WO2011038158A2

WO2011038158A2 - Methods, compositions, systems and apparatus for molecular array fabrication

Info

Publication number: WO2011038158A2
Application number: PCT/US2010/050070
Authority: WO
Inventors: George A. Fry; Sam Lee Woo; Christina E. Inman
Original assignee: Life Technologies Corp
Current assignee: Life Technologies Corp
Priority date: 2009-09-23
Filing date: 2010-09-23
Publication date: 2011-03-31
Anticipated expiration: 2012-03-23
Also published as: WO2011038158A3

Abstract

Provided herein are compositions, methods, systems and apparatuses for use in preparing arrays of molecules or other compounds. The systems include beads which are linked to the molecules or other compounds to be arrayed. The arrays are prepared by depositing the bead-molecule complexes onto a surface. The bead-molecule complexes can be used to prepare an array of single molecules, where each single molecule is attached to a surface at a single location on the surface. In one exemplary embodiment, the molecules to be deposited can include nucleic acid.

Description

METHODS, COMPOSITIONS, SYSTEMS AND APPARATUS

FOR MOLECULAR ARRAY FABRICATION

[0001] This application claims the benefit of U.S. Provisional Application Nos. 61/245,248, filed September 23, 2009 and 61/302,475, filed February 8, 2010, all of which applications are incorporated by reference in their entirety.

FIELD OF USE

[0002] In some embodiments, the disclosure relates generally to compositions, systems, methods and apparatuses for preparing arrays of molecules which are deposited onto a surface, optionally using beads, as well as arrays formed using such methods and apparatuses.

BACKGROUND

[0003] Molecular arrays are conventionally used for a wide range of laboratory procedures for diagnostic, screening, and characterization analyses. For example, nucleic acid arrays can be used for polymorphism analyses, elucidation of protein/nucleic acid interactions, screening of candidate pharmaceutical compounds, and sequencing. Arrays can be prepared by depositing one or more molecules or compounds to be arrayed onto a suitable surface in a desired pattern. Current procedures for deposition and patterning include drop projection, micro-contact printing, electron- beam lithography, and dip-pen nanolithography. In many assays, however, it can be desirable to deposit single members at discrete locations on the surface. For example, arrays of single nucleic acid or polymerase molecules, in which each of the single nucleic acid molecules or polymerases can be resolved from adjacent members of the array using a suitable detection system, are useful in single molecule sequencing applications.

[0004] One challenge of fabricating single molecule arrays using deposition techniques is to ensure suitable separation of each member of the array from adjacent members within the array. Frequently, two different members will attach to the array at locations not sufficiently spaced apart to permit individual detection and/or resolution of each member using the applicable detection system of choice. For example, many current fluorescence-based single molecule detection systems typically cannot resolve individual members that are spaced apart less than 1.5-0.5 microns apart from each other; although the resolution capability of current detection systems is expected to increase over time, analysis of single molecule arrays will always be limited by the constraints of such detection systems, as well as by the precision limits of the technology used to fabricate the single molecule array itself. Such challenges are enhanced by the desirability of fabricating arrays wherein the individual members are packed at high densities; increasing the density of the arrayed population (which necessarily decreases the spacing between individual members) is desirable because it permits increased throughput at lower cost. It is also desirable to use a deposition method that is easily adjustable to permit fine-tuning of the spacing distances between the deposited members. Most conventional methods, however, are not suitable for fabrication of such high-density single molecule arrays wherein the spacing between individual members (e.g., single molecules) within the array can be precisely controlled. It is therefore desirable to develop improved methods, compositions, systems and apparatuses for fabrication of single molecule arrays, including arrays of single nucleic acid molecules.

SUMMARY

[0005] In some embodiments, the disclosure relates generally to compositions, methods, systems and apparatuses useful for preparing molecular arrays via bead deposition.

[0006] In some embodiments the disclosure relates generally to compositions comprising a bead-molecule complex comprising at least one bead linked to one or more molecules. The one or more molecules optionally include one or more nucleic acid portions. Optionally, the one or more molecules can include a cleavable moiety, a reporter moiety, and/or an attachment moiety.

[0007] In some embodiments, the disclosure relates generally to a method for attaching a molecule to a surface, comprising: linking a bead to a molecule through a cleavable bond, thereby forming a cleavable bead-molecule complex where the complex includes the bead cleavably linked to the molecule; linking the molecule of the cleavable bead-molecule complex to a surface, thereby forming a surface-attached bead-molecule complex; and cleaving the cleavable bond, thereby releasing the bead of the cleavable bead-molecule complex and forming a surface- attached molecule. Optionally, a plurality of bead-molecule complexes can be attached to the surface simultaneously or sequentially, thereby forming an array of surface-attached molecules.

[0008] Optionally, the molecule is a linker that can bind or react with a target analyte of interest. In some embodiments, the method further includes contacting the surface-attached molecule (or linker) with a target analyte under conditions where the target analyte binds to, or reacts with, the surface-attached molecule, thereby attaching the target analyte to the surface and forming a surface-attached target analyte. Optionally, the method includes contacting a plurality of surface- attached molecules (or linkers) with a plurality of target analytes, thereby forming a plurality of surface-attached target analytes.

[0009] In some embodiments, the disclosure relates generally to a method for fabricating a molecular array, comprising: forming a cleavable bead-linker complex by binding a linker to a bead through a cleavable bond; binding the linker of the cleavable bead-linker complex to a surface; and cleaving the cleavable bond, thereby releasing the bead of the cleavable bead-linker complex and forming a surface-attached linker. Optionally, the method further includes forming a plurality of cleavable bead linker complexes by binding a linker to each of a plurality of beads, binding the linker of each complex of the plurality of cleavable bead linker complexes to the surface, and cleaving the cleavable bond of each complex, thereby forming a plurality of surface- attached linkers.

[0010] In some embodiments, the disclosure relates generally to a composition useful for fabricating a molecular array, comprising: a bead-linker complex including at least one bead attached to at least one linker through a cleavable bond. Optionally, the linker is an

oligonucleotide linker, which may include a cleavable moiety. Optionally, the linker can further include a rigid polymer. The linker can include a surface attachment moiety.

[0011] In some embodiments, the disclosure relates generally to a cleavable bead-linker system useful for preparing a molecular array, comprising: a linker including a cleavable moiety and a surface-reactive moiety; a bead linked to the linker through the cleavable moiety; and a surface; wherein the linker is attached to the surface through the surface-reactive moiety. Optionally, the linker is an oligonucleotide linker.

[0012] In some embodiments, the disclosure relates generally to a method for forming a molecular array, comprising: contacting a plurality of bead- linker complexes with a surface, where each bead-linker complex includes at least one bead linked to one or more linkers through a cleavable moiety and where the contacting is performed under conditions where at least one linker of each bead-linker complexes binds to the surface, thereby forming a plurality of surface-attached bead-linker complexes; and cleaving the cleavable moiety of each surface- attached bead- linker complex, thereby releasing the bead of each surface-attached bead-linker complex and forming a plurality of surface-attached linkers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows some embodiments of metal patterned spots.

[0014] FIG. 2 shows one embodiment of a design of blocks of metal patterned spots. [0015] FIG. 3 depicts one embodiment of a pattern of blank spots among a pattern of metal spots.

[0016] FIG. 4 depicts various exemplary configurations of arrays fabricated using the methods, compositions, systems and apparatuses of the disclosure. Panel (A) depicts a nanoarray block configuration; Panel (B) depicts a second nanoarray block configuration, and Panel (C) depicts a nanoarray die (i.e., chip) containing multiple nanoarray blocks.

[0017] FIG. 5 shows a microscope image of streptavidin functionalized quantum dots bound to biotin, which were loaded into 600-700 nm diameter spots on titanium coated slides using magnetic beads functionalized with oligonucleotides, in accordance with one embodiment.

[0018] FIG. 6 shows the oligonucleotides loaded into metal patterned spots after cleavage from the beads, in accordance with one embodiment.

[0019] FIG. 7 shows a graph depicting a determination of the number of quantum dots loaded per metal patterned spot. Long linkers have 4 HEO units and short linkers have 1 HEO unit, in accordance with one embodiment.

[0020] FIG. 8 depicts graphically the increase in array density obtained as the distribution of members within the array moves from random or semi-ordered.

[0021] FIG. 9 depicts an exemplary method of fabricating an array of linkers including template attachment moieties according to the present disclosure. Panel (A) depicts an overview of the fabrication process including deposition of a population of bead-linker complexes, followed by cleavage and removal of the beads of the complexes, leaving an array of surface-attached linkers. Panel (B) depicts a detailed view of a single bead-linker complex.

[0022] FIG. 10 depicts a detailed overview of an exemplary method of fabricating

oligonucleotide linker arrays according to the disclosure.

[0023] FIG. 11 depicts a schematic of an exemplary linker that can be used in bead-linker complexes according to the disclosure.

[0024] FIG. 12 depicts data obtained from an exemplary random oligonucleotide linker array prepared via direct deposition of linkers onto the surface using conventional deposition methods, and an exemplary semi-ordered array using the bead-based deposition techniques of the disclosure. Panel (A) depicts images obtained from the semi-ordered array and the random array before and after filtering. Panel (B) depicts a graph plotting the effect of loading density on the resulting number of useful, i.e., functional linkers in the array. Panel (C) depicts a histogram illustrating the effect of linker length on the average number of functional linkers (useful attachment sites) deposited into each discrete location on the surface.

DETAILED DESCRIPTION

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which these inventions belong. All patents, patent applications, published applications, treatises and other publications referred to herein, both supra and infra, are incorporated by reference in their entirety. If a definition and/or description is explicitly or implicitly set forth herein that is contrary to or otherwise inconsistent with any definition set forth in the patents, patent applications, published applications, and other publications that are herein incorporated by reference, the definition and/or description set forth herein prevails over the definition that is incorporated by reference.

[0026] In some embodiments, the disclosure relates generally to compositions, systems, methods and apparatuses for preparing one or more molecular arrays comprising a plurality of molecules (or any other composition desired to be arrayed) attached to a surface using deposition. In some embodiments, the array can be prepared by depositing one or more molecules onto a suitable surface. The one or more molecules can optionally attach to the surface following deposition. Each molecule optionally attaches to a distinct location on the surface, such that it is spaced apart from all other molecules attached to the surface. Optionally, the one or more molecules include a surface attachment moiety that facilitates binding of the one or more molecules to the surface.

[0027] The one or more molecules of the array can optionally include any molecule, molecular complex or compound desired to be attached to the surface within the array, including, for example, nucleic acid molecules, protein molecules {e.g., enzymes, hormones, antibodies and the like), carbohydrates, lipids, glycoproteins, or any other molecule or compound, including molecules of non-biological interest. In one exemplary embodiment, the molecule comprises nucleic acid, for example an oligonucleotide. In some embodiments, the molecules can be deposited onto the surface of a nanoarray.

[0028] In some embodiments, the disclosure relates generally to methods, systems, apparatuses and compositions for forming a molecular array comprising a plurality of molecules (or any other composition desired to be arrayed) attached to a surface using bead deposition techniques. In some embodiments, one or more molecules to be arrayed can be linked to one or more beads to form a bead-molecule complex. As used herein, the term "linked" and its variants refer to any fusion or bonding or association between a combination of different compounds or molecules. The fusion, bond or association is sufficiently stable to withstand conditions encountered in the methods described herein, including washing, flowing, temperature or pH changes, reagent changes, or is sufficiently photostable to withstand illumination with light.

[0029] Optionally, the molecules are deposited onto the surface by contacting one or more bead- molecule complexes with the surface, each bead-molecule complex comprising at least one bead linked to one or more molecules. Optionally, the contacting is performed under conditions wherein at least one molecule of the bead-molecule complex binds to the surface, thereby attaching the bead-molecule complex to a surface and forming a surface-attached bead-molecule complex. Optionally, the linkage between the bead and the molecule of the surface- attached bead-molecule complex can be cleaved, thereby releasing the bead from the surface- attached bead-molecule complex and forming a surface-attached molecule.

[0030] In some embodiments, the molecule of the bead- molecule complex is linked to the bead of the complex through a cleavable bond. Optionally, the bond is an enzymatically cleavable, photocleavable or chemically cleavable bond. In some embodiments, the bond can cleaved using conditions that do not denature or otherwise disrupt the structure or function of the molecule of the bead-molecule complex.

[0031] Use of beads to deposit the molecules to be arrayed onto the surface provides several advantages. For example, use of a population of beads having similar or identical dimensions can be helpful in ensuring relatively uniform distribution of single molecules to discrete locations in the array; in some embodiments, one bead delivers a single molecule (or a single linker molecule capable of binding a target molecule) to a discrete location in the array.

[0032] In some embodiments, the disclosure relates generally to methods, compositions, systems and apparatuses for forming a molecular array via deposition of bead-molecule complexes onto a surface. In one exemplary embodiment, a plurality of bead-molecule complexes are deposited onto (or contacted with) a surface under conditions where the molecule of each bead- molecule complex of the plurality of bead-molecule complexes attaches to the surface, thereby forming a population of surface-attached bead-molecule complexes. Optionally, the linkage between the bead and the molecule of each surface-attached bead-molecule complex in the population can be cleaved, thereby releasing the bead of each surface-attached bead-molecule complex and forming a population of surface-attached molecules. Optionally, all of the molecules in the population of surface-attached molecules are structurally the same; optionally, at least two of the molecules in the population of surface attached molecules are different from each other. The at least two molecules can differ structurally, chemically or functionally from each other. The disclosure also relates generally arrays of surface-attached molecules prepared using such methods, as well as to arrays of single molecules, where each single molecule is attached to a surface at a single location on the surface, that can be prepared using such methods. The arrays can be patterned or unpatterned. In some embodiments, the array can be random or organized (e.g., ordered or semi-ordered)

[0033] In some embodiments, the molecules to be deposited are nucleic acid molecules, and disclosure relates generally to a method for forming a nucleic acid array comprising: providing a surface with a plurality of active sites, each active site supporting a first functional group;

contacting the surface with at least one bead-molecule complex, the at least one bead-molecule complex including at least one bead linked to one or more nucleic acid molecules, each nucleic acid molecule of the complex having a second functional group at a free end thereof; coupling at least one active site of the surface to a nucleic acid molecule of the at least one bead-molecule complex by forming a bond between the first and the second functional groups and thereby providing at least one surface-attached bead-molecule complex; and cleaving the linkage between the at least one bead and the one or more nucleic acid molecules of the surface-attached bead- molecule complex to form a surface-attached nucleic acid molecule. The surface-attached nucleic acid molecule can include an active site having an open nucleic acid molecule attached thereto. The disclosure also relates generally arrays of surface-attached nucleic acid molecules prepared using such methods, as well as to arrays of single nucleic acid molecules, where each single nucleic acid molecule is attached to a surface at a single location on the surface, that can be prepared using such methods. The arrays can be patterned or unpatterned. In some embodiments, the array can be random or organized (e.g., ordered or semi-ordered).

[0034] In some embodiments, the linkage between the bead and the molecule of the bead- molecule complex is enzymatically cleavable, chemically cleavable or photocleavable.

Optionally, the cleavage is performed using conditions that do not denature or otherwise disrupt the structure and/or function of the molecule of the bead- molecule complex; and/or do not disrupt the attachment of the molecule of the bead-molecule complex to the surface.

[0035] In some embodiments, the molecule of the bead- molecule complex comprises one or more linkers that can bind to a target analyte of interest. In such embodiments, the complex can be referred to as a "bead-linker complex" instead of a "bead-molecule complex", although the two categories are not mutually exclusive. The use of bead-molecule complexes (including bead- linker complexes) to deposit single members of an array onto a surface can provide several advantages. For example, direct deposition of the molecules or linkers to be arrayed on the surface using conventional deposition techniques can frequently be inefficient between at high loadings several molecules can bind too close to each other to be resolved using the detection system of choice, and will therefore appear as "overlapping" points on the surface; the effective sample loss due to loading can be as high as 30% (data not shown). The use of beads can minimize such overlap and ensure greater uniformity in distribution of array members, thus allowing the creation of arrays with higher surface densities that permit increased throughput. For example, ensuring 1 : 1 correspondence between each bead and a linker moiety can be useful in ensuring that linker is deposited onto a discrete location on the surface and thus can be

individually detected and resolved from other, adjacent linkers. The use of bead deposition can also be helpful in ensuring that the linkers/molecules are spaced apart from adjacent deposited linkers/molecules by a desired average distance.

[0036] The bead can act to control the spacing distance that separates two adjacent members of the array. In some embodiments, the bead-linker complexes are deposited onto a patterned surface that includes physical features (e.g., cavities, sockets, wells and the like) that physically constrain and guide the bead-linker complex to a particular location on the surface. For example, the surface can include cavities into which the deposited bead-linker complex will settle. The cavity can be sized to ensure that only one bead-linker complex can enter the cavity. Use of a uniformly patterned surface thus ensures the formation of a uniformly patterned array.

[0037] Alternatively, the bead-linker complexes can be deposited onto a surface that lacks physical features to guide or constrain the position of the deposited complex. When such "open field" surfaces are used, the bead of the bead-linker complex can still act to exclude binding of adjacent members to the surface within a defined zone by physically excluding other bead- linker complexes from entering that zone. In some embodiments, the average distance between adjacent members of the array can be at least about the diameter of the bead. Each bead can act via size- exclusion to prevent binding of other linkers within a zone around the deposited linker

corresponding having a diameter that is roughly equal to the diameter of the bead itself. The bead itself acts as a structure that effectively excludes a specific area of the surface from access by other linkers or beads. In this manner, the average distance between adjacent members of the array can be increased or decreased by adjusting the diameter of the beads used to fabricate the array.

[0038] In some embodiments, the average diameter (or length in longest dimension) of the bead is at least about 0.1 micron, 0.25 micron, 0.50 micron, 0.75 micron, 1.0 micron, 1.25 microns, 1.50 microns, 1.75 microns, 2.0 microns, or greater. In some embodiments, the average diameter (or length in longest dimension) of the bead is no greater than about 0.1 micron, 0.25 micron, 0.50 micron, 0.75 micron, 1.0 micron, 1.25 microns, 1.50 microns, 1.75 microns, 2.0 microns, or 5.0 microns. In some embodiments, the average diameter (or length in longest dimension) of the bead is selected such that the average distance between adjacent members of the resulting array is at least about 0.1 micron, 0.25 micron, 0.50 micron, 0.75 micron, 1.0 micron, 1.25 microns, 1.50 microns, 1.75 microns, 2.0 microns, or greater. In some embodiments, the average diameter (or length in longest dimension) of the bead is selected to ensure that the average distance between adjacent members of the array is no greater than about 0.1 micron, 0.25 micron, 0.50 micron, 0.75 micron, 1.0 micron, 1.25 microns, 1.50 microns, 1.75 microns, 2.0 microns, or 5.0 microns. In one exemplary single molecule fluorescence-based detection system, the optimal spacing between adjacent members that provides for maximal throughput while ensuring individual resolvability of each member is on the order of about 1.2 microns.

[0039] In addition to permitting more uniform distribution of single molecules or linkers on a surface, the use of beads can also be useful in controlling the distribution and amount of single molecule present on the surface. Such distribution and/or amount can simply be increased by increasing the loading of the beads. In a typical assay, for example, beads deposited at low loading densities can adopt a random distribution on the surface. As the bead loading (i.e., the number of deposited bead-linker complexes) increases, the arrangement of beads on the surface moves from a random to semi-ordered and finally to an ordered arrangement on the surface. Use of semi-ordered or ordered arrangements typically provide arrays characterized by higher feature density (i.e., number of members per unit surface area). In a typical assay as depicted in Figure 8, a random distribution of beads exhibited a maximum surface density of 580 spots per FOV (field of view) whereas a semi-ordered distribution exhibited a maximum surface density of 2300 spots per FOV.

[0040] In some embodiments, the disclosure relates generally to bead-linker complexes, each such complex comprising one or more linkers linked to at least one bead. In a typical embodiment, the bead-linker complex includes at least one linker attached to a single bead.

Optionally, the complex includes one bead linked to 1, 2, 3, 4, or more linkers.

[0041] Optionally, the bead-linker complexes are cleavable bead-linker complexes wherein at least one linker of the complex is attached to the bead of the complex via a cleavable bond. In some embodiments, the cleavable bond is an enzymatically cleavable, chemically cleavable or photocleavable bond. Optionally, the bond can be cleaved using conditions that do not denature or otherwise disrupt the structure and/or function of the linker of the bead- linker complex; and/or do not disrupt the attachment of the linker of the bead-linker complex to the surface. In some embodiments, the cleavable bond is present within the linker itself. For example, the cleavable bond can be found in a cleavable moiety that is included within the linker. In some embodiments, the cleavable bond is a disulfide (-S-S-) bond, which can be cleaved upon treatment with dithiothreitol (DTT).

[0042] Optionally, the binding between the linker of the bead-linker complex and the target analyte can be selective or specific.

[0043] In some embodiments, the one or more linkers of the bead-linker complex can include one or more oligonucleotide linkers. An oligonucleotide linker is any molecule or compound that includes one or more nucleic acid portions, although such language is in no way intended to convey that the oligonucleotide linker is exclusively comprised of nucleic acid; an oligonucleotide linker can also optionally comprise one or more non-nucleic acid portions in addition to the one or more nucleic acid portions. The nucleic acid portion of the linker can include a linear or branched nucleic acid. The nucleic acid portion can include single- or double- stranded nucleic acid, including DNA, RNA, DNA/RNA hybrids, or analogs thereof. In some embodiments, the linker includes one or more nucleic acid portions of the linker is comprised of an oligonucleotide.

[0044] In some embodiments, the disclosure relates generally to methods, compositions, systems and apparatuses for forming an analyte array involving deposition of a plurality of bead- linker complexes onto a surface. Optionally, a plurality of bead-linker complexes are deposited onto (or contacted with) a surface under conditions where the linker of at least one bead-linker complex binds to the surface to form at least one surface-attached bead-linker complex.

Optionally, the cleavable bond linking the bead and the linker can be cleaved, thereby releasing the bead of the bead-linker complex and leaving a surface-attached linker. The surface- attached linker can optionally be contacted with a target analyte under conditions where the surface- attached linker binds to the analyte, thereby anchoring the analyte to the surface and forming a surface-attached linker. In some embodiments, a surface-attached linker is contacted with the analyte using conditions where the linker binds the target analyte, thereby anchoring the target to the surface and forming a surface-attached analyte.

[0045] The disclosure also relates generally arrays of surface-attached linkers (or surface- attached target analytes) prepared using such methods, as well as to arrays of single linkers (or analytes), where each single linker (or analyte) is attached to a surface at a single location on the surface, that can be prepared using such methods. The arrays can be patterned or unpatterned. In some embodiments, the array can be random or organized (e.g., ordered or semi- ordered).

[0046] In one exemplary embodiment, the linker and analyte bind to each other selectively or specifically. In one exemplary embodiment, the linker and/or the analyte comprises nucleic acid. Optionally, both the linker and the analyte comprise nucleic acid. The linker and the analyte can comprise nucleic acid sequences that are partially or wholly complementary to each other. In some embodiments, the linker and the analyte can bind to each other via nucleic acid

hybridization.

[0047] In some embodiments, the linker of the bead- linker complex can include one or more moieties ("attachment moieties") that can facilitates binding of the linker to the surface, the bead and/or to a target. The attachment moiety can include any moiety, for example a reactive group or a functional linking group, which can bind to the surface and/or the target under suitable conditions. In some embodiments, the attachment moiety includes an amino group, aldehyde group, NHS ester, or alkyne group, or a member of a binding partner pair.

[0048] In some embodiments, the disclosure relates generally to compositions, systems, methods and apparatuses for use in preparing nucleic acid arrays through delivery of an oligonucleotide linker to a surface using a bead. For example, one or more molecules of an oligonucleotide linker can each be linked to a bead to form a bead-linker complex; a plurality of bead-linker complexes can be deposited onto the surface under conditions where the

oligonucleotide linker of each complex binds to the surface, thereby forming a population of surface-attached bead-linker complexes; and the linkage between the oligonucleotide linker and the bead of each complex can be cleaved to release the bead from each such surface-attached bead-linker complex, thereby forming a population of surface-attached oligonucleotide linkers, i.e., an oligonucleotide array. The disclosure also relates generally arrays of surface- attached oligonucleotide linkers prepared using such methods, as well as to arrays of single molecules, where each single linker is attached to a surface at a single location on the surface, that can be prepared using such methods. The arrays can be patterned or unpatterned. In some embodiments, the array can be random or organized (e.g., ordered or semi-ordered)

[0049] In some embodiments, each oligonucleotide linker of the surface-attached population of oligonucleotide linkers can be used to attach a second nucleic acid molecule to the surface. For example, the oligonucleotide array can be contacted with a population of nucleic acid molecules under conditions where one or more of the surface-attached oligonucleotide linkers of the array each binds to a nucleic acid molecule within the population, thereby anchoring the nucleic acid molecule to the surface.

[0050] In some embodiments, a population of nucleic acid molecules can first be ligated to a linker sequence that is complementary to the surface-attached oligonucleotide. The linker-ligated population can then be contacted with the population of surface-attached oligonucleotides under conditions where one or more members of the linker-ligated population each hybridizes to a surface-attached oligonucleotide, thereby forming a population of surface-attached nucleic acid molecules. In some embodiments, a population of nucleic acid molecules can be contacted with a population of surface-attached oligonucleotides to form a nucleic acid array. The nucleic acid molecules can optionally comprise a unique sequence (for example a cDNA sequence) and a universal sequence (for example a sequence complementary to the oligonucleotide sequence) that facilitates binding of the nucleic acid molecule to the surface-attached linker. Optionally, at least two nucleic acid molecules in the population of nucleic acid molecules are different from each other (for example, include different nucleic acid sequences); alternatively, all of the nucleic acid molecules in the population of nucleic acid molecules are the same (for example, include the same nucleic acid sequence). In one exemplary embodiment, the population of nucleic acid molecules includes a library of cDNA or genomic DNA molecules.

[0051] Optionally, an array of deposited, single nucleic acid molecules can be formed through such deposition techniques. Such arrays can be used to bind/link other biological molecules or non-biological compounds to create an array for analytical procedures. Optionally, the array can be a nanoscale array. The methods for preparing the bead, oligonucleotide, and nucleic acid arrays, can optionally employ recombinant DNA technology and linking chemistries.

[0052] The beads used according to the disclosed methods, compositions, systems and apparatuses can be made from any suitable material, including para-magnetic core material, which can be coated or uncoated. The coating can be a plastic compound. The beads can be made from glass, silica, and the like. The beads can be spherical in shape; alternatively, the beads can be made of any other shape, including irregularly shaped beads. A population of the beads can be homogeneous or heterogeneous in shape and/or size. In one exemplary embodiment, the bead comprises a para-magnetic core which is coated with a plastic compound.

[0053] The oligonucleotide linker of the bead-linker complex can be prepared using any procedure, including: recombinant DNA technology and/or chemical synthesis. In some embodiments, the length of the oligonucleotide can be selected to optimize tethering, proximity, flexibility or rigidity of the linker of the bead-linker complex. In some embodiments, the oligonucleotide is selected to optimize the orientation of the attached bead. For example, the oligonucleotide can be about 5-50, or about 5-40, or about 5-30, or about 5-20, or about 5-10 nucleotides in length. In some embodiments, the oligonucleotide can be about 15-20 nucleotides in length. The length of the oligonucleotide can be adjustable. The sequence of the

oligonucleotide can include any nucleotide sequence, including homo-polymeric or hetero- polymeric sequences. For example, the oligonucleotide molecule can be homo-polymeric- A, homo-polymeric -G, homo-polymeric -C, homo-polymeric -T, homo-polymeric -U, or homo- polymeric-I. In another example, the oligonucleotide can include any restriction enzyme sequence. In another example, the oligonucleotide can have a hetero-polymeric sequence, such as: 5 ' -GGGCGGCGACCTGGGT-Biotin-dT-3 ' .

[0054] Without being bound to any particular theory of operation, it can be surmised that increased inker length increases the probability that the linker of the bead-linker complex will bind to the surface. In order to bind, any linker will need to contact the surface; when bead diameter is large relative to linker length, then only linkers that are bound to the bead at positions close to the point of contact between the bead and the surface will themselves be able to contact the surface. Figure 12C depicts the effect of linker length on the average number of linkers observed to be bound to the surface.

[0055] In some embodiments, the linker of the bead- linker complex can include a rigidity moiety that increases the rigidity of the linker. In some embodiments, the linker can include a flexibility moiety that increases the flexibility of the linker. The rigidity moiety can include a rigid polymer. Linker rigidity can be desirable for various reasons. For example, use of a rigid linker can facilitate attachment of the bead- linker complex to the surface, and/or binding of the surface-attached linker to a target analyte following cleavage of the bead-linker complex and release of the bead. Any suitable rigidity moiety can be used to increase the rigidity of the linker; in one exemplary embodiment, the rigid polymer can include a polyethylene oxide (PEO) polymer chain comprised of linked ethylene oxide (EO) units or a polyethylene glycol (PEG) polymer chain. The PEO polymer chain can optionally include one or more hexapolyethylene oxide (HEO) units. In some embodiments, the HEO units can be linked by, e.g., bisurethane tolyl linkages. In some embodiments, the linker includes 1, 2, 3, 4, or more HEO units. Some examples of HEO- comprising linkers can be found, for example, in U.S. Pat. No. 5,807,682 to Grossman et al.

[0056] Optionally, the linker of the bead-linker complex can include both a rigid polymer and a nucleic acid portion. For example, in some embodiments the linker includes an oligonucleotide comprising at least 5 linked nucleotides, optionally linked via phosphodiester bonds, as well as a rigid polymer including two or more EO, PEO, PEG and/or HEO units. The EO, PEO, PEG or HEO units can be linked to the nucleic acid portion of the linker using any suitable method, including, for example, the methods disclosed in U.S. Pat. No. 5,807,682 to Grossman et al.

[0057] Optionally, the linker of the bead-linker complex, or the molecule of the bead-molecule complex, can be labeled. The label of the complex can be an optically detectable label, a chemically detectable label, a magnetic label, a mass tag, and the like. In some embodiments, the label includes a reporter moiety. The reporter moiety can include a fluorescent or fluorogenic moiety, for example a fluorescent dye.

[0058] Use of a label can be useful in determining the location of the surface-attached linker, as well as to assess whether more than one linker is attached to a defined region ("spot") of the surface. For example, in some embodiments, the presence of multiple linkers within the defined region will be detected as an additive increase in signal from the label. The signal from each detected spot can be analyzed using, for example, aggregate signal analysis to determine the number of linker units present at each spot.

[0059] In some embodiments, the attachment moiety can include any one member of a binding partner pair. The surface and/or the target analyte can optionally include the other member of the binding pair. Examples of binding partners include: biotin or desthiobiotin or photoactivatable biotin and their binding partners avidin, streptavidin, NEUTRAVIDIN, or CAPTAVIDIN; His- tags which bind with nickel, cobalt or copper; cysteine, histidine, or histidine patch which bind Ni- NTA; maltose which binds with maltose binding protein (MBP); lectin-carbohydrate binding partners; calcium-calcium binding protein (CBP); acetylcholine and receptor-acetylcholine;

protein A and binding partner anti-FLAG antibody; GST and binding partner glutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor) protein; antigen or epitope tags which binds to antibody or antibody fragments, particularly antigens such as digoxigenin, fluorescein, dinitrophenol or bromodeoxyuridine and their respective antibodies; mouse immunoglobulin and goat anti-mouse immunoglobulin; IgG bound and protein A; receptor- receptor agonist or receptor antagonist; enzyme-enzyme cof actors; enzyme-enzyme inhibitors; and thyroxine-cortisol. Another binding partner for biotin is a biotin-binding protein from chicken (Hytonen, et al., BMC Structural Biology 7:8).

[0060] Other examples of binding partner pairs include: artificial biotin binding sequences, such as an AVI- TAG (Avidity LLC). In some embodiments, the artificial biotin binding sequence comprises a biotin ligase sequence. In another embodiment, the biotin binding sequence comprises the sequence (in single-letter amino acid symbols) GLNDIFEAQKIEWHE. The biotin can bind the lysine (K) residue within the artificial biotin binding sequence. The artificial biotin binding sequence can be used for site-specific and/or mono- biotinylation of proteins. See for example Chapmann- Smith and Cronan 1999 Trends Biochem Sci 24:359-363; M.A. Eisenberg, et al., 1982 J. Biol Chem 275: 15167-15173; J.E. Cronan 1990 J Biol Chem 265: 10327-10333; and P.J. Schatz 1993 Biotechnology 11: 1138-1143. In some embodiments, binding partner pairs can be used to link the linker of the bead-linker complex, the bead, reporter moiety, flexibility/rigidity moiety (e.g., rigid polymer), target analyte, surface and/or any other compound to each other in any combination. For example, in some embodiments the linker can include an oligonucleotide, one member of a binding partner pair can be linked to one end of the oligonucleotide, and the other member of the binding partner can be linked to the surface.

[0061] In some embodiments, the attachment moiety includes a "click" group that can react via "click" chemistry with another "click" group of the surface and/or the target (e.g., an azide-alkyne click reaction such as Huisgen cylcoaddition, Diels-Alder reaction or a "copperless" click reaction, Ruthenium-catalyzed azide-alkyne cycloaddition, and the like), as described further below.

[0062] The number of bead and oligonucleotide molecules linked to each other within a single bead-linker complex can vary. For example, one oligonucleotide can be linked to one bead, or multiple oligonucleotides can be linked to one bead, or one oligonucleotide can be linked to multiple beads. The bead and oligonucleotide are linked to each other using conventional linking methods including any type of linker moiety or linking chemistry, including: NHS ester chemistry; click chemistry; or aldehyde/hydrazide chemistry. Any other suitable linking chemistry scheme can optionally be used.

[0063] In some embodiments, the linker of the bead- linker complex can be adapted to various functions and/or array fabrication procedures since it can be modified to include different functional moieties. For example, in some embodiments the linker can include at least one attachment moiety, reporter moiety, binding partner moiety, extension moiety, and/or

flexibility/rigidity moiety. For example, the linker can include at least one suitable attachment moiety (e.g., functional groups) that can optionally be cleaved upon suitable treatment. In another example, the linker can include at least one suitable attachment moiety for linkage to the bead, to the surface, to a target (e.g., target analyte) or to other compounds. In another example, the linkers can include at least one suitable attachment moiety for linkage to reporter moieties, extension moiety, and/or binding partners. In another example, the linkers can include moieties that replace one or more nucleosides and can alter the flexibility or rigidity of the linker. The rigidity moieties can include polyimide or phenyl units. In another example, the linker can include at least one extension moiety which serves as an extender. In another example, the linkers can include at least one suitable attachment moiety, binding partner, reporter moiety, extension moiety, and/or flexibility/rigidity moiety, in any combination thereof and in any arrangement along the linker and at any position on the linker (e.g., in the case of an oligonucleotide linker, at the 5' or 3' ends, or at any internal position within the oligonucleotide linker). In another example, if the linker includes a double- stranded nucleic acid portion (e.g., DNA), then the linker can include a sequence that is recognized for cleavage by a restriction endonuclease enzyme.

[0064] In some embodiments, the linker is a linear linker that comprises a first end and a second end. The first end of the linker can include an amino, NHS ester, alkyne, or aldehyde functional group for linkage to the bead, surface and/or target analyte. The second end of the linker can optionally include an amino or aldehyde functional group for linkage to the bead, surface and/or target analyte.

[0065] In some embodiments, the linker can include polyethylene glycol or polyethylene oxide units, which have a polymer coil volume in aqueous environments that permit the units to extend into the environment rather than curl/coil. In some embodiments, the linker can include about 1- 12 PEG or PEO units. The linker comprising the PEG or PEO units can be prepared by employing phosphoramidite chemistry. In another embodiment, an amino-derivatized solid phase can be used to link the PEG or PEO units to the linker molecule (see the method disclosed by Woo in U.S. Patent No. 5,625,052). The linker comprising the PEG or PEO units can also include an amine group (e.g., aminohexyl group) at the first or second end.

[0066] In one exemplary, the linker is an oligonucleotide linker and successive PEO units can be added to the oligonucleotide linker using a base-modified deoxyuridine phosphoramidite with a TFA-protected amine (e.g., LAN from Molecular Biosystems, as disclosed by Grossman in U.S. Patent No. 5,807,682). An oligonucleotide linker comprising PEG or PEO units can be prepared starting from the first or second end of the nucleic acid molecule using solid phase synthesis methods and/or employing nucleic acid synthesis equipment (e.g., ABI 394 DNA synthesizer).

[0067] The first or second end of the linker can be linked to the bead. The first or second end of the linker can be linked to the surface.

[0068] In some embodiments the linker includes an oligonucleotide comprising the sequence: 5'-(ATCG)-S-S-TAT-biotin-(PEO)_N -amine -3', where the "ATGC" can include any nucleotide sequence, and the "S-S" can include a thiol linker or a photocleavable linker, and "N" can be about 1-12 PEO units. In some embodiments, the oligonucleotide includes five PEO units.

[0069] In some embodiments, the linkers comprise at least one suitable linking group or chemical bond that attaches the linker to the: beads, reporter moieties, binding partners, flexibility/rigidity moieties, surfaces, and/or other compounds.

[0070] The suitable linkers, reporter moieties, binding partners, and flexibility/rigidity moieties do not interfere with the function or activity of the bead or the linker, or with each other. The suitable linkers can be selected to optimize proximity, length, distance, orientation, charge, or flexibility or rigidity.

[0071] The suitable linker can be linked to the bead, reporter moieties, surfaces, and/or other compounds, via covalent bonding, non-covalent bonding, ionic bonding, hydrophobic interactions, or any combination thereof. Examples of non-covalent attachment includes: ionic, hydrogen bonding, dipole-dipole interactions, van der Waals interactions, ionic interactions, and

hydrophobic interactions. In particular, examples of non-covalent attachment includes: nucleic acid hybridization, protein aptamer-target binding, electrostatic interaction, hydrophobic interaction, non-specific adsorption, and solvent evaporation.

[0072] The suitable linker can include a short, long, extended, or hydrophilic. The suitable linker can be rigid or flexible. The suitable linker can be linear, non-linear, branched,

bifunctional, trifunctional, homofunctional, or heterofunctional. Many cleavable, and bifunctional (both homo- and hetero-bifunctional) bead arms with varying lengths are available commercially. Some linkers have pendant side chains or pendant functional groups, or both. The suitable linker can be resistant to heat, salts, acids, bases, light, chemicals, or shearing forces or flow. The suitable linker can include multiple amino acid residues, such as a poly-arginine linker. [0073] The suitable linker can be a cleavable, self-cleavable, or fragmentable linker. The linker can be cleavable or fragmentable using temperature, enzymatic activity, chemical agent, and/or electromagnetic radiation. In some embodiments, suitable cleavage techniques can be used to reverse the attachment of the linker to the bead, surface and/or target analyte.

[0074] The linker of the bead-linker complex can optionally include at least one suitable cleavable linking group to permit release of the bead. The cleavable linker can include a disulfide, silyl, amide, thioamide, ester, thioester, vicinal diol, phosphoramidite, or hemiacetal group. Other cleavable bonds include enzymatically-cleavable bonds, such as peptide bonds (cleaved by peptidases), phosphate bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases).

[0075] The photo-cleavable linkers include nitrobenzyl derivatives, phenacyl groups, and benzoin esters. Analogs of the 2-nitrobenzyl linker, and other photocleavable linkers including: 2- nitrobenzyloxycarbonyl; nitroveratryl; 1-pyrenylmethyl; 6-nitroveratryloxycarbonyl;

dimethyldimethoxybenzyloxyc arbonyl ; 5 -bromo-7 -nitroindolinyl ; O -hydroxy- alpha-methyl- cinnamoyl; methyl-6-nitroveratryloxycarbonyl; methyl-6-nitropiperonyloxycarbonyl; 2- oxymethylene anthraquinone; dimethoxybenzyloxy carbonyl; 5-bromo-7 -nitroindolinyl; O- hydroxy- alpha- methyl cinnamoyl; and 2-oxymethylene anthriquinone (see: McGall, U.S. Patent No. 5,412,087; Pirrung, U.S. Patent No. 5,143,854; and Conrad, U.S. Patent No. 5,773,308). The photocleavable linkers can be illuminated with an electromagnetic source at about 320-800 nm, depending on the particular linker, to achieve cleavage. The self-cleaving linker can be a trimethyl lock or a quinone methide linker.

[0076] Many cleavable groups are known in the art. See for example, J.W. Walker, et al., 1997 Bioorg. Med. Chem. Lett. 7: 1243-1248; R. S. Givens, et al., 1997 Journal of the American

Chemical Society 119:8369-8370; R. S. Givens, et al., 1997 Journal of the American Chemical Society 119:2453-2463; Jung et al., 1983 Biochem. Biophys. Acta, 761: 152-162; Joshi et al., 1990 J. Biol. Chem., 265: 14518-14525; Zarling et al., 1980 J. Immunol., 124: 913-920; Bouizar et al., 1986 Eur. J. Biochem., 155: 141-147; Park et al., 1986 J. Biol. Chem., 261: 205-210;

Browning et al., 1989 J. Immunol., 143: 1859-1867; and Korlach, U.S. Patent No. 7,033,764. The cleavable linker can be a commercially-available linker.

[0077] In some embodiments, the photocleavable linker is a phosphoramidite (e.g., Glen Research, catalog #10-4920-xx). In another embodiment, the cleavable disulfide linker is a thiol modifier (e.g., Glen Research, catalog #10-1936-xx). [0078] The fragmentable linker can be capable of fragmenting in an electronic cascade self- elimination reaction (Graham, U.S. published patent application No. 2006/0003383; and Lee, U.S. published patent application No. 2008/0050780). In some embodiments, the fragmentable linker comprises a trigger moiety. The trigger moiety comprises a substrate that can be cleaved or "activated" by a specified trigger agent. Activation of the trigger moiety initiates a spontaneous rearrangement that results in the fragmentation of the linker and release of the enjoined molecules (e.g., bead and/or target analyte). For example, the trigger moiety can initiate a ring closure mechanism or elimination reaction. Various elimination reactions, include 1,4-, 1,6- and 1,8- elimination reactions.

[0079] Any means of activating the trigger moiety may be used. Selection of a particular means of activation, and hence the trigger moiety, may depend, in part, on the particular fragmentation reaction desired. In some embodiments, activation is based upon cleavage of the trigger moiety. The trigger moiety can include a cleavage site that is cleavable by a chemical reagent or enzyme. For example, the trigger moiety can include be a cleavage recognition site that is cleavable by a sulfatase (e.g., S0₃ and analogs thereof), esterase, phosphatase, nuclease, glycosidase, lipase, esterase, protease, or catalytic antibody.

[0080] In some embodiments, the linker of the bead-linker complex comprises about 1-100 plural valent atoms. In some embodiments, the linker comprises about 1-40 plural valent atoms, or more, selected from the group consisting of C, N, O, S and P.

[0081] In some embodiments, the linker of the bead-linker complex can include any

combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds or carbon-sulfur bonds. Exemplary linking members include a moiety that includes -C(0)NH-, -C(0)0-, -NH-, -S-, -0-, and the like. The linkers can include a combination of moieties selected from amine, alkyl, alkylene, aryl, -C(0)NH- -C(0)0- -NH-, -S-, -0-, -C(O)-, -S(0)_n- , where n is 0, 1, 2, 3, 4, 5, or 6- membered monocyclic rings and optional pendant functional groups, for example sulfo, hydroxy and carboxy.

[0082] The linker can include a pendant side chain or pendant functional group, or both.

Examples of pendant moieties include hydrophilicity modifiers, for example solubilizing groups such as sulfo (-SO₃H- or -SO - ). The trifunctional linker can be linked to multiple reporter moieties (the same or different reporter moieties) for dendritic amplification of the signal emitted by the reporter moieties (Graham, U.S. published patent application Nos. 2006/0003383 and 2007/0009980).

[0083] The linker can include a rigid polymer which can be used, for example, to improve a FRET signal by optimizing the orientation of the energy transfer dye. Examples of rigid linkers include benzyl linkers, proline or poly-proline linkers (S. Flemer, et al., 2008 Journal Org. Chem. 73:7593-7602), bis-azide linkers (M.P.L. Werts, et al., 2003 Macromolecules 36:7004-7013), and rigid linkers synthesized by modifying the so-called "click" chemistry scheme that is described by Megiatto and Schuster (2008 Journal of the Am. Chem. Soc. 130: 12872-12873). For example, click chemistry can include azide alkyne Huisgen cycloaddition or alkynyl linkage.

[0084] The suitable linker can be capable of energy transfer, such as those disclosed by Ju in U.S. published patent application No. 2006/0057565.

[0085] In some embodiments, the linker of the bead- linker complex can include an NHS ester linkage, such as one provided by an NHS-carboxy-dT compound (e.g., Glen Research, catalog # 10-1535-xx). In another embodiment, the linker can include an aldehyde linkage, such as one provided by a 5-formylindole-CE phosphoramidite (e.g., Glen Research, catalog #10-1934-xx) or a 5 '-aldehyde-modifier C2 phosphoramidite compound (e.g., Glen Research, catalog #10-1933- xx). In yet another embodiment, the linker can include an alkynyl linkage such as one provided by a 5'-hexynyl phosphoramidite compound (e.g., Glen Research, catalog # 10-1908-xx). The alkynyl linkage can be used for click chemistry.

[0086] In another embodiment, the linker of the bead-linker complex is an oligonucleotide linker, and the 5' end of the oligonucleotide linker can include an amino group, such as one provided by a 3 '-amino-modifier C7 CPG 500 compound (e.g., Glen Research, catalog #20-2957- xx). In another embodiment, the 3' end of the oligonucleotide linker can include an amino group, such as one provided by a 5 '-amino-modifier C6 compound (e.g., Glen Research, catalog #10- 1906-xx).

[0087] In some embodiments, the linker can include non-natural nucleotides having a reactive group that will attached, bind or otherwise react with another reactive group located, for example, on the surface, the target analyte and/or the bead. For example, the non-natural nucleotides include peptide nucleic acids, locked nucleic acids, oligonucleotide phosphoramidates, and oligo- 2 ' - O- alkylribonucleotide s .

[0088] In some embodiments, the linker can be modified with one or more amino groups at the first or second ends, or internally, for attachment to modified surfaces. The amino group at the first end of the linker can optionally include: a simple amino group; a short or long tethering arm having one or more terminal amino groups; or an amino-modified thymidine or cytosine. The amino group at the second end of the linker can initially be protected by a

fluorenylmethylcarbamoyl (Fmoc) group. To expose the amino group, the protecting group can be removed and acylated with an appropriate succinimidyl ester, such as an N-hydroxy succinimidyl ester (NHS ester). In another embodiment, the oligonucleotide linker can include one or more internal amino groups for binding to the surface. For example, 2' amino modified oligonucleotide linkers can be produce by methoxyoxalamido (MOX) or succinyl (SUC) chemistry to produce nucleotide analogs having amino linkers attached at the 2' C of the sugar moiety.

[0089] In another embodiment, the linker can include succinylated nucleic acids which can be attached to aminophenyl- or aminopropyl-modified surfaces (B. Joos et al., 1997 Anal. Biochem. 247: 96-101).

[0090] In another embodiment, the linkers can include a thiol group which is placed at the first or second end of the linker. The thiol group can form reversible or irreversible disulfide bonds with the surface, bead and/or target analyte. For example, the thiol group can be attached to the 5' or 3' end of an oligonucleotide linker and can optionally include a phosphoramidate. The phosphoramidate can be attached to the 5' end of the oligonucleotide linker using S-trityl-6- mercaptohexyl derivatives.

[0091] In another embodiment, the linker can be reacted with modifying reagents such as:

carbodiimides (e.g., dicyclohexylcarbodiimide, DCC), carbonyldiimidazoles (e.g.,

carbonyldiimidazole, CDIz), or potassium periodate.

[0092] In another embodiment, the linker can have protective photoprotective caps (Fodor, U.S. Patent No. 5,510,270) capped with a photoremovable protective group. DMT-protected oligonucleotides can be immobilized to the surface via a carboxyl bond to the 3' hydroxyl of the nucleoside moiety (Pease, U.S. Pat. No. 5,599,695; Pease et al., 1994 Proc. Natl. Acad. Sci. USA 91:5022-5026).

[0093] In yet another embodiment, the oligonucleotides can be functionalized at their 5' ends with activated 1-O-mimethoxytrityl hexyl disulfide l'-[(2-cyanoethyl)-N,N-diisopropyl)] phosphoramidate (Rogers et al., 1999 Anal. Biochem. 266:23).

[0094] Any suitable linking chemistry scheme can be used to generate reactive groups for linking together the bead, linker (e.g., cleavable linker), target analyte, binding partners, reporter moieties, flexibility/rigidity moieties, or other compounds, and/or surfaces, in any combination and in any order. Typically, the reactive groups include: amine, aldehyde, hydroxyl, sulfate, carboxylate groups, and others.

[0095] For example, reacting activated esters, acyl azides, acyl halides, acyl nitriles, or carboxylic acids with amines or anilines to form carboxamide bonds. Reacting acrylamides, alkyl halides, alkyl sulfonates, aziridines, haloacetamides, or maleimides with thiols to form thioether bonds. Reacting acyl halides, acyl nitriles, anhydrides, or carboxylic acids with alcohols or phenols to form an ester bond. Reacting an aldehyde with an amine or aniline to form an imine bond. Reacting an aldehyde or ketone with a hydrazine to form a hydrazone bond. Reacting an aldehyde or ketone with a hydroxylamine to form an oxime bond. Reacting an alkyl halide with an amine or aniline to form an alkyl amine bond. Reacting alkyl halides, alkyl sulfonates, diazoalkanes, or epoxides with carboxylic acids to form an ester bond. Reacting an alkyl halides or alkyl sulfonates with an alcohol or phenol to form an ether bond. Reacting an anhydride with an amine or aniline to form a carboxamide or imide bond. Reacting an aryl halide with a thiol to form a thiophenol bond. Reacting an aryl halide with an amine to form an aryl amine bond.

Reacting a boronate with a glycol to form a boronate ester bond. Reacting a carboxylic acid with a hydrazine to form a hydrazide bond. Reacting a carbodiimide with a carboxylic acid to form an N-acylurea or anhydride bond. Reacting an epoxide with a thiol to form a thioether bond.

Reacting a haloplatinate with an amino or heterocyclic group to form a platinum complex.

Reacting a halotriazine with an amine or aniline to form an aminotriazine bond. Reacting a halotriazines with an alcohol or phenol to form a triazinyl ether bond. Reacting an imido ester with an amine or aniline to form an amidine bond. Reacting an isocyanate with an amine or aniline to form a urea. Reacting an isocyanate with an alcohol or phenol to form a urethane bond. Reacting an isothiocyanate with an amine or aniline to form a thiourea bond. Reacting a phosphoramidate with an alcohol to form a phosphite ester bond. Reacting a silyl halide with an alcohol to form a silyl ether bond. Reacting a sulfonate ester with an amine or aniline to form an alkyl amine bond. Reacting a sulfonyl halide with an amine or aniline to form a sulfonamide bond.

[0096] The linking chemistry scheme can include "click" chemistry schemes (Gheorghe, et al., 2008 Organic Letters 10:4171-4174).

[0097] The suitable linking scheme can include reacting the components to be linked in a suitable solvent in which both are soluble. Water-insoluble substances can be chemically modified in an aprotic solvent such as dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform. Similar modification of water-soluble materials can be accomplished using reactive compounds to make them more readily soluble in organic solvents.

[0098] In some embodiments, the linking reaction between the bead, linker (or molecule) and/or the surface can be inducible, i.e., designed to occur upon the occurrence of a defined triggering event or upon a defined change in environmental conditions. For example, the linking scheme can be photoinducible, wherein the linking reaction is triggered by exposure to electromagnetic radiation of a particular wavelength, or chemically inducible, when the linking reaction is triggered by the presence of a certain catalyst or by a certain set of chemical changes in the environment. In some embodiments, the linking reaction is pH-inducible, and can be triggered or induced to occur by simply flushing the system with different pH solutions. In one exemplary embodiment, the linking reaction between the linker of the bead-linker complex and the surface is pH-inducible. The surface includes an NHS ester and the linker of the bead-linker complex includes a terminal amine that is non-reactive with the NHS ester at pH values less than about 5- 5.5, but is reactive with the NHS ester group at pH values greater than about 8-8.5. The bead- linker complex is contacted with the surface at low pH conditions. The system can optionally be centrifuged to deposit the bead-linker complexes onto the surface, and then excess bead-linker complexes can be rinsed away. The surface including deposited bead-linker complexes can then be placed into a fresh solution having a pH of about 8.5, whereupon the terminal amine of the linker of each deposited bead-linker complex reacts with the surface NHS ester to covalently link the deposited complex to the surface.

[0099] In some embodiments, polymers of ethylene oxide (EO) can be used to attach the bead, linker, binding partners, reporter moieties, target analyte, flexibility/rigidity moieties, or other compounds, and/or surfaces, to each other in any combination. Examples of polymers of ethylene oxide include, but are not limited to: polyethylene glycol (PEG), such as short to very long PEG; branched PEG; amino-PEG-acids; PEG-amines; PEG-hydrazines; PEG-guanidines; PEG-azides; biotin-PEG; PEG-thiols; and PEG-maleinimides. In some embodiments, PEG includes: PEG- 1000, PEG-2000, PEG-12-OMe, PEG-8-OH, PEG-12-COOH, and PEG-12-NH₂.

[00100] In one exemplary embodiment, the surface can include one or more capture probes capable of binding selectively to the linker of the bead linker complex. For example, the surface may comprise capture probes including nucleic acid, the linker of the bead-linker complex is an oligonucleotide linker, and the capture probe can bind, e.g., via Watson-Crick base pairing, with the oligonucleotide linker. In some embodiments, the capture probe is a nucleic acid molecule complementary to some or the entire nucleic acid portion of the oligonucleotide linker. In some embodiments, a nucleic acid array is formed by contacting a population of bead-linker complexes with a surface, the surface including a capture probe, and the bead-linker complexes including an oligonucleotide linker, where the contacting is performed under conditions where the capture probe hybridizes to the oligonucleotide linker, thereby attaching the bead-linker complex to the surface. Optionally, the linkage between the oligonucleotide linker and the bead of the bead-linker complex is then cleaved, releasing the bead and leaving an oligonucleotide linker that is attached to the surface via hybridization to the capture probe. In some embodiments, the 5' or 3' end of the oligonucleotide linker can hybridize to the capture probe. Optionally, the surface can include one or more cavities, e.g., pores, wells or sockets, and the capture probe is present at the bottom of the cavity. The bead of the bead-linker complex can be sized to ensure that only one bead can enter the cavity at any given time, thus ensuring that only a single bead can enter the well at a given time and will exclude other beads from entering the well.

[00101] The capture probes can include oligonucleotide clamps (U.S. Pat. No. 5,473,060).

The parameters for selecting the length and sequence of the capture probes are well known (Wetmur 1991 Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259; Britten and Davidson, chapter 1 in: Nucleic Acid Hybridization: A Practical Approach, Hames et al, editors, IRL Press, Oxford, 1985). The length and sequence of the capture probes may be selected for sufficiently stability during low and/or high stringency wash steps. The length of the capture probes ranges from about 6 to 50 nucleotides, or from about 10 to 24 nucleotides, or longer.

[00102] The capture probes can be immobilized to the surface via a single or multiple biotin/avidin interactions. In some embodiments, a dual oligonucleotide can be used to

immobilize the capture probe and the oligonucleotide linker of the bead-linker complex to the surface. The 5' or 3' end of the oligonucleotide or capture probe can be linked to a biotin molecule. The surface can be linked to avidin-liked molecules (e.g., avidin). The avidin molecules are capable of binding up to four biotin molecules, permitting stable binding of a biotin end-labeled duplex (e.g., capture probe/oligonucleotide) (Buzby, U.S. Patent No. 7,220,549).

[00103] In some embodiments, the surface can be modified to bind amino-modified linkers. For example, 5' amino-modified linkers can be attached to surfaces modified with silane, such as epoxy silane derivatives (J. B. Lamture, et al., 1994 Nucleic Acids Res. 22:2121-2125; W. G. Beattie et al., 1995 Mol. Biotechnol. 4:213-225) or isothiocyanate (Z. Guo, et al., 1994 Nucleic Acids Res. 22:5456-5465). Acylating reagents can be used to modify the surface for attaching the amino-modified linkers. The acylating reagents include: isothiocyanates, succinimidyl ester, and sulfonyl chloride. In some embodiments, the amino-modified linkers can attach to surface amino groups which have been converted to amino reactive phenylisothiocyanate groups by treating the surface with p-phenylene 1,4 diisothiocyanate (PDC). In other methods, the surface amino groups can be reacted with homobifunctional crosslinking agents, such as disuccinimidylcaronate (DCS), disuccinimidyloxalate (DSO), phenylenediisothiocyanate (PDITC) or

dimethylsuberimidate (DMS) for attachment to the amino-modified linkers. In another example, metal and metal oxide surfaces can be modified with an alkoxysilane, such as 3- aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GOPMS).

[00104] In another embodiment, the surface can be treated with an alkylating agent such as iodoacetamide or maleimide for linking with thiol-modified nucleic acid molecules.

[00105] In another embodiment, thiol-modified linkers can be attached to silane-treated surfaces (e.g., glass) using succinimidyl 4-(malemidophenyl)butyrate (SMPB).

[00106] In another embodiment, thiol-modified surfaces can be used to attach linkers carrying disulfide groups (Y. H. Rogers et al., 1999 Anal. Biochem. 266:23-30).

[00107] In yet another embodiment, the surface can be coated with a polyelectrolyte multilayer (PEM) via light-directed attachment (U.S. Patent Nos. 5,599,695, 5,831,070, and 5,959,837) or via chemical attachment. The PEM chemical attachment can occur by sequential addition of polycations and polyanions (Decher, et al., 1992 Thin Solid Films 210:831-835). In some embodiments, the glass surface can be coated with a polyelectrolyte multilayer which terminated with polyanions or polycations. The polyelectrolyte multilayer can be coated with biotin and an avidin-like compound. Biotinylated molecules (nucleic acid molecules or nanocrystals) can be attached to the PEM/biotin/avidin coated surface (Quake, U.S. Patent Nos. 6,818,395, 6,911,345, and 7,501,245).

[00108] In still another embodiment, the surface can be coated with a compound that increases electrostatic interaction between the surface and linkers or capture probes. The surface can be coated with poly-D-lysine or 3-aminopropyltriethoxysilane (Schwartz, U.S. Patent Nos.

6,221,592, 6,294,136; and Schwartz, U.S. published patent application Nos. 2006/275806 and 2007/0161028).

[00109] The surface can be coated with one or more linking agents, including: symmetrical bifunctional reagents, such as bis succinimide (e.g., bis-N-hydroxy succinimide) and maleimide (bis-N-hydroxy maleimide) esters, or toluene diisocyanate. The linking agents can be heterobifunctional cross-linkers including: m-maleimido benzoyl-N-hydroxy succinimidyl ester (MBS); succinimidyl-4-(p-maleimido phenyl)-Butyrate (SMPB); and succinimidyl-4-(N- Maleimidomethyl)Cyclohexane-l-Carboxylate (SMCC) (L. A. Chrisey et al, 1996 Nucleic Acids Res. 24:3031-3039).

[00110] In one example, a glass surface can be coated with a layer of gold (e.g., about 2 nm thickness). The gold can be reacted with mercaptohexanoic acid. The mercaptohexanoic acid can be placed in a patterned array. The mercaptohexanoic acid can be reacted with PEG. The PEG can be reacted to bind the oligonucleotides. Any of these procedures can be used to link the surface to the linkers (or capture probes).

[00111] In some embodiments, the linker of the bead linker complex can be linked to at least one reporter moiety. The reporter moiety can optionally generate, or cause to be generated, a detectable signal. The reporter moiety can be used to locate the linker (e.g., locate the surface- attached linker on the surface).

[00112] Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent (including energy transfer), phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. The reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other.

[00113] The reporter moieties may be selected so that each absorbs excitation radiation and/or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction. Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles.

[00114] In one aspect, the signals from the different reporter moieties do not significantly overlap or interfere, by quenching, colorimetric interference, or spectral interference.

[00115] In some embodiments, the reporter moiety includes a chromophore moiety. The chromophore moiety may be 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, or p- nitrophenyl phosphate, and derivatives thereof.

[00116] In some embodiments, the reporter moiety includes a chemiluminescent moiety. The chemiluminescent moiety may be a phosphatase-activated 1,2-dioxetane compound. The 1,2- dioxetane compound includes disodium 2-chloro-5-(4-methoxyspiro[l,2-dioxetane-3,2'-(5-chloro-

)tricyclo[3,3,l-l 3 ' 7 ]-decan]-l-yl)-l -phenyl phosphate (e.g., CDP-STAR) , chloroadamant-2'- ylidenemethoxyphenoxy phosphorylated dioxetane (e.g., CSPD) , and 3-(2'-spiroadamantane)-4- methoxy-4-(3"-phosphoryloxy)phenyl-l,2-dioxetane (e.g., AMPPD).

[00117] In some embodiments, the reporter moiety includes a fluorescent moiety. The fluorescent moiety optionally includes one or more of the following fluorescent moieties: rhodols; resorufins; coumarins; xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins;

phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles; stilbenes; squarenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones; eosin; erythrosin; Malachite green; CY dyes (GE Biosciences), including Cy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS and DYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701, DY-734, DY-752, DY- 777 and DY-782; Lucifer Yellow; CASCADE BLUE ; TEXAS RED; BODIPY (boron- dipyrromethene) (Molecular Probes) dyes including BODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO 390, ATTO 425, ATTO 465, ATTO 610 61 IX, ATTO 610 (N-succinimidyl ester), ATTO 635 (NHS ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXA FLUOR 680 (Molecular Probes); DDAO (7-hydroxy-9H-(l,3-dichloro- 9,9-dimethylacridin-2-one or any derivatives thereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes (LiCor) including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester) and IRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes (Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED (Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED (Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED (phosphoramidate), EPOCH YAKIMA YELLOW (phosphoramidate), EPOCH GIG HARBOR GREEN (phosphoramidate); Tokyo green (M. Kamiya, et al., 2005 Angew. Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647 and CF555 (Biotium).

[00118] In some embodiments, the fluorescent moiety can be a quencher dye, including: ATTO 540Q, ATTO 580Q, and ATTO 612Q (Atto-Tec); QSY dyes including QSY 7, QSY 9, QSY 21, and QSY 35 (Molecular Probes); and EPOCH ECLIPSE QUENCHER (phosphoramidate) (Glen Research). The fluorescent moiety can be a 7-hydroxycoumarin-hemicyanine hybrid molecule which is a far-red emitting dye (Richard 2008 Org. Lett. 10:4175-4178). [00119] The fluorescent moiety may be a fluorescence-emitting metal such as a lanthanide complex, including those of Europium and Terbium.

[00120] A number of examples of fluorescent moieties are found in PCT publication

WO/2008/030115, and in Haugland, Molecular Probes Handbook, (Eugene, Oregon) 6th Edition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999).

[00121] In some embodiments, the linker of the bead-linker complex can be linked to an energy transfer donor and/or to an energy transfer acceptor moiety. For example, the energy transfer donor can be a nanocrystal (e.g., quantum dot) or fluorescent dye. In another example, the energy transfer acceptor moiety can be a fluorescent dye.

[00122] Typically, the energy transfer donor is capable of absorbing electromagnetic energy (e.g., light) at a first wavelength and emitting excitation energy in response. The energy acceptor is capable of absorbing excitation energy emitted by the donor and fluorescing at a second wavelength in response.

[00123] The energy transfer donor and acceptor moieties can interact with each other physically or optically in a manner that produces a detectable signal when the two moieties are in proximity with each other. A proximity event includes two different moieties (e.g., energy transfer donor and acceptor) approaching each other, or associating with each other, or binding each other.

[00124] The donor and acceptor moieties can transfer energy in various modes, including:

fluorescence resonance energy transfer (FRET) (L. Stryer 1978 Ann. Rev. Biochem. 47: 819- 846), scintillation proximity assays (SPA) (Hart and Greenwald 1979 Molecular Immunology 16:265-267; U.S. Pat. No. 4,658,649), luminescence resonance energy transfer (LRET) (G. Mathis 1995 Clin. Chem. 41: 1391-1397), direct quenching (Tyagi et al, 1998 Nature Biotechnology 16:49-53), chemiluminescence energy transfer (CRET) (Campbell and Patel 1983 Biochem.

Journal 216: 185-194), bioluminescence resonance energy transfer (BRET) (Y. Xu, et al., 1999 Proc. Natl. Acad. Sci. 96: 151-156), and excimer formation (J. R. Lakowicz 1999 "Principles of Fluorescence Spectroscopy", Kluwer Academic/Plenum Press, New York).

[00125] In one aspect, the energy transfer pair (donor and acceptor) can be FRET donor and acceptor moieties. FRET is a distance-dependent radiationless transmission of excitation energy from a donor moiety to an acceptor moiety. Typically, the efficiency of FRET energy

transmission is dependent on the inverse sixth-power of the separation distance between the donor and acceptor, which is approximately 10-100 Angstroms. FRET is useful for investigating changes in proximity between and/or within biological molecules. FRET efficiency may depend on donor-acceptor distance r as 1/r⁶. The distance where FRET efficiency is 50% is termed Ro, also known as the Forster distance. R₀ is unique for each donor-acceptor combination and may be about 5 to 10 nm. The efficiency of FRET energy transfer can sometimes be dependent on energy transfer from a point to a plane which varies by the fourth power of distance separation (E. Jares- Erijman, et al., 2003 Nat. Biotechnol. 21: 1387).

[00126] In some embodiments, the disclosure relates generally to bead-linker complexes comprising one or more oligonucleotide linkers linked to at least one bead. In some embodiments, the one or more linkers can be linked to the at least one bead through a cleavable bond. The linker can optionally include one or more members of binding partner pair, reporter moiety, and/or flexibility/rigidity moiety, in any combination thereof and in any arrangement on the adaptors. In some embodiments, the oligonucleotide linker comprises a 5' end and a 3' end; either end can optionally include a suitable linking moiety for attachment to the surface. In another embodiment, the linker is an oligonucleotide linker and either the 5' or 3' end of the oligonucleotide linker (or both the 5' and 3' ends) can be linked to the surface.

[00127] In some embodiments, the oligonucleotide linker of the bead- linker complex can include (listed in the 5' to 3' order): a reporter moiety, a cleavable moiety, a binding partner, and a 3' amino or aldehyde group. In another embodiment, the oligonucleotide linker of the bead- linker complex can include (listed in the 5' to 3' order): a 5' functional group (e.g., amino, NHS ester, alkyne, or aldehyde), binding partner, cleavable moiety, and reporter moiety. One skilled in the art will appreciate that other types of cleavable moieties, binding partners, reporter moieties, and/or linking moieties, are possible in any combination and in any order.

[00128] In one exemplary embodiment involving bead-linker complexes comprising an oligonucleotide linker, as depicted in Figure 9A, an array of single stranded nucleic acid templates is fabricated via deposition of bead- linker complexes onto a surface. Each bead- linker complex optionally comprises a single spherical bead linked to at least one linker that includes a surface attachment moiety and a template attachment moiety. The surface can optionally be

functionalized. Each bead-linker complex optionally deliver a single linker to a discrete location on the surface. A population of bead-linker complexes can be contacted with the surface under conditions where the at least one linker of the complex binds covalently to the surface. The beads of each complex can then be cleaved away to reveal an array of surface-attached linkers, each such surface-attached linker including a template-attachment moiety. The average distance between any two adjacent surface- attached linkers is typically at least about the diameter of the bead. The array of surface-attached linkers can then be contacted with a population of nucleic acid templates in solution under conditions where one or more templates binds to a surface-attached linker, but no two templates bind to the same linker.

[00129] Some optional features of this exemplary embodiment are further depicted in Figure 9B. For example, the linker of the bead-linker complex can include the following moieties along its length: a reactive terminal amine, a branched alkyl chain comprising a first end, a second end and a third end, the first end including the reactive terminal amine, the second end including a dye label or a biotin moiety, and the third end including a cleavable disulfide linkage and an oligonucleotide linker, the oligonucleotide linker including a first end and a second end, the first end of the oligonucleotide linker being attached to the third end of the branched alkyl chain through the cleavable disulfide linkage, and the second end of the oligonucleotide linker being attached to the bead of the bead- linker complex. Optionally, the functionalized surface includes a PEG moiety including a terminal reactive NHS ester that can react with the terminal amine of the linker to covalently link the bead-linker complex to the surface.

[00130] An exemplary method of using this exemplary bead-linker complex to fabricate an oligonucleotide linker array are depicted in Figure 10. The bead is first linked to at least one linker of the type depicted in Figure 9B including a reactive terminal amine, a branched alkyl chain, a dye label or biotin moiety, a cleavable disulfide linkage and an oligonucleotide linker to form a cleavable bead- linker complex. The bead-linker complex can be contacted with a functionalized surface including a PEG moiety having a terminal reactive NHS ester under conditions where the reactive terminal amine of the linker reacts with the NHS ester of the surface, forming an amide linkage that covalently links the bead-linker complex to the surface. The cleavable disulfide linkage can be cleaved via treatment with dithiothreitol (DTT) to release the bead and leave a surface- attached linker. The bead is optionally washed away, and the surface- attached linker can then be ligated or alternatively hybridized to a template to form a surface- attached template.

[00131] The structure of another exemplary linker that can be used according to the disclosure is depicted in Figure 11. The linker includes can include (listed in the 5' to 3' order): A terminal phosphate group, an oligonucleotide or other nucleic acid sequence (depicted in Figure 11 as the exemplary nucleic acid sequence 5'-GATTGTCAGATACAC-3'), a cleavable bond (depicted in Figure 11 as an exemplary cleavable disulfide linkage), a second nucleic acid (depicted in Figure 11 as the exemplary nucleic acid sequencing 5'-ATT-3'), a rigid polymer (depicted in Figure 11 as a polymer including one or more HEO units) and a 3' terminal amine. The 5' terminal phosphate can optionally be linked to the bead using any suitable linking methodology, and the 3' terminal amine can optionally be linked to the surface, e.g., via reaction with an NHS ester present on the surface.

[00132] The disclosed methods, compositions systems and apparatuses permit the use of bead- linker complexes to deliver single molecules to be arrayed (or, alternatively, single linkers that, once arrayed, can bind to target molecules to be arrayed) onto a surface. Three of the key variables that affect the array density and uniformity (and thus the level of throughput that can be achieved) include the average bead size, the average number of linkers present per bead, and the length of the rigid polymer included in the linker, if any. In some embodiments, the average bead size can be not greater than about 1000 nm (1 micron), 750 nm (0.75 micron) or 500 nm (0.5 micron). In some embodiments, the population of bead-linker complexes comprises an average of about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 250, 500, 750, 1000, 5000 or 10,000 linkers per bead. In some embodiments, the linker includes a rigid polymer including at least about 1, 2, 3, 4, 5, 7, 10, 15, 20, 25 or 50 units of EO, PEO, HEO or ethylene glycol.

[00133] In some embodiments, the disclosure relates generally to surfaces (e.g., solid surfaces) that can be linked to one or more linkers, molecules or other moieties (e.g., reporter moieties, energy transfer moieties, nanocrystals, proteins, etc.) using the linking methodologies described herein. The surface attached linkers can be attached to the surface at the first end, second end, along their length, or along their length with a first or second end exposed.

[00134] In some embodiments, the linkers of the bead-linker complexes can be attached to the surface in a manner that renders them resistant to removal or degradation during any particular environment conditions likely to be encountered in array-based analysis, including procedures that involve washing, flowing, temperature or pH changes, and reagent changes. In another aspect, the linkers of the bead-linker complexes and other moieties can be reversibly attached to the surface.

[00135] The surface may be a solid surface, and includes planar surfaces, as well as concave, convex, or any combination thereof. The surface may comprise texture (e.g., etched, cavitated or bumps). The surface includes a nanoscale device, a channel, a well, bead, particle, sphere, filter, gel, or the inner walls of a capillary. The surface can be optically transparent, minimally reflective, minimally absorptive, or exhibit low fluorescence. The surface may be non-porous. The surface may be made from materials such as glass, borosilicate glass, silica, quartz, fused quartz, mica, sapphire, polyacrylamide, plastic (e.g., polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics, silicon, semiconductor, high refractive index dielectrics, crystals, gels, polymers, or films (e.g., films of titanium, gold, silver, aluminum, or diamond).

[00136] Optionally, the surface can be treated or modified to include one or more functional groups that will facilitate attachment of the linkers, molecules or other members of the array to the surface. Optionally, the functionalized surface can include a reactive group capable of reacting with a second group in the molecule, linker or other member to be arrayed. The reactive group can optionally include a reactive amine, aldehyde, azide, alkyne, NHS ester group, and the like.

[00137] In one exemplary embodiment, the surface of a glass slide can be functionalized by exposing the patterned slide to a solution 0.2 mM poly(ethylene glycol) phosphonic acid in ethanol to passivate a titanium portion of the slide; removing the patterned slide and rinsing with ethanol and deionized water; functionalizing a plurality of exposed glass sockets by soaking the patterned slide in an aqueous solution of 5 mM zirconium acetylacetonate to form functionalized sockets; and exposing functionalized sockets in a 20 mM aqueous solution of 2- aminoethylphosphonic acid to define a plurality of amine-functionalized sockets surrounded by PEG passivated titanium. The surface can optionally be further treated by soaking the amine- functionalized sockets in a solution of 0.1 M glutaric anhydride, 10 mM ( para-N,N'- dimethylpyridin) DMAP and 20 mM diisopropylethylamine (DIEA) in anhydrous toluene; rinsing the slide with toluene and iso-propanol before drying the slide with nitrogen gas; soaking the dried slide in a solution of 0.5M TSTU and 0.25M DIEA in DMSO; and rinsing the slide with 1 mM HC1 and blow drying with nitrogen to produce NHS ester linking groups on the surface to form an ester linking group on at least one of the amine-functionalized sockets. The resulting

functionalized surface can be used for attachment of molecules, linkers or other members to be arrayed via reaction with the amine functional groups of the functionalized surface.

[00138] Some of the disclosed embodiments relate to arrays of surface-attached linkers, which can be prepared by employing bead-linker complexes, each such complex including at least one linker, to generate an array of linkers attached to the surface. Multiple bead-linker complexes can be used to deliver multiple linkers to the surface, thereby forming a linker array. Optionally, the surface-attached linkers can be further linked to target analytes, thereby forming an array of surface-attached analytes, each such analyte being tethered to the surface through a surface- attached linker. In some embodiments, each bead- linker complex delivers a linker to a particular site/location on the surface. Figure 6 discloses an exemplary embodiment involving the use of bead-linker complexes comprising oligonucleotide linkers to generate an oligonucleotide array.

[00139] In some embodiments, the arrangement and/or average distance between the linkers of the array can be facilitated by the size dimensions of the bead of the bead-linker complex. For example, the diameter of the bead can be selected to ensure that the linkers of the array are spaced apart at a desired average distance in the array. Thus, the distances between the rows or columns (e.g., pitch) of surface- attached linkers can be adjusted by using larger or smaller beads. For example, beads having dimensions of about 1-5 micron diameter can be used to deliver the linkers to the surface. In some embodiments, the average distance between any two linkers in the array is sufficient to ensure resolution of the individual linkers using any suitable detection system (including, e.g., optical and/or chemical detection systems).

[00140] The immobilized linkers may be arranged in a random, semi-ordered or ordered array on a surface. The ordered array can include rectilinear and hexagonal patterns. The surface can be uncoated or coated with an adhesive and/or resist layer which can be applied to the surface to create a patterned array. The linkers can be linked to the regions of the patterned array that include a functional linking moiety that bind to a functional linking moiety on the linker.

[00141] In the array, multiple linkers can be immobilized onto the surface. Each of the multiple immobilized linkers can optionally bind, capture, or react with at least one biomolecule (e.g., nucleic acid molecules, polypeptides, carbohydrates, lipids, or reagents or compounds).

Accordingly, an array of biomolecules can be prepared by binding, capturing, or reacting, the biomolecules with the multiple immobilized linkers.

[00142] In some embodiments, the bead- linker complex includes a linker attached to a bead via a cleavable bond. The cleavable bond can be cleaved to release the bead from the complex, leaving a surface-attached linker. In some embodiments, a population of bead-linker complexes can be employed, and cleavage and release of the beads can reveal an array of surface-attached linkers, which can optionally be used to bind other biomolecules or nanocrystals (e.g., quantum dots) and link these to the surface. In one exemplary embodiment, the linkers include single- stranded nucleic acid portions that can be used to capture (e.g., via hybridization) other nucleic acid molecules, such as target molecules for sequencing (e.g., single molecule sequencing). In another embodiment, the immobilized linkers include nucleic acid portions (single- or double-stranded) that can be used to bind to other biological molecules (e.g., polypeptides or antibodies), or bind chemical compounds, or bind drugs (e.g., candidate drugs), or bind to non-biological compounds. [00143] In some embodiments, the disclosure relates to arrays of beads, linkers, nanocrystals, protein, reporter moieties, or energy transfer moieties. Some embodiments include a surface (e.g., transparent surface) coated with a layer of metal. The metal can include an array of features (e.g., spots). The features can be produced using lift-off or etching technology. For example, Figure 3 shows a metal array of spots. In another example, Figure 5 shows a metal array of spots loaded with nanocrystals (i.e., dots).

[00144] One skilled in the art will appreciate that the spots can be arranged in any pattern, including columns and rows of spots (e.g., Figure 3). The spots on the array can be arranged to reduce the amount of light or signal cross-talk from a neighboring spot, or to reduce artifacts from the edge of the surface. One skilled in the art will know how to optimize the edge-to-edge spot separation distances. In another embodiment, the array can include any number of spots.

[00145] In another embodiment, the array can include any number of rows and columns of spots (Figure 3). The rows of spots can span X distance. The row of spots can span about 50 - 300 μιη, or up to millimeter distances, but other distances are possible. For example, the row of spots can span about 50- 100 μιη, or about 80 μιη. The column of spots can span Y distance. The column of spots can span about 20 - 300 μιη, or up to millimeter distances, but other distances are possible. For example, the column of spots can span about 20 - 100 μιη, or about 20 - 50 μιη, or about 24 μιη.

[00146] In another embodiment, any number of spots can be included in the rows and columns. The distance spanned by the spots in a row or column (including pitch distance between the spots), the total number of spots (e.g., n in Figure 3), and/or the size and number of the spots, can be dictated by optical constraints including imaging and resolution capabilities, laser spot size, and/or pixel sizes, of the optical set-up to be used to visualize the spots. For example, the array in Figure 3 includes 7 rows and 17 columns of spots, but other arrangements are possible. In another embodiment, some of the spot positions can be blank (e.g., Figure 3). In another embodiment, the blank spots can be in any position on the array and arranged in any shape (e.g., Figure 3). For example, the blank spots can be arranged in the shape of a cross having 2n-l spots (Figure 3), but other arrangements are possible. The blank spots can serve as a guide for orienting or aligning the array, for example aligning the spots on a microscope stage (e.g., a detection element). The array can include spots which are loaded with beads, linkers (e.g., oligonucleotide linkers), nanocrystals, protein, reporter moieties, or energy transfer moieties, or any combination thereof. [00147] In some embodiments, the surface can include one or more physical features that permit manipulation and/or analysis, of biological molecules at a microscale or nanoscale level. The microscopic features can be at the micro meter size level, nano meter size level, or pico meter size level, or smaller sized levels. The microscopic features can be prepared from organic and/or inorganic compounds.

[00148] The microscopic features can optionally include one or more of the following features: spots, channels, slits, cavities, pores, pillars, or loops. The microscopic features can have length, width, and height dimensions. The microscopic features can be linear or branched shaped, and/or be attached to inlet and/or outlet ports. The branched microscopic features can form a T or Y junction, or other shape and geometries.

[00149] The microscopic features can be used for delivering, binding, holding, confining, sorting, separating, enriching, mixing, reacting, streaming, flowing, washing, flushing, elongating, stretching, flushing, or washing the beads or oligonucleotides, or reagents that react with the beads or oligonucleotides.

[00150] The surface can have a coating, such as a metal coating. The metal coating can be about 50 nm to about 100 nm thick (Figure 1). The metal coating can be thick enough to provide an opaque barrier to light. The metal coating can provide a physical structure into which the nucleic acid/bead can be deposited. Thinner metal coatings of about 10-20 nm can provide optical enhancement.

[00151] The surface can include one or a plurality of microscopic features, typically more than 5, 10, 50, 100, 500, 1000, 10,000, 100,000, or 1,000,000, or more.

[00152] The dimensions of the microscopic features (e.g., trench width or depth) can be about 1 micron, or about 0.1 micron, or about 0.01 micron, or about 0.001 micron, or about 0.0001 micron. The dimensions of the microscopic features can be between about 10-25 nm, or about 25- 50 nm, or about 50-100 nm, or about 100-200 nm, or about 200-500 nm, or about 500-750 nm, or about 750-1000 nm. The microscopic features can have a trench width equal to or less than about 150 nanometers. The microscopic features can have a trench depth equal to or less than about 200 nanometers.

[00153] The features can be any shape including circular-like (e.g., circles, ovals, and the like), quadrilateral- shaped (e.g., squares, rectangles, rhombus, and the like); triangular; slits; trenches. The circle-like features can be referred to as "spots". The diameter of the spot openings can be about 50 to 10,000 nm (Figure 1). The center-to-center spacing of the spots in an array can be about 100 nm to about 10 μηι, but one skilled in the art will appreciate that other spacings can be produced.

[00154] Use of a surface including features (such as trenches, grooves, cavities and the like) is optional. When features are present, they can facilitate the fabrication of ordered and/or semi- ordered arrays by forming structures that physically "guide" or constrain the beads to occupy a particular location on the surface. For example, the surface can include cavities sized to ensure that they can be occupied by only one bead at any given time, thus ensuring that only a single bead occupies the cavity and deposits a linker onto the bottom surface of the cavity. The cavities can thus be used to ensure that the beads are uniformly distributed across the surface, thus resulting in the formation of a uniform array.

[00155] In some embodiments, the surface does not include any physical features, and can include an open field surface across which the bead population can be freely rolled or flowed. In some embodiments, the open field surface can include local areas, or "islands" that include distinctive chemical moieties or reactive groups capable of binding to the linker and/or bead of a bead-linker complex. For example, the surface, while devoid of physical features, can include local islands or defined regions including an NHS reactive moiety that reacts with a terminal amine group on the bead and/or linker.

[00156] Even in embodiments including open field surfaces that lack physical features, it is possible to achieve ordered or semi-ordered distribution of beads such that the resulting array density is higher than the densities that can be achieved through random distribution of beads across the surface. Such random distribution typically proceeds as a Poisson distribution.

[00157] In some embodiments, the surface can include one array, or more than one array of features (e.g., spots). For example, the surface can include multiple arrays (Figures 2, and 4A and B). The multiple arrays can be placed on the surface in any arrangement. In some embodiments, the multiple arrays can be in rows and columns of arrays. For example, Figure 4A depicts a surface having 4 rows and 2 columns of spot arrays (see also Figure 4B). The rows and columns of arrays can be spaced apart by X and Y distances, respectively (Figure 4A). The selection of X and Y can be dictated by optical constraints including imaging and resolution capabilities, laser spot size, and/or pixel sizes, of the optical set-up to be used to visualize the arrays of spots.

[00158] For example, the arrays can be spaced X = 400 μιη apart. In another example, the arrays can be spaced Y = 400 μιη apart. The edge-to-edge distance of the arrays can be s distance apart (Figure 4A). The various arrays can have different feature shapes, sizes, distances between the features, distances to the edge, and the like.

[00159] The surface can include markings, such as text or shapes. The marking can be place anywhere on the surface. For example, the marking can include text which can indicate the row or column number, or the size of the features (e.g., spots). For example, the text can have height (about 1 - 5 μιη) and width (about 1 - 1- μιη). The surface can include a fiducial, which can be placed anywhere on the surface. The fiducial can be spaced a distance Z from the arrays (Figure 4B). For example, the spacing between the spot arrays and the fiducial can be about Z = 400 μιη. The fiducial can be any shape such as a bulls eye or geometric shape (Figures 4A and B, respectively). The fiducial can serve as a guide for orienting or aligning the array, for example aligning the spots on a microscope stage (e.g., a detection element).

[00160] In another embodiment, the surface can include multiple blocks of arrays. For example, Figure 4C depicts a surface (e.g., a chip) having multiple blocks of arrays. The multiple blocks of arrays can be vertically and/or horizontally centered, or arranged close to one or more edges. The chip can have W height and Z length. Typically, the dimensions of W x Z are about 5x5 mm, 10x10 mm, or 12x12 mm, but other dimensions are possible. The distance between the blocks can be measured from the center of each block or from the edge of each block. The blocks can be spaced Y distance apart (Figure 4C). The chip can be installed in a fluidics set-up. The Y distance can be dictated by the dimensions of the fluidics set-up. For example, the distance Y can be about 6 mm. The distance from the edge of the chip to at least one of the blocks can be measured from the center or the edge of the block. For example, the distance from the center of the block to the edge of the chip can be spaced by X distance (Figure 4C). For example, the distance X can be about 2 mm. The chip can include information such as chip number, design name, date, and/or logo. The information can be included anywhere on the chip.

[00161] In some embodiments, the disclosure relates to a chip, comprising: a plurality of blocks each including one or more arrays, wherein each array includes one or more reactive surface spots having an affinity for a target moiety; and a calibration mark configured to align the sequencing chip with a detection element.

[00162] In some embodiments, the chip can include a glass substrate which can be about 170 μιη thick (e.g., Schott D263). The glass substrate can be coated with a metal layer which can be about 80 nm thick. The metal layer can be titanium or aluminum. The array can include spots which range in diameter from about 200 - 800 nm, or about 400 - 700 nm. The edge to edge spot separation distance can be about 320 or 640 nm.

[00163] To prepare the arrays and/or microscopic features, the surface can be uncoated or coated with an adhesive and/or resist layer which can be applied to the surface in any order. The adhesive layer can bind/link the linkers of the bead-linker complexes. The resist layer may not bind/link, or exhibits decreased binding/linking, to the oligonucleotides. The surface can be coated with a thin metal film (e.g., about 10-20 nm depending on the metal) for optical enhancement or quenching, or coated with a thick metal mask (e.g., up to about 100 nm depending on the metal) for opacity.

[00164] The microscopic features may be prepared/fabricated from any suitable organic or inorganic compound including: amine, silane, biotin, avidin (or avidin-like compounds), PEG, protein binding partners, silicon, carbon, glass, polymer (e.g., poly-dimethylsiloxane), metals, titanium, aluminum, gold, chromium, platinum, silver, nitrides (e.g., boron nitrides), chromium, gold, synthetic vesicles, silicone, or any combination thereof.

[00165] The arrays and/or microscopic features may be prepared/fabricated using any suitable method, including: lithography; photolithography; deep UV lithography; soft lithography;

diffraction gradient lithography (DGL); nanoimprint lithography (NIL); interference lithography; contact nanoprinting; self-assembled copolymer pattern transfer; spin coating; electron beam lithography; focused ion beam milling; plasma-enhanced chemical vapor deposition; electron beam evaporation; sputter deposition; bulk or surface micromachining; replication techniques such as embossing, printing, casting and injection molding; etching including nuclear track, chemical, or physical etching, reactive ion-etching, wet-etching; sacrificial layer etching; wafer bonding; channel sealing; and combinations thereof. The selection of the method for preparing the arrays or microscopic features may depend upon the desired size of the microscopic feature. For example, photolithography or deep UV lithography are typically selected to prepare microscopic features that are greater than about one micron. In another example, electron beam lithography is typically selected to prepare microscopic feature that are smaller than about one micron.

[00166] For example, microscopic features can be prepared on a surface by: applying a photoresist compound to a glass surface; passivating the glass surface with a metal (e.g., aluminum or titanium) using a metal evaporation procedure; and removing the photo-resist to produce a metal- passivated glass surface having islands of glass that can be functionalized for binding the bead- oligonucleotides. The glass islands can be functionalized with PEG, amines, biotin, and/or avidin- like compounds, to bind the bead-oligonucleotides. The methods for applying and removing resists, metal-passivation on a glass surface, and chemical functionalization, are well known in the art.

[00167] The surface can be coupled to a light source, detector (e.g., photon detector), camera, and/or various plumbing components such as microvalves, micropumps, connecting channels, and microreservoirs for controlled flow (in and/or out) of reagents.

[00168] In some embodiments, the nanoscale device includes: a flow cell; reservoirs for holding reagents; inlet ports in fluid communication with the reservoirs and flow cell for delivering the various reagents; outlet ports in fluid communication with the flow cell; photon detectors; and cameras for determining the location of a signal. The surface of the flow cell can be coated with PEM/biotin/avidin (U.S. Patent Nos. Quake, U.S. Patent Nos. 6,818,395, 6,911,345, and

7,501,245). The reagents can be pulled through the inlet or outlet ports via capillary action, or by vacuum (Lawson, U.S. published patent application No. 2008/0219890; and Harris, et al., 2008 Science 320: 106-109, and Supplemental Materials and Methods from the supporting online material), or moved via a pressure-driven fluidics system. In another embodiment, the reagents can be pulled through the inlet or outlet ports using a passive vacuum source (Ulmer, U.S. patent No. 7,276,720).

[00169] In yet another embodiment, the flow cell can be a two-sided multi-channel flow cell comprising multiple independently-addressable sample channels and removable loading blocks for sample loading (Lawson, U.S. published patent application No. 2008/0219888).

[00170] In still another embodiment, the surface can be enclosed by being surmounted with a sealing material using suitable methods. See, for example, U.S. Publication No. 2004/0197843. The surface can include a sample reservoir capable of releasing a fluid, and a waste reservoir capable of receiving a fluid, wherein both reservoirs are in fluid communication with a common reaction area. The surface can include a microfluidic area located adjacent to the nanofluidic area, and a gradient interface between the microfluidic and nanofluidic area that reduces the local entropic barrier for entry into a microscopic feature area (e.g., channels). See, for example, U.S. Patent No. 7,217,562. See also Cao, U.S. Patent No. 7,217,562 and U.S. published patent application No. 2007/0020772; and Han, U.S. Patent No. 6,635,163.

[00171] In another embodiment, the surface (e.g., glass) can be patterned using photo-resists, and/or photolithography or electron-beam lithography. The patterned surface can be passivated by metal evaporation procedures (e.g., aluminum). The photo-resists can be removed. The exposed glass can be functionalized with biotin or amine groups, and the non-functionalized areas can be coated with PEG.

[00172] In some embodiments, the disclosure relates generally to methods for: binding a linker (e.g., a nucleic acid molecule) to a surface; binding multiple linkers to a surface; and preparing an array of linkers. The methods can be practiced using the bead- linker complexes described herein. In general, the surface is contacted with the bead-linker complex to bind the linker to the surface. The surface can have a random or organized pattern of linking groups to bind to the linker of the complex.

[00173] The methods can be practiced using suitable conditions that permit binding of the oligonucleotide to the surface, including parameters such as: time, temperature, pH, buffers, reagents, salts, and concentrations of the bead-oligonucleotides.

[00174] For example, the bead-linker complex can be contacted with the surface for a time that is sufficient to permit binding of the linker of the complex to the surface, such as about 10 minutes to 48 hours.

[00175] In another example, the bead-linker complex can be contacted with the surface at a temperature that will permit binding of the linker to the surface, such as about 4 - 80°C.

[00176] In another example, the bead-linker complex can be contacted with the surface at a pH that will permit binding the oligonucleotide portion to the surface, such as about ph 4-12. The suitable pH will be dependent upon the type of linking chemistry between the oligonucleotide molecule and surface.

[00177] The buffer or reagents can include a source of monovalent or divalent ions. The buffer can include chelating agents such as EDTA and EGTA, and the like.

[00178] In one aspect, the disclosure relates to methods for binding a nucleic acid molecule to a surface, comprises: contacting a surface with a bead- linker complex comprising one or more oligonucleotide linkers linked to at least one bead.

[00179] In another aspect, methods for binding multiple nucleic acid molecules to a surface comprises: contacting the surface with multiple bead- linker complexes.

[00180] In some embodiments, the surface comprises a linking group that can facilitate or mediate binding of the linker of the bead-linker complex to the surface. The linking group can include an amino group, aldehyde group, NHS-ester group, alkyne group, or one member of a binding partner pair. In another embodiment, the surface comprises a linking group which is arranged on the surface as a random or organized pattern (e.g. array). [00181] In some embodiments, the linker of the bead-linker complex is an oligonucleotide linker that includes one or more nucleic acid portions. The one or more nucleic acid portions can include single-stranded or double-stranded nucleic acid. In some embodiments, the oligonucleotide linker includes an oligonucleotide that is about 5-50 nt or bp in length. The oligonucleotide linker can optionally include a cleavable linker moiety, a member of a binding partner pair, a reporter moiety, and/or a flexibility/rigidity moiety. In some embodiments, the linker includes a cleavable moiety, which can be a photocleavable linker moiety or a chemical-cleavable linker moiety. In one exemplary embodiment, the oligonucleotide linker includes a reporter moiety that is a fluorescent dye.

[00182] In some embodiments, the linker of the bead- linker complex includes a cleavable moiety. The cleavable moiety can be cleaved, for example, after the linker of the complex binds to the surface, forming a surface-attached bead-linker complex. Cleavage of the cleavable moiety and consequent release of the bead of the complex result in the formation of a surface-attached linker.

[00183] In one embodiment, the bead-linker complex includes a single bead linked to a single oligonucleotide linker. Therefore, a single bead-oligonucleotide can deposit a single

oligonucleotide to the surface. In some embodiments, the complex includes a single bead attached to a plurality of linkers, but the linkers may be spaced sufficiently far apart on the bead surface to ensure that an average of one linker binds to the surface when the complex is contacted with the surface; the remaining linkers attached to the bead will not bind to the surface because they do not contact the surface directly.

[00184] In some embodiments, the linker of the bead- linker complex includes a binding partner that can be contacted with the other member of the binding partner pair before, during or after the linker binds to the surface.

[00185] In some embodiments, the fluorescent dye can be excited with an electromagnetic excitation source before, during or after the linker binds to the surface.

[00186] In some embodiments, the surface is contacted with a homogeneous or heterogeneous population of bead- linker complexes. For example, the population of bead- linker complexes can be a homogeneous population, in which each complex in the population comprises the same type of linker and/or the same type of bead. In some embodiments, all of the bead-linker complexes in the population include the same type of linker. In some embodiments, all of the bead- linker complexes in the population include the same type of bead. Alternatively, the population of bead- linker complexes can be a heterogeneous population, wherein two or more complexes in the population comprise different types of linkers and/or different types of beads, from each other. The different types of linkers can include different structures, functions, sequences, lengths, cleavable moieties, members of a binding partner pair, reporter moieties, linking groups, and/or flexibility/rigidity moieties. The different types of beads can comprise different materials, sizes, or dimensions and linking groups.

[00187] In some embodiments, the surface can be contacted with a first population of bead- linker complexes (homogenous or heterogeneous), and subsequently contacted with a second population of bead-linker complexes (homogeneous or heterogeneous). The surface can be contacted repeatedly with homogeneous or heterogeneous populations of bead- linker complexes. The contacting can be performed using conditions where the linker of the bead-linker complexes bind to the surface. Optionally, the beads of the bead-linker complexes can be cleaved and released, thereby forming a population of surface-attached linkers. Such cleavage and release can optionally occur before or after the surface is contacted with the next population of bead- linker complexes.

[00188] In some embodiments, the surface can include multiple first linking groups which are arranged into a random or organized pattern (array). The surface can optionally include additional linking groups (e.g., 2^nd, 3^rd, 4^th, 5^th linking groups, or more) which differ from the first linking groups. In another embodiment, the surface can include two or more different types of linking groups which are arranged into a random or organized pattern.

[00189] In some embodiments, the surface is contacted with a homogeneous or heterogeneous population of multiple bead-linker complexes, each complex including a linker including one or more types of linking groups, so that each type of linking group in the linker binds to its cognate linking group on the surface.

[00190] In some embodiments, the surface comprises a chemical group that does not bind the first linking group. Optionally, such chemical group can serve to "mask" certain areas of the surface and prevent non-specific binding of the bead- linker complex to defined locations of the surface.

[00191] In some embodiments, the method further includes the step of: contacting the surface with a reagent that modifies the chemical group that does not bind the first linking group (e.g., masking, blocking, and the like).

[00192] In some embodiments, the methods can further comprise washing to remove the unbound bead-linker complexes, unbound linkers, or cleaved beads, or unbound binding partners, or binding reagents/buffers, or reagents that modify the chemical group that does not bind the first linking group.

[00193] In some embodiments, the methods further comprise contacting the surface-attached linkers with target analytes, for example biological molecules (e.g., nucleic acid molecules, polypeptides, or antibodies) or chemical compounds or drug candidate compounds. This step can be conducted before, during, or after the bead-linker complexes are linked to the surface and/or cleavage of the bead from the bead- linker complex.

[00194] In some embodiments, the linker of the bead-linker complex can be an oligonucleotide linker that comprises one or more nucleic acid portions. The oligonucleotide linker can include one or more nucleic acids from any suitable source, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, chemically synthesized, or any combination thereof. The oligonucleotide linker can include a nucleic acid can be isolated from any source including from: organisms such as phage, prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses, cells; tissues, body fluids, or synthesized nucleic acid molecules using recombinant DNA technology or chemical synthesis methods. The oligonucleotide linker can include nucleic acid from any commercially available source.

[00195] The oligonucleotide linker can include one or more nucleic acid portions comprising naturally-occurring nucleotides or nucleotide analogs, or any combination thereof. Any portion of the oligonucleotide linker can include a base, sugar, and/or phosphate group analog.

[00196] In some embodiments, the oligonucleotide linker includes a nucleic acid molecule comprised of nucleotides including a sugar analog, such as carbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7: 3013-3016), and other suitable sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124.; and U.S. Pat. No. 5,558,991). The sugar moiety can be: ribosyl, 2'-deoxyribosyl, 3'-deoxyribosyl, 2',3'- dideoxyribosyl, 2',3'-didehydrodideoxyribosyl, 2'-alkoxyribosyl, 2'-azidoribosyl, 2'-aminoribosyl, 2'-fluororibosyl, 2'-mercaptoriboxyl, 2'-alkylthioribosyl, 3'-alkoxyribosyl, 3'-azidoribosyl, 3'- aminoribosyl, 3'-fluororibosyl, 3'-mercaptoriboxyl, 3'-alkylthioribosyl carbocyclic, acyclic, or other modified sugars. [00197] In some embodiments, the oligonucleotide linker includes a nucleic acid molecule comprised of nucleotides including a hetero cyclic base which includes substituted or

unsubstituted nitrogen-containing parent heteroaromatic ring, including naturally- occurring, substituted, modified, or engineered variants, or analogs of the same. The base can be capable of forming Watson-Crick and/or Hoogstein hydrogen bonds with an appropriately complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2- aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N⁶-A²-isopentenyladenine (6iA), N⁶-A²-isopentenyl-2-methylthioadenine (2ms6iA), N⁶-methyladenine, guanine (G), isoguanine, N -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and 0⁶-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7- deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5, 6-dihydro thymine, 0⁴-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additional exemplary bases can be found in Fasman, 1989, Practical Handbook of Biochemistry and

Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Florida, and the references cited therein.

[00198] In some embodiments, the oligonucleotide linker includes a nucleic acid molecule comprised of nucleotides including phosphate group analogs, such as: phosphoramidate;

phosphorothioate; phosphorodithioate; O-methylphosphoroamidite linkages; and peptide nucleic acid backbones and linkages.

[00199] In some embodiments, the oligonucleotide linker includes a nucleic acid molecule comprised of nucleic acids analogs including those with bicyclic structures including locked nucleic acids; positive backbones; non-ionic backbones; and non-ribose backbones.

[00200] In some embodiments, the ends and/or interior of the oligonucleotide linkers or capture probes may be isolated and modified at their ends and/or the interior of the molecules using well known procedures, including: fragmentation, ligation, hybridization, enzymatic and/or chemical modification, conjugation with a reporter moiety, or linking to an energy transfer (donor or acceptor), or any combination of these procedures.

[00201] In some embodiments, the bead-linker complex comprises an oligonucleotide linker including one or more nucleic acid molecules fragmented at random or specific sites using any fragmentation procedure. The nucleic acid molecules can be fragmented using mechanical force, including: shear forces (e.g., small orifice or a needle); nebulization (S. Surzycki 1990 in: "The International Conference on the Status and Future of Research on the Human Genome. Human Genome Π", San Diego, CA, pp. 51; and S. J. Surzycki, 2000 in: "Basic Methods in Molecular Biology", New York, NY: Springer- Verlag); or sonication.

[00202] In some embodiments, the oligonucleotide linker is comprised of one or more nucleic acid molecules that are chemically fragmented using, for example: acid-catalyzed hydrolysis of the backbone and cleavage with piperidine; internucleosomal DNA fragmentation using a copper (II) complex of 1,10-phenanthroline (o-phenanthroline, OP), CuII(OP)₂ in the presence of ascorbic acid (Shui Ying Tsang 1996 Biochem. Journal 317: 13-16).

[00203] In some embodiments, the oligonucleotide linker is comprised of one or more nucleic acid molecules that are enzymatically fragmented using type I, II or III restriction endonucleases (N.E. Murray 2000 Microbiol. Mol. Biol. Rev. 64: 412-34; A. Pingoud and A. Jeltsch 2001 Nucleic Acids Res. 29: 3705-27; D. T. Dryden, et al., 2001 Nucleic Acids Res. 29: 3728-41; and A. Meisel, et al., 1992 Nature 355: 467-9). Enzymatic cleavage of DNA may include digestion using various ribo- and deoxyribonucleases or glycosylases. The nucleic acid molecules can be digested with DNase I or II. The nucleic acid fragments can be generated by enzymatically copying an RNA template. Fragments can be generated using processive enzymatic degradation (e.g., S I nuclease). The enzymatic reactions can be conducted in the presence or absence of salts (e.g., Mg²⁺, Mn²⁺, and/or Ca²⁺), and the pH and temperature conditions can be varied according to the desired rate of reaction and results, as is well known in the art.

[00204] In some embodiments, the 5' or 3' overhang ends of the nucleic acid molecules that comprise the oligonucleotide linker can be converted to blunt-ends using a "fill-in" procedure (e.g., dNTPS and DNA polymerase, Klenow, or Pfu or T4 polymerase) or using exonuclease procedure to digest away the protruding end.

[00205] In some embodiments, the oligonucleotide linker can be ligated to one or more other nucleic acid molecules using DNA ligase or RNA ligase. The nucleic acid molecules can be further hybridized to one or more additional oligonucleotides. The additional oligonucleotides can serve as linkers, oligonucleotides, bridges, clamps, oligonucleotides, or capture oligonucleotides.

[00206] The oligonucleotide linker can include sequences which are: enzyme recognition sequences (e.g., restriction endonuclease recognition sites, DNA or RNA polymerase recognition sites); hybridization sites; or can include a detachable portion. [00207] The oligonucleotide linker can linked to a protein-binding molecule such as biotin or strep tavidin.

[00208] The oligonucleotide linker can be methylated, for example, to confer resistance to restriction enzyme digestion (e.g., EcoRI). The ends of the oligonucleotide linker can be phosphorylated or dephosphorylated.

[00209] In some embodiments, a nick can be introduced into the oligonucleotide linker or into cognate nucleic acid molecules using, for example DNase I. A pre-designed nick site can be introduced in dsDNA using a double stranded probe, type II restriction enzyme, ligase, and dephosphorylation (Fu Dong-Jing, 1997 Nucleic Acids Research 25:677-679).

[00210] A nick can be repaired using polymerase (e.g., DNA pol I or phi29), ligase (e.g., T4 ligase) and kinase (polynucleotide kinase).

[00211] A poly tail can be added to the 3' end of the oligonucleotide linker using terminal transferase (e.g., polyA, polyG, polyC, polyT, or polyU).

[00212] In some embodiments, the oligonucleotide linker (or cognate nucleic acid molecule) can be modified using bisulfite treatment (e.g., disodium bisulfite) to convert unmethylated cytosines to uracils, which permits detection of methylated cytosines using, for example, methylation specific procedures (e.g., PCR or bisulfite genomic sequencing).

[00213] In some embodiments, the oligonucleotide linker is comprised of nucleic acid molecules that can be size selected, or separated from undesirable molecules, using any art-known methods, including gel electrophoresis, size exclusion chromatography (e.g., spin columns), sucrose sedimentation, or gradient centrifugation.

[00214] The oligonucleotide linker can optionally be amplified using methods, including:

polymerase chain reaction (PCR); ligation chain reaction, which is sometimes referred to as oligonucleotide ligase amplification (OLA); cycling probe technology (CPT); strand displacement assay (SDA); transcription mediated amplification (TMA); nucleic acid sequence based amplification (NASBA); rolling circle amplification (RCA); and invasive cleavage technology. In some embodiments, the bead-linker complex comprises a bead linked to a clonally amplified population of nucleic acid linkers.

[00215] Undesired compounds can be removed or separated from the desired nucleic acid molecules to facilitate enrichment of the desired molecules (e.g., oligonucleotides). Enrichment methods can be achieved using well known methods, including gel electrophoresis,

chromatography, or solid phase immobilization (reversible or non-reversible). For example, AMPURE beads (Agencourt) can bind DNA fragments but not bind unincorporated nucleotides, free primers, DNA polymerases, and salts, thereby facilitating enrichment of the desired DNA fragments.

[00216] The desired nucleic acid molecules can be enriched using a dialysis procedure, which can be conducted by employing a dialysis membrane having a suitable molecular weight cut-off (MWCO) limit, for a sufficient amount of time, and in a suitable exchange buffer. For example, the nucleic acid molecules can be enriched using dialysis membranes having about 2K, 3.5K, 7K, or 10K MWCO. The dialysis procedure can be conducted for about 2-48 hours. The exchange buffer can include Tris at a pH range of about pH 6-9.

EXAMPLES

[00217] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. In some cases, the compositions and methods of this invention have been described in terms of embodiments, however these embodiments are in no way intended to limit the scope of the claims, and it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain components which are both chemically and physiologically related may be substituted for the components described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[00218] EXAMPLE 1

[00219] Producing Metal Masked Nanoarrays

[00220] A.) Metal Mask Nanospot Process - Subtractive Procedure

[00221] The surface was a commercially-available transparent substrate (e.g., Schott D263 glass). The glass surface was global-coated with metal using by evaporation or sputtering procedures. The metals included titanium or aluminium (e.g., other metals could include Gold, Chrome, and Silver; other metals and opaque materials that could be used include: Nickel, Platinum, Copper, Tungsten, Titanium-Tungsten, carbon, carbon nanotubes, nanoparticles, and polymers). The surface was spin-coated with an imaging resist using e-beam or photo resist procedures. The exposure step was achieved using e-beam or photomask lithography, and developed. Oxygen plasma was used to clean the patterned features.

[00222] To expose the substrate spots, the metal was treated with plasma or was wet-etched. The imaging resist was stripped using wet or plasma chemistry. Passivation chemistry was used to coat (e.g., using dilute biotin-PEG:PEG).

[00223] Optional Process Flow - Metal Patterning Liftoff

[00224] Spin coat with e-beam resist. E-beam expose and develop. Oxygen plasma clean patterned features. Global coat aluminium by directional evaporation. Perform liftoff by soaking in acetone or more aggressive resist stripping agent. Coat with dilute biotin-PEG:PEG passivation chemistry.

[00225] B. Metal Mask Nanospot Process

[00226] E-beam was used to produce the pattern nanospot arrays

a) Spin coated imaging resist

b) De thinned (1 to 10 nm) metallization to make surface conductive / eliminate EBL charging

c) Exposed with EBL then developed

[00227] Evaporated titanium metal 80 nm +/- lOnm thick with lift off compatible parameters

[00228] Pattern metallization via lift off

a) Wet strip EBL resist

b) Plasma ash away any remaining contaminants

c) Package and ship

[00229] Figure 1 shows an example of metal patterned spots.

[00230] C.) Metal Mask Nanospot Process - Additive Procedure

[00231] Pattern Surface: For greater than 1 micron features, the surface was patterned with photolithography. For less than 1 micron features, surface was patterned with e-beam.

[00232] Metallize surface: Metal was evaporated on top of patterned surface. The metal included titanium or aluminium (e.g., other metals could include Gold, Chrome, and Silver; other metals and opaque materials that could be used include: Nickel, Platinum, Copper, Tungsten, Titanium- Tungsten, carbon, carbon nanotubes, nanoparticles, and polymers)

[00233] Lift Off: The photoresist was removed from the surface.

[00234] The glass surface was functionalized (e.g., with biotin and/or amine and/or PEG. [00235] The metal was functionalized (e.g., with PEG).

[00236] D.) Embodiments for Subtract! ve and Additive Methods

[00237] The procedures described in Example 1, sections A, B and C, above were used to produce: spot sizes of about Ιμιη to 10 μιη; array pitches of about 2 μιη to 8 μιη; metal thickness of about 50nm and about lOOnm; and pattern polarity: negative and positive

[00238] Other embodiments include:

[00239] The field is covered by opaque metal mask. Possible metals include: Titanium,

Aluminum, Gold, Chrome, and Silver, as well as other metals or opaque materials such as Nickel, Platinum, Copper, Tungsten, Titanium- Tungsten, carbon, carbon nanotubes, nanoparticles, and polymers.

[00240] The target thickness of the metal ranges from about 50 nm to about 100 nm (Figure 1). The metal serves to produce an opaque barrier to light, and to provide a physical structure into which the nucleic acid/bead can be deposited. Metal thickness of about 10-20 nm can provide optical enhancement. The spot openings can be any shape including circular-like (e.g., circles, ovals, and the like), quadrilateral- shaped (e.g., squares, rectangles, rhombus, and the like);

triangular; slits; trenches. The diameter of the spot openings can be about 50 to 10,000 nm (Figure 1). The center- to-center spacing of the spots in an array can be about 100 nm to about 10 μιη, or about 1.1, 1.2, 1.6, 2.4, or 4.8 um, but one skilled in the art will appreciate that other spacings can be produced (see Figure 2).

[00241] Nano features:

[00242] 2D arrays of spots ranging from 50 to 2000 nm (see below)

[00243] Titanium metallization 80 nm +/- 10 nm

[00244] 12 arrays per block

[00245] 18 blocks per die

[00246] Micron features:

[00247] Text over each array within each block uses 10 micron tall characters

[00248] Repeats of design blocks: (Figure 2)

[00249] X = column spacing = 6 mm

[00250] Y = row spacing = 1 mm

[00251] Millimeter features (ports and chip outline):

[00252] L = Chip side length = 22.0 mm

[00253] t = Glass thickness = 175 microns +/- 20 microns [00254] Each block:

[00255] Has 12 arrays of spots.

[00256] Spots in each array covers 100 microns by 100 microns.

[00257] Spots are spaced 4.8 microns in both X and Y axes.

[00258] Each of the 12 arrays has one sized spot diameter of either: 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 nm.

[00259] Text above each array is 10 microns tall and indicates the spot diameter for that area.

[00260] Example 2

[00261] Non-Metal Patterned Spots

[00262] Alternatively, non-metal patterned spots can be made by: coating a glass surface with a zirconium compound (e.g., Zr0₂), creating a patterned feature on the zirconium layer using an imaging resist, treating the exposed zirconium layer with a phosphonic compound (e.g., amino phosphonic acid), removing the patterned feature which leaves the phosphonic acid-treated spots on the zirconium layer, attaching to the phosphonic acid spots biotin/PEG/NHS, and coating the regions between the spots with PEG.

[00263] Example 3

[00264] Surface Chemistry

[00265] A.) Passivation Procedure Using PEG Phosphonic Acid

[00266] The titanium patterned slides as plasma-cleaned (300 watts, 5 minutes, 150 mtorr 0₂). The patterned slide was soaked overnight in a solution of 0.2 mM poly(ethylene glycol) phosphonic acid in ethanol. The patterned slide was removed, rinsed with copious amounts of ethanol and deionized water, and dried under vacuum. The exposed glass was functionalized by soaking the patterned slide in an aqueous solution of 5 mM zirconium acetylacetonate at 50 °C overnight. The patterned slide as removed, rinsed with copious amounts of deionized water, and dry under vacuum. The slide was soaked in a 20 mM aqueous solution of 2- aminoethylphosphonic acid overnight. The slide was removed from the solution and rinsed with deionized water and dry under vacuum. This process yielded amine functionalized glass islands surrounded by PEG passivated titanium.

[00267] The slides were soaked in a solution of 0.1 M glutaric anhydride, 10 mM (para-N,N'- dimethylpyridin) DMAP and 20 mM diisopropylethylamine (DIEA) in anhydrous toluene, overnight at room temperature. The slides were rinsed with toluene and iso-propanol thoroughly and blow dry with nitrogen gas. The slides were soaked in a solution of 0.5M TSTU and 0.25M DIEA in DMSO, for 30-60 minutes at room temperature. The slides were rinsed with 1 mM HCl repeatedly and blow dried with nitrogen gas. This procedure produced NHS ester linking groups on the surface for binding to amine linking groups of the nucleic acid molecules.

[00268] B.) Synthesis and purification of mPE5k-acetomidoethylphosphonic acid-DSC

[00269] Scheme:

FW = 5018 plus/minus (44)n

FW = 5130 plus/minus (44)n

[00270] MPEG5k acetic acid (1 gram, av. 0.0005mole) was co-evaporated with anhydrous acetonitrile three times (20 ml each) and redissolved in dry dichloromethane (10 ml). TEA (0.11 ml, 0.00075 mole) was added under dry nitrogen. DSC (0.3 gram, 0.001 moles) was added portion-wise under nitrogen with stirring. The reaction was left at room temperature for 3 hours. The solvent was removed on a rotavap. Anhydrous dioxane (2 ml) was added to dissolve the residual and was added dropwise to an aqueous solution (2 ml) of amnioethylphosphonic acid (2M, pH to 8 with NaOH) with vigorous stirring. The reaction was left for two hours at room temperature. Take 1 ml and add 1.5 ml water, desalted on P- 10 column twice. The left over was rotavap to dryness, added 10 ml ethyl acetate, heat to dissolve, filtered off undissolved residual through a short packaged silica column on a buckner funnel. The clear ethyl acetate was left at - 20 freezer. The product recrystalized. MS verified the mass value of the desired product. The product can be used to passivate titanium patterned surfaces. [00271] Example 4

[00272] Synthesis of Modified DNA Oligonucleotide for Attachment to Beads

[00273] 5' Phosphate-GATTGTC AGATAC AC- (S -S )- AT- (Biotin TEG)-GAG-HEO-HEO-HEO-

HEO-C6-Amine (Figure 11)

[00274] The oligonucleotide was synthesized using an Applied Biosystems Model 394

Synthesizer. The synthesis used standard protocols, reagents and nucleoside phosphoramidites (purchased from ABI). The synthesis also used modified phosphoramidites to introduce: the 5' phosphate, the disulfide linker, the Biotin TEG and the HEO linker (see for example methods for synthesis and use of the HEO linker disclosed in Grossman, U.S. patent 5,807,682, and methods for synthesis and use of 3' C6-amine disclosed in Woo, U.S. patent 5,625,052).

[00275] The disulfide linker (thio modifier C6-S-S; catalog No. 10-1936-xx), biotin-TEG linker (catalog No. 10-1955-xx), and Phosphorylation reagent (catalog No. 10-1900-xx), were obtained from Glen Research (Sterling, VA). The HEO linker (catalog No. CLP-9765) was obtained from ChemGenes (Wilmington, MA).

[00276] Example 5

[00277] Loading the Bead/Oligonucleotide complex into the Nanoarays

[00278] A.) Activation Amine surface group to NHS-Ester on glass cover slip slide

[00279] Converting the amine to Carboxyl:

(1) Made a solution containing 0.1 M glutaric anhydride, 10 mM (para-N,N'-dimethylpyridin) DMAP and 20 mM diisopropylethylamine (DIEA) in anhydrous toluene.

(2) Soaked the amine slides (silanized with aminopropyltriethoxysilane) overnight at room temperature.

(3) Rinsed the slides with toluene and iso-propanol thoroughly and nitrogen blow dry.

[00280] Converting the Carboxyl to NHS -ester group:

(1) Made a solution containing 0.5M TSTU and 0.25M DIEA in DMSO.

(2) Soaked the slide surface with the solution for 30-60 minutes at room temperature

room temperature 30 minutes

(3) Rinsed the slide surface with 1 mM HC1 repetitively

and blow dry with filtered Nitrogen Gas

[00281] B.) Building the Test Chamber

[00282] Prepared when ready to perform attachment.

[00283] Attached one side of the double side chamber tape to plastic support. [00284] Pressed gently and secure the tape chambers.

[00285] Removed exterior of the double side tape.

[00286] The tape was cut with well chambers.

[00287] Aligned the glass support, 4 well per each 20X20mm slide, or aligned the glass support,

2 well per each 10X10mm slide.

[00288] Pressed gently to secure glass support to tape.

[00289] Just before deposition ready the test chamber/slide.

[00290] Rinsed the chamber/slide with attachment buffer 100 mM NaOAc pH 5.5 and sonicate 60-seconds to pre-wet surface.

[00291] C.) Preparation of Covalent Oligo Magnetic Beads

Scheme:

(1) Beads from Dynal: 1.0, 0.75 um and 0.50 um magnetic beads with carboxylic functional

groups.

(2) Washed bead three times on a magnetic stand by pelleting and sonication resuspending with a buffer containing 50% DMSO, 0.2 M NaCl, 5 mM imidazole pH 5.0: Calculate the amount of buffer to make the wash suspension at 10 %. Sonicate to resuspend pellet and centrifuge to re- pellet each time. Sonication should generate well suspended beads without clumps or pellets.

(3) Activation / Coupling Procedure

Remove wash buffer from the beads and add the following (final volume 200 μί):

a. water 32 μϊ_^

b. 5M NaCl 8 μΐ.

c. DMSO 100 μΐ.

d. ImM NH2-P1 oligo 20 μΐ.

e. Dissolve weighed EDC in 5 mM imidazole-hydrochloride solution (pH 5) to a final 1M concentration (5x) and immediately add:

f. 5x EDC/Imidazole 40 μΐ. Mixed and vortexed well. Sonicated and place the tube on a rotator for incubation overnight.

(4) Added a buffer containing 50mM Tris, 1 mM EDTA, 5% EtOH, pH 8.0 to the reaction

suspension with mixing. The final suspension was 10 %.

(5) Washed beads three times to remove unreacted oligo and hydrolyzed NHS with 50mM Tris, 1 mM EDTA, 5% EtOH, pH 8.0.

[00292] D.) Ligation Coupling the Bead to Linker moiety to form a bead-linker complex

[00293] Dynal Beads to tube 1000, 750, 500 nm beads

[00294] Rinsed beads with 0.1% Tween-20 and lxSSPE solution

[00295] Magnetic isolated beads remove solution and repeat rinse

[00296] Re-suspend in linker, bridge solution

Linker oligo moiety

Bridge oligo

lxSSPE

Covaris solution:

G3 bead Deposit cycle:

Treatment-2

Cycle burst 0

Time 5 seconds

Duty cycle (%) 1.0

Intensity 5.0

Treatment- 1

Cycle burst 0

Time 5 seconds

Duty cycle (%) 5.0

Intensity 5.0

Temperature 15 degree Incubate 9700 thermal cycler

Hold step cycle

Heat 85 degrees

Slow ramp cool to 25

Hold at 15 degrees

[00297] Structure of the Linker moiety:

[00298] 5' phosphate complementary DNA strand for ligation to the bead

[00299] Cleavable disulfide unit

[00300] Biotin coupler mid-sequence unit

[00301] Terminal amine unit

[00302] Magnetic isolate beads remove solution and did not rinse

[00303] Add in Ligation mixture as follows:

NaOAc

T4 5xLigase buffer

20 x SSPE

Di-water

Covaris sonciate separate beads G3 bead Deposit cycle

Added T4 Ligase 1.0 μΐ,

Ligate in a 9700 thermal-cycler

Hold:

10 degree 5 min

Cycle: 45 cycles

25 deg 10 sec.

10 deg 5 min.

Hold :

15 deg. 7 min

4 deg. hold Collected sample. Spun briefly.

Magnetic isolation of beads, removed liquid and rinsed beads with IxTEX (100 mM Tris, 0.05mM EDTA, TritonX-100)

Repeat previous step and store beads in IxTEX buffer until ready to use.

[00304] E.) Loading the Chamber and Attaching bead-linker complex to the surface

[00305] Readied the test chamber/slide

[00306] Rinse the chamber/slide with attachment buffer 100 mM NaOAc pH 5.5 and a Sonicate 60-seconds to wet surface using Bransonic Table Top B3-R ultrasonic cleaner.

[00307] Prepared bead-linker complex for attachment:

Prepare enough bead-linker complexes to give a final concentration range

of 4-6 million bead- linker complexes per uL.

Magnetic isolate the bead-linker complex remove solution and rinse

Rinse bead-linker complex in 100 mM NaOAc pH 5.5 repeat twice

Final re-suspend in 100 mM NaOAc pH 5.5

32 uL of volume for 20x20mm slides

14-uL volume for 10x10mm slides

Sonicate the suspension briefly in Covaris G3 bead Deposit cycle

Spin-down briefly, to bring down the liquid not to pellet beads, use low force tabletop centrifuge (National LabNet mini-centrifuge C-1200)

[00308] Loaded the Chamber:

Vacuum remove NaOAc buffer solution from the chamber

Pipet in the prepared solution of bead- linker complexes including oligonucleotides linked to beads.

Split load the beads into two loading

First loading, store at room temperature for 15-minutes

Second loading, store at room temperature for 45-minutes

(The terminal amine of the bead-linker complex reacts with the NHS -ester slide surface forming a covalent attachment linking the bead-linker complex to the slide) Post attachment Rinse:

Rinse the chamber with 100 mM NaOAc pH 5.5, 4 times

Blocking Step

Pushed in the blocker solution , mPEG-12amine at concentration of 3mg in lOOOul 100- mM NaOAc pH 5.5 buffer into the chamber

Incubated for 30 minutes at room temperature (The mPEG solution reaction with the available NHS-ester site on the slide effectively blocking the surface.)

(m-dPEG12-amine PN- 10288 QuantaBioDesign LtD)

Rinsed the slide with 0.5M-Tris pH 7.4 2 x times

Incubated for slide with solution for 10 minutes

Cleavage Step to remove bead and leave the Linker Moiety on surface.

Added in 100 mM DTT solution and incubate for 10 minutes

Pushed in fresh 100 mM DTT solution and incubate for 10 minutes repeat 3 times Vacuumed to remove the excess liquid

Post incubation: place rinse the chamber 4 times with 100 mM DTT solution; vacuum clear the chamber of liquid and beads

Rinsed slide chamber with Distilled water 2 times vacuum remove the liquid

Rinsed slide chamber with PBS solution 2 times vacuum remove the liquid

Low sonication Bead separation step:

This step aids in the separation of the beads from the slide surface: perform a low energy sonication step, using a Bransonic Table Top B3-R ultrasonic cleaner.

Filled chamber with PBS solution, tape seal the holes and rest the slide/assembly chamber onto the surface of the water in the sonicator, sonicate for 60 seconds.

Continued rinsing the slide chambers with PBS repeat 2 times, vacuum assist the removal the liquid from chambers and fill the chamber with PBS.

Stored slide in PBS solution and tape seal the holes

[00309] The Linker moiety attached to surface and the Biotin coupler is available for binding. [00310] Prepared Qdots-605 Streptavidin label dots: (Invitrogen PN#Q10101MP)

[00311] Preformed a serial dilution to generate a final 500 pM concentration of dots in the Separation solution. Following the additions, Vortex the solution and dots to break cluster of Dots.

[00312] Separation solution: lOOmM NaCl, lOOmM Tris pH 7.4

[00313] Added in the Streptavidin dots in the separation solution.

[00314] Incubated at room temperature 20 minutes

[00315] Rinsed slide chamber with PBS solution 4 times

[00316] Rinsed slide chamber with Distilled water 2 times

[00317] Rinsed slide chamber with PBS solution 2 times and store

[00318] Imaged the side using the Image platform: Olympus Microscope, 100X lens, TIRF illumination, and excitation image used either a 405nm or 532-laser excitation.

[00319] Zeiss Immersol 518F optical oil is used to merge slide to lens.

[00320] The beads included a Dynal bead which is linked to at least one nucleic acid molecule (e.g., oligonucleotide) which includes a ligation sequence to attach to the bead, a cleavable disulfide linker moiety, a binding partner moiety (e.g., biotin), an adjustable length portion (e.g., HEO is 7 bases in length, and can include 0-4 or more HEO units), and a chemical group for attachment to the surface.

[00321] Figure 5 shows a microscope image of streptavidin functionalized quantum dots bound to biotin, which were loaded into 600-700 nm diameter spots on titanium coated slides using magnetic beads functionalized with oligonucleotides. The circles in right image are around dots detected by software. Figure 6 shows the oligonucleotides loaded into metal patterned spots after cleavage from the beads. Figure 7 shows a graph depicting a determination of the number of quantum dots loaded per metal patterned spot. Long linkers have 4 HEO units and short linkers have 1 HEO unit.

[00322] Example 6

[00323] Surface Chemistry for Titanium Patterned Slides

[00324] The titanium patterned slides included a glass substrate with multiple spot arrays (e.g., Figures 4A, B and C). Each array included spots having diameters of about 400, 500, 600, or 700 nm. The titanium was about 80 nm thick. The titanium patterned slide were plasma cleaned (300 watts, 5 minutes, 150 mtorr 0₂). The patterned slides were soaked overnight in a solution of 5 mM poly(ethylene glycol) phosphonic acid in ethanol. The patterned slides were rinsed with copious amounts of ethanol and deionized water, and dried under vacuum. The exposed glass in the patterned slides were functionalized by soaking in an aqueous solution of 5 mM zirconium acetylacetonate at 50 °C overnight. The patterned slides were removed, rinsed with copious amounts of deionized water, and dried under vacuum.

[00325] The patterned slides were soaked in a 20 mM aqueous solution of 2- aminoethylphosphonic acid overnight. The patterned slides were removed from the solution and rinse with deionized water and dry under vacuum. This process yielded amine functionalized glass islands surrounded by PEG passivated titanium. The patterned slides were soaked in a solution of biotin-PEG-NHS ester and m-PEG NHS ester (Laysan Bio, Inc.) in DMF (total concentration is 0.25 mM) for two hours to overnight. The patterned slides were removed and sonicated/rinsed with DMF, toluene, IPA, ethanol and deionized water and dried under vacuum.

[00326] Another method involves reacting the patterned slides with various compounds to adjust the ratios of functional groups density, where the functional groups can react with nucleic acid molecules, proteins (e.g., polymerases), nanocrystals, reporter moieties, and/or energy transfer moieties. For example, the patterned slides can be reacted with methoxy-PEG and biotin-PEG- monophosphonic acid, or reacted with methoxy-PEG and carboxy valery PEG-bis-phosphonic acid.

[00327] The patterned slides were plasma cleaned (300 watts, 5 minutes, 150 mtorr 0₂). The patterned slides were soaked overnight in a solution of 5 mM methoxy-poly(ethylene glycol) phosphonic acid in ethanol. The patterned slides were rinsed with copious amounts of ethanol and deionized water, and dried under vacuum. The exposed glass in the patterned slides were functionalized by soaking in an aqueous solution of 5 mM zirconium acetylacetonate at 50 °C overnight. The patterned slides were removed, rinsed with copious amounts of deionized water, and dried under vacuum.

[00328] The patterned slides were soaked in a solution of biotin-PEG-PC F and PEG-PO₃H₂ in 95% ethanol (total concentration 0.25 mM) overnight. The patterned slides were removed and sonicated in/rinsed with ethanol and deionized water and dried under vacuum.

[00329] Immobilizing Qdots

[00330] Streptavidin 605 Qdot attachment: The patterned slides were assembled with a 2-lane flow cell cartridge. Each lane is washed with 200 PEB (50 mM Tris pH 7.6, 50 mM NaCl, 0.5% BSA). The lane is incubated with 20 of a 10 pM-100 nM solution of Qdot 605- streptavidin conjugate (Invitrogen, part number QIOIOIMP) in PEB for 5-30 minutes. The lane is washed with 200 PEB.

[00331] Biotin 605 Qdot attachment: The patterned slide is assembled with a 2-lane flow cell cartridge. Each lane is washed with 200 PEB (50 mM Tris pH 7.6, 50 mM NaCl, 0.5% BSA). The lane is incubated with 20 μΐ_^ of a 10 pM-100 nM solution of streptavidin (Invitrogen, part number 43-4301) in PEB for 5-30 minutes. The lane is washed with 200 μΐ_^ PEB. The lane is incubated with 20 μΐ_^ of a 10 pM-100 nM solution of Q dot 605-biotin conjugate (Invitrogen, part number Q10301MP) in PEB for 5-30 minutes. The lane is washed with 200 PEB.

[00332] Synthesis of mPEG5k Acetamido-Ethyl-Fhosphonic Acid

[00333] MPEG5K-OH (250 gram, av. 0.02mole) was dissolved in 250 ml anhydrous

dichloromethane with NaOH (3g, 0.075 moles) added and chilled on ice under nitrogen.

Bromoacetic t-butyl ester (16 g, 0.08 moles) was added portion-wise with stirring. The final mixture was agitated on an orbital shaker (stirring became difficult due to formation of NaBr solid) at room temperature for 24-48 hours. A lot of solid precipitate formed (NaBr). It was removed by centrifugation or decanting. Removed most of DCM on a rotavap. Added hexane to extract out unreacted Bromoacetic t-butyl ester. The semi-solid after decanting hexane was adjusted to pH 1.0 with HCl (cone.) on ice and stirred overnight at room temperature. The product was extracted with DCM three times (total 800 ml), washed with brine, and dried with anhydrous NaS04. Removed most of the DCM. Added to hexane dropwise with stirring to precipitate the product. An oily product, which became white solid on ice, was collected and washed with hexane, and dried. MPEG5k acetic acid (10 gram, av. 0.002mole) was co-evaporated with anhydrous acetontirile three times (120 ml each) and redissolved in dry dichloromethane (60 ml). TEA (0.42 ml, 0.0030 mole) was added under dry nitrogen. DSC (1.2 gram, 0.004 moles) was added portion- wise under nitrogen with stirring. The reaction was left at room temperature for 3 hours. The solvent was removed on a rotavap. Anhydrous dioxane (50 ml) was added to dissolve the residual and was added dropwise to an aqueous solution of amnioethylphosphonic acid (40 ml, 1.0 M, adjusted to pH 8 with NaOH) with vigorous stirring. The reaction was left for two hours at room temperature. Rotavaped to dryness to remove dioxane. Redissolved the residual in 50 ml water, loaded to P-10 column to desalt. Fractions were monitored with TLC plate/12 chamber. Used MS verified the mass value of the desired product.

[00334] Synthesis Of M-PEG-2K Amine-Bis-Phosphonic Acid Using Phosphorous Acid And Formaldehyde [00335] mPEG2K-amine (1 gram, 0.5 mmole) was dissolved in 2.0 ml water, added 2.0 ml HC1 (cone), and H3P03 (phosphorus acid, 0.5 g) to obtain a clear solution. Warmed the solution up to 50 degree C. HCHO ( 37% aq solution, 2.0 ml) was added, refluxed gently for 4 to 5 hours.

Cooled to room temperature. Added 5.0 ml water to dilute. Desalted on P-10 column twice. Rotavaped and co-evaporate with water until dry or lyophilize to obtain a powder. Mass spectra verified the desired product was obtained. (See P Krassimira P. Guerra, Rita Delgado, Michael G. B. Drew, Vitor Felix, Dalton Trans., 2006, (34),4124-4133)

[00336] Synthesis Of Biotin-PEG5k-Monophosphonic Acid

[00337] Aminoethylphosphonic acid (1 gram ) was dissolved in 2 ml of water, pH was adjusted pH to 8 using 5 N NaOH on ice. You can calculate how much NaOH needed to avoid over shoot. If you overshoot, you can add more aminoethylphosphonic acid. Dissolved the Biotin-PEG5K- NHS ester (200 mg) in 0.5 ml anhydrous dioxane (keep NHS ester dry). Added the biotin- PEG5K-NHS dioxane solution dropwise (using a syringe) to the above solution with vigorous stirring at room temperature. Allowed the reaction to proceed for one more hour. Diluted the reaction by adding 2 ml of water. Desalted on P-10 column twice. MS indicated that desired product was obtained.

[00338] Synthesis of Carboxy Valery-PEG5K amino-Bis-phosphonic acid using phosphorous acid and formaldehyde

[00339] Carboxy valery PEG5K-amine (1 gram, 0.2 mmole) was dissolved in 1.0 ml water, added 1.0 ml HC1 (cone), and H3P03 (phosphorus acid, 0.25 g) to obtain a clear solution.

Warmed the solution up to 40 degree C. HCHO (37% aq solution, 1.0 ml) was added, and the reaction mixture was kept at 40 degree C for 12 hours. Cooled to room temperature. Added 2.0 ml water to dilute. Desalted on P-10 column twice. Rotavaped and co-evaporate with water until dry or lyophilize to obtain a powder. Mass spectra verified the desired product was obtained. (See Krassimira P. Guerra, Rita Delgado, Michael G. B. Drew, Vitor Felix, Dalton

Trans., 2006, (34),4124-4133)

[00340] Example 7

[00341] Surface Chemistry for Titanium Patterned Slides

[00342] An exemplary assay for fabricating semi-ordered arrays of single oligonucleotide molecules by depositing bead-linker complexes onto open field surfaces was performed. The bead of each bead-linker complex defined the size of the discrete location on the surface into which the complex was deposited. For each spot detected in the resulting array, the content and number of actual oligonucleotide linkers present in the spot was determined using aggregate signal analysis. Such methods were used to tune the bead-linker system to efficiently control the number of linker units deposited onto each spot on the surface.

[00343] As shown in Figure 12, Panel A, when linker molecules were randomly deposited onto the surface using conventional deposition techniques, the resulting deposited linkers exhibited significant overlap of adjacent linkers with each other and a relatively high degree of observed loss. As the loading density of oligonucleotide linker onto the array was increased, the observed loss increased to 30%. In contrast, when bead- linker complexes were distributed onto the array ("semi-ordered"), the observed loss even at high loadings was only 17%.

[00344] Figure 12, Panel B depicts a graph plotting the loading number, i.e., the number of oligonucleotide linkers deposited using conventional techniques, or the number of oligonucleotide linkers deposited using the bead-linker complexes of the disclosure, (X axis), against the observed number of useful attachment sites resulting from the deposition (Y axis). As the graph illustrates, when the oligonucleotide linker is directly deposited onto the surface using conventional deposition techniques (random distribution), the number of useful attachment sites initially increases as the linker loading density is increased, but eventually levels off in a Pois son- dependent fashion at higher linker loadings. In contrast, when the linkers are deposited using the bead-linker complexes of the present disclosure, the number of useful attachment sites increases proportionally with the linker loading density, and the increase persists well beyond the limits of Poisson distribution applicable in the case of direct linker loadings using conventional techniques.

[00345] Figure 12, Panel C depicts the influence of linker length on the number of functional linkers (useful attachment sites) transferred to the surface using the bead-linker complexes of the disclosure. Each linker was attached to a single quantum dot; the numbers of linkers present in each spot (X axis) was determined using aggregate signal analysis. The bead- linker complex includes a variety of tunable parameters for controlling the number of functional groups transferred to the surface, including: bead size, linker coverage on bead and linker length. As depicted in Figure 12, Panel C, use of the shorter linker shifted the distribution of linkers per spot to mostly one, while use of the longer linker expanded the distribution.

Claims

CLAIMS [00346] What is claimed:

1. A method for fabricating a molecular array, comprising:

forming a cleavable bead-linker complex by binding a linker to a bead through a cleavable bond;

binding the linker of the cleavable bead-linker complex to a surface; and

cleaving the cleavable bond, thereby releasing the bead from the cleavable bead-linker complex and forming a surface-attached linker.

2. The method of claim 1, further including binding the surface-attached linker to a target analyte, thereby anchoring the analyte to the surface.

3. The method of claim 1, wherein the linker of the cleavable bead-linker complex includes a terminal amine moiety, the surface includes an amine-reactive moiety, and wherein binding the linker of the cleavable bead- linker complex includes forming a bond between the terminal amine moiety of the linker and the amine-reactive moiety of the surface.

4. The method of claim 1, wherein the linker of the cleavable bead-linker complex includes a biotin moiety, the surface includes an avidin moiety, and wherein binding the linker of the cleavable bead- linker complex includes forming a bond between the biotin moiety of the linker and the avidin moiety of the surface.

5. The method of claim 1, wherein the linker of the cleavable bead-linker complex includes one or more nucleotides linked via phosphodiester bonds, a rigid linear polymer and a linking moiety selected from the group consisting of: an amine moiety and a biotin moiety.

6. The method of claim 1, wherein the binding includes contacting the complex with a surface under conditions where the linker of the complex binds to the surface.

7. The method of claim 1, further including forming a plurality of cleavable bead linker complexes by binding a linker to each of a plurality of beads, binding the linker of each complex of the plurality of cleavable bead linker complexes to the surface, and cleaving the cleavable bond of each complex, thereby forming a plurality of surface-attached linkers.

8. The method of claim 7, wherein two or more of the surface-attached linkers are different from each other.

9. The method of claim 7, wherein each of the plurality of surface-attached linkers are the same as each other.

10. The method of claim 7, wherein the plurality of surface- attached linkers are an average distance of no greater than about 1500 nm apart from each other.

11. A composition for fabricating a molecular array, comprising: a bead-linker complex including at least one bead attached to at least one linker through a cleavable bond.

12. The composition of claim 11, wherein the linker is an oligonucleotide linker.

13. The composition of claim 12, wherein the oligonucleotide linker includes a cleavable moiety.

14. The composition of claim 13, wherein the cleavable moiety includes a disulfide moiety.

15. The composition of claim 14, wherein the oligonucleotide linker further includes an attachment moiety selected from the group consisting of: a reactive amine, an NHS ester, an alkyne, an azide and an aldehyde.

16. A cleavable bead-linker system useful for preparing a molecular array, comprising:

an oligonucleotide linker including a cleavable moiety and a surface-reactive moiety; a bead linked to the oligonucleotide linker through the cleavable moiety; and

a surface;

wherein the oligonucleotide linker is attached to the surface through the surface-reactive moiety.

17. The system of claim 16, wherein the oligonucleotide linker includes one or more nucleotides linked via phosphodiester bonds, the rigid linear polymer includes polyethylene glycol, and the linking moiety is selected from the group consisting of: an amine moiety and a biotin moiety.

18. The system of claim 16, wherein the diameter of the bead is between about 500 nm and 1500 nm.

19. The system of claim 16, wherein the surface comprises cavities sufficiently large to accommodate the bead.

20. The system of claim 19, wherein the cavities are not large enough to accommodate more than one bead simultaneously.

21. The system of claim 20, wherein the cavities of the surface have a diameter that less than twice the diameter of the bead.

22. A method for forming a molecular array, comprising:

contacting a plurality of bead- linker complexes with a surface, where each bead-linker complex includes at least one bead linked to one or more linkers through a cleavable moiety and where the contacting is performed under conditions where at least one linker of each bead-linker complexes binds to the surface, thereby forming a plurality of surface- attached bead-linker complexes; and

cleaving the cleavable moiety of each surface- attached bead-linker complex, thereby releasing the bead of each surface-attached bead- linker complex and forming a plurality of surface-attached linkers.

23. The method of claim 22, wherein the one or more linkers of each complex are

oligonucleotide linkers.

24. The method of claim 23, wherein the cleavable moiety includes a disulfide moiety.

25. The method of claim 23, wherein the linker includes a first attachment moiety, the surface includes a second attachment moiety, and the linker binds to the surface via reaction of the first and second attachment moieties with each other.