Methods, systems, and compositions relating to boronic acid-treated surfaces

Abstract

Compositions, methods, kits, and systems related to surfaces such as glass and polymeric surfaces, preferably surfaces conjugated to biomolecules, wherein the surfaces are exposed to a boronic acid composition comprising a boronic acid or boronic acid derivative, preferably 4-hydroxyphenyl boronic acid, under conditions wherein the boronic acid or boronic acid derivative passivates at least a portion of the surface.

Description

ENMRA-43211.601METHODS, SYSTEMS, AND COMPOSITIONSRELATING TO BORONIC ACID-TREATED SURFACESThe present application claims priority to U.S. Provisional Application Serial No. 63 / 744,707, filed January 13, 2025, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTIONThe present invention relates to compositions and methods for determining numbers of copies of individual molecules, such as nucleic acid molecules, without digital sequencing. The technologies find use, for example, in analysis of variations in copy numbers of specific nucleic acids sequences that may arise, e.g., from variations in chromosome number, gene copy number, expression level, etc. The technologies find particular application in genetic screening, e.g., analysis of cell-free nucleic acid (cfDNA) from a subject that may be indicative of a fetal condition, a neoplasm, or another disease indication. Analysis of cell-free nucleic acid in blood can be used to assess the health of a subject, or of a fetus carried by the subject. The technology herein relates to methods, systems, and kits for detecting and quantifying molecules, particularly nucleic acid molecules, and for determining relative amounts of molecules, e.g., fetal, and maternal DNA, gene dosage, etc.BACKGROUND OF THE INVENTIONDetection of the presence of, or variations in the numbers of molecules in a sample is a useful way of characterizing the sample and the source of the sample. For example, variations in gene dosage are clinically significant indicators of disease states, e.g., in a subject from whom a sample is collected. Variations in gene dosage arise due to errors in DNA replication and can occur in germ line cells, leading to congenital defects and even embryonic demise, or in somatic cells, often resulting in cancer. These replication anomalies can cause deletion or duplication of parts of genes, full-length genes and their surrounding regulatory regions, megabase-long portions of chromosomes, or entire chromosomes.Analysis of other biomolecules is also clinically important. For example, variations in amounts of RNA or protein may indicate changes in expression of a gene associated with a disease state.Quantifying variations in numbers of molecules finds application broadly in analyzing nucleic acids for any purpose, e.g., for characterizing nucleic acids and nucleic acid mixtures,ENMRA-43211.601such as nucleic acids indicative of cancer or other disease in a subject, nucleic acids indicative of microbes, e.g., viral and bacterial microbes and mixtures of microbes in a sample, etc.Current methods for quantifying variations in numbers of molecules, for example performing aneuploidy screening, that rely on next generation sequencing (NGS) are often time-consuming, expensive, and require extensive bioinformatics analysis.SUMMARY OF THE INVENTIONThe technology provided herein relates to compositions, methods, kits, and systems related to surfaces, such as glass and polymeric surfaces, preferably surfaces conjugated to biomolecules such as nucleic acids, wherein the surfaces are exposed to a boronic acid composition comprising a boronic acid or boronic acid derivative, preferably 4-hydroxyphenyl boronic acid, under conditions wherein the boronic acid or boronic acid derivative passivates at least a portion of the surface. Embodiments of the technology include but are not limited to:1. A method of modifying a support, the method comprising treating a surface on the support with a boronic acid composition under conditions wherein boronic acid or a boronic acid derivative, preferably 4-hydroxyphenyl boronic acid, passivates at least a portion of the surface.2. The method of embodiment 1, wherein at least a portion of the surface comprises glass, wherein the treating comprises exposing glass directly to the boronic acid composition.3. The method of embodiment 1, wherein at least a portion of the surface is polymeric, wherein the treating comprises exposing a polymeric surface to the boronic acid composition.4. The method of embodiment 3, wherein the polymeric surface comprises polyphenols.5. The method of embodiment 3, wherein the polymeric surface comprises tannic acid.ENMRA-43211.6016. The method of embodiment 3, wherein the polymeric surface comprises polyacrylic acid.7. The method of any one of embodiments 3 to 6, wherein the polymeric surface is homopolymeric.8. The method of any one of embodiments 3 to 6, wherein the polymeric surface is heteropolymeric.9. The method of embodiment 8, wherein the heteropolymeric surface comprises polyacrylic acid and tannic acid.10. The method of any one of embodiments 1 to 9, wherein the boronic acid composition comprises 4-hydroxyphenyl boronic acid, and wherein passivating the surface comprises complexing 4-hydroxyphenyl boronic acid to cv.s-diols on the surface.11. A method, comprising:a) providing a support comprising a surface; andb) exposing the surface to a boronic acid composition comprising a boronic acid or boronic acid derivative, preferably 4-hydroxyphenyl boronic acid, under conditions wherein the boronic acid or boronic acid derivative passivates at least a portion of the surface.12. The method of embodiment 11, wherein at least a portion of the surface comprises glass, wherein the method comprises exposing glass directly to the boronic acid composition.13. The method of embodiment 11, wherein at least a portion of the surface is polymeric, wherein the method comprises exposing a polymeric surface to the boronic acid composition.ENMRA-43211.60114. The method of embodiment 13, wherein the polymeric surface comprises polyphenols.15. The method of embodiment 13, wherein the polymeric surface comprises tannic acid.16. The method of embodiment 13, wherein the polymeric surface comprises polyacrylic acid.17. The method of any one of embodiments 13 to 16, wherein the polymeric surface is homopolymeric.18. The method of any one of embodiments 13 to 16, wherein the polymeric surface is heteropolymeric.19. The method of embodiment 18, wherein the heteropolymeric surface comprises polyacrylic acid and tannic acid.20. The method of any one of embodiments 13 to 19, wherein the boronic acid composition comprises 4-hydroxyphenyl boronic acid, and wherein passivating comprises complexing 4-hydroxyphenyl boronic acid to c / .s-diols on the polymeric surface.21. The method of embodiment 20, wherein the polymeric surface comprises galloyl groups.22. A method, comprising:a) conjugating nucleic acid to a surface to form a nucleic acid-conjugated surface; andb) exposing the nucleic acid-conjugated surface to a boronic acid composition comprising a boronic acid or boronic acid derivative, preferably 4-hydroxyphenyl boronic acid, under conditions wherein the boronic acid or boronic acid derivative passivates at least a portion of the surface.ENMRA-43211.60123. The method of embodiment 22, wherein prior to step a), the method comprises exposing the surface to a mixture comprising a monomer, under conditions wherein the mixture forms a polymeric coating.24. The method of embodiment 23, wherein the mixture comprises an acrylic acid monomer.25. The method of embodiment 23 or embodiment 24, wherein the mixture comprises a polyphenolic monomer comprising a galloyl group.26. The method of embodiment 25, wherein the polyphenolic monomer comprises one or more of epigallocatechin-3 -gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and tannic acid.27. The method of embodiment 25 or embodiment 26, wherein the mixture further comprises a non-phenolic monomer.28. The method of embodiment 27, wherein the mixture comprises acrylic acid and tannic acid.29. The method of any one of embodiments 22-28, wherein the surface comprises glass.30. A composition comprising a plurality of complexes bound to a 4-hydroxyphenyl boronic acid-passivated surface, wherein the complexes comprise single or double-stranded nucleic acid.31. The composition of embodiment 30, wherein the 4-hydroxyphenyl boronic acid-passivated surface comprises a polymeric surface, preferably a polymeric surface comprising galloyl groups.32. The composition of embodiment 31, wherein the polymeric surface comprises tannic acid complexed with 4-hydroxyphenyl boronic acid.ENMRA-43211.60133. The composition of embodiment 30, wherein the 4-hydroxyphenyl boronic acid-passivated surface comprises 4-hydroxyphenyl boronic acid-passivated glass.DEFINITIONSTo facilitate an understanding of the present invention, a number of terms and phrases are defined below:Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.” The transitional phrase “consisting essentially of’ as used in claims in the present application limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel character! stic(s)” of the claimed invention, as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition “consisting essentially of’ recited elements may contain an unrecited contaminant at a level such that, though present, the contaminant does not alter the function of the recited composition as compared to a pure composition, i.e., a composition “consisting of’ the recited components.The term “sample” in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid foodENMRA-43211.601and feed products and ingredients such as dairy items, vegetables, meat and meat byproducts, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as canines, felines, ungulates, bear, fish, lagomorphs, rodents, marsupials, etc.Environmental samples include environmental material such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.The term “target” as used herein refers to a molecule sought to be sorted out from other molecules for assessment, measurement, or other characterization. For example, a target nucleic acid may be sorted from other nucleic acids in a sample, e.g., by probe binding, amplification, isolation, capture, etc. When used in reference to a hybridization-based detection, e.g., polymerase chain reaction, “target” refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction, while when used in an assay in which target DNA is not amplified, e.g., in capture by molecular inversion probes (MIPS), a target comprises the site bounded by the hybridization of the target-specific arms of the MIP, such that the MIP can be ligated and the presence of the target nucleic acid can be detected.The term “source of target nucleic acid” refers to any sample that contains nucleic acids (RNA or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to blood, plasma, serum, saliva, urine, feces, gastrointestinal fluid, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, and semen.As used herein, the “sensitivity” of a given assay (or set of assays used together) refers to the percentage of samples that report a particular form or variant, e.g., a mutation, gene duplication, chromosome duplication, above a threshold value that distinguishes between samples exhibiting a variant phenotype (e.g., cancerous cells, aneuploidy) and samples exhibiting a normal or wild-type phenotype (e.g., non-cancerous cells, euploidy). In some embodiments, a “positive” is defined as a clinically-confirmed variant that reports an assay result associated with the presence of the disease or condition to be detected, and a false negative is defined as a clinically-confirmed variant that reports an assay result associated with the absence of the disease or condition. The value of sensitivity, therefore, reflects the probability that a given diagnostic assay performed on a known variant or diseased sample will produce a result indicative of the presence of the variation or disease. AsENMRA-43211.601defined here, the clinical relevance of a calculated sensitivity value represents an estimation of the probability that a given assay would detect the presence of a clinical condition when applied to a subject with that condition. Using the technology described herein, it may be possible to achieve a certain level of accuracy without the need for generating sequence reads. The accuracy may refer to sensitivity, it may refer to specificity, or it may refer to some combination thereof. The desired level of accuracy may be between 90% and 95%; it may be between 95% and 98%; it may be between 98% and 99%; it may be between 99% and 99.5%; it may be between 99.5% and 99.9%; it may be between 99.9% and 99.99%; it may be between 99.99% and 99.999%, it may be between 99.999% and 100%. Levels of accuracy above 95% may be referred to as high accuracy.As used herein, the “specificity” of a given assay (or set of assays used together) refers to the percentage of normal samples that report an assay result associated with the presence of the disease or condition to be detected, and a false positive is defined as a clinically-confirmed normal sample that reports an assay result associated with the presence of the disease or condition. The value of specificity, therefore, reflects the probability that a given diagnostic assay performed on a known normal sample will produce a result indicative of the presence of the variation or disease. As defined here, the clinical relevance of the calculated specificity value represents an estimation of the probability that a given marker would detect the absence of a clinical condition when applied to a subject without that condition.The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide or a precursor. The RNA or polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.The term “chromosome-specific” as used herein refers to a sequence that is found only in that particular type of chromosome.As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization ( / .< ., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the Tmof the formed hybrid. “Hybridization” methods involve the annealing of oneENMRA-43211.601nucleic acid to another, complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the “hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology.The term “oligonucleotide” as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides and more preferably at least about 15 to 30 nucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof.Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the “3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. A first region along a nucleic acid strand is said to be upstream of another region if the 3' end of the first region is before the 5' end of the second region when moving along a strand of nucleic acid in a 5' to 3' direction.When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3' end of one oligonucleotide points towards the 5' end of the other, the former may be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide. Similarly, when two overlapping oligonucleotides are hybridized to the same linear complementary nucleic acid sequence, with the first oligonucleotide positioned such that its 5' end is upstream of the 5' end of the second oligonucleotide, and the 3' end of the first oligonucleotide is upstream of the 3' end of the second oligonucleotide, the first oligonucleotide may be called theENMRA-43211.601“upstream” oligonucleotide and the second oligonucleotide may be called the “downstream” oligonucleotide.The term “primer” refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated, e.g., in the presence of nucleotides and a suitable nucleic acid polymerase. An oligonucleotide “primer” may occur naturally, may be made using molecular biological methods, e.g., purification of a restriction digest, or may be produced synthetically. In preferred embodiments, a primer is composed of or comprises DNA.A primer is selected to be “substantially” complementary to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.The term “sequence variation” as used herein refers to differences in nucleic acid sequence between two nucleic acids. For example, a wild-type structural gene and a mutant form of this wild-type structural gene may vary in sequence by the presence of single base substitutions and / or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another. A second mutant form of the structural gene may exist. This second mutant form is said to vary in sequence from both the wild-type gene and the first mutant form of the gene.The term “nucleotide analog” as used herein refers to modified or non-naturally occurring nucleotides including but not limited to analogs that have altered stacking interactions such as 7-deaza purines ( / .< ., 7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogen bonding configurations (e.g., such as Iso-C and Iso-G and other non-standard base pairs described in U.S. Patent No. 6,001,983 to S. Benner); non-hydrogen bonding analogs (e.g., non-polar, aromatic nucleoside analogs such as 2,4-difluorotoluene, described by B.A. Schweitzer and E.T. Kool, J. Org. Chem., 1994, 59, 7238-7242, B.A. Schweitzer and E.T. Kool, J. Am. Chem. Soc., 1995, 117, 1863-1872); “universal” bases such as 5-nitroindole and 3 -nitropyrrole; and universal purines and pyrimidines (such as “K”ENMRA-43211.601and “P” nucleotides, respectively; P. Kong, etal., Nucleic Acids Res., 1989, 17, 10373-10383, P. Kong etal., Nucleic Acids Res., 1992, 20, 5149-5152). Nucleotide analogs include base analogs, and comprise modified forms of deoxyribonucleotides as well as ribonucleotides, and include but are not limited to modified bases and nucleotides described in U.S. Pat. Nos. 5,432,272; 6,001,983; 6,037,120; 6,140,496; 5,912,340; 6,127,121 and 6,143,877, each of which is incorporated herein by reference in their entireties; heterocyclic base analogs based on the purine or pyrimidine ring systems, and other heterocyclic bases.The term “duplex” refers to the state of nucleic acids in which the base portions of the nucleotides on one strand are bound through hydrogen bonding their complementary bases arrayed on a second strand. The condition of being in a duplex form reflects on the state of the bases of a nucleic acid. By virtue of base pairing, the strands of nucleic acid also generally assume the tertiary structure of a double helix, having a major and a minor groove. The assumption of the helical form is implicit in the act of becoming duplexed.The term “template” refers to a strand of nucleic acid on which a complementary copy is built from nucleoside triphosphates through the activity of a template-dependent nucleic acid polymerase. Within a duplex the template strand is, by convention, depicted and described as the “bottom” strand. Similarly, the non-template strand is often depicted and described as the “top” strand.As applied to polynucleotides, the term “substantial identity” denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence, which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence, for example, as a splice variant of the full-length sequences.As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions that are not identical differ byENMRA-43211.601conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.The term “label” as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein. Labels include but are not limited to dyes; radiolabels such as32P; binding moieties such as biotin; haptens such as digoxigenin; luminogenic, phosphorescent or fluorogenic moieties; mass tags; and fluorescent dyes alone or in combination with moieties that can suppress (“quench”) or shift emission spectra by fluorescence resonance energy transfer (FRET). FRET is a distance-dependent interaction between the electronic excited states of two molecules (e.g., two dye molecules, or a dye molecule and a non-fluorescing quencher molecule) in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. (Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300, each incorporated herein by reference). As used herein, the term “donor” refers to a fluorophore that absorbs at a first wavelength and emits at a second, longer wavelength. The term “acceptor” refers to a moiety such as a fluorophore, chromophore, or quencher that has an absorption spectrum that overlaps the donor’s emission spectrum, and that is able to absorb some or most of the emitted energy from the donor when it is near the donor group (typically between 1-100 nm). If the acceptor is a fluorophore, it generally then re-emits at a third, still longer wavelength; if it is a chromophore or quencher, it then releases the energy absorbed from the donor without emitting a photon. In some embodiments, changes in detectable emission from a donor dye (e.g., when an acceptor moiety is near or distant) are detected. In some embodiments, changes in detectable emission from an acceptor dye are detected. In preferred embodiments, the emission spectrum of the acceptor dye is distinct from the emission spectrum of the donor dye such that emissions from the dyes can be differentiated (e.g., spectrally resolved) from each other.ENMRA-43211.601In some embodiments, a donor dye is used in combination with multiple acceptor moieties. In a preferred embodiment, a donor dye is used in combination with a nonfluorescing quencher and with an acceptor dye, such that when the donor dye is close to the quencher, its excitation is transferred to the quencher rather than the acceptor dye, and when the quencher is removed (e.g., by cleavage of a probe), donor dye excitation is transferred to an acceptor dye. In particularly preferred embodiments, emission from the acceptor dye is detected. See, e.g., Tyagi, et aL, Nature Biotechnology 18:1191 (2000), which is incorporated herein by reference.Labels may provide signals detectable by fluorescence (e.g., simple fluorescence, FRET, time-resolved fluorescence, fluorescence polarization, etc.), radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, characteristics of mass or behavior affected by mass (e.g., MALDI time-of-flight mass spectrometry), and the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable.In some embodiment a label comprises a particle for detection. In preferred embodiments, the particle is a phosphor particle. In particularly preferred embodiments, the phosphor particle is an up-converting phosphor particle (see, e.g., Ostermayer, F.W.Preparation and properties of infrared-to-visible conversion phosphors. Metall. Trans. 752, 747-755

[1971] ). In some embodiments, rare earth-doped ceramic particles are used as phosphor particles. Phosphor particles may be detected by any suitable method, including but not limited to up-converting phosphor technology (UPT), in which up-converting phosphors transfer low energy infrared (IR) radiation to high-energy visible light. While the present invention is not limited to any particular mechanism, in some embodiments the UPT up-converts infrared light to visible light by multi-photon absorption and subsequent emission of dopant-dependent phosphorescence. See, e.g., U.S. Patent No. 6,399,397, Issued June 4, 2002 to Zarling, et al.,' van De Rijke, et al., Nature Biotechnol. 19(3):273-6

[2001] ; Corstjens, et al., IEE Proc. Nanobiotechnol. 152(2):64

[2005] , each incorporated by reference herein in its entirety.As used herein, the terms “solid support” or “support” refer to any material that provides a substrate structure to which another material can be attached. A support or substrate may be, but need not be, solid. Support materials include smooth solid supports (e.g., smooth metal, glass, quartz, plastic, silicon, wafers, carbon (e.g., diamond), and ceramicENMRA-43211.601surfaces, etc.), as well as textured and porous materials. Solid supports need not be flat. Supports include any type of shape, including spherical shapes (e.g., beads). Support materials also include, but are not limited to, gels, hydrogels, aerogels, rubbers, polymers, and other porous and / or non-rigid materials.As used herein, the terms “bead” and “particle” are used interchangeably, and refer to a small support, typically a solid support, that is capable of moving about when in a solution (e.g., it has dimensions smaller than those of the enclosure or container in which the solution resides). In some embodiments, beads may settle out of a solution when the solution is not mixed (e.g., by shaking, thermal mixing, vortexing), while in other embodiments, beads may be suspended in solution in a colloidal fashion. In some embodiments, beads are completely or partially spherical or cylindrical. However, beads are not limited to any particular three-dimensional shape. In some embodiments, beads or particles may be paramagnetic. For example, in some embodiments, beads and particles comprise a magnetic material, e.g., ferrous oxide.A bead or particle is not limited to any particular size, and in a preparation comprising a plurality of particles, the particles may be essentially uniform in size (e.g., in diameter) or may be a mixture of different sizes. In some embodiments, beads comprise or consist of nanoparticles, e.g., particles of less than about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, or 1 nm in diameter. In some embodiments, the nanoparticle beads between 5 and 20 nm average diameter.As used herein, the term “passivate” as used in reference to surfaces refers to making a surface less reactive or unreactive, e.g., by altering the surface and / or by coating the surface. Passivation of a surface may comprise covalent and / or non-covalent modifications of the surface, and the reactivity to be reduced or eliminated by a particular treatment may relate to reducing covalent and / or non-covalent reactivity, e.g., with an environmental element such as a reaction mixture, biomolecule, corrosive agent, etc., as compared to the reactivity of the surface if unpassivated.Materials attached to a solid support may be attached to any portion of the solid support (e.g., may be attached to an interior portion of a porous solid support material, or to an exterior portion, or to a flat portion on an otherwise non-flat support, or vice versa). In preferred embodiments of the technology, biological molecules such as nucleic acid or protein molecules are attached to solid supports. A biological material is “attached” to a solidENMRA-43211.601support when it is affixed to the solid support through chemical or physical interaction. In some embodiments, attachment is through a covalent bond. However, attachments need not be covalent and need not be permanent. In some embodiments, an attachment may be undone or disassociated by a change in condition, e.g, by temperature, ionic change, addition or removal of a chelating agent, or other changes in the solution conditions to which the surface and bound molecule are exposed.In some embodiments, materials are attached to a first support and are localized to the surface of a second support. For example, in some embodiments, materials that comprise a ferrous or magnetic particle may be magnetically localized to a surface or a region of a surface, such as a planar surface of a slide or well.As used herein in reference to a support or substrate, e.g., for a coating or for attachment of a molecule, the term “surface” broadly refers to a portion of a support or substrate that is accessible for a purpose. For example, a portion of a bead or vessel or plate that is accessible to be coated, functionalized, attached to a moiety, e.g., an oligonucleotide or other macromolecule, or otherwise treated, may be considered a “surface” of the bead or plate, even if the surface is on an interior portion of the bead or vessel (e.g., within a pore, within a sintered matrix, inside a well, etc. Similarly, a portion of a matrix that is flexible and / or porous (e.g., a hydrogel, aerogel, mesh, and that is accessible for a purpose, e.g., to be coated, functionalized, attached to a moiety, derivatized, etc., may be considered a surface of the matrix. In certain embodiments, a support may comprise a support surface, sometimes termed a first surface, which is the surface of the structural support material, e.g., in the absence of a coating or modifying layer, and may further comprise substrate surface, sometimes termed a second surface, which is the surface that is accessible for a purpose after the support surface is modified, e.g., by coating with a polymer or other coating. In some embodiments, the substrate surface comprises functional groups capable of complexing covalently or non-covalently with the one or more analytes, such as oligonucleotides or polypeptides that comprise reactive or binding groups suitable for complexing with the substrate surface functional groups.As used herein, the term “detergent” refers any of a group of synthetic, organic, liquid or water-soluble agents that have wetting-agent and emulsifying-agent properties, and include anionic agents (e.g, sodium dodecyl sulfate, sodium lauryl sulfate, ammonium lauryl sulfate, cationic (e.g., benzalkonium chloride, cetyltrimethylammonium bromide) linear alkylbenzene sulfonates (e.g., sodium dodecylbenzene sulfonate), non-ionic (e.g., a TWEEN (e.g.,ENMRA-43211.601polyoxyethylene (20) sorbitan -monolaurate, -monopalmitate, -monostearate, or -monooleate); TRITON (polyethylene glycol / ?-(l,l,3,3-tetramethylbutyl)-phenyl ether, steroid and steroidal al glycosides (e.g., saponin, digitonin); and zwitterionic (net neutral) agents such as 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), compounds, some embodiments, a “detergent” comprises a mixture of agents, e.g., TEEPOL® detergent, comprising sodium dodecylbenzene sulfonate, sodium C12-C15 alcohol ether sulfate.In some embodiments, a target molecule, e.g, a biological material, is attached to a solid support through a “spacer molecule” or “linker group.” Such spacer molecules are molecules that have a first portion that attaches to the biological material and a second portion that attaches to the solid support. Spacer molecules typically comprise a chain of atoms, e.g, carbon atoms, that provide additional distance between the first portion and the second portion. Thus, when attached to the solid support, the spacer molecule permits separation between the solid support and the biological material, but is attached to both. Examples of linkers and spacers include but are not limited to carbon chains, e.g., C3 and C6 (hexanediol), l',2'-dideoxyribose (dSpacer); photocl eavable (PC) spacers; tri ethylene glycol (TEG); and hexa-ethylene glycol spacers (Integrated DNA Technologies, Inc.).As used herein, the terms “array” and “microarray” refer a surface or vessel comprising a plurality of pre-defined loci that are addressable for analysis of the locus, e.g., to determine a result of an assay. Analysis at a locus in an array is not limited to any particular type of analysis and includes, e.g., analysis for detection of an atom, molecule, chemical reaction, light or fluorescence emission, suppression, or alteration (e.g., in intensity or wavelength) indicative of a result at that locus. Examples of pre-defined loci include a grid or any other pattern, wherein the locus to be analyzed is determined by its known position in the array pattern. Microarrays, for example, are described generally in Schena, “Microarray Biochip Technology,” Eaton Publishing, Natick, MA, 2000. Examples of arrays include but are not limited to supports with a plurality of molecules non-randomly bound to the surface (e.g., in a grid or other regular pattern) and vessels comprising a plurality of defined reaction loci (e.g., wells) in which molecules or signal -generating reactions may be detected. In some embodiments, an array comprises a patterned distribution of wells that receive beads, e.g., as described above for the SIMOA technology. See also U.S. Patent Nos. 9,057,730; 9,556,429; 9,481,883; and 9,376,677, each of which is incorporated herein by reference in its entirety, for all purposes.ENMRA-43211.601As used herein, the terms “dispersed” and “dispersal” as used in reference to loci or sites, e.g, on a support or surface, refers to a collection of loci or sites that are distributed or scattered on or about the surface, wherein at least some of the loci are sufficiently separated from other loci that they are individually detectable or resolvable, one from another, e.g., by a detector such as a microscope. Dispersed loci may be in an ordered array, or they may be in an irregular distribution or dispersal, as described below.As used herein, the term “irregular” as used in reference to a dispersal or distribution of loci or sites, e.g., on a solid support or surface, refers to distribution of loci on or in a surface in a non- arrayed manner. For example, molecules may be irregularly dispersed on a surface by application of a solution of a particular concentration that provides a desired approximate average distance between the molecules on the surface, but at sites that are not pre-defined by or addressable any pattern on the surface or by the means of applying the solution (e.g., inkjet printing). In such embodiments, analysis of the surface may comprise finding the locus of a molecule by detection of a signal wherever it may appear (e.g., scanning a whole surface to detect fluorescence anywhere on the surface). This contrasts to locating a signal by analysis of a surface or vessel only at predetermined loci (e.g., points in a grid array), to determine how much (or what type of) signal appears at each locus in the grid.As used herein, the term “distinct” in reference to signals refers to signals that can be differentiated one from another, e.g., by spectral properties such as fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, etc., or by capability of interaction with another moiety, such as with a chemical reagent, an enzyme, an antibody, etc.As used herein, the term “nucleic acid detection assay” refers to any method of determining the nucleotide composition of a nucleic acid of interest. Nucleic acid detection assay include but are not limited to, DNA sequencing methods, probe hybridization methods, structure specific cleavage assays (e.g., the INVADER assay, (Hologic, Inc.) and are described, e.g, in U.S. Patent Nos. 5,846,717; 5,985,557; 5,994,069; 6,001,567; 6,090,543; and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and US Pat. No. 9,096,893, each of which is herein incorporated by reference in its entirety for all purposes); enzyme mismatch cleavage methods (e.g, Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, herein incorporated by reference in their entireties); polymerase chain reaction (PCR), described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, andENMRA-43211.6015,624,802, herein incorporated by reference in their entireties); rolling circle amplification (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, herein incorporated by reference in their entireties); the variation of rolling circle amplification called “RAM amplification” (see, e.g., US 5,942,391, incorporated herein by reference in its entirety; NASBA (e.g., U.S. Pat. No. 5,409,818, herein incorporated by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, herein incorporated by reference in its entirety); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, herein incorporated by reference in their entireties); cycling probe technology (e.g., U.S. Pat. Nos.5,403,711, 5,011,769, and 5,660,988, herein incorporated by reference in their entireties); Dade Behring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, herein incorporated by reference in their entireties); ligase chain reaction (e.g., Barany Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Pat. No. 5,288,609, herein incorporated by reference in its entirety).In some embodiments, target nucleic acid is amplified (e.g., by PCR) and amplified nucleic acid is detected simultaneously using an invasive cleavage assay. Assays configured for performing a detection assay (e.g., invasive cleavage assay) in combination with an amplification assay are described in U.S. Pat. No. 9,096,893, incorporated herein by reference in its entirety for all purposes. Additional amplification plus invasive cleavage detection configurations, termed the QuARTS method, are described in, e.g., in U.S. Pat. Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392, each of which is incorporated herein by reference for all purposes. The term “invasive cleavage structure” as used herein refers to a cleavage structure comprising i) a target nucleic acid, ii) an upstream nucleic acid (e.g., an invasive or “INVADER” oligonucleotide), and iii) a downstream nucleic acid (e.g., a probe), where the upstream and downstream nucleic acids anneal to contiguous regions of the target nucleic acid, and where an overlap forms between the a 3' portion of the upstream nucleic acid and duplex formed between the downstream nucleic acid and the target nucleic acid. An overlap occurs where one or more bases from the upstream and downstream nucleic acids occupy the same position with respect to a target nucleic acid base, whether or not the overlapping base(s) of the upstream nucleic acid are complementary with the target nucleic acid, and whether or not those bases are natural bases or non-natural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps with the downstream duplex is a non-base chemical moiety such as an aromatic ring structure, e.g., as disclosed,ENMRA-43211.601for example, in U.S. Pat. No. 6,090,543, incorporated herein by reference in its entirety. In some embodiments, one or more of the nucleic acids may be attached to each other, e.g., through a covalent linkage such as nucleic acid stem-loop, or through a non-nucleic acid chemical linkage (e.g., a multi-carbon chain). As used herein, the term “flap endonuclease assay” includes “INVADER” invasive cleavage assays and QuARTS assays, as described above.As used herein, the terms “digital PCR,” “single molecule PCR” and “single molecule amplification” refer to PCR and other nucleic acid amplification methods that are configured to provide amplification product or signal from a single starting molecule. Typically, samples are divided, e.g., by serial dilution or by partition into small enough portions (e.g., in microchambers or in emulsions) such that each portion or dilution has, on average as assessed according to Poisson distribution, no more than a single copy of the target nucleic acid.Methods of single molecule PCR are described, e.g, in US 6,143,496, which relates to a method comprising dividing a sample into multiple chambers such that at least one chamber has at least one target, and amplifying the target to determine how many chambers had a target molecule; US 6,391,559; which relates to an assembly for containing and portioning fluid; and US 7,459,315, which relates to a method of dividing a sample into an assembly with sample chambers where the samples are partitioned by surface affinity to the chambers, then sealing the chambers with a curable “displacing fluid.” See also US 6,440,706 and US 6,753,147, and Vogelstein, etal., Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9236-9241, August 1999. See also US 20080254474, describing a combination of digital PCR combined with methylation detection.The term “sequencing,” as used herein, is used in a broad sense and may refer to any technique known in the art that allows the order of at least some consecutive nucleotides in at least part of a nucleic acid to be identified, including without limitation at least part of an extension product or a vector insert. In some embodiments, sequencing allows the distinguishing of sequence differences between different target sequences. Exemplary sequencing techniques include targeted sequencing, single molecule real-time sequencing, electron microscopy -based sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, targeted sequencing, exon sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-ENMRA-43211.601base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, realtime sequencing, reverse-terminator sequencing, ion semiconductor sequencing, nanoball sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, miSeq (Illumina), HiSeq 2000 (Illumina), HiSeq 2500 (Illumina), Illumina Genome Analyzer (Illumina), Ion Torrent PGM™ (Life Technologies), MinlON™ (Oxford Nanopore Technologies), real-time SMRT™ technology (Pacific Biosciences), the Probe- Anchor Ligation (cP AL™) (Complete Genomics / BGI), SOLiD® sequencing, MS-PET sequencing, mass spectrometry, and a combination thereof. In some embodiments, sequencing comprises detecting the sequencing product using an instrument, for example but not limited to an ABI PRISM® 377 DNA Sequencer, an ABI PRISM® 310, 3100, 3100-Avant, 3730, or 3730x1 Genetic Analyzer, an ABI PRISM® 3700 DNA Analyzer, or an Applied Biosystems SOLiD™ System (all from Applied Biosystems), a Genome Sequencer 20 System (Roche Applied Science), or a mass spectrometer. In certain embodiments, sequencing comprises emulsion PCR. In certain embodiments, sequencing comprises a high throughput sequencing technique, for example but not limited to, massively parallel signature sequencing (MPSS).As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably in reference to a chain of two or more amino acids linked together by peptide bonds. Polypeptides may be synthetic or naturally occurring, and may be short, e.g., between two about 30 amino acid residues, or may be hundreds or thousands of amino acid residues in length. Polypeptides may be composed of the 20 main naturally-occurring amino acids, or may comprise one or more non-natural amino acids, e.g., peptide nucleic acid residues, which comprise pyrimidine or purine bases on a peptide chain backbone, or modified versions of natural amino acids (e.g., modified in the structure of the side groups).As used herein, the term “lectins” refers to a class of non-antibody proteins that specifically binds to sugars and to sugar moi eties (e.g., sugar moi eties on glycoproteins and glucolipids, or within complex carbohydrates).As used herein, the terms “crowding agent” and “volume excluder,” as used in reference to a component of a fluid reaction mixture, are used interchangeably and refer toENMRA-43211.601compounds, generally polymeric compounds, that reduce available fluid volume in a reaction mixture, thereby increasing the effective concentration of reactant macromolecules (e.g., nucleic acids, enzymes, etc.) Crowding reagents include, e.g., glycerol, ethylene glycol, polyethylene glycol, ficoll, serum albumin, casein, and dextran.As used herein, the terms “digital sequencing,” “single-molecule sequencing,” and “next generation sequencing (NGS)” are used interchangeably and refer to determining the nucleotide sequence of individual nucleic acid molecules. Systems for individual molecule sequencing include but are not limited to the 454 FLX™ or 454 TITANIUM™ (Roche), the SOLEXA™ / Illumina Genome Analyzer (Illumina), the HELISCOPE™ Single Molecule Sequencer (Helicos Biosciences), and the SOLID™ DNA Sequencer (Life Technologies / Applied Biosystems) instruments), as well as other platforms still under development by companies such as Intelligent Biosystems and Pacific Biosystems. See also U.S. Patent No. 7,888,017, entitled “Non-invasive fetal genetic screening by digital analysis,” relating to digital analysis of maternal and fetal DNA, e.g, cfDNA.As used herein, the term “probe” or “hybridization probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing, at least in part, to another oligonucleotide of interest. A probe may be singlestranded or double-stranded. Probes are useful in the detection, identification and isolation of particular sequences. In some preferred embodiments, probes used in the present invention will be labeled with a “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.The term “MIP” as used herein, refers to a molecular inversion probe (or a circular capture probe). Molecular inversion probes (or circular capture probes) are nucleic acid molecules that comprise a pair of unique polynucleotide arms that hybridize to a target nucleic acid to form a nick or gap and a polynucleotide linker e.g., a universal backbone linker). In some embodiments, the unique polynucleotide arms hybridize to a target strand immediately adjacent to each other to form a ligatable nick (generally termed “padlock probes”) while in some embodiments, one the hybridized MIP must be further modified (e.g., by polymerase extension, base excision, and / or flap cleavage) to form a ligatable nick.ENMRA-43211.601Ligation of a MIP probe to form a circular nucleic acid is typically indicative of the presence of the complementary target strand. In some embodiments, MIPs comprise one or more unique molecular tags (or unique molecular identifiers). See, for example, Figure 1. In some embodiments, a MIP may comprise more than one unique molecular tags, such as, two unique molecular tags, three unique molecular tags, or more. In some embodiments, the unique polynucleotide arms in each MIP are located at the 5' and 3' ends of the MIP, while the unique molecular tag(s) and the polynucleotide linker are located internal to the 5' and 3' ends of the MIP. For example, the MIPs that are used in some embodiments of this disclosure comprise in sequence the following components: first unique polynucleotide arm - first unique molecular tag - polynucleotide linker - second unique molecular tag - second unique polynucleotide arm. In some embodiments, the MIP is a 5' phosphorylated single-stranded nucleic acid (e.g., DNA) molecule. See, for example, WO 2017 / 020023, filed July 29, 2016, and WO 2017 / 020024, filed July 29, 2016, each of which is incorporated by reference herein for all purposes.As used herein, the terms “circular nucleic acid” and “circularized nucleic acid” as used, for example, in reference to probe nucleic acids, refers to nucleic acid strands that are joined at the ends, e.g., by ligation, to form a continuous circular strand of nucleic acid.The unique molecular tag may be any tag that is detectable and can be incorporated into or attached to a nucleic acid (e.g., a polynucleotide) and allows detection and / or identification of nucleic acids that comprise the tag. In some embodiments the tag is incorporated into or attached to a nucleic acid during sequencing (e.g., by a polymerase). Non-limiting examples of tags include nucleic acid tags, nucleic acid indexes or barcodes, radiolabels (e.g., isotopes), metallic labels, fluorescent labels, chemiluminescent labels, phosphorescent labels, fluorophore quenchers, dyes, proteins (e.g., enzymes, antibodies or parts thereof, linkers, members of a binding pair), the like or combinations thereof. In some embodiments, particularly sequencing embodiments, the tag (e.g., a molecular tag) is a unique, known and / or identifiable sequence of nucleotides or nucleotide analogues (e.g., nucleotides comprising a nucleic acid analogue, a sugar and one to three phosphate groups). In some embodiments, tags are six or more contiguous nucleotides. A multitude of fluorophore-based tags are available with a variety of different excitation and emission spectra. Any suitable type and / or number of fluorophores can be used as a tag. In some embodiments 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500 orENMRA-43211.601more, 1000 or more, 10,000 or more, 100,000 or more different tags are utilized in a method described herein (e.g., a nucleic acid detection and / or sequencing method). In some embodiments, one or two types of tags (e.g., different fluorescent labels) are linked to each nucleic acid in a library. In some embodiments, chromosome-specific tags are used to make chromosomal counting faster or more efficient. Detection and / or quantification of a tag can be performed by a suitable method, machine or apparatus, non-limiting examples of which include flow cytometry, quantitative polymerase chain reaction (qPCR), gel electrophoresis, a luminometer, a fluorometer, a spectrophotometer, a suitable gene- chip or microarray analysis, Western blot, mass spectrometry, chromatography, cytofluorimetric analysis, fluorescence microscopy, a suitable fluorescence or digital imaging method, confocal laser scanning microscopy, laser scanning cytometry, affinity chromatography, manual batch mode separation, electric field suspension, a suitable nucleic acid sequencing method and / or nucleic acid sequencing apparatus, the like and combinations thereof.In the MIPs, the unique polynucleotide arms are designed to hybridize immediately upstream and downstream of a specific target sequence (or site) in a nucleic acid target, e.g., in an RNA, cfDNA, or genomic nucleic acid sample. In some embodiments, hybridization of a MIP to a target sequence produces a ligatable nick without a gap, / .< ., the two arms of the MIP hybridize to contiguous sequences in the target strand such that no overlap or gap is formed upon hybridization. Such zero-gap MIPs are generally termed “padlock” probes. See, e.g., M. Nilsson, et al. “Padlock probes: circularizing oligonucleotides for localized DNA detection.” Science. 265 (5181): 2085-2088 (1994); J. Baner, et al., Nucleic Acids Res. , 26 (22):5073-5078 (1998). In other embodiments the hybridized MIP / target nucleic acid complex requires modification to produce a ligatable nick. For example, in some embodiments, hybridization leaves a gap that is filled, e.g., by polymerase extending a 3' end of the MIP, prior to ligation, while in other embodiments, hybridization forms an overlapping flap structure that must be modified, e.g., by a flap endonuclease or a 3' exonuclease, to produce a ligatable nick. In some embodiments, MIPS comprise unique molecular tags are short nucleotide sequences that are randomly generated. In some embodiments, the unique molecular tags do not hybridize to any sequence or site located on a genomic nucleic acid fragment or in a genomic nucleic acid sample. In some embodiments, the polynucleotide linker (or the backbone linker) in the MIPs are universal in all the MIPs used in embodiments of this disclosure.ENMRA-43211.601In some embodiments, the MIPs are introduced to nucleic acid fragments derived from a test subject (or a reference subject) to perform capture of target sequences or sites (or control sequences or sites) located on a nucleic acid sample (e.g., a genomic DNA). In some embodiments, fragmenting aids in capture of target nucleic acid by molecular inversion probes. In some embodiments, for example, when the nucleic acid sample is comprised of cell free nucleic acid, fragmenting may not be necessary to improve capture of target nucleic acid by molecular inversion probes. For example, in some types of samples, cell free nucleic acid is fragmented in the sample such that further fragmentation is not necessary and may even be detrimental capture of the target nucleic acids. As described in greater detail herein, after capture of the target sequence (e.g., locus) of interest, the captured target may be subjected to enzymatic gap-filling and ligation steps, such that a copy of the target sequence is incorporated into a circle-like structure. In some embodiments, nucleic acid analogs, e.g, containing labels, haptens, etc., may be incorporated in the filled section, for use, e.g., in downstream detection, purification, or other processing steps. Capture efficiency of the MIP to the target sequence on the nucleic acid fragment can, in some embodiments, be improved by lengthening the hybridization and gap-filling incubation periods. (See, e.g., Turner E H, et al., Nat Methods. 2009 Apr. 6:1-2.).In some embodiments, the MIPs that are used according to the disclosure to capture a target site or target sequence comprise in sequence the following components:first targeting polynucleotide arm - first unique targeting molecular tag - polynucleotide linker - second unique targeting molecular tag - second targeting polynucleotide arm.In some embodiments, the MIPs that are used in the disclosure to capture a control site or control sequence comprise in sequence the following components:first control polynucleotide arm - first unique control molecular tag - polynucleotide linker -second unique control molecular tag - second control polynucleotide arm.MIP technology may be used to detect or amplify particular nucleic acid sequences in complex mixtures. One of the advantages of using the MIP technology is in its capacity for a high degree of multiplexing, which allows thousands of target sequences to be captured in a single reaction containing thousands of MIPs. Various aspects of MIP technology are described in, for example, Hardenbol etal., “Multiplexed genotyping with sequence-tagged molecular inversion probes,” Nature Biotechnology, 21(6): 673-678 (2003); Hardenbol etal., “Highly multiplexed molecular inversion probe genotyping: Over 10,000 targeted SNPs genotyped in a single tube assay,” Genome Research, 15: 269-275 (2005); Burmester et al.,ENMRA-43211.601“DMET microarray technology for pharmacogenomics-based personalized medicine,” Methods in Molecular Biology, 632: 99-124 (2010); Sissung et al., “Clinical pharmacology and pharmacogenetics in a genomics era: the DMET platform,” Pharmacogenomics, 11(1): 89-103 (2010); Deeken, “The Affymetrix DMET platform and pharmacogenetics in drug development,” Current Opinion in Molecular Therapeutics, 11(3): 260-268 (2009); Wang et al., “High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays,” BMC Medical Genomics, 2:8 (2009); Wang et al., “Analysis of molecular inversion probe performance for allele copy number determination,” Genome Biology, 8(11): R246 (2007); Ji et al., “Molecular inversion probe analysis of gene copy alternations reveals distinct categories of colorectal carcinoma,” Cancer Research, 66(16): 7910-7919 (2006); and Wang etal., “Allele quantification using molecular inversion probes (MIP),” Nucleic Acids Research, 33(21): el83 (2005), each of which is hereby incorporated by reference in its entirety for all purposes. See also in U.S. Pat. Nos. 6,858,412; 5,817,921; 6,558,928; 7,320,860; 7,351,528; 5,866,337; 6,027,889 and 6,852,487, each of which is hereby incorporated by reference in its entirety for all purposes.MIP technology has previously been successfully applied to other areas of research, including the novel identification and subclassification of biomarkers in cancers. See, e.g., Brewster et al., “Copy number imbalances between screen- and symptom-detected breast cancers and impact on disease-free survival,” Cancer Prevention Research, 4(10): 1609-1616 (2011); Geiersbach et al., “Unknown partner for USP6 and unusual SS18 rearrangement detected by fluorescence in situ hybridization in a solid aneurysmal bone cyst,” Cancer Genetics, 204(4): 195-202 (2011); Schiffman et al., “Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas,” Cancer Research, 70(2): 512-519 (2010); Schiffman et al., “Molecular inversion probes reveal patterns of 9p21 deletion and copy number aberrations in childhood leukemia,” Cancer Genetics and Cytogenetics, 193(1): 9-18 (2009); Press etal., “Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities,” BMC Cancer, 8:17 (2008); and Deeken et al. , “A pharmacogenetic study of docetaxel and thalidomide in patients with castration-resistant prostate cancer using the DMET genotyping platform,” Pharmacogenomics, 10(3): 191-199 (2009), each of which is hereby incorporated by reference in its entirety for all purposes.MIP technology has also been applied to the identification of new drug-related biomarkers. See, e.g., Caldwell etal., “CYP4F2 genetic variant alters required warfarinENMRA-43211.601dose,” Blood, 111(8): 4106-4112 (2008); and McDonald et al., “CYP4F2 Is a Vitamin KI Oxidase: An Explanation for Altered Warfarin Dose in Carriers of the V433M Variant,” Molecular Pharmacology, 75: 1337-1346 (2009), each of which is hereby incorporated by reference in its entirety for all purposes. Other MIP applications include drug development and safety research. See, e.g., Mega etal., “Cytochrome P-450 Polymorphisms and Response to Clopidogrel,” New England Journal of Medicine, 360(4): 354-362 (2009); Dumaual etal., “Comprehensive assessment of metabolic enzyme and transporter genes using the Affymetrix Targeted Genotyping System,” Pharmacogenomics, 8(3): 293-305 (2007); and Daly etal., “Multiplex assay for comprehensive genotyping of genes involved in drug metabolism, excretion, and transport,” Clinical Chemistry, 53(7): 1222-1230 (2007), each of which is hereby incorporated by reference in its entirety for all purposes. Further applications of MIP technology include genotype and phenotype databasing. See, e.g., Man et al. , “Genetic Variation in Metabolizing Enzyme and Transporter Genes: Comprehensive Assessment in 3 Major East Asian Subpopulations with Comparison to Caucasians and Africans,” Journal of Clinical Pharmacology, 50(8): 929-940 (2010), which is hereby incorporated by reference in its entirety for all purposes.The term “capture” or “capturing,” as used herein, refers to the binding or hybridization reaction between a molecular inversion probe and its corresponding targeting site. In some embodiments, upon capturing, a circular replicon or a MIP replicon is produced or formed. In some embodiments, the targeting site is a deletion (e.g., partial, or full deletion of one or more exons). In some embodiments, a target MIP is designed to bind to or hybridize with a naturally-occurring (e.g., wild-type) genomic region of interest where a target deletion is expected to be located. The target MIP is designed to not bind to a genomic region exhibiting the deletion. In these embodiments, binding or hybridization between a target MIP and the target site of deletion is expected to not occur. The absence of such binding or hybridization indicates the presence of the target deletion. In these embodiments, the phrase “capturing a target site” or the phrase “capturing a target sequence” refers to detection of a target deletion by detecting the absence of such binding or hybridization. As used in reference to other oligonucleotides, e.g., “capture oligonucleotide” the term refers to a binding or hybridization reaction between the capture oligonucleotide and a nucleic acid to be captured, e.g., to be immobilized, removed from solution, or otherwise be manipulated by hybridization to the capture oligonucleotide.ENMRA-43211.601The term “MIP replicon” or “circular replicon,” as used herein, refers to a circular nucleic acid molecule generated via a capturing reaction (e.g., a binding or hybridization reaction between a MIP and its targeted sequence). In some embodiments, the MIP replicon is a single-stranded circular nucleic acid molecule. In some embodiments, a targeting MIP captures or hybridizes to a target sequence or site. After the capturing reaction or hybridization, in some embodiments, a ligation reaction mixture is introduced to ligate the nick formed by hybridization of the two targeting polynucleotide arms to form singlestranded circular nucleotide molecules, i.e., a targeting MIP replicon, while in some embodiments, hybridization of the MIP leaves a gap, and a ligation / extension mixture is introduced to extend and ligate the gap region between the two targeting polynucleotide arms to form a targeting MIP replicon. In some embodiments, a control MIP captures or hybridizes to a control sequence or site. After the capturing reaction or hybridization, a ligation reaction mixture is introduced to ligate the nick formed by hybridization of the two control polynucleotide arms, or a ligation / extension mixture is introduced to extend and ligate the gap region between the two control polynucleotide arms to form single-stranded circular nucleotide molecules, i.e., a control MIP replicon. MIP replicons may be amplified through a polymerase chain reaction (PCR) to produce a plurality of targeting MIP amplicons, which are double-stranded nucleic acid molecules. MIP replicons find particular application in rolling circle amplification, or RCA. RCA is an isothermal nucleic acidamplification technique where a DNA polymerase continuously adds single nucleotides to a primer annealed to a circular template, which results in a long concatemer of single stranded DNA that contains tens to hundreds to thousands of tandem repeats (complementary to the circular template). See, e.g., M. Ali, et al. “Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine.” Chemical Society Reviews. 43 (10): 3324-3341, which is incorporated herein by reference in its entirety, for all purposes. See also WO 2015 / 083002, which is incorporated herein by reference in its entirety, for all purposes.Polymerases typically used in RCA for DNA amplification are Phi29, Bst, and Vent exo-DNA polymerases, with Phi29 DNA polymerase being preferred in view of its superior processivity and strand displacement abilityThe term “amplicon,” as used herein, refers to a nucleic acid generated via amplification reaction (e.g., a PCR reaction). In some embodiments, the amplicon is a singlestranded nucleic acid molecule. In some embodiments, the amplicon is a double-strandedENMRA-43211.601nucleic acid molecule. In some embodiments, a targeting MIP replicon is amplified using conventional techniques to produce a plurality of targeting MIP amplicons, which are doublestranded nucleotide molecules. In some embodiments, a control MIP replicon is amplified using conventional techniques to produce a plurality of control MIP amplicons, which are double-stranded nucleotide molecules.As used herein, the phrase “not substantially complementary” as used in reference to a probe flap or arm means that the flap portion is sufficiently non-complementary not to hybridize selectively to a nucleic acid sequence, e.g, a target nucleic acid or amplified DNA, under the designated annealing conditions or stringent conditions, encompassing the terms “substantially non-complementary” and “perfectly non-complementary.”The term “signal” as used herein refers to any detectable effect, such as would be caused or provided by a label or by action or accumulation of a component or product in an assay reaction.As used herein, the term “detector” refers to a system or component of a system, e.g, an instrument (e.g. a camera, fluorimeter, charge-coupled device, scintillation counter, solid state nanopore device, etc..) or a reactive medium (X-ray or camera film, pH indicator, etc.), that can convey to a user or to another component of a system (e.g., a computer or controller) the presence of a signal or effect. A detector is not limited to a particular type of signal detected, and can be a photometric or spectrophotometric system, which can detect ultraviolet, visible or infrared light, including fluorescence or chemiluminescence; a radiation detection system; a charge detection system; a system for detection of an electronic signal, e.g., a current or charge perturbation; a spectroscopic system such as nuclear magnetic resonance spectroscopy, mass spectrometry or surface enhanced Raman spectrometry; a system such as gel or capillary electrophoresis or gel exclusion chromatography; or other detection system known in the art, or combinations thereof.The term “detection” as used herein refers to quantitatively or qualitatively identifying an analyte (e.g., DNA, RNA, or a protein), e.g., within a sample. The term “detection assay” as used herein refers to a kit, test, or procedure performed for the purpose of detecting an analyte within a sample. Detection assays produce a detectable signal or effect when performed in the presence of the target analyte, and include but are not limited to assays incorporating the processes of hybridization, nucleic acid cleavage (e.g., exo- or endonuclease), nucleic acid amplification, nucleotide sequencing, primer extension, nucleic acid ligation, antigen- antibody binding, interaction of a primary antibody with a secondaryENMRA-43211.601antibody, and / or conformational change in a nucleic acid (e.g., an oligonucleotide) or polypeptide e.g., a protein or small peptide).As used herein, the term “prenatal or pregnancy-related disease or condition” refers to any disease, disorder, or condition affecting a pregnant woman, embryo, or fetus. Prenatal or pregnancy-related conditions can also refer to any disease, disorder, or condition that is associated with or arises, either directly or indirectly, as a result of pregnancy. These diseases or conditions can include any and all birth defects, congenital conditions, or hereditary diseases or conditions. Examples of prenatal or pregnancy-related diseases include, but are not limited to, Rhesus disease, hemolytic disease of the newborn, beta-thalassemia, sex determination, determination of pregnancy, a hereditary Mendelian genetic disorder, chromosomal aberrations, a fetal chromosomal aneuploidy, fetal chromosomal trisomy, fetal chromosomal monosomy, trisomy 8, trisomy 13 (Patau Syndrome), trisomy 16, trisomy 18 (Edwards syndrome), trisomy 21 (Down syndrome), X-chromosome linked disorders, trisomy X (XXX syndrome), monosomy X (Turner syndrome), XXY syndrome, XYY syndrome, XYY syndrome, XXXY syndrome, XXYY syndrome, XYYY syndrome, XXXXX syndrome, XXXXY syndrome, XXXYY syndrome, XXYYY syndrome, Fragile X Syndrome, fetal growth restriction, cystic fibrosis, a hemoglobinopathy, fetal death, fetal alcohol syndrome, sickle cell anemia, hemophilia, Klinefelter syndrome, dup(17)(pl 1.2pl.2) syndrome, endometriosis, Pelizaeus-Merzbacher disease, dup(22)(ql 1.2ql 1.2) syndrome, cat eye syndrome, cri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease, neuropathy with liability to pressure palsies, Smith-Magenis syndrome, neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome, DiGeorge syndrome, steroid sulfatase deficiency, Prader-Willi syndrome, Kallmann syndrome, microphthalmia with linear skin defects, adrenal hypoplasia, glycerol kinase deficiency, Pelizaeus-Merzbacher disease, testis-determining factor on Y, azospermia (factor a), azospermia (factorb), azospermia (factor c), lp36 deletion, phenylketonuria, Tay-Sachs disease, adrenal hyperplasia, Fanconi anemia, spinal muscular atrophy, Duchenne’s muscular dystrophy, Huntington’s disease, myotonic dystrophy, Robertsonian translocation, Angelman syndrome, tuberous sclerosis, ataxia telangieltasia, open spina bifida, neural tube defects, ventral wall defects, small-for-gestational-age, congenital cytomegalovirus, achondroplasia, Marfan’s syndrome, congenital hypothyroidism, congenital toxoplasmosis, biotinidase deficiency, galactosemia, maple syrup urine disease, homocystinuria, medium-chain acyl Co-A dehydrogenase deficiency, structural birth defects, heart defects, abnormal limbs, club foot,ENMRA-43211.601anencephaly, arhinencephaly / holoprosencephaly, hydrocephaly, anophthalmos / microphthalmos, anotia / microtia, transposition of great vessels, tetralogy of Fallot, hypoplastic left heart syndrome, coarctation of aorta, cleft palate without cleft lip, cleft lip with or without cleft palate, oesophageal atresia / stenosis with or without fistula, small intestine atresia / stenosis, anorectal atresia / stenosis, hypospadias, indeterminate sex, renal agenesis, cystic kidney, preaxial polydactyly, limb reduction defects, diaphragmatic hernia, blindness, cataracts, visual problems, hearing loss, deafness, X-linked adrenoleukodystrophy, Rett syndrome, lysosomal disorders, cerebral palsy, autism, aglossia, albinism, ocular albinism, oculocutaneous albinism, gestational diabetes, Arnold-Chiari malformation, CHARGE syndrome, congenital diaphragmatic hernia, brachydactlia, aniridia, cleft foot and hand, heterochromia, Dwarnian ear, Ehlers Danlos syndrome, epidermolysis bullosa, Gorham’s disease, Hashimoto’s syndrome, hydrops fetalis, hypotonia, Klippel-Feil syndrome, muscular dystrophy, osteogenesis imperfecta, progeria, Smith Lemli Opitz symdrom, chromatelopsia, X-linked lymphoproliferative disease, omphalocele, gastroschisis, pre-eclampsia, eclampsia, pre-term labor, premature birth, miscarriage, delayed intrauterine growth, ectopic pregnancy, hyperemesis gravidarum, morning sickness, or likelihood for successful induction of labor.In some NIPT embodiments, the technology described herein further includes estimating a fetal fraction for a sample, wherein the fetal fraction is used to aid in the determination of whether the genetic data from the test subject is indicative of an aneuploidy. Methods for determining or calculating fetal fraction are known in the art.As used herein, the term “valid detection assay” refers to a detection assay that has been shown to accurately predict an association between the detection of a target and a phenotype (e.g., medical condition). Examples of valid detection assays include, but are not limited to, detection assays that, when a target is detected, accurately predict the phenotype medical 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% of the time. Other examples of valid detection assays include, but are not limited to, detection assays that qualify as and / or are marketed as Analyte-Specific Reagents (i.e., as defined by FDA regulations) or In-Vitro Diagnostics (i.e., approved by the FDA).As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions forENMRA-43211.601performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR’s) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.As used herein, the term “system” refers to two or more items, components, or articles, e.g., samples, reagents, instruments, containers, instruction information, used together for a particular purpose. In some embodiments, some, or all of the components of a system are provided together, e.g., in a kit, while in some embodiments, at least some of the components of the system are collected for use individually, e.g., by a user, for use together for a particular purpose.As used herein, the term “information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term “information related to a subject” refers to facts or data pertaining to a subject (e.g., a human, plant, or animal). The term “genomic information” refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, allele frequencies, RNA expression levels, protein expression, phenotypes correlating to genotypes, etc. “Allele frequency information” refers to facts or data pertaining allele frequencies, including, but not limited to, allele identities, statistical correlations between the presence of an allele and a characteristic of a subject (e.g., a human subject), the presence or absence of an allele in an individual or population, the percentage likelihood of an allele being present in an individual having one or more particular characteristics, etc.ENMRA-43211.601As used herein, the term “assay validation information” refers to genomic information and / or allele frequency information resulting from processing of test result data (e.g., processing with the aid of a computer). Assay validation information may be used, for example, to identify a particular candidate detection assay as a valid detection assay.BRIEF DESCRIPTION OF THE DRAWINGSThe patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.Fig. 1 provides a schematic diagram of a rolling circle amplification reaction.Fig. 2 provides a schematic diagram of a rolling circle amplification reaction using a primer conjugated to a surface.Fig. 3 A provides a schematic illustration of the architecture of a tannic acid (TA) and polyacrylic acid (PAA) polymeric surface on glass. After treatment with 4-hydroxyphenylboronic acid, the 4-HPBA complexes with tannic acid c / .s-diol groups, and reactive hydroxyl groups of TA are minimized in number and the added phenyl boronic acid derivatives confer passivation properties to the surface. Exposed glass also becomes coated with 4HPB A, further conferring passivation properties to the system.Fig. 3B provides a schematic illustration of the architecture of a polyacrylic acid (PAA) polymeric surface on glass, without tannic acid. After contact with 4-hydroxyphenylboronic acid the polymeric surface, its covalent linkers, and any exposed glass become coated with the molecule, conferring passivation properties to the surface.Fig. 4A shows the structure of 4-hydroxyphenylboronic acid (4-HPBA), and shows that 4-HPBA passivation of an acrylic acid / tannic acid polymer surface results in increased surface capture, amplification, and detection of DNA targets, as compared to an acrylic acid / tannic acid polymer surface without boronic passivation (“SOP”).Fig. 4B shows the structure of methyl boronic acid, and shows that passivation of an acrylic acid / tannic acid polymer surface on glass with methyl boronic acid exhibits no benefit compared to the same acrylic acid / tannic acid surface without any boronic acid coupling (SOP). These data show that not all boronic acid derivatives confer passivation effects and / or increase function of the acrylic acid / tannic acid polymeric surface function.Fig. 5 provides images and data showing that combining tannic acid with 4-HPBA protects tannic acid from oxidation by iron. As shown in panel A, tube 1 contains tannic acidENMRA-43211.601alone (control) and tube 2 contains tannic acid combined with Fe(III), which results in oxidation of the tannic acid, which is visible as a browning of the tannic acid. Tube 3 contains tannic acid combined with 4-HPB A (control) and tube 4 contains tannic acid combined with 4-HPB A, and subsequently treated with Fe(III), which shows a significant reduction in the oxidation of the tannic acid that is combined with 4-HPBA. Panel B shows UV visible spectra of the samples shown in Panel A.Fig. 6A provides graphs showing signal measured in 3 channels, comparing acrylic acid / tannic acid polymeric surfaces passivated by treatment with p-tolyl boronic acid (p-TBA), phenylboronic acid (PB A), 4-hydroxyphenylboronic acid (4-HPBA), or methyl boronic acid (Mb A), as compared to untreated acrylic acid / tannic acid surfaces (SOP). The surface able to bind the most DNA targets was provided by treatment with 100 mM 4-HPBA. Some derivatives were found not to improve target detection at similar concentrations, and other boronic acid derivatives were found to either improve or obtain similar amounts of target counts as SOP. “BA Conc” indicates the boronic acid derivative concentration during the surface passivation step.Fig. 6B provides graphs showing signal measured in 3 channels, comparing passivation effects on acrylic acid / tannic acid polymer surfaces using 50 or 100 mM 4-carb oxy phenylboronic acid (4cPBA), or 100 mM 4-hydroxyphenylboronic acid (4-HPBA), in Tris buffer or standard sodium citrate (SSC) buffer, as indicated in the legend. Although treatment with 50 mM 4cPBA in Tris buffer (gray) resulted in more targets detected than use of 100 mM 4-HPBA in SSC buffer (blue), use of 4-HPBA in Tris buffer (orange) gave the highest level of target detection.Fig. 7 provides graphs illustrating the effects of storing 4-HPBA-modified acrylic acid / tannic acid glass surfaces under Tris buffer at different pHs, and under Tris pH 7.5 with added 4-HPBA, as shown by DNA target detection levels, and with or without a mixture of inhibitors known to prevent DNA binding on acrylic acid / tannic acid glass surfaces (“CLE”). Panel A shows that PAA / TA polymeric surfaces passivated with 4-HPBA and stored in the absence of 4-HPBA at pH 7.4 (orange), 7.5 (green), or 8.0 (red) lose approximately 25%, 60%, and 75% of their ability to detect DNA targets, respectively, after 7 days of storage. PAA / TA polymeric surfaces treated with 4-HPBA and stored in pH 7.5 Tris buffer with fresh 4-HPBA (purple) lost approximately 8% of their ability to detect DNA targets after 7 days of storage. Panel B shows results of plates stored as described for A, but in which DNA targetsENMRA-43211.601were added to the stored plates, with the mixture of inhibitors (CLE) added in the same hybridization reaction.Fig. 8 provides graphs showing detection in 4 different channels with 0.01 and 0.1 frnol of synthetic DNA control circles on an unmodified PAA / TA polymeric surface (orange) and a 4-HPB A-modified PAA / TA polymeric surface (red), in the presence of spiked inhibitors (e.g. BSA, TX-100, enzymes, metal ions, etc.). Detection of 10 ng of genomic DNA that has been circularized using a probe capture, ligation, and exonuclease cleanup process and contains a multitude of inhibitors typically found in reaction carry-over (e.g. BSA, detergents, enzymes, salts, metal ions, etc.) is shown on an unmodified (blue) and 4-HPBA-modified (green) PAA / TA polymeric surface. These data show that modification of the PAA / TA polymeric surface with 4-HPBA mitigates inhibition caused by complex reaction chemistry components interacting with the detection surface.Fig. 9A compares conditions for conjugating tannic acid surfaces with 4-HPBA. Tris (T) and phosphate (P) buffers at various pH were compared for use in the 4-HPBA conjugation reaction with TA. Object counts indicate surface hybridization, amplification, and detection of synthetic control circles in pure buffer (blue) and synthetic control circles in buffer spiked with a multitude of inhibitors commonly found in enzyme reaction chemistry (e.g. BSA, TX-100, enzymes, metal ions, etc.) (gold), respectively.Fig. 9B compares oxidation of tannic acid in pH 8 buffer, and reduction of oxidation by addition of TCEP (tris(2-carboxyethyl)phosphine) reducing agent at pH 8 or pH 4. The 4-HPBA complexation reaction requires a basic pH, but tannic acid is shown to quickly oxidize at pH 8, e.g., within Ih. Preferred reaction conditions for modifying a PAA / TA polymeric surface with 4-HPBA were determined to be at about pH 8 in Tris buffer, and in the presence of a reducing agent (e.g., TCEP), for 4 h.Fig. 10A provides a graph comparing non-specific binding of protein (fluorescent-dye-labeled bovine serum albumin) on bare glass, glass coated with PAA / TA polymer with conjugated primers, and glass coated with PAA / TA polymer, each either treated with (blue) or without (red) 4-HPBA. These data show that treatment of uncoated glass and the primer-conjugated polymer surface with 4-HBPA provides a significant reduction in non-specific protein binding.Fig. 10B is an image showing 4-HPB A-treated and untreated glass after contact with fluorescent dye-labeled BSA, showing that treatment of the glass with 4-HPBA reduces BSA binding to the glass.ENMRA-43211.601Fig. 10C shows detection of fluorescent dye-labeled BSA on glass, when the glass is pre-treated with 4-HPBA, with unlabeled BSA, with nothing, or with Pluronic (PF 127) nonionic copolymer surfactant. While BSA does a better job than 4-HPBA at blocking bare glass, use of 4-HPBA nonetheless has an advantage over BSA in that, in many applications, it is desirable to avoid adding protein to the surface as a blocking agent. For example, the size of BSA molecules means that it may render immobilized primers on the surface inaccessible, and may thus block downstream DNA manipulation and detection steps.Figs. 11 A-l 1C show examination of the effects of 4HPBA on the performance of polymeric surfaces, with or without an ethanol wash step. Fig. 11 A provides a graph comparing counts of well objects of glass surfaces coated with silane and polyacrylic acid (PAA) with conjugated primers (a tannic-acid free surface) with or without an ethanol wash step, and with or without use of 4HPB A. Ethanol washing of silane / PAA surface resulted in greater surface uniformity (see Fig. 1 IB), but exhibited diminished counts when 4HPBA was absent (red data in Fig. 11 A). Counts were greater when there was neither an ethanol wash nor 4HPBA treatment (purple data in Fig. 11 A) and further improved when both ethanol wash and 4HPBA treatments were used (blue). Omitting ethanol washing combined with 4HPBA treatment resulted in the highest counts (Fig. 11 A, green) but the surface had poor uniformity. Only when the surfaces were ethanol washed, followed by 4HPBA treatment did counts recover to the highest levels (Fig. 11 A green data, and Fig. 11C).DETAILED DESCRIPTION OF THE INVENTIONAlthough the technology is discussed in reference to particular e.g., passivating glass and polymeric surfaces with or without tannic acid using 4-hydroxyphenylboronic acid, e.g., for DNA capture and detection reactions, the technology is not limited to any particular type of uncoated or polymer-coated surface, or any particular boronic acid derivative for passivating such surfaces, or any particular type of assay to be performed in the presence of the modified uncoated or polymeric surface.Provided herein are methods, compositions, systems, and kits for using or creating surfaces, e.g., on solid supports, that are compatible with specific conjugation or grafting of macromolecules, and compatible with complex mixtures, e.g., assay reaction mixtures that may be subject to inhibition or interference by properties of glass or polymeric surfaces, particularly polymeric surfaces that comprise polyphenols on the surface.ENMRA-43211.601Provided herein is technology in which at least some active or interfering moieties are rendered inert, such that, upon contact with reactive components found, for example, in nucleic acid modification reactions, reactivity between the reaction components and the surface is minimized. The technology finds particular application in reactions in which oligonucleotide primers used in the reaction are attached to the polymeric surface. In the absence of treatment using the technology described herein, a significant portion of surfacebound primers may interact with the surface in undesirable ways and are thus unavailable to participate in the nucleic acid modification reaction. Upon treatment as described herein, the surface is rendered more nearly inert, or is passivated, such that the bound oligonucleotides are unobstructed and are free to hybridize to nucleic acid targets, improving the sensitivity of detection (see, e.g., Fig. 4A).A number of different “front-end” methods for recognizing target nucleic acid and producing a new product are described, for example, in WO 2019 / 195346 and WO 2020 / 206170, which are both incorporated herein by reference in their entireties for all purposes. In some embodiments, such methods comprise use of a “back end” technology for detecting and / or measuring production of the new product from the front-end method. In certain embodiments, back-end technologies are based on immobilizing nucleic acid on a surface, preferably a surface coated with organic polymer. In preferred embodiments, technologies that comprise binding MIPs on a surface by hybridization to surface-immobilized oligonucleotide primers, and detecting the bound MIPs, e.g., using rolling circle amplification. The technology described herein finds application in technologies that comprise a step of immobilizing molecules, e.g., oligonucleotides, on a surface, e.g., a glass surface, in particular a glass surface that comprises a surface treatment comprising polymerized polyphenolic compounds, e.g., tannic acid.The technology is not limited to any particular underlying substrate or solid support, and can be configured for passivation of any polymer-coated substrate, e.g., a glass, gold, or carbon (e.g., diamond) substrate.Fig. 1 shows a schematic diagram of rolling circle amplification in which a primer specifically hybridizes to a circular DNA template, e.g., a MIP, and is then extended on the template to form a long strand of single-stranded DNA that contains a concatemer of the sequence complementary to the circular template. In this embodiment, the RCA product binds to a plurality of FRET-labeled probes, e.g., molecular beacon probes that have aENMRA-43211.601fluorophore and a quencher. Hybridization of the probes to the RCA product separates the quencher from the fluorophore, allowing detection of fluorescence from the beacon.In the illustrative embodiment shown in Fig. 2, primer oligonucleotides are immobilized on a surface, preferably by covalent attachment at their 5' ends, such that the 3' end of the primer immobilized primer is available to hybridize to a circular template and to be extended by a polymerase during RCA The resulting RCA product strand is immobilized on the surface.In one aspect, the technology provided herein provides a surface, e.g., on a solid support substrate, that is modified to comprise an organic coating, e.g., a polymeric coating comprising one or more natural or synthetic polyphenols, pyrogallol, or gallic acid, e.g., as disclosed in WO 2014 / 116812 and WO 2020 / 206170, each of which is incorporated herein by reference for all purposes, and which provide additional monomers suitable for use in forming polymeric coatings on surfaces. In preferred embodiments, the one or more polyphenols comprise one or more compounds selected from epigallocatechin-3 -gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and tannic acid. In certain preferred embodiments, polymeric coatings or surfaces of the technology comprise polyacrylic acid (PAA), with or without tannic acid (TA).A recognized problem of polyphenolic and polymeric surfaces for use proximal to biomolecules e.g., in diagnostic applications, is the capacity of these surfaces to interact with non-target molecules, such as proteins and other electrostatically sensitive molecules, (see, e.g., F. Poncin-Epaillard, Surface treatment of polymeric materials controlling the adhesion of biomolecules. J Funct Biomater. 2012 Aug 7;3(3):528-43. doi: 10.3390 / jfb3030528). During development of the present technology, it was recognized that undesirable interactions of PAA / TA surfaces may be caused by reactivity of tannic acid with macromolecules and reaction reagents. While not being limited to any particular theory of operation, due to their multiple aliphatic hydroxyls and phenolic hydroxyl groups, tannins have the ability to form complexes with proteins and polysaccharides as well as to interact with inorganic salts, interactions that may result in strong adsorption of nucleic acids, and even their cleavage. See, e.g., Khan NS, Ahmad A, Hadi SM. Anti-oxidant, pro-oxidant properties of tannic acid and its binding to DNA. Chem Biol Interact. 2000 Mar15; 125(3): 177-89. doi: 10.1016 / s0009-2797(00)00143-5, which is incorporated herein by reference in its entirety, for all purposes. Proteins can also adsorb to many surfaces, sometimes nearly irreversibly. Bovine serum albumin (BSA), for example, is oftenENMRA-43211.601intentionally added to enzyme preparations as a “carrier,” to prevent the enzymes from sticking to surfaces, such as the interior surfaces of plastic tubes and pipet tips generally used for enzyme storage, delivery, and measuring, rendering them unavailable for performing their enzymatic function.During development of the technology, it was determined that untreated PAA / TA surfaces inhibit nucleic acids, c.g, DNA molecules such as MIPs or target DNAs, from hybridizing to surface-bound oligonucleotide primers and / or from amplifying in reaction mixtures on the surfaces. These surfaces have shown reductions in detection sensitivity by >60% to 90%. Through application of the present technology, inhibition of detection reactions by the surfaces has been reduced to less than 15%.The technology provides a means of increasing sensitivity of detection of nucleic acid targets by modifying polyphenol groups on the surfaces, in particular by treating the PAA / TA surface with phenyl boronic acid and boronic acid derivatives, thereby reducing the inhibitory effects of the surface.Surprisingly, it was determined that undesirable non-specific binding to PAA / TA surfaces can be substantially reduced by modifying the PAA / TA surfaces using boronic acid compounds described herein, preferably phenyl boronic acids, such as hydroxyphenyl boronic acids. In preferred embodiments, a surface such as PAA / TA polymeric surface is treated with a preparation comprising 4-hydroxyphenylboronic acid. While not being limited to any particular mechanism of action, the methods of the technology take advantage of the vicinal cv.s-diols located on the galloyl units of tannic acid to react with phenylboronic acid, thus creating a covalently modified, passivated polymer surface having reduced undesirable adsorption properties and reduced reaction interference properties. The modified PAA / TA polymer coating is rendered substantially more suitable for hybridization of surface-bound oligonucleotides to a nucleic acid of interest, and for enzymatic reactions, c.g., nucleic acid modification and detection reactions.Combining 4-HPB A with tannic acid under conditions described herein results in a covalent bond between the 4-HPB A and cv.s-diol groups on tannic acid, c.g., as shown in Fig.3 A. While not being limited to any particular mechanism or mode of action, treatment of a PAA / TA polymeric surface with 4-HPBA can form similar covalent modifications, e.g., as shown in Fig. 3B, reducing the reactivity of the cv.s-diols with downstream assay nucleic acids and other reaction components, / . ., passivating the surface. Conditions are provided herein toENMRA-43211.601increase the robustness of the 4-HPBA modification of PAA / TA surfaces, such that high levels of passivation can be achieved.4-HPBA does not need to form a covalent bond with the molecules it associates with. An advantage of this technology is that is also provides a means to fill gaps or holes in the surface polymer coating (such as a polyacrylic acid surface or a polyacrylic acid-tannic acid surface), e.g., by associating directly with the glass substrate, to prevent non-specific binding of dyes and proteins to any exposed glass, e.g., when gaps or holes expose and underlying glass substrate to a reaction mixture, or when no polymeric coating is used at all. See Figs.10A-10C.Exemplary methodProvided hereinbelow is an exemplary protocol for chemical modification of assay plates using acrylic acid and tannic acid, attachment of oligonucleotides, and passivation with -HPBA. Creating a PAA / TA surface with passivating the surface by modifying TA with PBA. Modifying TA by the use of this technology preferably occurs after polymerization and deposition of PAA, after deposition of TA, and after grafting the surface with functional primers.First, tannic acid is mixed with acrylic acid, generally with a higher molar amount of acrylic acid, e.g., at a molar ratio of 1 :800. Initiators are present whereby polymerization on a glass surface with the AA / TA mixture is effectuated through incubation at low pH, a pH not amenable to TA modification with PBA.Second, oligonucleotides are conjugated to the surface to serve, e.g., as capture primers for nucleic acid targets.Third, phenylboronic acid (PBA) or boronic acid (BA), and / or their derivatives, are contacted to the surface, where they undergo a complexation reaction with the 1,2-diols of the galloyl units of tannic acid (see Fig. 3 A). There are approximately 10 galloyl units per TA molecule displaying 15 cv.s-diols per TA molecule, with each having the potential to react with PBA. Due to these large numbers, formation of PBA-TA covalent complexes result in effectively changing fundamental properties of the surface (see Fig. 4A). After successful modification, the resultant surface contains more singular hydroxyl moieties on the phenyl rings; whereas prior to the modification the PAA / TA surface presented a multitude ofENMRA-43211.601reactive hydroxyl groups on each phenyl ring. Surprisingly, despite the molar ratio of 800: 1 acrylic acid:tannic acid used in forming the polymer coating, it was found that modification of the tannic acid alone is sufficient to passivate the surface and render it compatible with complex reaction mixtures.During development of the technology, results were obtained that clearly indicate that both the particular boronic acid structure and solution pH used in the reaction affect the result. In particular, the diol-binding constants differ based on the boronic acid derivatives and their pKa. For example, comparing Figs. 4A and 4B, data shows that that 4-hydroxyphenylboronic acid is particularly effective in passivating the PAA / TA surface, while methyl boronic acid did not improve performance of the PAA / TA surface.Fourth, the surfaces are preferably washed with and / or stored in specialized buffers to prevent reversal of the reaction. In particular, buffers that contain free phenylboronic acid provide a reactant for any free c / .s-diol -containing substrates, e.g, galloyl groups on the surface that have lost their initial PBA complex due, for example, to reversal or damage. Fifth, the surfaces can be stored in a specialized buffer developed to prevent reversal of the reaction and loss of the PBA modification before use, e.g, Tris buffer with 4-HPBA (see Figs. 6B and 7).Sixth, during use, a nucleic acid sample containing targets, e.g., nucleic acid targets, especially small circular targets, are contacted with the surface and any targets that contain complementary sequence to the surface-grafted primers are hybridized to the modified PAA / TA surface. Surprisingly and unexpectedly, oligonucleotides attached to 4-HPBA-modified surfaces exhibit enhanced ability to hybridize to, amplify, and detect target nucleic acids (see, e.g., Figs 4A, 6A and 8). Surface-hybridized targets are amplified by any number of known amplification methods in the art (e.g. RCA); and the amplified targets are detected by any number of known labeling methods in the art (e.g. hybridization (“staining”) with dye-labeled oligonucleotide probes, followed by fluorescence imaging).ENMRA-43211.601EXAMPLE4-hydroxyphenyboronic acid modification of a polyacrylic / tannic acid polymeric surfacea. Polyacrylic / tannic Acid Plate Preparation1. Measure tannic acid and ammonium persulfate into 15ml Tube (or container) 2. Add nuclease-free H2O to container3. Add acrylic acid to solution4. Add TEMED to solution5. Vortex to mix6. Pipette 50pL to each well of the plate7. Incubate at room temperature (25°C) overnight (protected from UV and other light)b. Reagent removal and Washing1. Remove the tannic acid solution from wells and dispose into chemical waste 2. Wash the plate with nuclease-free water three (3) times using lOOpL per well.Remove the final wash fluid.3. Allow plates to dry completely (e.g., overnight incubation at room temperature (25°C), or in a biological hood in Pre for at least 3 hours before continuing to the oligo conjugation.ENMRA-43211.601c. Conjugation of oligonucleotide to prepared assay plate1. Prepare a lOOmM solution of EDC by measuring out 98mg of EDC and adding ImL ofH2O.2. Prepare a lOOmM solution of NHS by measuring out 57mg of NHS and adding ImL ofH2O.3. Combine the following into a 15ml conical tube (or container)a. 100 pL of lOOpM amine-modified oligonucleotideb. 1000 pL of lOOmM EDCc. 1000 pL of lOOmM NHSd. 7900 pL ofH204. Vortex to mix5. Dispense 50 pL of this solution into each well of the plate6. Incubate the plate at 37°C for one (1) hour7. Wash the plate with nuclease-free water three (3) times using 100 pL per well. Remove the final wash fluid.d. Phenylboronic acid (PBA) or Boronic acid (BA) modificationPrepare a solution of 85 mM 4-hydroxyphenylboronic acid in 100 mM Tris-Cl pH 8, with 0.5 mM TCEP.Dispense 50 pL of this solution into each well of the plate;Incubate the plate at 37°C for four (4) hours;ENMRA-43211.601Wash the plate three (3) times using 100 pL per well of 20 mM Tris-Cl, pH 7.5 with 5 mM HPB A;Store the plate with 100 pL per well of 20 mM Tris-Cl, pH 7.5 with 5 mM HPB A in each well.Additional materials incorporated herein by reference1. Poncin-Epaillard F, Vrlinic T, Debarnot D, Mozetic M, Coudreuse A, Legeay G, El Moualij B, Zorzi W. Surface treatment of polymeric materials controlling the adhesion of biomolecules. J Funct Biomater. 2012 Aug 7;3(3):528-43. doi:10.3390 / jfb3030528. PMID: 24955631; PMCID: PMC4030997.2. Czeslik, Claus & Jackler, Guido & Steitz, Roland & Griinberg, Hagen. (2004). The Journal of Physical Chemistry B. 108. 10.1021 / jp0488766.3. M. F. Delcroix, S. Demoustier-Champagne, and C. C. Dupont-Gillain Langmuir, 2014 30 (1), 268-277 DOI: 10.1021 / la403891k4. Alison E. Contreras, Zvi Steiner, Jing Miao, Roni Kasher, and Qilin Li, Environmental Science & Technology 2011 45 (15), 6309-6315, DOI:10.1021 / es200570t5. William L. A. Brooks and Brent S. Sumerlin, Chemical Reviews 2016 116 (3), 1375- 1397 DOI: 10.102 l / acs. chemrev.5b003006. Chen Chen, Hao Yang, Xiao Yang, and Qihghai Ma. Tannic acid: a crosslinker leading to versatile functional polymeric networks: a review. RSC Adv., 12:7689 (2022)7. Jumi Kang, Yuejin Kim, Hyeona Park, and Kyueui Lee. Boronic acid conjugated polyacrylate coating: A strategy for material-independent surface functionalization. Appl. Surf. Sci. v657: 159793, 1 June 2024,All literature and similar materials cited in this application, including the publications described in the Bibliography above, and including but not limited to patents, patent applications, articles, books, treatises, and internet web pages, are expressly incorporated by reference in their entireties for any purpose. Unless defined otherwise, all technical andENMRA-43211.601scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control.Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology, molecular diagnostics, nucleic acid’s structure, biochemistry, medical science, or related fields are intended to be within the scope of the claims.

Claims

ENMRA-43211.601CLAIMSWe claim:

1. A method of modifying a support, the method comprising treating a surface on the support with a boronic acid composition under conditions wherein boronic acid or a boronic acid derivative, preferably 4-hydroxyphenyl boronic acid, passivates at least a portion of the surface.

2. The method of claim 1, wherein at least a portion of the surface comprises glass, wherein the treating comprises exposing glass directly to the boronic acid composition.

3. The method of claim 1, wherein at least a portion of the surface is polymeric, wherein the treating comprises exposing a polymeric surface to the boronic acid composition.

4. The method of claim 3, wherein the polymeric surface comprises polyphenols.

5. The method of claim 3, wherein the polymeric surface comprises tannic acid.

6. The method of claim 3, wherein the polymeric surface comprises polyacrylic acid.

7. The method of any one of claims 3 to 6, wherein the polymeric surface is homopolymeric.

8. The method of any one of claims 3 to 6, wherein the polymeric surface is heteropolymeric.

9. The method of claim 8, wherein the heteropolymeric surface comprises polyacrylic acid and tannic acid.

10. The method of any one of claims 1 to 9, wherein the boronic acid composition comprises 4-hydroxyphenyl boronic acid, and wherein passivating the surface comprises complexing 4-hydroxyphenyl boronic acid to c / .s-diols on the surface.ENMRA-43211.60111. A method, comprising:a) providing a support comprising a surface; andb) exposing the surface to a boronic acid composition comprising a boronic acid or boronic acid derivative, preferably 4-hydroxyphenyl boronic acid, under conditions wherein the boronic acid or boronic acid derivative passivates at least a portion of the surface.

12. The method of claim 11, wherein at least a portion of the surface comprises glass, wherein the method comprises exposing glass directly to the boronic acid composition.

13. The method of claim 11, wherein at least a portion of the surface is polymeric, wherein the method comprises exposing a polymeric surface to the boronic acid composition.

14. The method of claim 13, wherein the polymeric surface comprises polyphenols.

15. The method of claim 13, wherein the polymeric surface comprises tannic acid.

16. The method of claim 13, wherein the polymeric surface comprises polyacrylic acid.

17. The method of any one of claims 13 to 16, wherein the polymeric surface is homopolymeric.

18. The method of any one of claims 13 to 16, wherein the polymeric surface is heteropolymeric.

19. The method of claim 18, wherein the heteropolymeric surface comprises polyacrylic acid and tannic acid.

20. The method of any one of claims 13 to 19, wherein the boronic acid composition comprises 4-hydroxyphenyl boronic acid, and wherein passivating comprises complexing 4-hydroxyphenyl boronic acid to cv.s-diols on the polymeric surface.

21. The method of claim 20, wherein the polymeric surface comprises galloyl groups.ENMRA-43211.60122. A method, comprising:a) conjugating nucleic acid to a surface to form a nucleic acid-conjugated surface; andb) exposing the nucleic acid-conjugated surface to a boronic acid composition comprising a boronic acid or boronic acid derivative, preferably 4-hydroxyphenyl boronic acid, under conditions wherein the boronic acid or boronic acid derivative passivates at least a portion of the surface.

23. The method of claim 22, wherein prior to step a), the method comprises exposing the surface to a mixture comprising a monomer, under conditions wherein the mixture forms a polymeric coating.

24. The method of claim 23, wherein the mixture comprises an acrylic acid monomer.

25. The method of claim 23 or claim 24, wherein the mixture comprises a polyphenolic monomer comprising a galloyl group.

26. The method of claim 25, wherein the polyphenolic monomer comprises one or more of epigallocatechin-3 -gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and tannic acid.

27. The method of claim 25 or claim 26, wherein the mixture further comprises a nonphenolic monomer.

28. The method of claim 27, wherein the mixture comprises acrylic acid and tannic acid.

29. The method of any one of claims 22-28, wherein the surface comprises glass.

30. A composition comprising a plurality of complexes bound to a 4-hydroxyphenyl boronic acid-passivated surface, wherein the complexes comprise single or double-stranded nucleic acid.ENMRA-43211.60131. The composition of claim 30, wherein the 4-hydroxyphenyl boronic acid-passivated surface comprises a polymeric surface, preferably a polymeric surface comprising galloyl groups.

32. The composition of claim 31, wherein the polymeric surface comprises tannic acid complexed with 4-hydroxyphenyl boronic acid.

33. The composition of claim 30, wherein the 4-hydroxyphenyl boronic acid-passivated surface comprises 4-hydroxyphenyl boronic acid-passivated glass.

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