the microarray with a sample of analytes such that analytes are immobilized on the surface via direct binding to one or more reactive groups or via indirect binding to one or more reactive groups via the linking moiety, wherein the surface density (surface coverage) of bound analytes within a given area of ​​the surface is accordingly less than about 20%.

[0109] In a fourth aspect, the present invention provides a method comprising: i) increasing the analyte signal to background noise ratio of the microarray; ii) increasing the rotational and conformational freedom of the analytes immobilized on the microarray; and / or iii) increasing the rate of physiologically relevant interactions between analytes immobilized on a microarray and test molecules applied to said microarray; wherein the method comprises providing a microarray comprising a surface having a plurality of reactive groups attached thereto, wherein analytes are immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface density (or surface coverage) of immobilized analytes within a given area of ​​the surface is less than about 20%.

[0110] In one embodiment of this fourth aspect of the invention, the microarray is a low density microarray.In one embodiment of this fourth aspect of the invention, the microarray is a low coverage microarray.

[0111] In one embodiment of this fourth aspect of the invention, the increased signal-to-noise ratio achieved corresponds to (i) an increase in physiologically relevant signal and (ii) a decrease in nonspecific (i.e., physiologically irrelevant) background. Without being bound by theory, it is believed that the low surface density (or low surface coverage) of analytes minimizes nonspecific aggregation of immobilized analytes, thereby reducing nonspecific background binding. Such aggregation promotes nonspecific binding of test molecules (applied to the microarray to determine whether they specifically bind to the immobilized analytes) to these aggregates. Nonspecific background binding is also reduced to non-aggregated analytes by reducing molecular crowding effects (which promote nonspecific interactions). Furthermore, it is believed that the increased spacing between immobilized antigen molecules, combined with the increased rotational and conformational freedom of the immobilized analyte molecules, increases the rate of physiologically relevant incorporation with test molecules (applied to the microarray to determine whether they specifically bind to the immobilized analytes), thereby increasing specific binding.

[0112] Features of embodiments of the first aspect of the invention (and, where appropriate, feature definitions discussed in the context of the first aspect of the invention) may be combined with or applied to features of embodiments of the second, third, or fourth aspects of the invention. Thus, for example, microarray features described in the context of the first aspect of the invention may be equally applied to method embodiments of the second, third, or fourth aspects of the invention.

[0113] The present invention further provides a microarray preparable by an embodiment of the method of the second, third or fourth aspect of the invention.

[0114] In a fifth aspect, the present invention relates to the use of a surface of a microarray as a low density (or low coverage) surface, where the surface density (or surface coverage) of immobilized analytes within a given area of ​​said surface is less than 20%, wherein the analytes are optionally immobilized to said microarray via a linking moiety.

[0115] In a sixth aspect, the present invention relates to the use of a surface for the manufacture of a low density (or low coverage) microarray comprising immobilized analytes, wherein the surface density (or surface coverage) of immobilized analytes within a given area of ​​said surface is less than 20%, and the analytes are optionally immobilized to said microarray via a linking moiety.

[0116] In a seventh aspect, the present invention relates to the use of reactive surface groups suitable for forming low density (or low coverage) microarrays to reduce the density of analytes immobilized on said surface, wherein some of said reactive groups are incapable of reacting with said analytes, and said analytes are optionally immobilized on said surface via a linking moiety, such that the surface density (or surface coverage) of analytes immobilized on a given area of ​​said surface is less than 20%.

[0117] In an eighth aspect, the present invention relates to the use of reactive groups on a surface suitable for forming a low-density (or low-coverage) microarray for the manufacture of a low-density (or low-coverage) protein microarray, wherein some of the reactive groups on the surface are incapable of reacting with an analyte, and the analyte is optionally immobilized to the surface via a linking moiety, such that the surface density (or surface coverage) of the analyte immobilized in a given area of ​​the surface is less than 20%.

[0118] Features of embodiments of the first aspect of the invention (and, where appropriate, feature definitions discussed in the context of the first aspect of the invention) may be combined with or applied to features of embodiments of the fifth, sixth, seventh, or eighth aspects of the invention. Thus, for example, microarray features described in the context of the first aspect of the invention may be equally applied to use embodiments of the fifth, sixth, seventh, or eighth aspects of the invention.

[0119] In a ninth aspect, the present invention relates to the use of a microarray as defined in the first aspect for: Identifying interactions between the analyte and a test molecule applied to the analyte; determining the antibody profile of the subject; Identifying a biomolecule that specifically binds to the immobilized analyte; Identification of an antibody that specifically binds to the immobilized analyte and is suitable for diagnosing or treating disease; or Identification of a biomolecule that specifically binds to the immobilized analyte and is capable of treating a disease mediated by the immobilized analyte.

[0120] In a tenth aspect of the present invention, there is provided a method of manufacturing a microarray, the method comprising: The method includes the steps of (i) providing a surface to which a plurality of reactive groups are attached; (ii) contacting the reactive groups with a linking moiety comprising a biotin-binding molecule under conditions in which the linking moiety reacts with one or more of the reactive groups to which it is capable of reacting, thereby binding the linking moiety to the surface; (iii) contacting the microarray with a sample of biotinylated analyte such that the analyte is immobilized on the surface via indirect binding to the reactive groups via the linking moiety; and then (iv) applying a solution of biotin to the surface of the microarray.

[0121] In an eleventh aspect of the present invention, there is provided a method for increasing the analyte signal-to-background noise ratio of a microarray, the method comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached, wherein no more than about 20% of the reactive groups are reactive at any one time; (ii) contacting the reactive groups with a linking moiety that is a biotin-binding moiety under conditions in which the linking moiety reacts with one or more of the reactive groups to thereby bind the linking moiety to the surface; (iii) depositing a sample of biotinylated analytes onto the surface in at least one discrete predetermined region of the surface such that the analytes are immobilized on the surface via indirect binding to the reactive groups via the linking moiety, wherein accordingly, the surface coverage (or surface density) of bound analytes in the at least one discrete predetermined region of the surface is less than about 20%; and thereafter (iv) applying a solution of biotin to the surface of the microarray.

[0122] In a further embodiment of this eleventh aspect of the invention, there is provided a method of increasing the analyte signal to background noise ratio of a microarray, comprising the steps of: (i) providing a surface having attached thereto a plurality of reactive groups, wherein no more than about 20% of the reactive groups are capable of reacting at any one time; (ii) contacting the reactive groups with a linking moiety that is a biotin-binding molecule selected from the group consisting of: (a) an anti-biotin antibody, (b) avidin, (c) neutravidin, (d) streptavidin, and (e) a fragment, variant, or analog of streptavidin, avidin, neutravidin, or the anti-biotin antibody that retains biotin-binding ability, wherein the contacting is performed under conditions in which the linking moiety reacts with the reactive group or groups, thereby binding the linking moiety to the surface; (iii) depositing a sample of biotinylated polypeptides onto the surface within at least one predetermined region of the surface, wherein biotin is attached to the polypeptide via a biotin carboxyl carrier protein (BCCP) motif, or an Avi tag (SEQ ID NO: 1), such that the polypeptide is immobilized on the surface via indirect binding to a reactive group via the linking moiety, and accordingly, the surface coverage (or surface density) of the immobilized polypeptides within at least one respective predetermined region of the surface is less than about 20%; and thereafter, (iv) Applying a solution of biotin to the surface of the microarray.

[0123] In a further embodiment of this eleventh aspect of the invention, the reactive group is attached to the surface via a hydrophilic organic polymer, e.g., polyethylene glycol, which may be selected from the group consisting of polyacrylamide, polyurethane, polyethyleneimine, and polyethylene glycol.

[0124] In embodiments of the invention where a solution of biotin is applied to the surface of a microarray, the solution of biotin is applied to the surface at a concentration of 10-100 μM, for example, 25-75 μM, for example, 25-75 μM, for example, about 50 μM.

[0125] Features of embodiments of the first aspect of the invention (and, where appropriate, feature definitions discussed in the context of the first aspect of the invention) may be combined with or applied to features of embodiments of the tenth, eleventh, twelfth or thirteenth aspects of the invention. Thus, for example, microarray features described in the context of the first aspect of the invention may be equally applied to method embodiments of the tenth, eleventh or thirteenth aspects of the invention, or to microarray embodiments of the twelfth aspect of the invention.

[0126] In a twelfth aspect of the present invention, there is provided a microarray comprising a surface having a plurality of reactive groups attached thereto, wherein analytes are immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the number density of the analytes within at least one individual defined area of ​​the surface is less than about 300 per square micrometer.

[0127] In one embodiment of this twelfth aspect, the number density of immobilized analyte molecules, optionally when analyzed by AFM or optionally when analyzed by SEM, is less than about 275 per square micrometer, e.g., less than about 250 per square micrometer, less than about 225 per square micrometer, less than about 200 per square micrometer, less than about 175 per square micrometer, less than about 150 per square micrometer, less than about 125 per square micrometer, less than about 100 per square micrometer, less than about 75 per square micrometer, less than about 50 per square micrometer, less than about 25 per square micrometer, less than about 20 per square micrometer, less than about 15 per square micrometer, less than about 10 per square micrometer, or less than about 5 per square micrometer. Number density refers to the number of analyte molecules immobilized on the surface per unit area.

[0128] In a thirteenth aspect of the present invention, there is provided a method of manufacturing a microarray, the method comprising: The method includes the steps of: (i) providing a surface to which a plurality of reactive groups are attached; and (ii) contacting the reactive groups with a solution of linking moieties, wherein the concentration of the linking moieties in the solution is less than 1 mg / ml, and the linking moieties react with one or more of the reactive groups to thereby attach the linking moieties to the surface. In this context, binding can refer to covalent binding. Alternatively, binding can refer to non-covalent binding, such as the interaction between an antibody and an antigen or a ligand and a receptor.

[0129] In one embodiment of this thirteenth aspect of the invention, the method comprises the further step (iii) of depositing a sample of the analyte on the surface such that the analyte is immobilized on the surface via indirect binding to one or more reactive groups via the linking moiety. In one embodiment of this thirteenth aspect of the invention, the concentration of the linking moiety, e.g., streptavidin, in the solution is 0.8 mg / ml or less, 0.6 mg / ml or less, 0.5 mg / ml or less, 0.4 mg / ml or less, 0.3 mg / ml or less, 0.25 mg / ml or less, 0.24 mg / ml or less, 0.2 mg / ml or less, 0.15 mg / ml or less, 0.1 mg / ml or less, 0.05 mg / ml or less, or 0.01 mg / ml or less. In one embodiment of this thirteenth aspect of the invention, the concentration of the linking moiety, e.g., streptavidin, in the solution is about 0.5 mg / ml, about 0.25 mg / ml, about 0.125 mg / ml, about 0.1 mg / ml, about 0.08 mg / ml, about 0.06 mg / ml, about 0.03 mg / ml, or about 0.01 mg / ml. In one embodiment of this thirteenth aspect of the invention, the linking moiety is a biotin-binding molecule. In one embodiment of this thirteenth aspect of the invention, the linking moiety is a biotin-binding molecule and the analyte is biotinylated. In one embodiment of this thirteenth aspect of the invention, the biotin-binding molecule is a protein. In one embodiment of this thirteenth aspect of the invention, the biotin-binding molecule is selected from the group consisting of (i) an anti-biotin antibody, (ii) avidin, (iii) neutravidin, (iv) streptavidin, or (iii) a fragment, variant, or analog of streptavidin, avidin, neutravidin, or the anti-biotin antibody, which retains biotin-binding ability. In one embodiment of this thirteenth aspect of the invention, the biotin-binding molecule comprises a sequence having at least 80% sequence identity, preferably at least 90% sequence identity, and more preferably 100% sequence identity, to SEQ ID NO: 3. In one embodiment of this thirteenth aspect of the invention, the biotin-binding molecule, e.g., streptavidin, is a tetramer (i.e., in tetrameric form) and is not aggregated.

[0130] In one embodiment of this thirteenth aspect of the invention, the analyte comprises a tag that allows for immobilization of the analyte to the surface. In one embodiment of this thirteenth aspect of the invention, the analyte comprises one or more of a polypeptide, a nucleic acid, a lipid, and a carbohydrate. In one embodiment of this thirteenth aspect of the invention, the analyte comprises one or more of a polypeptide and a nucleic acid. In one embodiment of this thirteenth aspect of the invention, the analyte is a polypeptide, e.g., a glycoprotein. In one embodiment of this thirteenth aspect of the invention, the analyte is a polypeptide. In one embodiment of this thirteenth aspect of the invention, the analyte is a polypeptide, and the tag that allows for immobilization of the polypeptide to the surface is fused to the N-terminus or C-terminus of the polypeptide. In one embodiment of this thirteenth aspect of the invention, the polypeptide is properly folded. In one embodiment of this thirteenth aspect of the invention, the analyte is biotinylated. In one embodiment of this thirteenth aspect of the invention, the biotinylated analyte is chemically biotinylated or enzymatically biotinylated. In one embodiment of this thirteenth aspect of the invention, the tag enabling immobilization of the analyte to the surface comprises a biotin carboxyl carrier protein (BCCP) motif or Avi tag (SEQ ID NO: 1). In one embodiment of this thirteenth aspect of the invention, the BCCP motif has at least 80% sequence identity, preferably at least 90% sequence identity, and more preferably 100% sequence identity, with SEQ ID NO: 2. In one embodiment of this thirteenth aspect of the invention, the BCCP motif is properly folded. In one embodiment of this thirteenth aspect of the invention, biotin is attached to (i) a biotin-binding domain within the BCCP motif or (ii) the Avi tag. In one embodiment of this thirteenth aspect of the invention, biotin is attached to the biotin-binding domain or the Avi tag via enzymatic biotinylation, preferably via a biotin ligase.

[0131] In one embodiment of this thirteenth aspect of the invention, the analyte is present in an amount of about 1×10 -12 Less than mol / L, e.g., about 1 × 10 -13Less than mol / L, e.g., about 1 × 10 -14 In one embodiment of this thirteenth aspect of the invention, the analyte comprises a biotin carboxyl carrier protein (BCCP) motif, or Avi tag (SEQ ID NO: 1).

[0132] In one embodiment of this thirteenth aspect of the invention, the method comprises the further step (iv) of applying a solution of a blocking agent to the surface of the microarray. In one embodiment of this thirteenth aspect of the invention, the blocking agent is selected from the group consisting of non-fat dry milk, bovine serum albumin (BSA), casein, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), gelatin, serum (e.g., fetal bovine serum), and biotin. In one embodiment of this thirteenth aspect of the invention, the blocking agent is about 1×10 -12 Less than mol / L, e.g., about 1 × 10 -13 Less than mol / L, e.g., about 1 × 10 -14 In one embodiment of this thirteenth aspect of the present invention, the blocking agent is biotin. In one embodiment of this thirteenth aspect of the present invention, the concentration of the blocking agent, e.g., biotin, in the solution to be applied is 1 mM to 100 mM, for example, 10 mM to 80 mM, for example, 20 mM to 70 mM, for example, 40 mM to 60 mM, for example, about 50 mM.

[0133] In one embodiment of this thirteenth aspect of the invention, the biotin-binding molecule is selected from the group consisting of (i) an anti-biotin antibody, (ii) avidin, (iii) neutravidin, (iv) streptavidin, or (iii) a fragment, variant, or analog of streptavidin, avidin, neutravidin, or the anti-biotin antibody, which retains biotin-binding ability. In one embodiment of this thirteenth aspect of the invention, the biotin-binding molecule comprises a sequence having at least 80% sequence identity, preferably at least 90% sequence identity, and more preferably 100% sequence identity, to SEQ ID NO:3.

[0134] In one embodiment of this thirteenth aspect of the invention, the reactive groups on the surface are selected from carboxylic acid groups, activated carboxylic acid groups, amine groups, imidoester groups, maleimide groups, haloacetyl groups, pyridyldithiol groups, azide groups, hydrazide groups, alkoxyamine groups, thiol groups, arylazide groups, and diazirine groups. In one embodiment of this thirteenth aspect of the invention, the reactive groups on the surface are selected from carboxylic acid groups, activated carboxylic acid groups, amine groups, and maleimide groups. In one embodiment of this thirteenth aspect of the invention, the reactive groups on the surface are activated carboxylic acid groups, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-activated carboxylic acid groups or N-hydroxysuccinimide (NHS)-activated carboxylic acid groups. In one embodiment of this thirteenth aspect of the invention, the reactive groups are attached to the surface via a hydrophilic organic polymer. In one embodiment of this thirteenth aspect of the invention, the hydrophilic organic polymer is selected from the group consisting of polyacrylamide, polyurethane, polyethyleneimine, and polyethylene glycol, preferably polyethylene glycol, and in one embodiment of this thirteenth aspect of the invention, the polyethylene glycol has an average molecular weight in the range of 500 to 20,000.

[0135] In one embodiment of this thirteenth aspect of the invention, the solution of linking moiety, e.g., streptavidin, further comprises a competitor molecule capable of competing with the linking moiety for reaction with the reactive group. In one embodiment of this thirteenth aspect of the invention, the competitor molecule comprises a free amine group. In one embodiment of this thirteenth aspect of the invention, the competitor molecule is selected from the group consisting of amino acids and alkanolamines. In one embodiment of this thirteenth aspect of the invention, the competitor molecule is selected from the group consisting of glycine, alanine, serine, lysine, arginine, histidine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, methanolamine, and ethanolamine. In one embodiment of this thirteenth aspect of the invention, the competitor molecule is selected from the group consisting of glycine and ethanolamine. In one embodiment of this thirteenth aspect of the invention, the competitor molecule is glycine. In one embodiment of this thirteenth aspect of the invention, the competitor molecule is present in said solution of linking moiety, e.g., streptavidin, at a concentration of 1 mM to 100 mM, such as 10 mM to 80 mM, for example, 20 mM to 70 mM, such as 40 mM to 60 mM, for example, about 50 mM.

[0136] In one embodiment of this thirteenth aspect of the invention, the pH of the solution of the linking moiety, e.g., streptavidin, is about 7.5 to about 11, e.g., about 8 to about 9.5 or about 8.5 to about 9, e.g., about 8.5. In one embodiment of this thirteenth aspect of the invention, the pH of the solution of the linking moiety, e.g., streptavidin, is about 4 to about 7.5, e.g., about 4.5 to about 6, e.g., about 4.5.

[0137] In one embodiment of this thirteenth aspect of the invention, the reactive group is contacted with a solution of a linking moiety (e.g., streptavidin) for a limited period of time (also known as an "incubation time"). In one embodiment of this thirteenth aspect of the invention, the incubation time is between 1 minute and 24 hours, e.g., between 10 minutes and 12 hours, between 15 minutes and 5 hours, between 20 minutes and 2 hours, between 30 minutes and 1.5 hours, or about 10 minutes, 20 minutes, 30 minutes, or 1 hour. In one embodiment of this thirteenth aspect of the invention, the incubation time is less than 1 hour, e.g., about 45 minutes.

[0138] In one embodiment of this thirteenth aspect of the invention, the reactive group is contacted with a solution of a linking moiety (e.g., streptavidin) at a temperature (also known as the "incubation temperature"). In one embodiment of this thirteenth aspect of the invention, the incubation temperature is between 4°C and 50°C, e.g., between 4°C and 15°C, between 10°C and 40°C, between 15°C and 30°C, or about 4°C, or about 20°C (room temperature).

[0139] In one embodiment of this thirteenth aspect of the invention, the analyte sample is deposited on the surface in the form of a solution, e.g., an aqueous solution. In one embodiment of this thirteenth aspect of the invention, the analyte solution is diluted, e.g., at least 2 dilution, at least 3 dilution, at least 4 dilution, at least 5 dilution, at least 1 / 10 dilution, at least 1 / 20 dilution, at least 1 / 25 dilution, at least 1 / 50 dilution, or 1 / 100 dilution, with a suitable aqueous buffer, e.g., prior to deposition on the surface.

[0140] In one embodiment of this thirteenth aspect of the invention, the method results in a density of said linking moieties within at least one discrete predetermined area of ​​said surface of less than about 300 linking moieties per square micrometer, e.g., less than about 275 per square micrometer, less than about 250 per square micrometer, less than about 225 per square micrometer, less than about 200 per square micrometer, less than about 175 per square micrometer, less than about 150 per square micrometer, less than about 125 per square micrometer, less than about 100 per square micrometer, less than about 75 per square micrometer, less than about 50 per square micrometer, less than about 25 per square micrometer, less than about 20 per square micrometer, less than about 15 per square micrometer, less than about 10 per square micrometer, or less than about 5 per square micrometer.

[0141] In one embodiment of this thirteenth aspect of the invention, the method results in a density of the analyte within at least one discrete predetermined area of ​​the surface of less than about 300 analyte molecules per square micrometer, e.g., less than about 275 per square micrometer, less than about 250 per square micrometer, less than about 225 per square micrometer, less than about 200 per square micrometer, less than about 175 per square micrometer, less than about 150 per square micrometer, less than about 125 per square micrometer, less than about 100 per square micrometer, less than about 75 per square micrometer, less than about 50 per square micrometer, less than about 25 per square micrometer, less than about 20 per square micrometer, less than about 15 per square micrometer, less than about 10 per square micrometer, or less than about 5 analyte molecules per square micrometer.

[0142] In a fourteenth aspect of the invention, the invention provides a microarray obtainable by either the method of the tenth or thirteenth aspects of the invention.

[0143] While the present invention has been described and illustrated with respect to specific embodiments, those skilled in the art will recognize that the invention itself is susceptible to many different variations not specifically illustrated herein.

[0144] Where the foregoing description refers to integers or elements that have known, obvious, or foreseeable equivalents, such equivalents are incorporated herein as if individually set forth. Reference should be made to the claims to determine the true scope of the invention, which should be construed to encompass all such equivalents. The reader will also understand that integers or features of the invention described as preferred, advantageous, convenient, or the like are optional and do not limit the scope of the independent claims. Furthermore, it should be understood that such optional integers or features, while a possible advantage in some embodiments of the invention, may be undesirable in other embodiments and, therefore, may not be present. [Example]

[0145] Example 1: Preparation of streptavidin-coated microarray slides Microarray fabrication is performed using streptavidin-coated, long-chain polyethylene glycol (PEG)-based glass slides (Nexterion H slides (Schott, Germany)). The PEG hydrogel coating acts as a long, flexible spacer, allowing significantly higher levels of rotational and conformational freedom for immobilized analytes, such as proteins, thereby enabling quantitative measurements of biomolecular interactions with the immobilized analytes. In addition, PEG is significantly superior to proteinaceous blocking agents, such as bovine serum albumin or milk powder, in reducing nonspecific background in surface-based assays. The polymeric structure of PEG also creates an aqueous-like environment, similar to amorphous water, thus helping to retain water and maintain the folding and functionality of immobilized proteins.

[0146] The first step in preparing the microarray surface involves coating a hydrogel-PEG slide with a uniformly low-density (<10% surface coverage) streptavidin layer. To reduce coating variability (typically less than 15%), multiple hydrogel slides can be coated with streptavidin in parallel per batch. Quality control testing of coating variability between slides within a batch is performed by incubating the coated slides with biotinylated bovine serum albumin (BSA) complexed with the fluorescent dye biotin (Cy5). The coefficient of variation is then measured from data extrapolated from the fluorescence intensity across the entire surface (see Figure 1A).

[0147] To ensure uniform slide coating, Nexterion H slides were equilibrated at room temperature before being unpacked. The batch procedure began with incubation of N-hydroxysuccinimide (NHS)-activated Nexterion H slides with a 1 mg / ml streptavidin (PROSPEC, pro-283) solution in HEPES buffer (200 mM KCl, 0.02% Triton X-100, 50 mM HEPES, pH 8.5) for 1 hour at room temperature, followed by a blocking step in ethanolamine (50 mM ethanolamine in 50 mM potassium phosphate buffer, pH 8.0) for an additional hour. The slides were then washed three times in 100 mM potassium phosphate buffer (100 mM K2PO4 (94% K2HPO4, 6% KH2PO4, pH 8.0)) and once in dH2O, followed by drying by centrifugation at 2000 g for 5 minutes. The coated slides were then stored dry in a dehydrated environment at −20° C. until use for microarray fabrication.

[0148] Example 2: Expression of CYP450 proteins containing a BCCP tag and subsequent biotinylation The recombinant proteins used for array fabrication are expressed as fusions to a compactly folded, approximately 80-residue domain from E. coli called biotin carboxyl carrier protein (BCCP) (Athappilly, FK & Hendrickson, WA, Structure 3, 1407-19 (1995), and Chapman-Smith, A. & Cronan, JE, Trends Biochem. Sci. 24, 359-63 (1999)). The BCCP domain consists of amino acids 74-156 of the E. coli AccB protein and is disclosed herein as SEQ ID NO: 2. The BCCP domain is fused to the C-terminus of a CYP450 protein. A His tag is fused to the C-terminus of the BCCP domain.

[0149] Expression in insect cells The synthetic gene for the target protein was cloned into the proprietary E. coli / Spodoptera frugiperda transfer vector, pPRO8 or pRO30, such that the construct encoded the full-length target protein as an in-frame fusion to three consecutive tags: biotin carboxyl carrier protein (BCCP), c-Myc tag, and hexahistidine tag. pPRO8 was used to generate the fusion protein, with BCCP-cmyc fused to the C-terminus of the antigen. For pRO30, the tag was fused to the N-terminal domain of the antigen, with the hexahistidine tag followed by c-Myc and BCCP. If necessary, a signal peptide to direct the protein into the secretory pathway was included in the pRO30 vector between the hexahistidine tag and the c-Myc tag.

[0150] pPRO8 and pRO30 consist of a viral polyhedrin promoter and cloning site. The polh-BCCP expression cassette is flanked by the baculovirus 603 and 1629 genes, allowing for subsequent homologous recombination of the construct into a replication-deficient baculovirus genome (Blackburn, Shoko, & Beeton-Kempen, 2012).

[0151] After cotransfection of S. frugiperda Sf9 cells with the appropriate transfer vector and a linearized, replication-deficient bacmid vector (Autographa californica baculovirus vector pBAC10:KO1629), baculovirus was amplified and recombinant proteins were expressed in S. frugiperda super strain Sf9-3 (Oxford Expression Technologies, Oxford, UK).

[0152] Three milliliters of Spodoptera frugiperda Sf9 cells were cultured in Sf-900 III SFM medium (Gibco) expressing individual C-terminally BCCP-tagged recombinant human CYP450 enzymes (CYP3A4, CYP2C9, and CYP2D6). Cells were grown at 27°C for 3 days. The BCCP tag is cross-recognized by insect cell biotin ligase, allowing efficient biotinylation without the need for co-expression of E. coli biotin ligase. Clarified cell lysates were prepared in insect lysis buffer (25 mM Hepes, 50 mM KCl, 20% glycerol, 0.1% Triton X100, 1x Halt™ protease inhibitor cocktail, EDTA-free (Thermo Scientific, Waltham, MA, USA), 0.25% sodium deoxycholate, Pierce Universal Nuclease (Thermo Scientific), pH 8.5). Expression yield and in vivo biotinylation of each antigen were assessed by Western blot using a streptavidin-HRP conjugate probe (GE Healthcare, Chicago, IL, USA). Crude lysates were diluted 1:1 with 40% (w / v) sucrose in PBS and stored at -80°C until microarray printing.

[0153] 3mL of Spodoptera For recombinant expression of target proteins in E. coli, synthetic genes were cloned into either pMDOO4 or pAN101. pMDOO4 is for expression of N-terminal hexahistidine BCCP fusion proteins, while pAN101 is for expression of C-terminal BCCP hexahistidine fusion proteins. Expression is driven by the T5 promoter / lac operator element using IPTG induction or lactose / glucose autoinduction conditions. E. coli cells are lysed using bacterial lysis buffer (Bugbuster), and in vivo biotinylation is assessed as previously described. Clarified lysates are stored in 20% glycerol at -80°C until microarray printing.

[0154] Example 3: Biotinylation of peptides, nucleic acids, carbohydrates, or lipids Biotinylation of peptides Biotinylated peptides are commercially available. During Fmoc solid-phase synthesis, peptides are synthesized with a biotin moiety linked to their N-terminus. At the same time, a C6 spacer is incorporated between the biotin moiety and the peptide to avoid downstream steric hindrance of the biotin-streptavidin interaction. Alternatively, the spacer can consist of a hydrocarbon chain (e.g., via aminohexanoic acid) or polyethylene oxide. The latter has the added advantage of improved solubility.

[0155] Biotinylation of nucleic acids Biotinylated DNA is commercially available. Single-stranded (ss) DNA is synthesized with a biotin moiety at either the 5' or 3' end. A C6 spacer is inserted between the biotin moiety and the DNA sequence to avoid downstream steric hindrance of the biotin-streptavidin interaction. Alternatively, a triethylene glycol (TEG) spacer extends the oligo-DNA-biotin distance by up to 15 atoms. In the case of double-stranded DNA, the complementary strand is annealed to an already biotinylated ssDNA molecule.

[0156] Carbohydrate biotinylation (a) Commercial kits are available for biotinylating carbohydrate molecules. Oxidation of carbohydrate chains to aldehydes facilitates their biotinylation. For example, sodium metaperiodate (NaIO) oxidizes sialic acid (commonly found at the ends of glycan chains) to form aldehydes that are sensitive to biotin and spacer-linked alkoxyamines (e.g., commercially available alkoxyamine-PEG spacer-biotin).

[0157] (b) The reactive biotin-LC-hydrazine can be easily attached to the reducing end of any carbohydrate (e.g., oligosaccharide) chain without interfering with the glycan's ability to cognitively bind to its lectin partner (Grun et al., 2006, Analytical Biochemistry, 354(1), 54-63, https: / / doi.org / 10.1016 / j.ab.2006.03.055).

[0158] (c) Poly(2-methylacrylic acid) (pMAA), which has multiple carboxyl groups for multivalent carbohydrate binding, can be biotinylated to enhance surface carbohydrate capture (Liu et al., 2022, New Journal of Chemistry, 46(9), 4300-4306, https: / / doi.org / 10.1039 / D1NJ0575 8H).

[0159] (d) Biotinylated lectins (carbohydrate-binding proteins) can be immobilized on the array surface to capture specific carbohydrates (Rosenfeld et al., 2007, Journal of Biochemical and Biophysical Methods, 70(3), 415-426, https: / / doi.org / https: / / doi.org / 10.1016 / j.jbbm.2006.09.008).

[0160] 3.4 Biotinylation of lipids Lipids can be indirectly biotinylated via an intervening lipid tether (oligo(ethylene glycol)-stearyl moiety, telechelic, or DphyTL) that also serves as a spacer between the lipid and the array surface. (Girard-Egrot OfeliaTI, 2021, Applied Sciences, Vol. 11, https: / / doi.org / 10.3390 / app11114876); (Sumino et al., 2011, Biomacromolecules, 12(7), 2850-2858, https: / / doi.org / 10.1021 / bm200585y); (Lahiri, Jonas, Frutos, Kalal, & Fang, 2001, Biomedical Microdevices, 3(2), 157-164, https: / / doi.org / 10.1023 / A:1011406511454).

[0161] Example 4: Printing biotinylated CYP450 proteins onto streptavidin-coated slides Crude lysates prepared from Spodoptera frugiperda cells as described in Example 2 were printed directly onto streptavidin-coated PEG hydrogel slides as prepared in Example 1. Specifically, 50 μL aliquots of lysed insect cells expressing individual biotinylated, BCCP-tagged recombinant proteins provided sufficient source material to print 25 replica slides in a 4-plex format. Each protein was printed in triplicate on each subarray using solid pin printing (300 μm flat-tipped pins). Thus, each 3 mL recombinant insect cell culture contained sufficient expressed biotinylated, BCCP-tagged protein to fabricate at least 700 replica subarrays of each protein, or over 2000 replica spots. The printed slides were washed with phosphate-buffered saline (PBS) (pH 7.5; 0.2% Tween-20) to remove unbound proteins, dried by centrifugation at 2000 g, and maintained under a gentle airflow for 5 minutes to completely dry. The slide was secured to the piezoelectric stage using double-sided tape and confirmed to be flat using a spirit level. An example of the uniformity of spots achievable with the described method is shown in Figure 1B, which demonstrates fluorescence from Cy5-biotin BSA spots. Figure 2 shows confirmation of His-tagged CYP450 binding to the slide using an anti-6xHis probe.

[0162] Example 5: Use of free biotin to block unoccupied biotin binding sites on streptavidin tetramers After binding of biotinylated BCCP-tagged proteins to the streptavidin-coated surface, the slides were treated for 30 minutes with a solution of 50 μM free biotin in 25 mM HEPES, pH 7.6, 50 mM KCl, 0.1% Triton X100, 20% glycerol, and 1 mM DTT. Without being bound by theory, we believe that biotin binds cooperatively to streptavidin tetramers, with each successive binding event to a subunit of the tetramer inducing a conformational change in the other subunits, thereby increasing the affinity of the remaining binding sites for free biotin. We demonstrated that treatment with free biotin reduced background, nonspecific background binding to the underlying streptavidin surface by 60-fold compared to blocking the slides with BSA treatment, further improving the signal-to-noise ratio. A flowchart illustrating the experimental method for comparing blocking of slides with free biotin or BSA is shown in Figure 3.

[0163] A comparison of microarrays blocked using the two blocking techniques is shown in Figure 4. The proteins on the array were BCCP-tagged full-length or truncated SARS-CoV-2 spike protein or nucleocapsid protein antigens. Figure 4A shows the layout of the microarray, where "403" indicates the full-length SARS-CoV-2 spike ectodomain protein-BCCP-His6 fusion protein; "406" indicates the SARS-CoV-2 nucleocapsid protein core domain-BCCP-His6 fusion protein; "407" indicates the SARS-CoV-2 nucleocapsid protein C-terminal domain-BCCP-His6 fusion protein; and "408" indicates the SARS-CoV-2 nucleocapsid protein N-terminal domain-BCCP-His6 fusion protein. FIG. 4B shows array images of the biotin block array scanned at 532 and 635 nm, and FIG. 4C shows array images of the BSA block array scanned at 532 and 635 nm.

[0164] A comparison of the foreground signal intensity (635 nm) of printed spots is shown in Figure 5A (biotin block array) and Figure 5B (BSA block array). Note: The foreground signal is proportional to the concentration of antigen printed on the array surface and is expected to follow a sigmoidal curve. This is clearly observed for the biotin block array but not for the BSA block array. Note: The SARS-CoV-2 nucleocapsid protein C-terminal domain-BCCP-His6 fusion protein ("407"), printed undiluted, had a low median RFU (<10,000) due to signal aggregation at the edge of the spot, resulting in a "coffee ring" effect with high signal intensity at the edge and low signal intensity toward the center of the spot. However, this effect was reduced by diluting the lysate two-fold with lysis buffer, resulting in an increase in median RFU and was further mitigated at higher lysis dilutions, resulting in the expected linear decrease in signal with increasing dilution. Slides blocked with BSA after printing also had lower median signals in the replica spots of undiluted protein (again, showing a "coffee ring" morphology), but in contrast to blocking with biotin, the signal did not decrease linearly even at high lysis dilutions.

[0165] The coefficient of variation (CV) values ​​of the foreground signal intensity (635 nm) of the replica spots of the same antigen are shown in the table below. [Table 1] The CV of the foreground signal of the replica spots is generally lower for the biotin block array than for the BSA block array.

[0166] A comparison of the signal intensities (at 635 nm) of the surrounding background areas adjacent to the printed spots for the biotin block array (FIG. 6A) and the BSA block array (FIG. 6B) is shown in Figure 6. The background signal intensity is over 60-fold lower and more uniform for the biotin block array compared to the BSA block array (note the different y-axis scales of the graphs).

[0167] The CV values ​​of background signal intensity (635 nm) of replica spots of the same antigen are shown in the table below. [Table 2] The CV of the background signal of the replica spots is significantly lower for the biotin block array than for the BSA block array.

[0168] These data also illustrate an additional method for controlling the final density of polypeptide analytes on a surface: the amount of immobilized analyte can be controlled by varying the concentration of the analyte in the printing solution. However, the inventors unexpectedly observed that this only worked as expected when performed in conjunction with a biotin-blocking treatment. Therefore, without being bound by theory, this is believed to indicate that in the absence of a biotin-blocking treatment, a portion of the analyte in each spot binds nonspecifically to the surface. Thus, the use of a biotin-blocking step advantageously reduces or avoids nonspecific binding of BCCP-tagged analytes to the microarray surface.

[0169] Example 6: Investigation of the surface density of immobilized proteins A scanning atomic force microscope (AFM) (Easyscan 2 AFM, Nanosurf) was used in contact mode with a 2 μm thick AFM pin (Nanosensors, Silicon SPM Sensors). An area of ​​the spot containing immobilized protein was randomly selected, the tip was approached, and the tip scan was performed at 50 μm. 2 or 1 μm 2The images were analyzed in scan mode using either a 200 μm diameter microarray spot or a 200 μm diameter microarray spot. To measure the distance between the immobilized recombinant proteins, the images were analyzed using Easyscan 2 software. The results (see Figure 7) revealed that the surface coverage of the immobilized recombinant proteins within each spot was less than 10%. Further analysis using AFM indicated that there were an average of 202.67 ± 33.31 streptavidin tetramer units per square micrometer of surface. Thus, a single 200 μm diameter microarray spot contains approximately 6,000,000 streptavidin binding units, and the average distance between CYP450-bound streptavidin tetramers is 52.87 ± 3.11 nm.

[0170] Example 7: Comparison of microarrays containing analytes bound at low surface density with microarrays containing analytes bound at high surface density Low-density microarray slides prepared according to the present invention are compared to alternative microarray slides with high analyte binding densities (i.e., short intermolecular distances between individual bound analyte molecules), such as commercially available HuProt slides (CDI Laboratories, Inc., Puerto Rico, USA).

[0171] In particular, the microscopic features and signal-to-noise performance of the two slides are compared and contrasted using experimental methods disclosed elsewhere in the experimental examples of this disclosure.

[0172] In further experiments, several microarrays with various surface densities of bound analytes were generated. This was achieved by preparing slides with different levels of NHS derivatization of the PEG moiety (e.g., 20:80, 50:50, and 100:0 derivatized:underivatized). The microscopic features and signal-to-noise performance of these slides were compared as described above.

[0173] Example 8: Investigating the effect of different conditions on the binding of molecules to microarrays In this example, the effects of varying the concentration of linking moiety (e.g., streptavidin), varying the pH with a competitor molecule (e.g., a molecule containing a free amine group), or using aged slides were investigated. In particular, the effect on the density of linking moiety / analyte binding was investigated.

[0174] 1. Methods and Materials 1.1 Coating of H-slides with streptavidin All microarray incubations and washing steps were performed at room temperature (RT) at 150 revolutions per minute (RPM) protected from light.

[0175] Lyophilized streptavidin (Prospec) was equilibrated at room temperature and resuspended in slide-coating buffer (NaPO4 with 0.001% Tween-20) at 10 mg / ml, pH 8.5. Streptavidin was further diluted to 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg / ml in slide-coating buffer or slide-coating buffer containing 50 mM glycine. Streptavidin was also diluted to 1 mg / ml in slide-coating buffer at pH 9 or pH 4.5. Negative controls (not derivatized with streptavidin) included slide surfaces containing slide buffer only, slide surfaces containing slide buffer containing 50 mM glycine, or empty wells. Nexterion H slides (Schott) were equilibrated at room temperature for 1 hour. The equilibrated H slides were mounted in clean 24-plex gaskets (GraceBio). 90 μl of diluted streptavidin or control was added to each corresponding gasket well and incubated for 1 hour. The gasket wells were briefly rinsed three times with 200 μl of buffer (KPO4), and the slides were removed from the gasket and placed in a quadriperm dish containing 4 ml of wash buffer. The slides were washed for 5 minutes, and this step was repeated twice. The slides were then blocked for 1 hour with 50 mM glycine-containing slide coating buffer. The slides were washed three times with wash buffer for 5 minutes per wash, and finally with 4 ml of water for 5 minutes, after which they were dried at 1200 × g for 2 minutes at 23°C. The dried slides were placed in a slide container, closed, sealed with parafilm, and stored at -20°C until needed.

[0176] 1.2 Detection of Streptavidin on Slide Surfaces Using Cy3-Labeled Biotin-BSA. Slides derivatized with streptavidin under various conditions (see Section 1.1) and Nexterion HS slides (Schott) were equilibrated at room temperature for 1 hour and then incubated with 3 ml of 40 μg / ml Cy3-labeled biotinylated BSA (Cy3-biotin-BSA) in phosphate-buffered saline (PBS) pH 7.5 (PBS T) containing 0.2% Tween-20 for 30 minutes. Slides were washed twice with PBS T for 5 minutes per wash, followed by two washes with PBS for 5 minutes per wash. Slides were dried by centrifugation at 1200 × g for 2 minutes at 23°C and scanned using an Innoscan 710 (Innopsys, Carbonne, France) at 1% gain, 10 mW power, and a pixel size of 10 μm.

[0177] Data were extracted using MAPIX software (Innopsys) and then processed using the i-Ome IA application to filter the data. KD and Bmax values ​​were obtained using GraphPad Prism (version 10.1.1).

[0178] 1.3 Scanning electron microscopy H-slides derivatized with streptavidin under various conditions (see section 1.1) and detected with Cy3-biotin-BSA were coated with a 5 nm thick layer of carbon using a Quorum Q150VEplus combined carbon / sputter coater and then scanned by scanning electron microscopy (SEM) using a Tescan MIRA3 electron microscope at 100,000x magnification and 10 kV.

[0179] 1.4 Gene synthesis and cloning of SARS-CoV-2 nucleocapsid (N) and spike (S) protein variants The SARS-CoV-2 spike (S) gene cDNA (positions 1–1273) and the SARS-CoV-2 nucleocapsid (N) gene cDNA of MN908947.3 (Wuhan-hu-1, China) were chemically synthesized (GeneArt, ThermoFisher Scientific). For recombinant expression of properly assembled, intact S protein in vivo, the R682S mutation was included in the synthesized cDNA sequence to prevent furin-mediated S1 / S2 cleavage within the S protein. The cDNA sequences were cloned into the unique Escherichia coli / Spodoptera frugiperda transfer vector pPRO8 so that each construct encoded the S protein and the full-length N protein C-terminal domain (CTD) as in-frame fusions with the C-terminal biotin carboxyl carrier protein (BCCP), c-Myc, followed by a hexahistidine sequence. pPRO8 is a derivative of pTriEx1.1 (Sigma, St. Louis, MO, USA) and encodes the E. coli BCCP domain (amino acids 74 to 156 of the E. coli accB gene) downstream of a viral polyhedrin promoter and cloning site. The polh-BCCP expression cassette is flanked by the baculovirus 603 and 1629 genes, allowing for subsequent homologous recombination of the construct into a replication-deficient baculovirus genome (Blackburn, JM; Shoko, A. Protein Function Microarrays for Customized Systems-Oriented Proteome Analysis. In Protein Microarrays; Korf, U., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2011; Vol. 785, pp. 305-330, https: / / doi.org / 10.1007 / 978-1-61 779-286-1_21).

[0180] Cell pellets from 3 ml cultures of Spodoptera frugiperda Sf9 III cells were harvested and resuspended in 400 μl of insect cell lysis buffer (25 mM Hepes, 50 mM KCl, 20% glycerol, 0.1% Triton X-100, 0.25% NaDeoxycholate, 0.5 mM TCEP, HALT protease inhibitor cocktail (EDTA-free) (ThermoScientific), and 10 U of Pierce Universal Nuclease (ThermoScientific) for cell lysis). Cells were lysed at room temperature for 30 min with shaking at 100 rpm. The lysate was further clarified at 15,000 × g for 15 min at 4°C, aliquoted into fresh Eppendorf tubes, and stored at -80°C until use.

[0181] 1.5 Printing Undiluted S protein lysate or undiluted C-terminal domain (CTD) constructs of N protein lysate were printed in triplicate in a 24-plex format onto streptavidin-derivatized Nexterion H slides (see section 1.1) under various conditions. CTD protein lysates were also printed undiluted or diluted 1:1, 1:3, 1:7, 1:15, or 1:31 in insect cell lysis buffer and printed in triplicate in a 16-plex format onto Nexterion HS slides. The printed slides were blocked with a biotin-containing buffer (25 mM HEPES, 50 mM KCl, 20% glycerol, 0.1% Triton X100, 1 mM DTT, and 50 mM biotin) for 30 minutes. One CTD-printed slide blocked with a BSA-containing buffer (25 mM HEPES, 50 mM KCl, 20% glycerol, 0.1% Triton X100, 1 mM DTT, and 0.1% BSA) was then transferred to a pap jar containing storage buffer (12.5 mM HEPES, 25 mM KCl, 1 mM CaCl, 5 mM MgCl, 0.05% BSA, 0.05% Triton X100, and 50% glycerol) and stored at -20°C until needed.

[0182] 1.6 Detection of printed proteins using anti-c-Myc or anti-His antibodies Printed slides containing undiluted S and CTD protein lysates or serially diluted CTD protein lysates were removed from the storage buffer and washed three times with ice-cold PBS for 5 minutes per wash. Next, the S and CTD protein slides were incubated with 3 ml of Cy3-labeled anti-c-Myc antibody (Invitrogen) diluted 1:400 in PBST for 30 minutes, while slides containing diluted CTD protein were incubated with 3 ml of Alexa Fluor™ 647-labeled anti-His (Invitrogen) diluted 1:50 in PBST for 30 minutes. The slides were washed twice with PBST for 5 minutes per wash, followed by two washes with PBS for 5 minutes per wash. The slides were dried at 1200 × g at 23 °C for 2 minutes and scanned using a 532 nm laser at 70% gain, 10 mW power, and a 10 μm pixel size. Data were extracted using MAPIX software and filtered using i-Ome AI software.

[0183] 1.7 Patient Assays for Detecting Anti-S Protein Antibodies The printed slides containing S protein were removed from the storage buffer and washed three times with ice-cold PBS for 5 minutes per wash. Each slide was then incubated with a serum sample from a participant who had previously tested positive for anti-S protein antibodies (positive control) and a serum sample from the same participant who had previously tested negative for anti-S protein antibodies (negative control). The serum was incubated for 1 hour, after which the slides were washed with PBST for 5 minutes per wash. The slides were incubated with 1.25 μg / ml Alexa Fluor™ 647-labeled anti-human IgG for 30 minutes, washed twice with PBST for 5 minutes per wash, and then washed twice with PBS for 5 minutes per wash. The slides were dried at 1200 × g at 23 °C for 2 minutes and scanned using a 635 nm laser at 10% gain, 10 mW power, and a 10 μm pixel size. Data were extracted using MAPIX software and filtered using i-Ome AI software.

[0184] result 2.1 Uniformity of Streptavidin Coating on Nexterion H Slides. H slides were coated with various concentrations of streptavidin alone (Figure 9A), co-incubated with 50 mM glycine to assess competitive binding (Figure 9B), or allowed to hydrolyze (Figure 9C). Additional experiments included derivatization with 1 mg / ml streptavidin at pH 9 or pH 4.5, as well as various controls, including slides incubated with slide coating buffer alone, slide coating buffer containing 50 mM glycine, or empty gasket wells (Figure 9D). Streptavidin was visualized on the slide surface using Cy3-labeled biotin-BSA, as described in section 1.2.

[0185] Figure 9E shows the binding curves of streptavidin in the absence or presence of 50 mM glycine on fresh slides, or in the absence of glycine on aged slides. The KD and BMAX were higher for the fresh H slides (KD = 0.04; BMAX = 5724) compared with the aged H slides (KD = 0.03; BMAX = 5509). However, the KD of the fresh H slides was lower and the BMAX of the fresh H slides was higher compared with the H slides derivatized with streptavidin co-incubated with 50 mM glycine (KD = 0.2386; BMAX = 2684). Coating with 1 mg / ml streptavidin at pH 9 resulted in a higher average intensity, suggesting that more streptavidin was coated onto the slide surface at pH 9, whereas slides coated with 1 mg / ml streptavidin at pH 4.5 resulted in a lower average intensity (Figure 9F).

[0186] In another experiment, H-slides were coated with 8, 4, 2, 1, 0.5, 0.25, or 0.125 mg / ml streptavidin, then coated with Cy3-biotin-BSA, and finally blocked with 50 mM biotin or a buffer-only control (i.e., no biotin). Relative fluorescence unit (RFU) readings were lower in the blocked streptavidin-coated areas compared to the unblocked areas. For a typical binding curve, when all binding sites are occupied, the signal saturates, resulting in a sigmoidal binding curve. However, due to nonspecific binding, the binding curve does not plateau; rather, the signal continues to increase even when all binding sites are occupied due to nonspecific binding. This nonspecific binding is demonstrated in microarrays blocked with and without biotin (Figure 10), where a second binding curve, starting with 1 mg / ml streptavidin, shows nonspecific binding of streptavidin and / or Cy3-biotin-BSA to the slide surface.

[0187] Scanning electron microscope (SEM) images of slide areas derivatized with 8 mg / ml streptavidin (Figure 11A), 0.25 mg / ml streptavidin (Figure 11B), and coating buffer control (Figure 11C) and incubated with Cy3-biotin-BSA (BSA chemically modified with biotin and Cy3) show average spot diameters of 38 ± 10 nm, 14 ± 4 nm, and 77 ± 15 nm, respectively. The average distances between spots were 159 ± 47 nm, 193 ± 90 nm, and 763 ± 368 nm, respectively. The 1 μm images of slide areas coated with 8 mg / ml streptavidin, 0.25 mg / ml streptavidin, and coating buffer control show average spot diameters of 38 ± 10 nm, 14 ± 4 nm, and 77 ± 15 nm, respectively. The average distances between spots were 159 ± 47 nm, 193 ± 90 nm, and 763 ± 368 nm, respectively. 2 The number of spots per slide was 23, 18, and 2, respectively. Note: References to "spots" in this paragraph refer to streptavidin moieties, e.g., streptavidin tetramers, that bind to Cy3-biotin-BSA. The "spots" seen on slides derivatized with the coating buffer control may have been salt crystal artifacts.

[0188] 2.2 Detection of recombinant proteins on streptavidin-coated H-slides using anti-c-Myc antibodies Biotin carboxyl carrier protein (BCCP)-tagged recombinant SARS-CoV-2 proteins, including the full-length spike (S) protein and the C-terminal domain (CTD) of the N protein, were printed in triplicate on streptavidin-coated H-slides (Figure 12). At higher streptavidin concentrations (0.5–2 mg / ml), the S protein aggregated at the edge of the spot, forming high signal intensity at the edge and decreasing signal intensity toward the center of the spot, resulting in a "coffee ring effect." This effect was alleviated as the streptavidin concentration decreased to the range of 0.25–0.03125 mg / ml, suggesting that the S protein did not aggregate at lower streptavidin concentrations (Figure 13A). The CTD protein also formed spots with the "coffee ring" effect. In both the glycine-treated areas (Figure 13B) and the aged H slide (Figure 13C), the S protein did not exhibit a "coffee ring" effect, although signal intensity appeared to increase in slide areas coated with lower concentrations of streptavidin. The "coffee ring" effect of the CTD protein was reduced in areas coated with 0.125 to 0.03215 mg / ml streptavidin (50 mM glycine). At pH 9 and pH 4.5, the S protein and CTD protein formed spots in the streptavidin-coated areas as well as in slide areas not derivatized with streptavidin (Figure 13D). At pH 9, the "coffee ring" effect of the S protein and CTD protein is evident in the streptavidin-derivatized areas. At pH 4.5, a similar spot morphology is evident for proteins printed on the streptavidin-derivatized areas, although the signal intensity is lower.Proteins formed spots on the slide surface not derivatized with streptavidin, including slide coating buffer (SCB) (SCB only), slide coating buffer containing 50 mM glycine (SCB + 50 mM glycine), and no buffer (empty) (Figure 13D), suggesting that lysate proteins bind to the slide surface despite blocking of free PEG NHS esters by 50 mM glycine. Spots printed in the buffer control region did not exhibit the "coffee ring effect," supporting the argument that the "coffee ring" effect corresponds to the presence of a high concentration of streptavidin on the slide surface.

[0189] 2.3 Anti-human IgG antibody assay The spot morphology of the anti-c-Myc assay was also observed in the anti-human IgG antibody assay, including a "coffee ring" effect with BCCP-labeled S protein printed in areas with higher streptavidin levels (which decreased in areas derivatized with lower concentrations of streptavidin) (Figure 14A), in the presence of glycine (Figure 14B), or on aged H slides (Figure 14C). Although higher RFUs were detected in patient samples than in control samples in slide areas derivatized with pH 9 or pH 4.5, the "coffee ring" effect was still observed. Even when the slide surface was blocked with glycine, proteins distinguishing between patient and control samples (i.e., anti-S antibodies) were still observed on the slide surface, suggesting that recombinant S protein formed spots in the glycine-blocked areas without streptavidin (Figure 14D). Despite the "coffee ring" effect, stronger signal intensity was observed for S protein exposed to patient plasma than to control plasma in all conditions except for the assay on the aged H slide.

[0190] The median pixel intensity for each spot reflects the signal distribution of the spotted protein. Larger RFU differences were detected between patient and control samples on areas coated with lower concentrations of streptavidin, except for 2 mg / ml streptavidin, which produced the largest difference between patient and control samples (Figure 15A). The higher signal likely results from nonspecific "coffee ring" binding rather than specific antibody signal directed toward the native S protein. Areas derivatized with streptavidin in the presence of 50 mM glycine also showed larger differences between patient and control samples at lower concentrations of streptavidin (Figure 15B). However, the binding curves for patient and control sample assays on aged streptavidin-derivatized slides were similar (Figure 15C). The difference in RFU between patient and control samples is large in the streptavidin-derivatized region at pH 9 and pH 4.5 (Figure 15D), but the "coffee ring" spot morphology is still observed, so the increased signal intensity may still be driven by nonspecific binding.

[0191] 3. Discussion Nexterion H slides are glass slides coated with NHS-activated polyethylene glycol (NHS-PEG), which reacts with lysine residues and primary amines found at the N-terminus of proteins. The distribution of native streptavidin on the H slide surface depends on several slide coating conditions, including, but not limited to, streptavidin concentration, the presence or absence of competing molecules, the pH of the coating buffer during slide derivatization, and the time course of H slide coating. Our starting conditions in this experiment were to derivatize fresh H slides with 1 mg / ml streptavidin at pH 8.5. Streptavidin binding curve kinetic analysis showed a KD of 0.04; therefore, H slides were expected to be fully coated with 0.08 mg / ml streptavidin, indicating that a streptavidin concentration of 1 mg / ml may be too high for slide coating. Coating slides with streptavidin in the presence of 50 mM glycine, a primary amine-containing amino acid, increased the KD to 0.24 due to competitive binding between streptavidin and glycine to the PEG-NHS ester. The BMAX was 2684 for slides coated with streptavidin in the presence of glycine compared to 5724 for slides coated with streptavidin alone, likely due to competitive binding between streptavidin and glycine, resulting in fewer free PEG-NHS ester groups available for streptavidin binding. Aged and fresh slides were stored at -20°C for 2 years and 1 month, respectively. Thus, the aged slides had more time for spontaneous hydrolysis to occur, resulting in fewer free PEG-NHS ester molecules available for streptavidin binding. The resulting KD of 0.03 on the aged slides indicates similar affinity of streptavidin and PEG-NHS on fresh slides, but the BMAX decreased slightly from 5724 to 5509, likely due to less available PEG-NHS ester during derivatization due to spontaneous hydrolysis.Coating with 1 mg / ml streptavidin at pH 9 resulted in a higher average intensity, suggesting that more streptavidin was coated onto the slide surface at pH 9. Lysine has a pKa of 10.54, and therefore pH 9 is better than pH 8.5 for driving streptavidin derivatization on the slide surface. This is because the lysine residues on streptavidin are deprotonated and therefore more readily bind to NHS-PEG. Coating with 1 mg / ml streptavidin at pH 4.5 resulted in a lower average intensity, indicating that fewer streptavidin molecules bound to the slide surface compared to pH 8.5 or pH 9. At lower pHs, the primary amine groups on streptavidin are expected to be protonated and not bind to NHS-PEG, but signal detection at pH 4.5 suggests that not all primary amine groups were protonated. Additionally, the lower pH drives hydrolysis of the NHS ester groups on the PEG molecules, further reducing the extent of streptavidin derivatization on the slide surface.

[0192] Streptavidin has a high affinity for biotin, with a KD of approximately 1×10 mol / L (Green, NM Avidin. In Advances in Protein Chemistry; Anfinsen, CB, Edsall, JT, Richard S, FM, Eds.; Academic Press, 1975; Vol. 29, pp. 85-133, https: / / doi.org / 10.1016 / S0065-3233(08)60411-8). Biotin-bound streptavidin complexes exhibit low nonspecific binding, high thermal stability, and resistance to denaturants, organic solvents, proteases, detergents, and extremes of pH. A biotin-blocking step can be incorporated into the assays of the present invention after printing to bind remaining free streptavidin molecules, thereby reducing nonspecific binding from macromolecules in biological samples (e.g., serum, plasma, or saliva) or detection antibodies in antibody assays. Performing a biotin blocking step after incubation with Cy3-biotin-BSA reduces the signal (Figure 10). The higher signal of the unblocked slide may be due to nonspecific binding of Cy3-biotin-BSA or macromolecules to the streptavidin surface. However, biotin blocking may displace and subsequently reduce nonspecific interactions.

[0193] As shown in Example 5, printing diluted CTD lysates onto streptavidin-coated slides followed by biotin or BSA blocking resulted in a decrease in signal (but an improved signal-to-noise ratio) on biotin-blocked slides compared to BSA-blocked slides. Furthermore, the linear decrease in signal at higher lysate dilutions (above 1:3 dilution) when a post-printing biotin blocking step was utilized indicates that the BCCP-tagged CTD protein specifically binds to streptavidin under those conditions. The same effect is not seen at higher lysate dilutions when a post-printing BSA blocking step is utilized.

[0194] A streptavidin tetramer has a diameter of approximately 5 nm (Kuzuya, A., Nucleic Acids Symp. Ser. 2008, 52(1), 681-682, https: / / doi.org / 10.1093 / nass / nrn344), and the diameter of BSA is approximately 7.3 nm (Ahmad, Md. W., Colloids Surf. Physicochem. Eng. Asp. 2014, 450, 67-75, https: / / doi.org / 10.1016 / j.colsurfa.2014.03.011). Each streptavidin tetramer can bind to four biotin molecules, but steric hindrance may allow only two Cy3-biotin-BSA molecules to bind to each streptavidin molecule. Therefore, the expected overall size of Cy3-biotin-BSA-conjugated streptavidin is predicted to be less than 19.2 nm. SEM analysis of H-slides coated with 0.25 mg / ml streptavidin resulted in an average spot size of 14 ± 4 nm, which is within the expected size range of Cy3-biotin-BSA-conjugated streptavidin. However, H-slides derivatized with 8 mg / ml streptavidin resulted in an average spot size of 38 ± 10 nm. An estimated 9.8 × 10 PEG NHS ester molecules are present in a 7 × 7 mm gasket well, while the number of streptavidin tetramers at 8 mg / ml is approximately 9.3 × 10 16 molecules and at 0.03 mg / ml is 3.5 × 10 14This indicates that streptavidin was in molar excess over the entire range tested on the H slide. Previous reports have shown that 2D streptavidin crystalline structures can form after 1 hour at 7-8 mg / ml streptavidin, which also increases the adsorptive properties of streptavidin (Calvert, T.L.; Leckband, D. Two-Dimensional Protein Crystallization at Solid-Liquid Interfaces. Langmuir 1997, 13(25), 6737-6745, https: / / doi.org / 10.1021 / la970590n). This suggests that the larger structures seen on the slide surface at 8 mg / ml may represent crystallized streptavidin structures bound specifically and / or nonspecifically to Cy3-biotin-BSA. Streptavidin forms a sticky 2D S-crystal layer at higher streptavidin concentrations (Calvert, TL; Leckband, D. Two-Dimensional Protein Crystallization at Solid-Liquid Interfaces. Langmuir 1997, 13(25), 6737-6745, https: / / doi.org / 10.1021 / la970590n). The lysate forming the spot containing the recombinant protein evaporates most rapidly at the slide-spot interface at the edge of the spot, resulting in increased recombinant protein concentration and increased adsorption of the recombinant protein at the edge of the spot. The higher signal intensity observed at the edge of the spot may be further enhanced by adsorption of the detection antibody in serum protein and / or antibody assays, resulting in a coffee ring effect. The distance between streptavidin spots at 8 mg / ml and 0.25 mg / ml was 159 ± 47 nm and 193 ± 90 nm, respectively. These results indicate that even at high concentrations, only a small number of streptavidin molecules are bound to the slide surface.In summary, the SEM results show that both the spot density and spot size vary based on the experimental conditions, and higher streptavidin concentrations appear to cause streptavidin aggregation, which promotes nonspecific binding on the slide surface.

[0195] Optimal slide-coating conditions should promote appropriately spaced and uniform distribution of recombinant proteins within printed spots on the slide surface, which in turn contributes to a highly sensitive and specific assay. To identify optimal slide-coating conditions, H slides were derivatized with streptavidin and printed with BCCP-labeled SARS-CoV-2 S protein. Patient and control plasma samples were evaluated for anti-S antibodies to determine the assay conditions that best distinguished patients from controls. With the exception of 2 mg / ml streptavidin, which produced the largest difference between the positive and negative control samples, larger RFU differences were detected between the positive and negative control samples on areas coated with lower concentrations of streptavidin; however, the higher signal likely resulted from nonspecific "coffee ring" binding rather than specific antibody signal directed toward the native S protein. Streptavidin-derivatized regions in the presence of 50 mM glycine also showed greater differences between the positive and negative control samples at lower concentrations of streptavidin, but the binding curves were similar across sample types for the streptavidin-derivatized, aged slide assays. Slides in which the PEG-NHS moieties were not derivatized with streptavidin but were blocked with glycine did not appear to bind Cy3-biotin-BSA (see Figure 9D), although lysates containing recombinant proteins did bind to the slide surface, suggesting that lysate proteins may bind nonspecifically to the slide surface.

[0196] In conclusion, the results of this example demonstrate that the binding density and structure of linking moieties and / or analytes on the microarray surface can be successfully tuned by varying the conditions used during coating of the microarray, including the concentration of linking moieties in the coating buffer, the presence of competitor molecules in the coating buffer, or the pH of the slide coating buffer. The results of this example further demonstrate that lower concentrations of linking moieties (e.g., <1 mg / ml, <0.5 mg / ml, or <0.25 mg / ml) and / or the presence of competitor molecules, and / or changes in pH can be used to avoid or reduce nonspecific binding and / or undesired aggregation of linking moieties and / or immobilized analytes on the surface of the microarray.

Claims

1. 1. A microarray comprising a surface having a plurality of reactive groups attached thereto, wherein an analyte is immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface density of the analyte within at least one discrete, defined region of the surface is less than about 20%.

2. 1. A microarray comprising a surface having a plurality of reactive groups attached thereto, wherein an analyte is immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface coverage of the analyte within at least one discrete, defined region of the surface is less than about 20%.

3. 10. The microarray of claim 1, wherein the surface density of the analyte within the at least one predetermined region of the surface is less than about 10%.

4. 4. The microarray of claim 1 or claim 3, wherein the surface density of the analyte within the at least one predetermined region of the surface is at least about 0.05%, preferably at least about 0.5%, more preferably at least about 1%.

5. 3. The microarray of claim 2, wherein the surface coverage of the analyte within the at least one predetermined area of ​​the surface is less than about 10%.

6. 6. The microarray of claim 2 or claim 5, wherein the surface coverage of the analyte within the at least one predetermined area of ​​the surface is at least about 0.05%, preferably at least about 0.5%, more preferably at least about 1%.

7. 7. The microarray of claim 1, wherein the number of immobilized analyte molecules in said at least one predetermined region is less than about 300 per square micrometer.

8. 8. The microarray of any of claims 1-7, wherein the number of immobilized analyte molecules in the at least one predetermined region is less than about 275 per micrometer square, less than about 250 per micrometer square, less than about 225 per micrometer square, less than about 200 per micrometer square, less than about 175 per micrometer square, less than about 150 per micrometer square, less than about 125 per micrometer square, less than about 100 per micrometer square, less than about 75 per micrometer square, less than about 50 per micrometer square, less than about 25 per micrometer square, less than about 20 per micrometer square, less than about 15 per micrometer square, less than about 10 per micrometer square, or less than about 5 per micrometer square.

9. 1. A microarray comprising a surface having a plurality of reactive groups attached thereto, wherein an analyte is immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the number density of immobilized analyte molecules within at least one discrete, defined area of ​​the surface is less than about 300 per square micrometer.

10. 10. The microarray of claim 9, wherein the number of immobilized analyte molecules in the at least one predetermined region is less than about 275 per micrometer square, less than about 250 per micrometer square, less than about 225 per micrometer square, less than about 200 per micrometer square, less than about 175 per micrometer square, less than about 150 per micrometer square, less than about 125 per micrometer square, less than about 100 per micrometer square, less than about 75 per micrometer square, less than about 50 per micrometer square, less than about 25 per micrometer square, less than about 20 per micrometer square, less than about 15 per micrometer square, less than about 10 per micrometer square, or less than about 5 per micrometer square.

11. 10. The microarray of any preceding claim, wherein the analyte is immobilized on the surface via indirect binding to one or more of the reactive groups via a linking moiety.

12. 10. A microarray according to any preceding claim, wherein the analytes comprise tags that allow for immobilisation of the analytes to the surface.

13. 10. The microarray of any preceding claim, wherein the analytes comprise one or more of polypeptides, nucleic acids, lipids, and carbohydrates.

14. 10. The microarray of any preceding claim, wherein the analytes comprise one or more of polypeptides and nucleic acids.

15. 10. A microarray according to any preceding claim, wherein the analyte is a polypeptide, such as a glycoprotein.

16. 10. The microarray of any preceding claim, wherein the analyte is a polypeptide.

17. The microarray of any one of claims 12 to 16, wherein the analyte is a polypeptide and the tag enabling immobilization of the polypeptide on the surface is fused to the N-terminus or C-terminus of the polypeptide.

18. The microarray of any one of claims 13 to 17, wherein the polypeptides are properly folded.

19. 10. The microarray of any preceding claim, wherein the analytes are biotinylated.

20. 20. The microarray of claim 19, wherein the biotinylated analytes are chemically biotinylated or enzymatically biotinylated.

21. 21. The microarray of any one of claims 12 to 20, wherein the tag that allows for immobilization of the analyte to the surface comprises a biotin carboxyl carrier protein (BCCP) motif or an Avi tag (SEQ ID NO: 1).

22. 22. The microarray of claim 21, wherein the BCCP motif has at least 80% sequence identity, preferably at least 90% sequence identity, more preferably 100% sequence identity with SEQ ID NO:

2.

23. 23. The microarray of claim 21 or claim 22, wherein the BCCP motif is properly folded.

24. The microarray of any one of claims 21 to 23, wherein biotin is bound to (i) a biotin-binding domain within the BCCP motif or (ii) the Avi tag.

25. 25. The microarray of claim 24, wherein biotin is attached to the biotin-binding domain or the Avi tag via enzymatic biotinylation, preferably via a biotin ligase.

26. 10. The microarray of any preceding claim, wherein the linking moiety is a biotin-binding molecule and the analyte is biotinylated.

27. 27. The microarray of claim 26, wherein the biotin-binding molecule is a protein.

28. 28. The microarray of claim 26 or claim 27, wherein the biotin-binding molecule is selected from the group consisting of: (i) an anti-biotin antibody, (ii) avidin, (iii) neutravidin, (iv) streptavidin, or (iii) a fragment, variant, or analog of streptavidin, avidin, neutravidin, or the anti-biotin antibody that retains biotin-binding ability.

29. The microarray of any one of claims 26 to 28, wherein the biotin-binding molecules comprise a sequence having at least 80% sequence identity, preferably at least 90% sequence identity, more preferably 100% sequence identity to SEQ ID NO:

3.

30. The microarray of any one of claims 26 to 29, wherein the biotin-binding molecules are in a homotetrameric form and do not aggregate.

31. 31. The microarray of claim 26, wherein the microarray comprises a plurality of binding sites for biotin via the biotin-binding molecules, some of the biotin-binding sites do not bind to biotinylated analytes, and the biotin-binding sites that do not bind to biotinylated analytes bind to free biotin.

32. 32. The microarray of claim 31 , wherein substantially all of the biotin binding sites that do not bind to biotinylated analyte are occupied by free biotin, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the biotin binding sites that do not bind to analyte are bound to free biotin.

33. 1. A microarray comprising a surface having a plurality of reactive groups attached thereto, wherein linking moieties are immobilized on the surface via binding to one or more of the reactive groups, and wherein the surface coverage of the linking moieties within at least one discrete, predetermined region of the surface is less than about 20%.

34. 1. A microarray comprising a surface having a plurality of reactive groups attached thereto, wherein linking moieties are immobilized on the surface via binding to one or more of the reactive groups, and the number of linking moieties immobilized within at least one distinct predetermined region of the surface is less than about 300 per square micrometer, e.g., less than about 275 per square micrometer, less than about 250 per square micrometer, less than about 225 per square micrometer, less than about 200 per square micrometer, less than about 175 per square micrometer, less than about 150 per square micrometer, less than about 125 per square micrometer, less than about 100 per square micrometer, less than about 75 per square micrometer, less than about 50 per square micrometer, less than about 25 per square micrometer, less than about 20 per square micrometer, less than about 15 per square micrometer, less than about 10 per square micrometer, or less than about 5 per square micrometer.

35. 35. The microarray of claim 33 or 34, wherein the linking moiety is a biotin-binding molecule.

36. The microarray of claim 35, wherein the biotin-binding molecule is as defined in any one of claims 27 to 30.

37. 10. The microarray of any preceding claim, wherein the surface density or the surface coverage is determined by microscopy.

38. 38. The microarray of claim 37, wherein the microscopy is selected from the group consisting of atomic force microscopy (AFM), electron microscopy, and super-resolution microscopy.

39. 10. The microarray of any of the preceding claims, wherein the reactive groups on the surface are selected from carboxylic acid groups, activated carboxylic acid groups, amine groups, imidoester groups, maleimide groups, haloacetyl groups, pyridyldithiol groups, azide groups, hydrazide groups, alkoxyamine groups, thiol groups, arylazide groups, and diazirine groups.

40. 10. The microarray of any preceding claim, wherein the reactive groups on the surface are selected from carboxylic acid groups, activated carboxylic acid groups, amine groups, and maleimide groups.

41. 10. The microarray of any preceding claim, wherein the reactive groups on the surface are activated carboxylic acid groups, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-activated carboxylic acid groups or N-hydroxysuccinimide (NHS)-activated carboxylic acid groups.

42. 10. A microarray according to any preceding claim, wherein the reactive groups are attached to the surface via a hydrophilic organic polymer.

43. 43. The microarray of claim 42, wherein the hydrophilic organic polymer is selected from the group consisting of polyacrylamide, polyurethane, polyethyleneimine, and polyethylene glycol, preferably polyethylene glycol.

44. The microarray of claim 43, wherein the polyethylene glycol has an average molecular weight in the range of 500 to 20,000.

45. 10. The microarray of any preceding claim, wherein the immobilized analytes, or the linking moieties, or the linking moieties that bind to immobilized analytes on the surface, are spaced at least 50 nm, such as at least 100 nm, or at least 150 nm apart from one another as measured by AFM.

46. The at least one predetermined area is 0.2 mm ２、 For example, 0.8 μm ２ ~0.2 mm ２ 10. The microarray of claim 1, having an area of

47. 10. A microarray according to any preceding claim, having a plurality of individual predetermined regions, e.g. a plurality of analyte spots, on its surface.

48. 1. A method for manufacturing a microarray, comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached, wherein only about 20% or less of the reactive groups are capable of reacting at any one time; (ii) optionally contacting the reactive groups with a linking moiety under conditions under which the linking moiety reacts with one or more of the reactive groups capable of reacting, thereby binding the linking moiety to the surface; and (iii) depositing a sample of the analyte on the surface in at least one discrete predetermined area of ​​the surface such that the analyte is immobilized on the surface via direct binding to one or more of the reactive groups capable of reacting or via indirect binding to a reactive group via the linking moiety, wherein correspondingly, the surface coverage of the bound analyte in the at least one discrete predetermined area of ​​the surface is less than about 20%.

49. 1. A method for reducing the density of analyte binding to a surface of a microarray, comprising: (i) providing a surface to which a plurality of reactive groups are attached; and (ii) deactivating or rendering inaccessible a portion of the reactive groups such that no more than about 20% of the reactive groups are available to react at any one time. (iii) optionally, contacting the reactive groups with a linking moiety under conditions that allow one or more reactive groups that retain reactivity to react with the linking moiety, resulting in binding of the linking moiety to the surface; and (iv) depositing a sample of the analyte on the surface in at least one discrete, predetermined area of ​​the surface such that the analyte is immobilized on the surface via direct binding to one or more reactive groups or via indirect binding to one or more reactive groups via the linking moiety, wherein accordingly, the surface coverage of the bound analyte in the at least one discrete, predetermined area of ​​the surface is less than about 20%.

50. i) increasing the analyte signal to background noise ratio of the microarray; ii) increasing the rotational and conformational freedom of analytes immobilized on the microarray; and / or iii) a method for increasing the rate of physiologically relevant interactions between analytes immobilized on a microarray and test molecules applied to said microarray, comprising: The method includes providing a microarray comprising a surface having a plurality of reactive groups attached thereto, wherein the analytes are immobilized on the surface via direct binding to one or more of the reactive groups or via indirect binding to one or more of the reactive groups via a linking moiety, and wherein the surface coverage of the analytes immobilized within at least one discrete, predetermined region of the surface is less than about 20%.

51. The method of any one of claims 48 to 50, wherein the microarray is further as defined in any one of claims 1 to 47.

52. 1. Use of the surface of a microarray as a low-density surface having a surface density of immobilized analytes within at least one individual predetermined region of the surface of less than about 20%, wherein the analytes are optionally immobilized to the microarray via a linking moiety.

53. 1. Use of a surface for the manufacture of a low-density microarray comprising immobilized analytes, wherein the surface density of immobilized analytes within at least one individual predetermined region of the surface is less than about 20%, and the analytes are optionally immobilized to the microarray via a linking moiety.

54. 1. Use of reactive groups on a surface suitable for forming a low-density microarray to reduce the density of analytes immobilized on the surface, wherein some of the reactive groups are incapable of reacting with the analytes such that the surface density of analytes immobilized on at least one discrete predetermined region of the surface is less than about 20%, and the analytes are optionally immobilized on the surface via a linking moiety.

55. 1. Use of reactive groups on a surface suitable for forming a low-density microarray for the manufacture of a low-density protein microarray, wherein a portion of the reactive groups on the surface are incapable of reacting with an analyte such that the surface density of the analyte immobilized on at least one individual predetermined region of the surface is less than about 20%, and the analyte is optionally immobilized on the surface via a linking moiety.

56. i) identifying interactions between the analyte and a test molecule applied to the analyte; ii) determining the antibody profile of the subject; iii) identification of biomolecules that specifically bind to the immobilized analyte; iv) identification of an antibody that specifically binds to the immobilized analyte and is suitable for diagnosing or treating a disease; or v) Use of the microarray according to any one of claims 1 to 47 for identifying biomolecules that specifically bind to the immobilized analyte and are capable of treating a disease mediated by the immobilized analyte.

57. The use according to any one of claims 52 to 56, wherein the low-density microarray is as defined in any one of claims 1 to 47.

58. 1. A method for manufacturing a microarray, comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached; (ii) contacting the reactive groups with a linking moiety comprising a biotin-binding molecule under conditions in which the linking moiety reacts with one or more of the reactive groups to which it is capable of reacting, thereby binding the linking moiety to the surface; (iii) depositing a sample of the biotinylated analyte on the surface such that the analyte is immobilized on the surface via indirect binding to the reactive groups via the linking moiety; and then (iv) applying a solution of biotin to the surface of the microarray.

59. 1. A method for manufacturing a microarray, comprising the steps of: (i) providing a surface to which a plurality of reactive groups are attached; and (ii) contacting the reactive groups with a solution of linking moieties, wherein the concentration of the linking moieties in the solution is less than 1 mg / ml, and wherein the linking moieties react with one or more reactive groups capable of reacting, thereby binding the linking moieties to the surface.

60. 60. The method of claim 59, wherein the method comprises the further step (iii) of depositing a sample of the analyte on the surface such that the analyte is immobilized on the surface via indirect binding to the one or more reactive groups via the linking moiety.

61. 61. The method of claim 59 or claim 60, wherein the concentration of the linking moiety in the solution is 0.8 mg / ml or less, 0.6 mg / ml or less, 0.5 mg / ml or less, 0.4 mg / ml or less, 0.3 mg / ml or less, 0.25 mg / ml or less, 0.15 mg / ml or less, 0.1 mg / ml or less, 0.05 mg / ml or less, or 0.01 mg / ml or less.

62. 62. The method of any one of claims 59 to 61, wherein the linking moiety is a biotin-binding molecule.

63. The method of any of claims 58 to 62, wherein the linking moiety is as defined in any of claims 26 to 30.

64. The method of any of claims 58 to 63, wherein the analyte is as defined in any of claims 12 to 20.

65. The analyte is present in an amount of about 1×10 －１２ mol / L, e.g., less than about 1×10 －１３ mol / L, e.g., less than about 1×10 －１４ The method of any of claims 58 to 64, wherein the antibody is capable of binding to the linking moiety with a KD of 100 mol / L.

66. The method of any of claims 58 to 65, wherein the analyte is biotinylated, for example chemically or enzymatically biotinylated.

67. 67. The method of any of claims 58 to 66, wherein the analyte comprises a biotin carboxyl carrier protein (BCCP) motif or an Avi tag (SEQ ID NO: 1).

68. 68. The method of claim 67, wherein the BCCP motif has at least 80% sequence identity, at least 90% sequence identity, or 100% sequence identity to SEQ ID NO:

2.

69. 69. The method of claim 67 or claim 68, wherein the BCCP motif is properly folded.

70. 70. The method of any of claims 67 to 69, wherein biotin is bound to (i) a biotin-binding domain within the BCCP motif or (ii) the Avi tag.

71. 71. The method of claim 70, wherein biotin is attached to the biotin-binding domain or the Avi tag via enzymatic biotinylation, e.g., via a biotin ligase.

72. The method of any of claims 60 to 71, wherein the method comprises the further step (iv) of applying a solution of a blocking agent to the surface of the microarray.

73. 73. The method of claim 72, wherein the blocking agent is selected from the group consisting of nonfat dry milk, bovine serum albumin (BSA), casein, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), gelatin, serum, and biotin.

74. The blocking agent is about 1×10 －１２ mol / L, e.g., less than about 1×10 －１３ mol / L, e.g., less than about 1×10 －１４ 74. The method of claim 72 or claim 73, wherein the antibody is capable of binding to the linking moiety with a KD of 100 mol / L.

75. The method of any one of claims 72 to 74, wherein the blocking agent is biotin.

76. The method of any of claims 72 to 75, wherein the linking moiety is as defined in any of claims 26 to 30.

77. The method of any of claims 58 to 76, wherein the reactive group is as defined in any of claims 39 to 44.

78. 78. The method of any of claims 58-77, wherein the solution of linking moieties further comprises a competitor molecule capable of competing with the linking moiety for reaction with the reactive group.

79. 79. The method of claim 78, wherein the competitor molecule comprises a free amine group.

80. 80. The method of claim 78 or claim 79, wherein the competitor molecule is selected from the group consisting of amino acids and alkanolamines.

81. 81. The method of any of claims 78-80, wherein the competitor molecule is selected from the group consisting of glycine, alanine, serine, lysine, arginine, histidine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, methanolamine, and ethanolamine.

82. 82. The method of any one of claims 78 to 81, wherein the competitor molecule is selected from the group consisting of glycine and ethanolamine.

83. 83. The method of any of claims 58 to 82, wherein the pH of the solution of the linking moiety is from about 7.5 to about 11, such as from about 8 to about 9.5 or from about 8.5 to about 9, such as about 8.

5.

84. 83. The method of any of claims 58 to 82, wherein the pH of the solution of the linking moiety is from about 4 to about 7.5, such as from about 4.5 to 6, such as 4.

5.

85. A method according to any one of claims 58 and 60 to 84, wherein the sample of analyte is deposited on the surface in the form of a solution.

86. 86. The method of claim 85, wherein the analyte solution is diluted prior to deposition onto the surface, e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 1 / 10-fold, at least 1 / 20-fold, at least 1 / 25-fold, at least 1 / 50-fold, or 1 / 100-fold.

87. 87. The method of any of claims 58-86, resulting in a density of said linking moieties within at least one discrete predetermined area of ​​said surface of less than about 300 linking moieties per square micrometer, e.g., less than about 275 per square micrometer, less than about 250 per square micrometer, less than about 225 per square micrometer, less than about 200 per square micrometer, less than about 175 per square micrometer, less than about 150 per square micrometer, less than about 125 per square micrometer, less than about 100 per square micrometer, less than about 75 per square micrometer, less than about 50 per square micrometer, less than about 25 per square micrometer, less than about 20 per square micrometer, less than about 15 per square micrometer, less than about 10 per square micrometer, or less than about 5 linking moieties per square micrometer.

88. 88. The method of any of claims 58 and 60-87, resulting in a density of the analyte within at least one discrete predetermined area of ​​the surface of less than about 300 analyte molecules per square micrometer, e.g., less than about 275 per square micrometer, less than about 250 per square micrometer, less than about 225 per square micrometer, less than about 200 per square micrometer, less than about 175 per square micrometer, less than about 150 per square micrometer, less than about 125 per square micrometer, less than about 100 per square micrometer, less than about 75 per square micrometer, less than about 50 per square micrometer, less than about 25 per square micrometer, less than about 20 per square micrometer, less than about 15 per square micrometer, less than about 10 per square micrometer, or less than about 5 analyte molecules per square micrometer.

89. A microarray obtained by the method of any one of claims 58 to 88.

Images