Nucleic acid isolation and related methods

Abstract

A solid support modified with a pectin derivative is provided. The solid support is useful for a method of isolating, separating, and detecting nucleic acids.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 765,149 of August 17, 2018, the entirety of which is incorporated herein by reference.

[0003] Field of invention

[0004] The present invention relates to a solid support comprising modified pectin and a method of using the same. Background Technology

[0005] Molecular diagnostic analysis, which utilizes the amplification and / or detection of nucleic acids by various automated analytical techniques such as polymerase chain reaction (PCR), provides rapid and accurate results in a short time compared to conventional diagnostic methods and can be easily automated. However, to perform molecular diagnostic analysis on biological samples, nucleic acids must be isolated from the biological material to remove components that, for example, inhibit polymerase activity and affect the accuracy of the analysis. While various methods for nucleic acid extraction exist, currently available methods generally involve lengthy steps and are not easily adaptable for automation. Consequently, preparing nucleic acid samples prior to the amplification and detection of specific targets is the most challenging step in molecular diagnostics.

[0006] To produce high-quality nucleic acids free from amplification inhibitors, there is a need for a simple and rapid nucleic acid isolation method that does not require extensive sample processing and can be applied to clinical laboratory automation. There is also a need for reagents capable of facilitating the isolation of nucleic acids from nucleic acid-containing biological samples in a manner compatible with rapid and automated nucleic acid detection methods. The present invention meets these requirements and provides additional related benefits.

[0007] In one embodiment, the present specification provides a solid support comprising a plurality of modified pectin molecules covalently bonded to the solid support. In some embodiments, the modified pectin comprises a plurality of amino groups. In some embodiments, the modified pectin is amidated pectin. In some embodiments, the amidated pectin is a unit represented by one or more of the following chemical formulas:

[0008] ,

[0009] It includes its isomers, salts, or tautomers,

[0010] Among the chemical formulas

[0011] n is 0, 1, 2, or 3, and;

[0012] R 1 is H or C1-C3 alkyl;

[0013] X is, in each case, independently a C2-C4 alkylene or a C4-C6 heteroalkylene;

[0014] Y is a C2-C3 alkylene or a C4-C6 heteroalkylene;

[0015] R 2 and R 3 is independently H or C1-C3 alkyl.

[0016] In some embodiments, the amidated pectin is C4-C 20 Polyamine is amided pectin. In some embodiments, the polyamine is ethylenediamine, putrescine, cadaverine, spermine, or spermidine.

[0017] In some embodiments, the amidated pectin is a unit having one or more of the following structures:

[0018] ,

[0019] It includes its isomers, salts, or tautomers,

[0020] Among the chemical formulas

[0021] n is 0, 1, 2, or 3, and;

[0022] m is 2, 3, or 4, and;

[0023] p is 2, 3, or 4, and;

[0024] R 1 , R 2 , and R 3 is independently H or C1-C3 alkyl.

[0025] In some embodiments, the amidated pectin is a unit having one or more of the following structures:

[0026] , ,

[0027] It includes its isomers, salts, or tautomers.

[0028] In some embodiments, the amidated pectin is amide citrus pectin or amide apple pectin. In some embodiments, the amidated pectin has a molecular weight of about 4,000 Da to about 500,000 Da, about 5,000 Da to about 300,000 Da, about 100,000 Da to about 300,000 Da, or about 50,000 Da to about 200,000 Da.

[0029] In some embodiments, the solid support comprises a material selected from polystyrene, glass, ceramic, polypropylene, polyethylene, silica, zirconia, titania, alumina, polycarbonate, latex, polyethersulfone, PMMA, carboxymethylcellulose, zeolite, and cellulose.

[0030] In some embodiments, the solid support is a magnetic bead, a glass bead, a polystyrene bead, a polystyrene filter, a polycarbonate filter, a polyethersulfone, or a glass filter.

[0031] In another aspect, the present specification provides a method for isolating nucleic acid from a nucleic acid-containing sample, said method comprising the following steps:

[0032] (a) a step of contacting a sample with a solid support disclosed herein to bind nucleic acid to the solid support;

[0033] (b) a step of selectively washing nucleic acids bound to a solid support; and

[0034] (c) A step of eluting nucleic acid from a solid support with an elution reagent.

[0035] In some embodiments, the eluent comprises ammonia or an alkali metal hydroxide. In some embodiments, the eluent has a pH of about 9 or higher, about 10 or higher, or about 11 or higher. In some embodiments, the eluent has a pH of about 9 to about 12, about 9.5 to about 12, about 10 to about 12, or about 9 to about 11. In some embodiments, the eluent comprises a polyanion. In some embodiments, the polyanion is carrageenan or a carrier nucleic acid. In some embodiments, the eluent comprises a polyanion and a base, for example, an alkali hydroxide. In some embodiments, the eluent comprises i-carrageenan and KOH. In some embodiments, the method comprises contacting a sample with a dissolution solution and then contacting the sample with a solid support to release the nucleic acid into the solution. In some embodiments, the dissolution solution comprises a chaotropic agent. In some embodiments, the disorder-inducing agent is selected from guanidium thiocyanate, guanidium hydrochloride, alkali perchlorate, alkali iodide, urea, formamide, or a combination thereof. In some embodiments, the disorder-inducing agent is guanidium thiocyanate or guanidium hydrochloride. In some embodiments, the dissolution solution contains a salt. In some embodiments, the salt is sodium chloride or calcium chloride. In some embodiments, the dissolution solution does not contain a disorder-inducing agent. In some embodiments, the dissolution solution contains a buffer. In some embodiments, the buffer is Tris. In some embodiments, the dissolution solution contains a surfactant. In some embodiments, the dissolution solution contains a defoaming agent.

[0036] In some embodiments, the step of contacting the sample with a solid support is performed without the presence of a disorder-inducing reagent.

[0037] In some embodiments, the sample is selected from blood, plasma, serum, semen, tissue biopsy, urine, stool, saliva, smear preparation, bacterial culture, cell culture, viral culture, PCR reaction mixture, or in vitro nucleic acid modification reaction mixture. In some embodiments, the tissue biopsy is paraffin-embedded tissue. In some embodiments, the nucleic acid comprises genomic DNA. In some embodiments, the nucleic acid comprises total RNA. In some embodiments, the nucleic acid comprises microbial nucleic acid or viral nucleic acid. In some embodiments, the viral nucleic acid is HBV DNA. In some embodiments, the nucleic acid is circulating nucleic acid.

[0038] In some embodiments, the method is performed in an automated cartridge.

[0039] In another aspect, the present specification provides a method for detecting nucleic acids in a sample, said method comprising the following steps:

[0040] (a) a step of contacting a nucleic acid-containing sample with a solid support disclosed herein to bind the nucleic acid to the solid support;

[0041] (b) a step of selectively washing nucleic acids bound to a solid support;

[0042] (c) a step of eluting nucleic acids; and

[0043] (d) Step for detecting nucleic acids.

[0044] In some embodiments, the nucleic acid detection step involves amplifying the nucleic acid by a polymerase chain reaction (PCR). In some embodiments, the polymerase chain reaction is nested PCR, isothermal PCR, or RT-PCR.

[0045] In another aspect, the present specification provides a separation material for chromatography comprising a solid support comprising covalently bonded amidated pectin.

[0046] In some embodiments, the amidated pectin is a unit represented by one or more of the following chemical formulas:

[0047] ,

[0048] It includes its isomers, salts, or tautomers,

[0049] R 2 and R 3 is H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C 20 It is independently selected from heteroalkyls.

[0050] In some embodiments, the solid support is silica, alumina, titania, zirconia, or a hybrid silica material. Specific details for implementing the invention

[0051] In one embodiment, the present specification provides a solid support for purifying nucleic acid from a nucleic acid-containing sample comprising one or more modified pectin molecules covalently bonded to a surface. In some embodiments, the modified pectin comprises a plurality of amino groups. In some embodiments, the modified pectin is amidated pectin. As used herein, the term “solid support” refers to any substrate including paramagnetic particles, gels, controlled pore glass, magnetic beads, microspheres, nanospheres, capillaries, filter membranes, columns, cloths, wipes, paper, flat supports, multi-well plates, porous membranes, porous monomers, wafers, combs, or any combination thereof. The solid support may comprise any suitable material including, but not limited to, glass, silica, titanium oxide, iron oxide, ethylene backbone polymer, polypropylene, polyethylene, polystyrene, ceramic, cellulose, nitrocellulose, and divinylbenzene. Preferably, the solid support comprises a material selected from polystyrene, glass, ceramic, polypropylene, polyethylene, silica, polycarbonate, latex, PMMA, zeolite, polyethersulfone, carboxymethylcellulose, cellulose, and combinations thereof. In some embodiments, the solid support is not pectin, for example, unmodified pectin or modified pectin.

[0052] In some embodiments, the solid support is a magnetic bead, a glass bead, a polystyrene bead, a polystyrene filter, a polycarbonate filter, a polyethersulfone filter, or a glass filter. Preferably, a material suitable for manufacturing the solid support disclosed herein has low non-specific binding, and, for example, in the absence of the pectin modification described herein, such a material does not bind to the nucleic acid, protein, or other component of the sample in which the nucleic acid is isolated.

[0053] Modified pectin

[0054] In some embodiments, the modified pectin is amidated pectin. Pectin is a naturally occurring complex polysaccharide generally found in plant cell walls. Pectin comprises an alpha 1-4 linked polygalacturonic acid backbone interposed by rhamnose residues and modified into neutral sugar side chains, and non-sugar components such as acetylene, methyl, and ferulic acid groups. The galacturonic acid residues of pectin are partially esterified and exist as methyl esters. The degree of esterification is defined as the percentage of esterified carboxyl groups. For example, pectin with a degree of esterification of 50% or more is classified as high-methyl ester ("HM") pectin or high-ester pectin, while pectin with a degree of esterification of less than 50% is referred to as low-methyl ester ("LM") pectin or low-ester pectin. Most pectin found in fruits and vegetables is HM pectin.

[0055] As used herein, “amidated pectin” refers to any naturally occurring pectin that has been structurally modified, for example, by chemical, physical, or biological (including enzymatic) means, or by any combination thereof, wherein the ester or acid group is converted to an amide group. Amidated pectin may be prepared by contacting unmodified pectin with a suitable amine solution to convert the ester group of the unmodified pectin to an amide.

[0056]

[0057] Alternatively, unmodified pectin or hydrolyzed pectin, including partially hydrolyzed pectin, may react with an amine in the presence of a suitable coupling agent to form amidated pectin. Examples of suitable coupling agents include, but are not limited to, carbodiimide coupling agents such as DCC and EDCI, and phosphonium and imonium type reagents such as BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH.

[0058]

[0059] In some embodiments, the modified pectin is modified pectin obtained by the reductive amination of periodate-oxidized pectin. Methods for the reductive amination of carbohydrates such as pectin are known within the art.

[0060] Modified pectin can be obtained from unmodified pectin by any method described herein. Starting materials particularly useful for the synthesis of modified pectin are apple and citrus pectin. In some embodiments, the starting pectin has a molecular weight of about 4,000 Da to about 500,000 Da, about 5,000 Da to about 300,000 Da, about 10,000 Da to about 150,000 Da, or about 10,000 Da to about 100,000 Da.

[0061] In some embodiments, the amidated pectin comprises a plurality of uronic acid units and one or more additional monomer units. Uronic acids comprise sugar acids containing both carbonyl (e.g., aldehyde or keto group) and carboxylic acid (-COOH) functional groups. Generally, uronic acids are derived from sugars in which the terminal hydroxyl group is oxidized to a carboxylic acid, and are generally named according to the parent sugar; for example, galacturonic acid (glucuronic acid) is a uronic acid derived from glucose. Uronic acids derived from hexose are known as hexuronic acid, and uronic acids derived from pentose are known as penturonic acid.

[0062] In some embodiments, in addition to one or more uronic acid units, the amidated pectin is:

[0063] (I), (II),

[0064] It further comprises one or more units selected from its isomers, salts, tautomers, and combinations thereof, and

[0065] R in the chemical formula 1 is optionally substituted C1-C8 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 heterocycloalkyl, and optionally substituted C2-C 20 Selected from heteroalkyls;

[0066] R 2 and R 3 is H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C 20 It is independently selected from heteroalkyls.

[0067] In some implementations, R 3 In some embodiments of an optionally substituted C1-C6 alkyl, R3 is an optionally substituted C4-C 20 It is a heteroalkyl, for example, a short PEG chain arbitrarily substituted with one or more amino groups.

[0068] In some implementations, R 1 , R 2 , and R 3 Each comprises one or fewer amino groups. In some embodiments, R 1 , R 2 , and R 3 Each does not contain an amino group. In some embodiments, R 2 and R 3 Each includes one or more amino groups. In some embodiments, R 2 is H and R 3 is an arbitrarily substituted C4-C 20 It is an oligomeric ethylene glycol comprising a 2-6 ethylene glycol unit optionally substituted with a heteroalkyl, for example, a polyamine or one or more amino groups.

[0069] In some implementations, R 1 is methyl, ethyl, or propyl. In some embodiments, R 2 and R3 All are H. In some embodiments, R 2 is H, and R 3 is an optionally substituted C1-C8 alkyl. In some embodiments, R 2 is H, and R 3 is H, CH3, CH2CH2NH2, CH2CH2N(CH3)2, CH2CH2OH, or CH2CH2NHCH2CH2NH2. In some embodiments, R 2 and R 3 All are CH3.

[0070] In some embodiments, the amidated pectin additionally comprises one or more units of formula (III):

[0071] (III), or

[0072] Includes isomers, salts, tautomers, or combinations thereof, wherein in the chemical formula:

[0073] R 3 is H, CH3, CH2CH2NH2, CH2CH2N(CH3)2, CH2CH2OH, (CH2)2O(CH2)2NH2, or CH2CH2NHCH2CH2NH2.

[0074] If the polysaccharide contains two or more units of chemical formula (II) or (III), R 3 It is understood that it may be the same or different within polysaccharides.

[0075] In some embodiments, the amidated pectin disclosed herein comprises one or more monomer units having at least one amino group. In some embodiments, the amidated pectin comprises one or more monomer units having the structure of Formula VI:

[0076] (IV),

[0077] Includes isomers, salts, tautomers, or combinations thereof, wherein in the chemical formula:

[0078] n is 0, 1, 2, or 3, and;

[0079] R4 is H or C1-C3 alkyl;

[0080] X is, in each case, independently a C2-C4 alkylene or a C4-C6 heteroalkylene;

[0081] Y is a C2-C3 alkylene or a C4-C6 heteroalkylene;

[0082] R 5 and R 6 is independently H or C1-C3 alkyl.

[0083] In some embodiments, the amidated pectin disclosed herein is a monomer unit having one or more chemical formula V structures:

[0084] (V),

[0085] Includes isomers, salts, tautomers, or combinations thereof, wherein in the chemical formula:

[0086] n is 0, 1, 2, or 3, and;

[0087] m is, in each case, independently 2, 3, or 4;

[0088] p is 2, 3, or 4, and;

[0089] R 4 is H, or C1-C3 alkyl;

[0090] R 5 and R 6 is independently H or C1-C3 alkyl.

[0091] In some embodiments, the amidated pectin comprises one or more monomer units comprising a primary amino group. In some embodiments, the amidated pectin comprises one or more monomer units comprising a quaternary ammonium group. In some embodiments, the amidated pectin is an amide amidated with a polyamine. As used herein, a polyamine is a compound comprising two or more amino groups. Polyamines that may be used for pectin modification of solid supports disclosed herein include both synthetic polyamines and naturally occurring polyamines, e.g., spermidine, spermine, and putrescine. In some embodiments, the polyamine is selected from the group consisting of spermine, spermidine, cadaverine, ethylenediamine, and putrescine. In some embodiments, the polyamine is spermine or spermidine.

[0092] In some embodiments, the amidated pectin has the structure of formula VI, formula VII, or formula VIII:

[0093] (VI), (VII), or

[0094] (VIII),

[0095] It also includes one or more units of its isomers, salts, and tautomers.

[0096] In some embodiments, the amidated pectin comprises a plurality of additional monomer units represented by the structure of formula I-VIII. As used herein, the term "plural" means one or more. For example, a plurality of monomer units means at least two monomer units, at least three monomer units, or at least monomer units, etc. Where an embodiment of the present invention comprises one or more monomer units, they may be referred to as a first monomer unit, a second monomer unit, a third monomer unit, etc.

[0097] As used herein, the terms “alkyl,” “alkenyl,” and “alkynyl” comprise straight-chain, branched-chain, and cyclic monovalent hydrocarbyl radicals, and combinations thereof, which contain only C and H when unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butinyl, etc. The total number of carbon atoms of each of these groups is often described herein; for example, if the group may contain up to 10 carbon atoms, 1-10C, C1-C 10 , C 1-10 Alternatively, it may be represented as C1-10. As used herein, the terms “heteroalkyl,” “heteroalkenyl,” and “heteroalkynyl” refer to the corresponding hydrocarbons in which one or more chain carbon atoms are replaced by heteroatoms. Exemplary heteroatoms include N, O, S, and P. Where heteroatoms can replace, for example, carbon atoms within the heteroalkyl group, the number describing the group is denoted, for example, as C3-C10, but this represents the sum of the number of carbon atoms in the ring or chain and the number of heteroatoms included as replacements for the described carbon atoms in the ring or chain.

[0098] A single group may include one or more types of multiple bonds or one or more multiple bonds; such groups are included within the definition of the term "alkenyl" if they contain at least one carbon-carbon double bond, and are included in the term "alkynyl" if they contain at least one carbon-carbon triple bond.

[0099] Alkyl, alkenyl, and alkynyl groups may be arbitrarily substituted to the extent that such substitution is chemically reasonable. Common substituents include, but are not limited to, the following: halogens (F, Cl, Br, I), =O, =NCN, =NOR, =NR, or, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R, and NO2, where each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C 10 Aryl, or C5-C 10 It is heteroaryl, and each R is optionally substituted with halogen (F, Cl, Br, I), =O, =NCN, =NOR', =NR', or', NR'2, SR', SO2R', SO2NR'2, NR'SO2R', NR'CONR'2, NR'C(O)OR', NR'C(O)R', CN, C(O)OR', C(O)NR'2, OC(O)R', C(O)R', and NO2, and each R' is independently substituted with H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl or C5-C 10 It is a heteroaryl. Alkyl, alkenyl, and alkynyl groups are also C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl, or C5-C 10 It can be substituted with a heteroaryl group, and each of the above can be substituted with a suitable substituent for a specific group.

[0100] As used herein, “alkyl” includes cycloalkyl and cycloalkylalkyl groups; the term “cycloalkyl” is used herein to describe a carbocyclic non-aromatic group connected via a ring carbon atom, and “cycloalkylalkyl” is used to describe a carbocyclic non-aromatic group connected to a molecule via an alkyl linker. Similarly, “heterocyclil” is used to identify a non-aromatic cyclic group comprising at least one heteroatom as a ring member and connected to a molecule via a ring atom (which may be C or N); and “heterocyclylalkyl” may be used to describe a group connected to another molecule via an alkylene linker. As used herein, these terms also include a double bond or a ring containing two, provided that the ring is not aromatic.

[0101] "Aromatic" or "aryl" substituents or moietyes refer to monocyclic or conjugated bicyclic moietyes having well-known aromatic properties; examples of aryls include phenyl and naphthyl. Similarly, "heteroaromatic" and "heteroaryl" refer to monocyclic or conjugated bicyclic ring systems containing one or more heteroatoms as ring members. Suitable heteroatoms include N, O, and S, which allow for the aromaticity of 5-membered as well as 6-membered rings. General heteroaromatic systems include monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl, and conjugated bicyclic moiety formed by conjugating one of these monocyclic groups to a phenyl ring or any heteroaromatic monocyclic group to form a C8-C10 bicyclic group, such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, sinolinyl, etc. Any monocyclic or conjugated ring bicyclic system having aromatic characteristics in terms of electron distribution across the entire ring system is included in this definition. It also includes a bicyclic group that is directly attached to the rest of the molecule having aromatic characteristics. Generally, the ring system contains 5 to 14 ring member atoms. Generally, monocyclic heteroaryls contain 5 to 6 ring members, and bicyclic heteroaryls contain 8 to 10 ring members.

[0102] Aryl and heteroaryl moiety are composed of various substituents, e.g., C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C 12It may be substituted with aryl, C1-C8 acyl, and their heteroforms, each of which may be further substituted itself; other substituents for the aryl and heteroaryl moiety include: halogens (F, Cl, Br, I), or, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R, and NO2, wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C7-C 12 Arylalkyl, or C6-C 12 It is a heteroarylalkyl, and each R is optionally substituted for the alkyl group as described above. Substituents on the aryl or heteroaryl group may be further substituted with groups described herein as suitable for each type of substituent or each component of the substituent. Thus, for example, the arylalkyl substituent may be substituted in the aryl portion with a substituent described herein as typical for the aryl group, and the alkyl portion may be further substituted with a substituent described herein as typical or suitable for the alkyl group.

[0103] As used herein, “optional substitution” indicates that the specific group described may have one or more hydrogen substituents replaced by non-hydrogen substituents. In some optionally substituted groups or moietyes, all hydrogen substituents are replaced by non-hydrogen substituents (e.g., polyfluoroalkyls such as trifluoromethyl). Unless otherwise specified, the total number of such substituents that may be present is equal to the number of H atoms present in the unsubstituted form of the group described. If the optional substituent is attached via a double bond, such as a carbonyl oxygen or an oxo (=O), the group occupies two available valencies, so the total number of substituents that may be included decreases according to the number of available valencies.

[0104] As used herein, unless otherwise specified, the term “amino group” includes primary, secondary, and tertiary amino groups.

[0105] Covalent attachment of amidated pectin to a solid support can be achieved in any suitable manner, for example, by reacting polyamine-amidated pectin with a solid support comprising an amine-reactive group, e.g., an epoxide, aldehyde, ketone, or activated ester. Amidated pectin comprising a primary or secondary amino group can also be attached to a solid support, e.g., an amino-modified solid surface, by crosslinking. As used herein, crosslinking refers to the process of chemically linking two or more molecules by covalent bonds. In some examples, a crosslinking agent may be used to attach amidated pectin to a solid support to form a pectin-modified solid support. As used herein, a crosslinking agent (or crosslinker) is a molecule comprising two or more functional ends capable of chemically attaching specific functional groups (e.g., primary amine, carboxyl, sulfhydryl, etc.) onto a molecule and / or a solid support. Methods for covalently linking molecules containing amino groups to functionalized surfaces and solid supports are known in the art.

[0106] In some embodiments, the amidated pectin of the present invention is covalently attached to a solid support through an amide bond, for example, an amide bond formed between a carboxyl group of a solid support and an amino group of the amidated pectin. The formation of the amide bond can be carried out by any suitable method. For example, an amidated pectin comprising one or more primary amino groups may react with a substrate comprising one or more carboxylic acid groups in the presence of a suitable coupling agent. Examples of suitable coupling agents include, but are not limited to, carbodiimide coupling agents such as DCC and EDCI, and phosphonium and imonium type reagents such as BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH. In some preferred embodiments, the carboxylic acid group of the solid substrate may be converted to an activated ester and then react with the amino group of the amidated pectin.

[0107] In some embodiments, the solid support comprises an amidated pectin having one or more units represented by any one of the formulas (II)-(VIII), and the amidated pectin is covalently attached to the solid support.

[0108] In another aspect, the present specification provides a method for isolating nucleic acid from a nucleic acid-containing sample, said method comprising the following steps:

[0109] (a) a step of contacting a sample with a solid support disclosed herein to bind nucleic acid to the solid support;

[0110] (b) a step of optionally washing nucleic acids bound to a solid support; and

[0111] (c) A step of bringing the nucleic acid bound to the solid support into contact with an elution reagent to elute the nucleic acid from the solid support.

[0112] Dissolution solution

[0113] In some embodiments, a nucleic acid-containing sample contacts a solid support after contact with a lysis solution to lyse the cells contained within the sample and release the nucleic acid into the solution. After the sample is lysed, the nucleic acid may be bound to a solid substrate, such as a silica or glass substrate covalently modified with the amidated pectin described herein. In some embodiments, the solid support is an automated cartridge, such as GenXpert ® It is integrated into a cartridge. After binding, the supernatant is removed, and the nucleic acid is eluted from the substrate with an elution buffer, for example, an alkaline solution as described above. The eluent can be processed in the cartridge to detect the target gene of interest. In some embodiments, at least a portion of the PCR reagent present in the cartridge is used to reconstitute it as lyophilized particles. In some embodiments, the PCR uses a Taq polymerase with a hot start function, such as AptaTaq (Roche, Switzerland).

[0114] In some embodiments, the dissolving solution comprises a disorder-inducing agent, such as guanidium thiocyanate, guanidium hydrochloride, alkali perchlorate, alkali iodide, urea, formamide, and combinations thereof. In some embodiments, the dissolving solution comprises a salt. Preferably, the salt is sodium chloride or calcium chloride.

[0115] In some embodiments, the method disclosed herein does not require the use of a disorder-inducing reagent or a high concentration of salt to bind nucleic acid to the solid support of the present invention.

[0116] In some embodiments, the sample is dissolved by adding a polysaccharide reagent solution and then contacting the sample with a lysis buffer prior to the precipitation of nucleic acid. In some embodiments, the lysis reagent is added to a solution of nucleic acid-precipitating polysaccharide reagent. In some embodiments, the polysaccharide reagent described herein is dissolved in the lysis solution. In some examples, the lysis solution comprises one or more proteases. Suitable proteases include, but are not limited to, serine proteases, threonine proteases, cysteine ​​proteases, aspartate proteases, metalloproteases, glutamic acid proteases, metalloproteases, and combinations thereof. Exemplary suitable proteases include, but are not limited to, Protenase k (broad-spectrum serine protease), subtilisin trypsin, chymotrypsin, pepsin, papain, etc. Using the teachings and examples provided herein, other proteases will be available to those skilled in the art.

[0117] In some embodiments, the method described herein is used in the steps of: isolating nucleic acids (e.g., DNA, RNA) from a fixed paraffin-embedded biological tissue sample according to any method described herein; amplifying the precipitated nucleic acids using a pair of oligonucleotide primers capable of amplifying a region of the target nucleic acid to obtain an amplified sample; and determining the presence and / or amount of the target nucleic acid. In some embodiments, the target nucleic acid is DNA (e.g., a gene). In some embodiments, the target nucleic acid is RNA (e.g., mRNA, non-coding RNA, etc.). In some embodiments, the nucleic acids isolated using the method described herein are highly suitable for use in diagnostic methods, preventive methods, therapeutic (e.g., cancer treatment) monitoring methods, etc. Thus, in some exemplary and non-limiting embodiments, nucleic acids extracted from a fixed paraffin-embedded sample (e.g., from an FFPET sample) may be used to identify the presence and / or expression level of a gene and / or the mutation status of the gene. This method is particularly well suited for identifying the presence and / or expression levels and / or mutation status of one or more cancer markers. Accordingly, in some embodiments, nucleic acids isolated using the method described herein are used to detect the presence, and / or copy number, and / or expression levels and / or mutation status of one or more cancer markers.

[0118] Washing and elution

[0119] The detection and isolation method disclosed herein may optionally include a washing step, that is, the precipitated nucleic acid may optionally be washed on a solid support, for example, to remove components of the lysis buffer. Generally, the concentrated, for example, precipitated nucleic acid is lysed before detection. In some embodiments, the concentrated nucleic acid is lysed in a buffer compatible with the PCR reaction.

[0120] In some embodiments, for example, when a polyamine-modified polysaccharide is used to precipitate a nucleic acid, the precipitated nucleic acid may be eluted from the polyamine by contacting it with a suitable eluent. In some embodiments, the eluent comprises ammonia or an alkali metal hydroxide. In some embodiments, the eluent has a basic pH. In some embodiments, the eluent has a pH of about 9 to about 12, about 9.5 to about 12, about 10 to about 12, or about 9 to about 11. Preferably, the pH of the eluent is 10 or higher. Preferably, the eluent comprises ammonium hydroxide, NaOH, or KOH at a concentration sufficient to prevent the binding of the nucleic acid and the polysaccharide reagent. An exemplary eluent comprises 1% ammonia, 15 mM KOH, or 15 mM NaOH.

[0121] In some embodiments, the eluent comprises a polyanion. In some embodiments, the polyanion is a polymer comprising a plurality of anionic groups. In some embodiments, the anionic group is a phosphate, phosphonate, sulfate, or sulfonate group, or a combination thereof. In some embodiments, the polyanion is a polymer that is negatively charged at a pH of about 7 or higher. Both synthetic polyanions and naturally occurring polyanions may be used in the methods disclosed herein. In some embodiments, the polyanion is carrageenan. In other embodiments, the polyanion is a carrier nucleic acid. As used herein, the carrier nucleic acid is, for example, a nucleic acid that does not interfere with the subsequent detection of nucleic acid enriched by PCR. Exemplary carrier nucleic acids include poly rA, poly dA, herring sperm DNA, salmon sperm DNA, and others well known to those skilled in the art. In some embodiments, the eluent comprises carrageenan and an alkali metal hydroxide, e.g., NaOH or KOH.

[0122] nucleic acids

[0123] In some embodiments, the method described herein is used to isolate nucleic acids from a nucleic acid-containing solution. A nucleic acid-containing solution may be obtained by dissolution from a nucleic acid-containing material. The nucleic acid-containing material is generally selected from the group comprising blood, tissue biopsies such as paraffin-embedded tissue, smear preparations, bacterial cultures, viral embeddings, urine, semen, cell suspensions and adherent cells, PCR reaction mixtures, and in vitro nucleic acid modification reaction mixtures. The nucleic acid-containing material may include human, bacterial, fungal, animal, or plant material. In other embodiments, a nucleic acid-containing solution may be obtained from a nucleic acid modification reaction or a nucleic acid synthesis reaction. In other embodiments, a nucleic acid-containing solution may be obtained from a nucleic acid modification reaction or a nucleic acid synthesis reaction.

[0124] As used herein, the term “nucleic acid” refers to any synthetic or naturally occurring nucleic acid, such as DNA or RNA, in the form of any possible configuration, namely double-stranded nucleic acid, single-stranded nucleic acid, aptamer, or any combination thereof. The nucleic acid may be DNA, for example, genomic DNA. The nucleic acid may also be RNA, for example, whole RNA. The nucleic acid may be single-stranded or double-stranded nucleic acid, for example, a short double-stranded DNA fragment. The nucleic acid may be synthetic nucleic acid. In some embodiments, the nucleic acid is cyclic nucleic acid.

[0125] The nucleic acid isolated using the method described herein and the solid support described herein are of a quality suitable for amplification to detect and / or quantify one or more target nucleic acid sequences in a sample. The nucleic acid isolation method and solid support described herein may be used in basic research aimed at discovering gene expression profiles related to the diagnosis and prognosis of diseases. The method may also be used for the diagnosis and / or prognosis of diseases, the determination of specific therapeutic regimens, and / or monitoring of therapeutic effects.

[0126] In some embodiments, the method described herein is used to precipitate nucleic acids from nucleic acid-containing samples. Nucleic acid-containing materials generally include blood, serum, tissue biopsies such as paraffin-embedded tissue, oral fluids, smear preparations, bacterial cultures, viral embeddings, urine, semen, cell suspensions and adherent cells, PCR reaction mixtures, and In a test tube It may be selected from the group comprising nucleic acid modification reaction mixtures. The nucleic acid-containing material may include human, animal, or plant material. In some embodiments, the nucleic acid is in a solution. The nucleic acid-containing solution includes an extracellular nucleic acid solution and a solution obtained by lysis of a nucleic acid-containing cell. In other embodiments, the nucleic acid-containing solution may be obtained from a nucleic acid modification reaction or a nucleic acid synthesis reaction.

[0127] Amplification method

[0128] The method described herein simplifies the isolation of nucleic acids from biological samples and efficiently produces isolated nucleic acids that are highly suitable for use in RT-PCR systems. In some embodiments, nucleic acids isolated from nucleic acid-containing samples using the method described herein may be detected by any suitable known nucleic acid detection method. In some embodiments, the extracted nucleic acids are used in amplification reactions, but other uses are also considered. Accordingly, for example, the isolated nucleic acids or their amplification product(s) may be used in various sequencing or hybridization protocols, including but not limited to nucleic acid-based microarrays and next-generation sequencing.

[0129] In one embodiment, the present specification provides a method for detecting nucleic acids, said method comprising the following steps:

[0130] (a) a step of contacting a nucleic acid-containing sample with a solid support disclosed herein to bind the nucleic acid to the solid support;

[0131] (b) a step of selectively washing nucleic acids bound to a solid support;

[0132] (c) a step of bringing the nucleic acid bound to the solid support into contact with an elution reagent to elute the nucleic acid from the solid support; and

[0133] (d) Step for detecting nucleic acids.

[0134] In some embodiments, the detection method includes nucleic acid amplification. Suitable, non-limiting, exemplary amplification methods include polymerase chain reaction (PCR), reverse transcriptase PCR, real-time PCR, nested PCR, multiplex PCR, quantitative PCR (Q-PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), rolling circle amplification (RCA), and strand displacement amplification (SDA).

[0135] In some embodiments, the amplification method comprises a cycle comprising initial denaturation at about 90 °C to about 10 °C for about 1 to about 10 minutes, denaturation at about 90 °C to about 100 °C for about 1 to about 30 seconds, annealing at about 55 °C to about 75 °C for about 1 to about 30 seconds, and extension at about 55 °C to about 75 °C for about 5 to about 60 seconds. In some embodiments, during the first cycle after the initial denaturation, the cycle denaturation step is omitted. Specific times and temperatures depend on the specific nucleic acid sequence being amplified and can be easily determined by a person skilled in the art.

[0136] In some embodiments, the isolation and detection of nucleic acids are performed on an automated sample handling and / or analysis platform. In some embodiments, a commercially available automated analysis platform is used. For example, in some embodiments, the GeneXpert system (Cepheid, Sunnyvale, California) is used. However, the present invention is not limited to a specific detection method or analysis platform. Those skilled in the art recognize that any number of platforms and methods may be used.

[0137] The GeneXpert system uses self-contained, disposable cartridges. Sample extraction, amplification, and detection of nucleic acids can all be performed within these self-contained "cartridge laboratories." For example, reference is made to U.S. Patent 6,374,684, the entirety of which is incorporated herein by reference. Components of the cartridge include, but are not limited to, a processing chamber containing reagents, filters, and capture techniques useful for extracting, purifying, and amplifying target nucleic acids. Valves enable fluid movement from chamber to chamber and include nucleic acid lysis and filtration components. Real-time optical detection is possible through optical windows. Reaction tubes enable very rapid thermal cycling. In some embodiments, the GenXpert system includes multiple modules for scalability. Each module includes multiple cartridges along with sample handling and analysis components.

[0138] Solid phase for chromatography

[0139] In some embodiments, the present specification discloses a separation material for chromatography comprising a solid support to which a polysaccharide is bound. In some embodiments, the polysaccharide is a polyuronic acid or an amidated pectin. In some embodiments, the polysaccharide is an amidated pectin adsorbed on the surface of a solid support. In other embodiments, the amidated pectin is immobilized on the surface of a solid support through covalent, non-covalent, or a combination of covalent and non-covalent interactions.

[0140] In some embodiments, the separation material comprises a polysaccharide bonded to a solid support, said polysaccharide being a unit represented by one or more of Chemical Formula II:

[0141] (II),

[0142] Includes isomers, salts, tautomers, or combinations thereof, among the chemical formulas

[0143] R 2 and R 3 is H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C 20 It is independently selected from heteroalkyls.

[0144] In some embodiments, the amidated pectin is a pectin comprising one or more units having the structure of formula II-VIII.

[0145] Solid supports suitable for the manufacture of separation materials include silica gel and other inorganic materials, such as Al2O3 (alumina), TiO2 (titania), or ZrO2 (zirconia). Organic polymer resins may also be used in the manufacture of the separation materials disclosed herein. Certain materials having hybrid particle technology (HPT), for example, hybrid organic / inorganic materials, such as Waters BEH Technology™ materials, are suitable for the manufacture of the separation materials disclosed herein. HPT materials retain the major advantages of silica, such as purity, mechanical strength, high spherical shape, particle size control ability, pore diameter, surface area, and surface chemistry. At the same time, these hybrid materials are stable at basic pH, for example, pH 8 or higher.

[0146] Preferably, the solid support used in the preparation of the separation material is porous. In some embodiments, the separation material is a porous particle having amidated pectin bonded through covalent or non-covalent interactions, and in other embodiments, the separation material is a porous monolithic support having amidated pectin bonded through covalent or non-covalent interactions.

[0147] In some embodiments, the solid support used in the preparation of the separation material disclosed herein is silica gel or silica. The silica is characterized by a pore diameter, particle size, and / or specific surface area. The silica gel-based separation material preferably has a pore diameter of about 30 to about 1000 Angstroms, a particle size of about 2 to about 300 microns, and a specific surface area of ​​about 35 m² 2 / g to about 1000 m 2 / g. In some embodiments, the silica gel has a pore diameter of about 40 Angstroms to about 500 Angstroms, about 60 Angstroms to about 500 Angstroms, about 100 Angstroms to about 300 Angstroms, and about 150 Angstroms to about 500 Angstroms. In some embodiments, the silica gel has a particle size of about 2 to about 25 microns, about 5 to about 25 microns, about 15 microns, about 63 to about 200 microns, and about 75 to about 200 microns; and a specific surface area of ​​about 100 m² 2 / g to about 350 m 2 / g, approximately 100 m 2 / g to about 500 m 2 / g, approx. 65 m 2 / g to about 550 m 2 / g, approximately 100 m 2 / g to about 675 m 2 / g, and about 35 to about 750 m 2 / g is.

[0148] In some embodiments, the chromatographic material according to the present invention comprises magnetic silica particles. The magnetic silica particles comprise a superparamagnetic core coated with a hydrated silica oxide adsorption surface (i.e., a surface having silanol or Si-OH groups). Suitable commercially available magnetic silica particles include MagneSil™ particles available from Promega Corporation (Madison, Wisconsin).

[0149] In some embodiments, the solid support is aluminum oxide. Exemplary aluminum oxide solid supports include, but are not limited to, Brockmann aluminum oxide with a mesh size of about 150 and 58 angstroms.

[0150] In some embodiments, the amidated pectin is chemically bonded to a solid support through a linker. The linker between the solid support and the amidated pectin may comprise an alkylene or heteroalkylene chain. Preferably, the linker comprises 2 to 20 carbon atoms and may include nitrogen and oxygen atoms in addition to carbon atoms. In some embodiments, the linker is an oligoethylene linker, for example, a PEG oligomer.

[0151] The preparation of the separation material can be achieved in any suitable manner. For example, the solid support may react with a surface modifier. As used herein, the surface modifier is a moiety that imparts some chromatographic function to the base solid support. Surface modifiers such as the amidated pectin disclosed herein may be attached to the base solid support via derivatization reactions, non-covalent coating, or a combination thereof. In some embodiments, the organic groups of such solid support form covalent bonds with the surface modifier, such as the amidated pectin, which contains reactive groups. Such covalent attachment of the amidated pectin may be achieved through a number of mechanisms well known in the art, such as cyclization addition and hydrophilic and electrophilic substitution.

[0152] In some embodiments, the base solid support is a silica gel containing silanol groups. Such silica gel solid support may react with a modifier containing silanizing groups to obtain the separation material disclosed herein. For example, the silanol groups are surface-modified with a silanizing reagent having the formula XaRbSi-LZ, wherein X is Cl, Br, I, C1-C5 alkoxy, dialkylamino, or trifluoromethanesulfonate; a and b are each integers from 0 to 3, and the sum of a and b is 3; R is a C1-C6 linear, branched, or cyclic alkyl; L is any C1-C20 alkylene or any heteroalkylene linker group that can be substituted; and Z is a functional group.

[0153] In some embodiments, Z comprises amidated pectin. In other embodiments, Z comprises functional groups, such as amino, carbonyl, or carboxyl groups, which can be further functionalized into amidated pectin. Examples of silanizing agents include amino silanizing agents, such as 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, aminoalkylsilatran, 3-(2-aminoethyl)aminopropyl-triethoxysilane, and 3-(2-aminoethyl)aminopropyltriethoxysilane. The reaction of the silica gel with the amino silanizing agent provides a silica gel comprising a surface amino group that can be further modified into amidated pectin comprising one or more reactive groups and / or react with it. In other embodiments, the silica gel reacts with an amidated pectin derivative comprising a silica-reactive group, such as a silatran or trialkoxysilane derivative.

[0154] In some embodiments, the present specification discloses a column, capillary, or cartridge comprising a solid support having a surface and one or more amidated pectin molecules attached to the surface, as an adsorbent or support.

[0155] In some embodiments, the separation material and chromatography column disclosed herein are useful for isolating, separating, and purifying nucleic acids from, for example, biological samples or chemical reaction mixtures. In some embodiments, separation is achieved by high-performance liquid chromatography (HPLC), size exclusion chromatography, or electrophoresis.

[0156] In some embodiments, the separation material disclosed herein is suitable for the separation of nucleic acids, including but not limited to dsDNA, ssDNA, RNA, and hybrids thereof. The elution of nucleic acids from the separation material and the separation thereof can be achieved by increasing the ionic strength of the eluent mobile phase or by increasing the concentration of the eluent in a stepwise or gradient manner. The mobile phase may optionally comprise an organic solvent suitable for HPLC separation, such as acetonitrile or methanol. An increase in ionic strength can be achieved by increasing the concentration of a suitable salt, such as sodium chloride or guanidium salt.

[0157] Although each element of the present invention is described herein as comprising various embodiments, it should be understood that, unless otherwise indicated, each embodiment of a given element of the present invention may be used together with each embodiment of another element of the present invention, and that each such use is intended to form different embodiments of the present invention.

[0158] The present invention is further explained by the following examples, which are merely for additional illustrative purposes and should not be construed as limiting.

[0159] Examples

[0160] Example 1: Preparation of Amidated Pectin-Modified Solid Support (EDC Route)

[0161] A. Preparation of Amidated Pectin-Modified Beads

[0162] All reagents were obtained from commercial sources unless otherwise specified.

[0163] Spermine-amidated pectin was prepared according to the procedure described below. Other amidated pectins were prepared in a similar manner.

[0164] (A) Apple pectin (2.5 g) was partially added to 250 mL of deionized water under magnetic stirring until completely dissolved. To the solution, 2.5 mL of 5 M NaOH was added and stirred for 20 minutes, after which 1 M HCl was added until the pH stabilized at ~4.5 (~12 mL of 1 M HCl was added). 1-ethyl-3-(3-dimethylaminopropyl)-carboimide hydrochloride (EDC.HCl, 2.5 g) and 0.75 g of N-hydroxysuccinimide (NHS) were added and stirred for 1 hour for activation. Spermin (Sigma, 18.63 g, 7 eq) was added all at once. The solution became gel-like and was shaken until everything dissolved, and then incubated for an additional 20 hours at room temperature.

[0165] (B) The reaction mixture of (A) was poured into 500 mL of MeOH while stirring to form a gel-like precipitate. The mixture was stirred for 30 minutes and filtered through a 500 mL plastic disposable filter fitted with a polyethylene frit (Opti-Chem, OP-6602-18). The collected gel-like cake was washed with methanol (100 mL) and filtered further overnight to form a dried brown gel mass. Subsequently, the material was washed with another 150 mL of MeOH and dried in a vacuum oven at 50 °C for 18 hours. The formed solid pellet was ground into a powder in a mortar and pestle.

[0166] (C) Washing

[0167] substance:

[0168] A. Acidic Wash. The following mixture was prepared in a 1000 mL bottle: IPA (550 mL, graduated cylinder), deionized water (345 mL), and concentrated HCl (105 mL).

[0169] B. Neutral washing. The following mixture was prepared in a 1000 mL flask: 590 mL IPA containing 410 mL deionized water.

[0170] The product of step (B) was loaded into a 125 mL flask, and 110 mL of washing solution was added to the powdered material. The suspension was stirred at room temperature for 30 minutes, filtered through a glass funnel, followed by 5 x 15-20 mL acid washes, 5 x 15-20 mL neutral washes, and 2 x 35 mL MeOH washes. The material was further air-dried for 60 minutes, and then dried at 0.15 mbar for 17 hours.

[0171] B. Preparation of Amidated Pectin-Modified Beads

[0172] The following solid support (bead) material was modified into amidated pectin according to the procedure described below:

[0173] Silica microspheres, carboxyl, 1.0 μm (Polysciences, Warrington, Pennsylvania, 24754-1)

[0174] Carboxyl-polystyrene particles, 5.11 μm (Spherotech, Germany, CP-50-10)

[0175] NHS-activated Cephalos 4 Fast Flow (GE Healthcare, Chicago, Illinois, Cephalos Bead, 17-0906-01); and

[0176] Carboxyl-modified magnetic bead, 5.7 μm (Spherotech, Germany).

[0177] For Cephalos beads, the NHS activated bead form was used, and the EDC / NHS activation step was omitted. Hydrolyzed NHS-Cephalos beads were used for the measurement of unmodified beads.

[0178] In these embodiments, a procedure for functionalizing carboxyl-modified beads using amine-containing amidated pectin, such as the product of Example 1, is provided.

[0179] Polystyrene beads modified with carboxyl groups (Spherotech, CP-50-10) (~5 microns, 5 wt% suspension in 2 mL) were diluted with deionized water (4 mL) and sonicated for 15 minutes. To the bead suspension, 40 mg of EDC.HCl and 40 mg of NHS were added. For activation, the suspension was stirred for 24 hours, briefly centrifuged at 4000 rpm for 5 minutes, and the supernatant was separated. The beads were resuspended in 5 mL of deionized water, to which a 1% solution of amidated pectin (5 mL) was added. The amidated pectin solution was prepared by infusing the amidated pectin in deionized water for 18 hours, and then centrifuged at 9000 rpm for 30 minutes to remove the dissolved material. The resulting suspension was stirred for 18 hours, then centrifuged at 9000 rpm for 30 minutes, diluted with 45 mL of water, and washed in the same manner. The process was repeated with 0.1 M NaOH (1x), 0.1 M HCl (1x), and deionized water (2x). The beads were resuspended in 5 mL of deionized water and sonicated for 30 minutes. After drying a 150 μL aliquot under vacuum in a SpeedVac, the concentration was determined by measuring the amount of remaining beads.

[0180] Example 2: Preparation of Amidated Pectin-Modified Solid Support (Reductive Amination Route)

[0181] In the above embodiment, a general procedure for modifying polysaccharides, e.g., pectin, using various polyamines through reductive amination after oxidation is provided.

[0182] (A). Oxidation. Apple pectin (2.5 g) was partially added to 250 mL of deionized water under magnetic stirring until completely dissolved. To this, potassium periodate (2.43 g) was partially added under stirring and stirred for 18 hours. The reaction mixture was then dialyzed against water through an 8 kDa MWCO dialysis tube over 3 days. The resulting desalted polymer was subsequently freeze-dried to obtain oxidized pectin as a grayish-white solid. The concentration of the aldehyde can be easily measured via hydroxyamine titration (e.g., Zhao, H.; Heindel, ND). J. Pharm. Res. See 8(3), 400-402. The aldehyde content was measured to be 4.9 mmol / g (~1 equivalent of aldehyde per polymer unit).

[0183] (B). Reductive amination. The oxidized pectin from step A (1.0 g) was suspended in 100 mL of deionized water, spermine (1.32 g, 1.25 eq) was added, and the mixture was stirred at room temperature for 18 hours. Sodium borohydride pellets (1.0 g) were added to the reaction, and the mixture was stirred for 18 hours. The reaction mixture was then dialyzed against water through an 8 kDa MWCO dialysis tube over 3 days and freeze-dried to obtain 200 mg of amidated pectin as a grayish-white, fluffy solid.

[0184] The product from the above reaction was used to modify a solid support as described in Example 1.

[0185] Example 3: Evaluation of nucleic acid capture by modified beads in filters

[0186] The above experiment demonstrated that an exemplary solid support, for example, an amidated pectin-modified bead prepared as described in Example 1, can capture DNA or RNA in a filter.

[0187] ingredient

[0188] In the examples, the following materials were used: genomic DNA (Promega Cat#G3041 ~202 ng / μL); RNA control (Life Tech Cat# 4307281, 50 ng / μL); quantitative fluorescent Picogreen DNA dye (Thermo); quantitative fluorescent Ribogreen RNA dye (Thermo); Biotek fluorescence system and black assay plates suitable for the fluorescence quantification of nucleic acids; calibrated pipettes and pipette tips; 1x TE buffer (Thermo: EnzChek®Reverse Transcriptase Assay Kit, P / N E22064 according to manufacturer's instructions), or 20 mM Tris prepared to pH ~8.5; Whatman GF / F filters and Pall Supor 0.2 micron filters; filter holders

[0189] method

[0190] A test solution of DNA or RNA was prepared in 1x TE buffer to the desired final concentration (e.g., 100 ng / mL). Modified beads were added to the test solution along with the DNA or RNA in TE. As a control, a DNA or RNA solution without added beads was prepared. Exemplary test solution:

[0191] TE buffer containing 0.1-1.5 mg of modified beads and nucleic acid;

[0192] TE buffer containing nucleic acids without beads (negative control);

[0193] TE buffer containing nucleic acids without beads, unfiltered;

[0194] A 1 mL sample of the nucleic acid solution was mixed with modified beads for 15 seconds to promote mixing and binding of the nucleic acid to the bead surface. The sample was aspirated with a 1 mL syringe; it was passed through a GF / F or other filter of interest using a syringe filter device or a pre-made filter. The eluent was collected in a 2 mL Eppendorf tube. Since the captured nucleic acid was retained on the beads of the filter, the amount of captured nucleic acid can be indirectly evaluated by the absence of nucleic acid in the eluent as follows.

[0195] Standard curves for DNA or RNA were prepared according to the manufacturer's instructions; 500 μL of each standard and blank were prepared in a total of 8 tubes. The working dye solution was prepared by diluting the dye 1:200 in TE buffer and protected from light. The fluorescence intensity of the standard curve samples and each eluent sample was measured according to the manufacturer's instructions for the Biotek plate reader. The standard curves were used to calculate the concentration of nucleic acids in the eluent samples and to calculate the capture percentage relative to the theoretical concentration. To determine the recovery rate of nucleic acids, the test samples were compared to a 100% unfiltered control. To evaluate minimal background filter capture, the control sample containing no beads was filtered. The 100% control was not filtered.

[0196] Table 1-5 shows the results of filtration experiments demonstrating that a solid support modified with amidated pectin can efficiently capture nucleic acids.

[0197] Table 1. Capture of hgRNA and hgDNA by modified glass beads on a Pall Supor 0.2 micron filter.

[0198] Bead type Modifier hgRNA capture (% of 100 ng) 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads glass 1 μm doesn't exist 0% 12% 6% 8% 1% glass 1 μm Spermin 11% 7% 12% 18% 9% glass 1 μm Spermidin 9% 12% 15% 10% 19% 1 µm glass Pectin-spermin 11% 8% 12% 10% 19% glass 1 μm Pectin-Spermidin 12% 9% 15% 22% 25% glass 1 μm Pectin ethylenediamine 12% 16% 14% 25% 27% doesn't exist N / A 0% 0% 0% 0% 0% hgRNA capture (% of 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads glass 1 μm doesn't exist 13% 58% 64% 80% 85% glass 1 μm Spermin 49% 64% 72% 84% 86% glass 1 μm Spermidin 48% 68% 72% 81% 85% glass 1 μm Pectin-spermin 37% 55% 68% 80% 86% glass 1 μm Pectin-Spermidin 53% 51% 62% 78% 75% glass 1 μm Pectin-ethylenediamine 45% 66% 65% 77% 75% doesn't exist N / A 16% 16% 16% 16% 16%

[0199] Table 2. Capture of hgRNA and hgDNA by modified Sepharose beads in Whatman GF / F filters.

[0200] hgRNA capture (% of 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads Sepharos doesn't exist 16% 13% 18% 33% 34% Sepharos Spermin 34% 51% 52% 60% 71% Sepharos Spermidin 7% 30% 41% 62% 66% Sepharos Pectin-spermin 0% 14% 38% 55% 52% Sepharos Pectin-Spermidin 38% 54% 65% 65% 64% Sepharos Pectin-ethylenediamine 23% 14% 19% 35% 42% doesn't exist N / A 38% 38% 38% 38% 38% hgDNA capture (per 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads Sepharos doesn't exist 20% 7% 8% 19% 12% Sepharos Spermin 47% 84% 91% 97% 93% Sepharos Spermidin 52% 40% 36% 38% 47% Sepharos Pectin-spermin Not detected 15% 37% 68% 75% Sepharos Pectin-Spermidin 5% 32% 44% 64% 71% Sepharos Pectin-ethylenediamine 18% 15% 26% 13% 31% doesn't exist N / A 38% 38% 38% 38% 38%

[0201] Table 3. Capture of hgRNA and hgDNA by modified polystyrene beads in Whatman GF / F filters.

[0202] hgRNA capture (% of 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads polystyrene doesn't exist 35% 35% 47% 41% 21% polystyrene Spermin 42% 59% 58% 74% 85% polystyrene Spermidin 42% 37% 44% 70% 76% polystyrene Pectin-spermin 29% 41% 63% 83% 86% polystyrene Pectin-Spermidin 38% 56% 72% 86% 81% polystyrene Pectin-ethylenediamine 8% 12% 28% 39% 46% doesn't exist N / A 38% 38% 38% 38% 38% hgDNA capture (per 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads polystyrene doesn't exist 21% 24% 32% 36% 17% polystyrene Spermin 98% 99% 94% 100% 100% polystyrene Spermidin 16% 17% 26% 35% 35% polystyrene Pectin-spermin 35% 53% 68% 84% 90% polystyrene Pectin-Spermidin 47% 74% 82% 94% 89% polystyrene Pectin-ethylenediamine 29% 35% 23% 30% 27% doesn't exist N / A 38% 38% 38% 38% 38%

[0203] Table 4. Capture of hgRNA and hgDNA by modified polystyrene beads on a Pall Supor 0.2 micron filter.

[0204] hgRNA capture (% of 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads Sepharos doesn't exist 16% 17% 18% 17% 18% Sepharos Spermin 29% 41% 51% 65% 83% Sepharos Spermidin 26% 37% 49% 69% 69% Sepharos Pectin-spermin 30% 38% 42% 70% 90% Sepharos Pectin-Spermidin 15% 11% 13% 28% 28% Sepharos Pectin-ethylenediamine 19% 23% 25% 35% 50% doesn't exist N / A 10% 10% 10% 10% 10% hgRNA capture (% of 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads Sepharos doesn't exist 15% 18% 17% 16% 16% Sepharos Spermin 10% 34% 27% 42% 49% Sepharos Spermidin 15% 18% 29% 52% 56% Sepharos Pectin-spermin 19% 23% 48% 68% 83% Sepharos Pectin-Spermidin 17% 21% 37% 35% 51% Sepharos Pectin-ethylenediamine 18% 15% 26% 13% 31% doesn't exist N / A 26% 26% 26% 26% 26%

[0205] Table 5. Capture of hgRNA and hgDNA by modified polystyrene beads on a Pall Supor 0.2 micron filter.

[0206] hgRNA capture (% of 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads polystyrene doesn't exist 0% 0% 0% 8% 8% polystyrene Spermin 33% 61% 88% 104% 105% polystyrene Spermidin 15% 21% 19% 24% 17% polystyrene Pectin-spermin 35% 40% 67% 87% 93% polystyrene Pectin-Spermidin 12% 36% 48% 66% 77% polystyrene Pectin-ethylenediamine 18% 11% 11% 7% 9% doesn't exist N / A 10% 10% 10% 10% 10% hgDNA capture (per 100 ng) Bead type Modifier 0.1 mg / mL beads 0.25 mg / mL beads 0.5 mg / mL beads 1 mg / mL bead 1.5 mg / mL beads polystyrene doesn't exist 10% 10% 24% 17% 22% polystyrene Spermin 21% 44% 54% 79% 95% polystyrene Spermidin 18% 36% 18% 32% 35% polystyrene Pectin-spermin 33% 43% 57% 82% 90% polystyrene Pectin-Spermidin 37% 58% 70% 83% 91% polystyrene Pectin-ethylenediamine 27% 27% 17% 20% 30% doesn't exist N / A 26% 26% 26% 26% 26%

[0207] Example 4. Extraction of nucleic acids from urine and feces

[0208] The above experiment demonstrates that the solid support disclosed herein can be used to extract nucleic acids from stool and urine samples, and that the isolated DNA can be detected by PCR amplification.

[0209] Preparation of urine or stool samples

[0210] Fragmented MTB DNA (fMTB DNA 200-400 bp) was spiked into various volumes of urine or stool samples as indicated below. A control group for the experiment was prepared by directly spiked the same amount of fMTB DNA into a separate RT-PCR reaction to provide a comparison with 100% extraction and recovery efficiency.

[0211] Extraction of fragmented MTB DNA from urine or feces using exemplary microspheres modified with amidated pectin .

[0212] 1–10 mL of urine / stool samples were added to centrifuge tubes or Eppendorf tubes of appropriate size. Exemplary microparticles modified with amidated pectin were added to the samples. The optimal amount to add depends on the bead lot, sample type, and sample volume selected for each experiment. The samples were thoroughly mixed and optionally incubated for up to 60 minutes to increase nucleic acid binding, after which the microparticles were precipitated by high-speed centrifugation on a benchtop centrifuge for 2 minutes. The supernatant was carefully separated to avoid disturbing the microparticle pellet. The bead pellet was washed with 1 mL of water and gently mixed, and then the microparticles were precipitated by high-speed centrifugation on a benchtop centrifuge for 2 minutes. The supernatant was carefully separated to avoid disturbing the microparticle pellet. 100 μL of low-salt elution buffer consisting of 10 mM KOH with 0.01% i-carrageenan (Sigma) was added to the bead pellet. The pellet was gently mixed and optionally incubated for up to 60 minutes to increase elution from the microparticles. The supernatant containing the eluted nucleic acid was carefully removed to avoid disturbing the bead pellet. The eluent was then used directly for the RT-PCR reaction. PCR was performed as described by Chakravorty et al. regarding the Xpert MTB / RIF Ultra Assay in [mBio, July / August 2017 Volume 8 Issue 4 e00812-17].

[0213] The results are shown in Table 6-8.

[0214] Table 6. PCR analysis of DNA extracted from 10 mL of urine sample. Performance of microparticles modified with spermine-aminated (EDC-coupled or reductive amide) pectin for extracting MTB DNA from 10 mL of urine is shown. ΔCt is calculated as the difference in extraction Ct obtained from 100% spikes of the control group.

[0215] Pectin Modifier / Procedure Base beads (2.5 mL of 2.5% solution) EDC / NHS (mg) Compound (mg) Time, Date purifying μL particles per mL (2.5%) Δ from 100% control group Spermin / EDC coupling Carboxylated iron oxide nanoparticles 50 / 200 10 1 10 mM KOH, 0.01% Tween 10-20 1.5-2.5 Spermin / EDC coupling Thermo magnetic beads (1-4 μm) 31 / 156 18.75 1 0.1 M HCl, 0.01% Tween 10-15 5.5-5.9 Spermin / Reducing Amination Spherotech magnetic beads (5.7 μm) 25 / 100 20 3 10 mM KOH, 0.01% Tween 10-20 1.2-2.8 Spermin / Reducing Amination Spherotech magnetic beads (5.7 μm) 25 / 100 20 3 10 mM KOH, 0.01% Tween 20-40 2.5-2.8 Spermin / Reducing Amination Spherotech magnetic beads (5.7 μm) 25 / 50 4 1 10 mM KOH, 0.01% Tween 15-20 2.8-5.3

[0216] Table 7. PCR analysis of DNA extracted from 1 mL of urine sample. Performance of microparticles modified with spermine-aminated (EDC-coupled or reductive amide) pectin in extracting MTB DNA from 1 mL of urine is shown. Δ is calculated as the difference in extraction Ct obtained from 100% spikes of the control group.

[0217] Pectin Modifier / Procedure Base beads (2.5 mL of 2.5% solution) EDC / NHS (mg) Amidated pectin (mg) Time, Date purifying uL particles per mL (2.5%) Δ from 100% control group Spermin / Reducing Amination (NaBH4) Spherotech magnetic beads (5.7 μm) 25 / 100 20 3 20 mM KOH, 0.01% Tween 10-100 1.5-4 Spermin / Reducing Amination (NaBH4) Spherotech magnetic beads (5.7 μm) 25 / 100 20 3 10 mM KOH, 0.01% Tween 10-100 0.7-4.7 Spermin / Reducing Amination (NaBH4) Spherotech magnetic beads (5.7 μm) 25 / 100 20 3 10 mM KOH, 0.01% Tween 10-100 1-3.5 Spermin / Reductive Amination (STABH) Spherotech magnetic beads (5.7 μm) 25 / 100 20 3 20 mM KOH, 0.01% Tween 10-100 No Ct -3

[0218] Table 8. PCR analysis of DNA extracted from 1 mL of stool sample. Different microparticle modification formulations for extracting MTB DNA from 1 mL of stool sample and their performance. The presence of a reducing agent indicates polymer modification via the reductive amination pathway. The absence of a reducing agent (N / A) indicates polymer modification via EDC / NHS. Δ is calculated as the extraction Ct difference obtained from 100% spikes of the control group.

[0219] Pectin Modifier / Procedure Base beads (2.5 mL of 2.5% solution) EDC / NHS(mg) CP (mg) Time; Date purifying uL particles per mL (2.5%) Δ from 100% control group Spermin / EDC coupling Spherotech magnetic beads (2.8 μm) 50 / 200 20 1 10 mM KOH, 0.01% Tween 10.00 3.2 Spermin / EDC coupling Carboxylated iron oxide nanoparticles 50 / 200 10 1 10 mM KOH, 0.01% Tween 100-200 2-2.4 Spermin / EDC coupling Thermo magnetic beads (1-4 μm) 31 / 156 6.25 1 0.1 M HCl, 0.01% Tween 10 1.7 Spermin / EDC coupling Thermo magnetic beads (1-4 μm) 31 / 156 12.5 1 0.1 M HCl, 0.01% Tween 10 0.8 Spermin / EDC coupling Thermo magnetic beads (1-4 μm) 31 / 156 18.75 1 0.1 M HCl, 0.01% Tween 10 0.2 Spermin / EDC coupling Thermo magnetic beads (1-4 μm) 31 / 156 25 1 0.1 M HCl, 0.01% Tween 10 3.7 Spermin / Reducing Amination (NaBH4) Spherotech magnetic beads (5.7 μm) 25 / 100 20 3 10 mM KOH, 0.01% Tween 10-50 1.7-3.5 Spermin / Reducing Amination (NaBH4) Spherotech magnetic beads (5.7 μm) 25 / 100 20 3 10 mM KOH, 0.01% Tween 10 3.8 Spermin / Reducing Amination (NaBH4) Spherotech magnetic beads (5.1 μm) 25 / 100 5 3 10 mM KOH, 0.01% Tween 10 3.6

[0220] Although exemplary embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention.

Claims

Claim 1 A solid support comprising a plurality of modified pectin molecules covalently bonded to a solid support, wherein the modified pectin comprises a plurality of amino groups, and the modified pectin has the following chemical formula: It is an amidated pectin having one or more units represented by , or isomers thereof, salts, tautomers, or combinations thereof, wherein R in the chemical formula 2 and R 3 is H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C 20 A solid support independently selected from heteroalkyls. Claim 2 In claim 1, the amidated pectin has the following chemical formula: It comprises one or more units represented by , or isomers thereof, salts, tautomers, or combinations thereof, wherein n in the chemical formula is 0-3; R 1 is H or C1-C3 alkyl; X is, in each case, independently a C2-C4 alkylene or a C4-C6 heteroalkylene; Y is a C2-C3 alkylene or a C4-C6 heteroalkylene; and R 2 and R 3 A solid support that is independently H or C1-C3 alkyl. Claim 3 In claim 1, the amidated pectin is C4-C 20 A solid support that is pectin aided to polyamine. Claim 4 A solid support according to paragraph 3, wherein the polyamine is ethylenediamine, putrescine, cadaverine, spermine, or spermidine. Claim 5 In claim 1, the amidated pectin has the following structure: It comprises one or more units having , or isomers thereof, salts, tautomers, or combinations thereof, where n is 0, 1, 2, or 3; m is 2, 3, or 4; p is 2, 3, or 4; and R 1 , R 2 , and R 3 A solid support that is independently H or C1-C3 alkyl. Claim 6 In claim 1, the amidated pectin has the following structure: , , A solid support comprising one or more units having , or isomers thereof, salts, or tautomers. Claim 7 A solid support according to claim 1, wherein the amidated pectin is amide citrus pectin or amide apple pectin. Claim 8 A solid support according to claim 1, wherein the amidated pectin has a molecular weight of 4,000 Da to 500,000 Da, 5,000 Da to 300,000 Da, 100,000 Da to 300,000 Da, or 50,000 Da to 200,000 Da. Claim 9 A solid support according to claim 1, wherein the solid support comprises a material selected from polystyrene, glass, ceramic, polypropylene, polyethylene, silica, zirconia, titania, alumina, polycarbonate, latex, PMMA, zeolite, polyethersulfone, carboxymethylcellulose, and cellulose. Claim 10 A solid support according to claim 1, wherein the solid support is a magnetic bead, a glass bead, a polystyrene bead, a polystyrene filter, a polycarbonate filter, a polyethersulfone, or a glass filter. Claim 11 A method for isolating nucleic acid from a nucleic acid-containing sample, comprising the following steps: (a) contacting the sample with the solid support of claim 1 to bind the nucleic acid to the solid support; (b) optionally washing the nucleic acid bound to the solid support; and (c) eluting the nucleic acid from the solid support with an eluent. Claim 12 A method according to claim 11, wherein the eluent comprises ammonia or an alkali metal hydroxide. Claim 13 In claim 11, the method wherein the eluent has a pH of 9 or higher, 10 or higher, or 11 or higher. Claim 14 In claim 11, the method wherein the eluent has a pH of 9 to 12, 9.5 to 12, 10 to 12, or 9 to 11. Claim 15 A method according to claim 11, wherein the eluent comprises a polyanion. Claim 16 A method according to claim 15, wherein the multiple anions are carrageenan. Claim 17 In paragraph 15, the method wherein the multiple anions are carrier nucleic acids. Claim 18 A method according to claim 11, wherein the eluent comprises carrageenan and KOH. Claim 19 A method according to claim 11, wherein the method comprises contacting a sample with a dissolving solution, then contacting the sample with a solid support to release nucleic acid into a solution. Claim 20 A method according to claim 19, wherein the dissolving solution comprises a chaotropic agent. Claim 21 A method according to claim 20, wherein the disorder-causing agent is selected from guanidium thiocyanate, guanidium hydrochloride, alkali perchlorate, alkali iodide, urea, formamide, or a combination thereof. Claim 22 A method according to claim 20, wherein the disorder-causing agent is guanidium thiocyanate or guanidium hydrochloride. Claim 23 In paragraph 19, the method wherein the dissolving solution comprises a salt. Claim 24 In paragraph 23, the method wherein the salt is sodium chloride or calcium chloride. Claim 25 A method according to paragraph 19, wherein the dissolving solution does not contain a disorder-inducing agent. Claim 26 In paragraph 19, the method wherein the dissolution solution comprises a buffer. Claim 27 In paragraph 26, the method wherein the buffer is Tris. Claim 28 A method according to claim 19, wherein the dissolving solution comprises a surfactant. Claim 29 In paragraph 19, the method wherein the dissolving solution comprises an antifoaming agent. Claim 30 A method according to claim 11, wherein the contact step between the sample and the solid support is performed without the presence of a disorder-inducing reagent. Claim 31 A method according to claim 11, wherein the sample is selected from blood, plasma, serum, semen, tissue biopsy, urine, stool, saliva, smear preparation, bacterial culture, cell culture, viral culture, PCR reaction mixture, or in vitro nucleic acid modification reaction mixture. Claim 32 In paragraph 31, the method wherein the tissue biopsy is a paraffin-embedded tissue. Claim 33 A method according to claim 11, wherein the nucleic acid comprises genomic DNA. Claim 34 In paragraph 11, the method wherein the nucleic acid comprises whole RNA. Claim 35 A method according to claim 11, wherein the nucleic acid comprises microbial nucleic acid or viral nucleic acid. Claim 36 In paragraph 35, the method wherein the viral nucleic acid is HBV DNA. Claim 37 In paragraph 11, the above nucleic acid is a circulating nucleic acid. Claim 38 In paragraph 11, the above method is performed in an automated cartridge. Claim 39 A method for detecting nucleic acid in a sample, comprising the following steps: (a) contacting a nucleic acid-containing sample with a solid support according to any one of claims 1 to 10 to bind the nucleic acid to the solid support; (b) optionally washing the nucleic acid bound to the solid support; (c) eluting the nucleic acid; and (d) detecting the nucleic acid. Claim 40 A method according to claim 39, wherein the step of detecting the nucleic acid comprises amplifying the nucleic acid by a polymerase chain reaction (PCR). Claim 41 A method according to claim 40, wherein the polymerase chain reaction is nested PCR, isothermal PCR, or RT-PCR. Claim 42 A separation material for chromatography comprising a solid support comprising amidated pectin chemically bonded to a solid support, wherein the amidated pectin has the following chemical formula: Having a unit represented by , or an isomer thereof, a salt, a tautomer, or a combination thereof, and R in the chemical formula 2 and R 3 is H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C 20 A separated material that is independently selected from heteroalkyls. Claim 43 In paragraph 42, the separation material is a solid support that is silica, alumina, titania, zirconia, or a hybrid silica material. Claim 44 delete Claim 45 delete Claim 46 delete

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