Compositions and methods for selective extraction of oligonucleotides from complex matrices

Abstract

Compositions and methods for isolating and detecting nucleic acids in a biological sample are provided. The compositions and methods utilize a solid support and an affinity reagent to isolate and detect the nucleic acids.

Description

[0001] Cross-references to related applications

[0002] This application, filed as a PCT international application on March 21, 2025, claims the benefit of U.S. Provisional Application No. 63 / 568,277, filed on March 21, 2024, the disclosure of which is hereby incorporated herein by reference in its entirety. Priority to the above-disclosed application is claimed to the extent appropriate. Technical Field

[0003] This invention generally relates to the field of molecular biology. In some embodiments, this invention provides apparatus, kits, and methods related to the isolation and detection of nucleic acids. Background Technology

[0004] Some existing nucleic acid sample preparation methods involve using automated or semi-automated cartridges to handle and move the samples and reagents used. Typically, after lysis of the biological sample, nucleic acids bind to the filter material in the cartridge, are optionally washed, and eluted for detection. The buffers used in these cartridges often contain substances such as PEG, guanidine, or other eluting agents, which can be carried into the final detection reaction, affecting its efficacy even when present at low concentrations. It is desirable to avoid co-eluting such substances into the eluent, and specifically, to eliminate the need for their use in the cartridge in the first place.

[0005] Furthermore, enhanced control over the properties (e.g., density or binding capacity) of filter materials used for nucleic acid separation is desired. To date, control over the surface properties of materials has typically been achieved by altering the chemical conditions of the liquid-phase or gas-phase methods used to prepare such materials. However, these methods can be cumbersome and expensive to implement on a manufacturing scale, and the degree of customization allowed is limited by the increasing manufacturing complexity. Certain embodiments of the invention described herein address these and other needs. Other embodiments of the invention described herein provide apparatus and kits for isolating nucleic acids from samples. Still other embodiments of the invention provide for the detection of nucleic acids in samples. Summary of the Invention

[0006] This document describes compositions, methods, and apparatus for isolating and purifying nucleic acids from samples. These compositions, methods, and apparatus utilize optionally modified solid supports and affinity reagents comprising a solid support binding portion and a DNA binding portion. Additionally, methods for modifying and controlling the properties of solid support materials used for nucleic acid extraction and isolation are provided. These methods, by utilizing changes to the assay and solid support chemistry, allow for greater flexibility during assay development and offer significant cost savings in manufacturing, as a single solid support can be used for many different assays.

[0007] In some aspects, methods for isolating nucleic acids from a sample are disclosed, comprising: contacting a solid support with an affinity reagent and a sample, wherein the affinity reagent comprises a first portion that interacts with the solid support and a second portion that interacts with the nucleic acids in the sample; and concentrating the nucleic acids onto the solid support. The affinity reagent may also comprise a linker that interacts with a solvent and increases the solubility of the affinity reagent. The solid support may comprise functional surface groups that interact with the first portion of the affinity reagent. In some embodiments, the functional surface groups may interact with the first portion of the affinity reagent covalently (e.g., click / SPAAC reaction) or non-covalently (e.g., charged interactions such as ionic bonding or polar interactions, hydrophobic or van der Waals interactions, biotin-streptavidin interactions), or combinations thereof. In some instances, functional surface groups include hydrophobic binding groups, negatively charged binding groups (e.g., sulfonic acid, sulfate, phosphate, phosphonic acid, or carboxyl groups), positively charged binding groups (e.g., primary, secondary, tertiary, and quaternary ammonium, heterocyclic amines, such as pyridine, pyrimidine, pyridinium, piperazine), polar binding groups (e.g., chemical moieties containing polarized chemical bonds, such as C—O, C═O, C—N, C═N, C═N, N—H, O—H, C—F, C—Cl, C—Br, C—S, S—H, S—O, S═O, C—P, P—O, P═O, P—H, more specifically carboxyl, alcohol, thiol, amide, halide, amine, ester, ether, or thioester) or combinations thereof. In some instances, the functional surface groups include hydrophobic binding groups selected from alkyl groups, cycloalkyl groups, haloalkyl groups (e.g., fluoroalkyl groups), aryl groups, or combinations thereof, for interaction with affinity reagents. In more specific instances, the hydrophobic binding groups may include straight-chain alkyl groups, optionally wherein the straight-chain alkyl groups are selected from C4-C6. 20 Alkyl groups, C4-C 12 alkyl groups or C4-C 10 Alkyl groups. In one embodiment, the first portion of the functional surface group and the affinity reagent comprises an azide group (e.g., azide silane) and a cycloalkyne (e.g., cyclooctyne), wherein the azide group and the cycloalkyne react to form a nitrogen-containing heterocycle.

[0008] Functional surface groups can be bound to a solid support in any suitable manner, such as covalently (e.g., click / SPAAC reaction) or non-covalently (e.g., charged interactions such as ionic bonding or polar interactions, hydrophobic or van der Waals interactions, biotin-streptavidin interactions) or combinations thereof. In some embodiments, the functional surface groups can be bound to the solid support via triazole groups, triazine groups, imidazole groups, indole groups, silane groups, heteroazosilane tricyclic groups, siloxane groups, cyclic siloxane groups, silsesquioxane groups, silazane groups, or combinations thereof. In one embodiment, the functional surface groups can be bound to the solid support via silane groups.

[0009] Solid supports can be derived from any suitable material used to capture cells or nucleic acids during nucleic acid extraction and separation. In some embodiments, the solid support can be a porous material, such as a fibrous porous material. Porous materials can be derived from silica, glass (e.g., glass beads or glass filters), cellulose (e.g., cellulose filters), vinyl backbone polymers, mica, polycarbonate (e.g., polycarbonate filters), zeolite, titanium dioxide, magnetic materials (e.g., magnetic beads), polyethersulfone (e.g., polyethersulfone filters), polytetrafluoroethylene (e.g., polytetrafluoroethylene filters), polyvinylpyrrolidone (e.g., polyvinylpyrrolidone filters), or combinations thereof. In one embodiment, the solid support is a porous material derived from a glass fiber filter. The glass fiber filter can be modified with functional surface groups as described herein, optionally including hydrophobic silane compounds.

[0010] As described herein, the method includes contacting a solid support with an affinity reagent and a sample, wherein the affinity reagent comprises a first portion that interacts with the solid support and a second portion that interacts with nucleic acids in the sample. The first portion of the affinity reagent may interact with the solid support in any suitable manner, such as covalently (e.g., click / SPAAC reaction) or non-covalently (e.g., charged interactions such as ionic bonding or polar interactions, hydrophobic or van der Waals interactions, biotin-streptavidin interactions) or combinations thereof. In some embodiments, the first portion of the affinity reagent may interact with the solid support via a hydrophobic group, a negatively charged binding group, a positively charged binding group, a polar group, or a combination thereof. For example, the first portion of the affinity reagent may contain a hydrophobic group for interacting with the solid support, wherein the hydrophobic group is selected from alkyl groups, cycloalkyl groups, haloalkyl groups (e.g., fluoroalkyl), aryl groups, or combinations thereof. In some embodiments, the first portion of the affinity reagent contains a straight-chain alkyl group, optionally wherein the straight-chain alkyl group is selected from C4-C6. 20 Alkyl groups, C4-C 12 alkyl groups or C4-C10 Alkyl group. In some embodiments, the first portion of the affinity reagent comprises a branched alkyl group, optionally wherein the branched alkyl group is selected from C6-C6. 20 Alkyl groups, C8-C 12 alkyl groups or C8-C 10 Alkyl groups.

[0011] The second part of the affinity reagent can interact with nucleic acids in any suitable manner, such as covalently (e.g., click / SPAAC reaction) or non-covalently (e.g., charged interactions such as ionic bonding or polar interactions, hydrophobic or van der Waals interactions, biotin-streptavidin interactions) or combinations thereof. For example, the second part of the affinity reagent may comprise an amine, a heterocycle (such as a triazole, imidazole, or indole), an intercalator, a DNA groove binder, a peptide, an amino acid, a protein, or a combination thereof for interacting with nucleic acids. In one embodiment, the second part of the affinity reagent comprises an amine, optionally selected from alkylamines, cycloalkylamines, branched amines, alkoxyamines, polyamine moieties, arylamines, or combinations thereof for interacting with nucleic acids. In another embodiment, the second part of the affinity reagent comprises an alkylamine, an imidazole, a bisbenzylimine small groove binder, a polycyclic intercalator, or a combination thereof for interacting with nucleic acids. In some instances, the second part of the affinity reagent comprises spermine, methylamine, ethylamine, propylamine, ethylenediamine, diethylenetriamine, 1,3-dimethyldipropylenediamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium chloride, tris(2-aminoethyl)amine, or combinations thereof, for interaction with nucleic acids.

[0012] The affinity reagent may also include a linker that interacts with the solvent and increases the solubility of the affinity reagent. In some embodiments, the linker may include a hydrophilic group selected from polyethylene glycol linkers, ester-based linkers (such as branched ester-based linkers), amide-based linkers (such as branched amide-based linkers, including N,N-dihexyl-3-imidazol-2-oxoamine linkers), amine-based linkers (such as 3-dibutylaminopropylamine linkers, N,N-dihexyl-3-aminopropylamine linkers), organophosphorus-based linkers (such as organophosphorus-based linkers, organophosphorus oxide-based linkers, organophosphonates-based linkers, organophosphoramide-based linkers, organophosphate-based linkers, organophosphonamide-based linkers, or organophosphonate-based linkers), glucuronic acid-based linkers, disulfide linkers, cathepsin B linkers, or combinations thereof.

[0013] Affinity reagents may also contain photodegradable groups for releasing nucleic acids concentrated on a solid carrier.

[0014] As described in this method, contacting the solid support with the sample and affinity reagent can be performed under conditions that effectively allow interactions between a) the nucleic acid and the affinity reagent, and b) the solid support and the affinity reagent. These conditions may include a pH less than 7. The method may also include contacting the sample with a lysis buffer before or simultaneously with contacting the solid support with the sample. The lysis buffer may contain one or more of a dissociation agent, salt, buffer, surfactant, defoamer, and binding agent.

[0015] The method may also include releasing nucleic acids concentrated on a solid carrier.

[0016] This method includes releasing nucleic acids concentrated on a solid support, which can be performed in the presence of an eluent, heat, sonication, photochemical lysis conditions, or a combination thereof. In some embodiments, the release of nucleic acids concentrated on the solid support can be performed in the presence of an eluent, optionally wherein the eluent has a pH greater than about 7, greater than about 8, greater than about 9, and / or a salt concentration higher than that of the sample. In some embodiments, the release of nucleic acids concentrated on the solid support can be performed in the presence of heat, optionally wherein the nucleic acids, affinity reagent, and solid support are heated to temperatures of 35°C or higher, 45°C or higher, 55°C or higher, 65°C or higher, 75°C or higher, 85°C or higher, 95°C or higher, or up to 100°C or higher. In some embodiments, the release of nucleic acids concentrated on the solid support can be performed in the presence of sonication. In some embodiments, the release of nucleic acids concentrated on the solid support can be performed in the presence of photochemical lysis conditions of an affinity reagent, optionally wherein the photochemical lysis conditions include exposing the affinity reagent to UV light.

[0017] The method described herein may also include washing the solid support with a washing solution. The washing solution may have a pH less than 7.

[0018] Methods for detecting nucleic acids in biological samples are also described. Methods for detecting nucleic acids in biological samples may include (a) isolating nucleic acids from the sample using methods disclosed herein; (b) releasing nucleic acids from a solid carrier using an eluent; and (c) detecting the nucleic acids.

[0019] The disclosed methods can be performed in a box, or optionally in an automated box. This paper describes sample boxes and automated boxes.

[0020] A sample cartridge for isolating and detecting nucleic acids from biological samples may include: a cartridge having multiple chambers, including a sample chamber configured to receive a biological sample; a reaction vessel fluidly connected to the multiple chambers and configured to amplify nucleic acids and detect multiple amplification products; a filter disposed in a fluid path between the multiple chambers and the reaction vessel, wherein the filter includes a solid carrier having a surface capable of binding an affinity reagent disposed in one of the multiple chambers, the affinity reagent comprising a first portion interacting with the solid carrier and a second portion interacting with nucleic acids from the biological sample; and primers and / or probes disposed in one or more of the multiple chambers or the reaction vessel for detecting nucleic acids. The primers and / or probes may be immobilized on a surface (e.g., a biosensor surface) in one or more of the multiple chambers or the reaction vessel for detecting nucleic acids. The affinity reagent may also include a connector that interacts with a solvent and increases the solubility of the affinity reagent.

[0021] The sample cartridge may include multiple chambers, including a lysis chamber in fluid communication with the sample chamber, wherein the lysis chamber contains lysis reagents for releasing nucleic acids. In some instances, the sample chamber and the lysis chamber are identical. The multiple chambers may also contain binding reagents, filtration buffers, washing reagents, elution buffers, or combinations thereof.

[0022] As described in the sample cassette, the filter comprises a solid support having a surface capable of binding an affinity reagent. The solid support, as described herein, includes functional surface groups that interact with a first portion of the affinity reagent, optionally wherein the functional surface groups interact covalently (e.g., click / SPAAC reaction) or non-covalently (e.g., charged interactions such as ionic bonding or polar interactions, hydrophobic or van der Waals interactions, biotin-streptavidin interactions), or combinations thereof, with the first portion of the affinity reagent. The solid support may be a porous material, such as a glass fiber filter.

[0023] Based on solid supports, affinity reagents can be applied at 10 nmol / cm³. 2 Or larger, 20 nmol / cm 2 Or larger, 35 nmol / cm 2 Or larger or 30-100 nmol / cm 2 The surface density exists on the solid support within the cartridge. In some embodiments, based on the solid support, the affinity reagent can be expressed at 3,000 nmol / cm³. 2 Or smaller, 2,500 nmol / cm 2 Or smaller, 2,000 nmol / cm 2 Or smaller, 1,000 nmol / cm2 or smaller, 500 nmol / cm 2 or smaller, 400 nmol / cm 2 or smaller, 300 nmol / cm 2 or smaller, 200 nmol / cm 2 Or smaller, or 100 nmol / cm 2 Or even lower surface density. The solid support, together with the affinity reagent, has a surface density of at least 10 µg / cm³. 2 20 µg / cm 2 or larger, 35 µg / cm 2 Or larger or 30-100 µg / cm 2 DNA binding ability.

[0024] The solid support within the sample cartridge may have a pore size of 0.2 µm to 3 µm, 0.2 µm to 2 µm, 0.5 µm to 1.0 µm, or 0.6 µm to 0.8 µm. The solid support (e.g., a glass fiber filter) may also contain beads to facilitate mechanical lysis, wherein the beads are selected from glass beads, silica beads, or combinations thereof. The solid support (e.g., a glass fiber filter) may have a pore size of 35 g / m³. 2 Up to 100 g / m 2 Preferably 50 g / m 2 Up to 85 g / m 2 or 70 g / m 2 Up to 85 g / m 2 The basis weight. The solid carrier (e.g., a glass fiber filter) may have a fiber diameter of 1 µm to 100 µm, preferably 1 µm to 50 µm or 1 µm to 25 µm. The solid carrier (e.g., a glass fiber filter) may have a thickness of 250 µm to 2,000 µm, 300 µm to 1,500 µm, 300 µm to 1,000 µm, 300 µm to 750 µm or 350 µm to 500 µm.

[0025] The sample cartridge, together with the reagent, allows flow rates up to approximately 100 µL per second, such as approximately 10 µL to approximately 100 µL. In some embodiments, the sample cartridge, together with the reagent, allows pressures below 100 psi, below 80 psi, or below 60 psi. As described herein, the sample cartridge can be an automated cartridge.

[0026] A method is provided for detecting nucleic acids in biological samples obtained from a subject within a sample cassette disclosed herein. The method may include a) contacting nucleic acids from the biological sample with a set of primers and optional probes as provided in the sample cassette disclosed herein; b) subjecting the nucleic acids, primer pairs, and optional probes to amplification conditions; c) optionally detecting the presence of amplification products via real-time PCR, melting curve analysis, or a combination thereof; and d) detecting the presence of nucleic acids in the biological sample based on the detection of the amplification products. In some embodiments, the method does not include the use of a dissociation agent, lysis buffer, or binding agent.

[0027] Certain aspects of this disclosure generally relate to compositions, apparatus, and methods for identifying viruses, such as coronaviruses. For example, some aspects relate to dispensing biological samples containing viruses and free nucleic acids using the apparatus disclosed herein for isolating and purifying nucleic acids, and identifying the virus within the apparatus. In some cases, at least 95% of the free nucleic acid is dispensed onto a solid carrier of the apparatus.

[0028] Biological samples may be selected from blood, blood cultures, plasma, serum, semen, cerebrospinal fluid, tissue, tears, urine, feces, saliva, smear preparations, respiratory samples, nasopharyngeal swab samples, anterior nasal samples, middle turbinate nasal samples, oropharyngeal samples, vaginal swabs, vaginal mucus samples, vaginal tissue samples, vaginal cell samples, bacterial cultures, mammalian cell cultures, viral cultures, human cells, bacteria, extracellular fluid, pancreatic juice, cell lysates, PCR reaction mixtures, or in vitro nucleic acid modification reaction mixtures. Preferably, the biological sample is blood, plasma, respiratory samples, or vaginal swabs. In some instances, the biological sample contains nucleic acids selected from: genomic DNA, total RNA, short DNA, small DNA, tumor-derived nucleic acids, methylated DNA, microbial nucleic acids, bacterial nucleic acids, viral nucleic acids, cell-free nucleic acids, or combinations thereof.

[0029] The methods and sample kits disclosed herein can be used to amplify nucleic acids via nested PCR, gradient PCR, isothermal PCR, qPCR, or RT-PCR. In one embodiment, amplification is performed via real-time PCR multiplex assay. Attached Figure Description

[0030] Figure 1 This is a schematic diagram showing the extraction and separation of nucleic acids using a modified solid carrier in the presence of an affinity reagent.

[0031] Figure 2 This is a schematic diagram illustrating nucleic acid extraction and separation via a modified solid carrier in the presence of an affinity reagent containing cleavable groups.

[0032] Figure 3This is a schematic diagram showing nucleic acid extraction and separation via a modified solid carrier in the presence of an affinity reagent, as well as nucleic acid separation via heating.

[0033] Figure 4 This is a schematic diagram showing the modified solid support. The solid support is modified with hydrophobic surface groups, which are covalently bonded to the solid support via triazole linkers.

[0034] Figure 5 An overview view (left) of the sample box with a reaction vessel and an exploded view (right) of the sample box valve assembly are shown.

[0035] Figures 6A to 6C Various valve assemblies and components are shown.

[0036] Figures 7A to 7E Valve assemblies and components according to some embodiments are shown.

[0037] Figures 8A to 8E Box plots show the levels of nucleic acid and free RNA from the CoV2 virus, measured using glass fiber filters (GFF) modified with an amino affinity group (GFF 3) and unmodified glass fiber filters (Reg), expressed as circulation threshold (Ct) values.

[0038] Figure 9A and Figure 9B The graph shows DNA binding and elution at different buffers and pH levels, as measured by fluorescence assay.

[0039] Figure 10A and Figure 10B The effects of salt on DNA binding and elution, as measured by fluorescence assay, are demonstrated.

[0040] Figure 11A and Figure 11B The effects of washing with KCl, NaCl, LiCl, NaPi, and GuHCl on DNA binding and elution are demonstrated.

[0041] Figure 12 DNA binding and elution were compared using affinity reagents 3DP and DAP. Detailed Implementation

[0042] definition

[0043] To facilitate understanding of this disclosure, numerous terms and phrases are defined below:

[0044] As used in the specification and appended claims, the singular forms “a / an” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, reference to “a compound” includes a mixture of compounds, and reference to “an amine group” includes a mixture of two or more such amine groups, etc.

[0045] A range may be expressed herein as from “about” a specific value and / or to “about” another specific value. When expressing such a range, another embodiment includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the specific value forms another embodiment. It should also be understood that each endpoint of a range is meaningful both relative to and independent of another endpoint.

[0046] Throughout this specification and the following claims, numerous terms will be used, which should be defined as having the following meanings:

[0047] The term "alkyl" includes both "unsubstituted alkyl" and "substituted alkyl," the latter referring to an alkyl moiety having one or more substituents replacing hydrogen on one or more carbons of a hydrocarbon skeleton. Such substituents include, but are not limited to, halogens, hydroxyl groups, carbonyl groups (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (such as thioesters, thioacetates, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate groups, phosphonates, hypophosphonates, amino groups, amide groups, amido groups, imino groups, cyano groups, nitro groups, azide groups, mercapto groups, alkylthio groups, sulfate groups, sulfonate groups, aminosulfonyl groups, sulfonamide groups, sulfonyl groups, heterocyclic groups, aralkyl groups, or aromatic or heteroaromatic moieties.

[0048] Unless the number of carbon atoms is otherwise specified, “alkyl” as used herein refers to an alkyl group as defined above, but with a skeletal structure having one to twenty carbon atoms, more preferably one to ten carbon atoms. Similarly, “alkenyl” and “alkynyl” have similar chain lengths.

[0049] Alkyl groups may also contain one or more heteroatoms within the carbon skeleton. Examples include oxygen, nitrogen, sulfur, and combinations thereof. In some embodiments, the alkyl group contains between one and four heteroatoms.

[0050] As used herein, the term "heteroalkyl" refers to a straight-chain, branched, or cyclic carbonaceous group or combination thereof containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein phosphorus and sulfur atoms may optionally be oxidized, and nitrogen heteroatoms may optionally be quaternized. Heteroalkyl groups may be substituted as defined above for alkyl groups.

[0051] As used herein, "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups containing one or more double or triple bonds, the length of which (e.g., C2-C5) is... 30 ) and possible substitutions similar to the alkyl groups mentioned above.

[0052] As used herein, “aryl” refers to 5-, 6-, and 7-membered aromatic rings. The ring can be a carbocyclic ring, heterocyclic ring, fused carbocyclic ring, fused heterocyclic ring, two-carbocyclic ring, or a di-heterocyclic ring system, optionally substituted as described above for alkyl groups. More broadly, as used herein, “Ar” includes 5-, 6-, and 7-membered monocyclic aromatic groups, which may include zero to four heteroatoms. Examples include, but are not limited to, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Those aryl groups having heteroatoms in their ring structure may also be referred to as “heteroaryl,” “aryl heterocyclic,” or “heteroaromatic.” The aromatic ring may be substituted at one or more ring positions with substituents as described above, such as halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, mercapto, imino, amide, phosphonate, phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclic, aromatic or heteroaromatic moiety, --CF3, and --CN. The term "Ar" also includes polycyclic systems having two or more rings, wherein two or more carbons are shared by two adjacent rings (the rings are "fused rings"), wherein at least one ring is aromatic; for example, other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclic, or both rings may be aromatic.

[0053] As used herein, "alkylaryl" or "aryl-alkyl" refers to an alkyl group that has been replaced by an aryl group (e.g., an aromatic or heteroaromatic group).

[0054] As used herein, "heterocyclic" or "heterocyclic" refers to a cyclic group linked by a monocyclic or bicyclic ring of carbon or nitrogen atoms, the monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably 5-6 ring atoms, containing carbon and one to four heteroatoms, each heteroatom selected from non-peroxide oxygen, sulfur, and N(Y), wherein Y is absent or H, O, (C 1-4The alkyl, phenyl, or benzyl group may optionally contain one or more double or triple bonds and may optionally be substituted with one or more substituents. The term "heterocycle" also encompasses substituted and unsubstituted heteroaryl rings. Examples of heterocycles include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenel, benzooxazolyl, benzooxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzoisooxazolyl, benzoisothiazolyl, benzimidazolyl, carbazolyl, 4aH-carbazolyl, carbazolyl, chromanyl, chromenyl, terpineyl, decahydroquinolinyl, 2H,6H-1,5,2-diathiazinyl, dihydrofurano[2,3-b]tetrahydrofuran, and furanyl. Furazanyl, imidazolinyl, imidazolinyl, imidazolyl, 1H-indazole, indolenyl, dihydroindolyl, indoleyl, 3H-indolyl, indoleyl, isobenzofuranyl, isochoryl, isoindazole, isodihydroindolyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthidyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl Azolyl, 1,3,4-oxadiazolyl, oxazolylalkyl, oxazolyl, hydroxyindolyl, pyrimidinyl, phenanthridine, phenanthrololinyl, phenazinyl, phenothiazinyl, phenothiazinyl, phenothiazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidinoneyl, 4-piperidinoneyl, piperinyl, pteridinyl, purine, pyranyl, pyrazinyl, pyrazolylalkyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridinyl, pyrrolylalkyl, pyrrololinyl 2H-pyrrole, pyrrole, quinazolinyl, quinolinyl, 4H-quinazinyl, quinoxalinyl, quininecycloyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl and xanthonyl.

[0055] As used herein, “heteroaryl” refers to a monocyclic aromatic ring containing five or six ring atoms, each containing carbon and one, two, three, or four heteroatoms selected from non-peroxide oxygen, sulfur, and N(Y), wherein Y is absent or is H, O, (C1-C8)alkyl, phenyl, or benzyl. Non-limiting examples of heteroaryl include furanyl, imidazolyl, triazolyl, triazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrroleyl, pyrazinyl, tetrazolyl, pyridinyl (or its N-oxide), thiophene, pyrimidinyl (or its N-oxide), indolyl, isoquinolinyl (or its N-oxide), quinolinyl (or its N-oxide), etc. The term “heteroaryl” may include groups of about eight to ten ring atoms of ortho-fused bicyclic heterocycles derived therefrom, particularly benzo[a] derivatives or groups derived by fusion with propylene, trimethylene, or tetramethylene bimethyl groups. Examples of heteroaryl groups include, but are not limited to, furanyl, imidazolyl, triazolyl, triazinyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrroleyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide), thiophenyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolinyl (or its N-oxide), quinolinyl (or its N-oxide), etc.

[0056] As used herein, the term "alkoxyl" or "alkoxy" refers to an alkyl group as defined above, having an oxygen group attached to it. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, etc. An "ether" is two hydrocarbons covalently linked by oxygen. Therefore, the substituents of the alkyl group that make it an ether are or similar to alkoxy groups, such as those represented by -O-alkyl, -O-alkenyl, and -O-ynyl. Aryloxy groups can be represented by –O-aryl or O-heteroaryl, where aryl and heteroaryl are defined below. Alkoxy and aryloxy groups may be substituted as described above for alkyl groups.

[0057] The terms "amine" and "amino" are recognized in the art and refer to unsubstituted and substituted amines, for example, portions that can be represented by the general formula: -NR9R 10 Or NR9R 10 R' 10 Among them, R9, R 10 and R' 10 Each can independently represent hydrogen, alkyl, alkenyl, or -(CH2). m -R'8, or R9 and R 10 Together with the N atoms they are attached to, they form a heterocycle having 4 to 8 atoms in the ring structure; R'8 represents aryl, cycloalkyl, cycloalkenyl, heterocyclic, or polycyclic; and m is zero or an integer in the range of 1 to 8. In some embodiments, R9 or R 10 Only one of them can be a carbonyl group, for example, R9, R10 It does not form an imide with nitrogen. In some embodiments, the term "amine" does not cover amides, for example, where R9 and R 10 One of them represents a carbonyl group. In some embodiments, R9 and R 10 (and optional R') 10 Each of these groups independently represents hydrogen, alkyl or cycloalkyl, alkenyl or cycloalkenyl, or ynyl. Therefore, the term "alkylamine" as used herein refers to an amine group as defined above, having a substituted (as described above for alkyl) or unsubstituted alkyl group attached to it, i.e., R9 and R1. 10 At least one of them is an alkyl group.

[0058] The term "amide" or "amide" is generally understood in the art to refer to an amino-substituted carbonyl group, and includes groups that can be derived from the general formula -CONR9R. 10 The part that is represented, where R9 and R 10 As defined above.

[0059] As used in this article, "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0060] As used in this article, "hydroxyl group" refers to -OH.

[0061] The term "carbonyl" is recognized in the art and includes, for example, groups that can be derived from the general formula -CO-XR. 11 Or –X-CO- R' 11 The part represents X, where X is a bond or represents oxygen or sulfur, and R... 11 R' indicates hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, or ynyl. 11 Indicates hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, or alkynyl. When X is oxygen and R... 11 or R' 11 When X is not hydrogen, the formula represents "ester". When X is oxygen and R is oxygen, the formula represents "ester". 11 As defined above, this part is referred to herein as a carboxyl group, and especially when R 11 When X is hydrogen, the formula represents "carboxylic acid". When X is oxygen and R' 11 When X is hydrogen, this formula represents a "formate ester". Typically, when the oxygen atom in the above formula is replaced by sulfur, this formula represents a "thiocarbonyl" group. When X is sulfur and R... 11 When R'11 is not hydrogen, the formula represents "thioester". When X is sulfur and R... 11 When X is hydrogen, the formula represents "thiocarboxylic acid". When X is sulfur and R' 11 When X is hydrogen, the formula represents "thiocarbamate". On the other hand, when X is a bond and R... 11 When X is not hydrogen, the above formula represents a "ketone" group. When X is a bond, and R... 11 When it is hydrogen, the above formula represents an "aldehyde" group.

[0062] As used herein, the term "substituted" refers to all permissible substituents in the compounds described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic group containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally including one or more heteroatoms, such as oxygen, sulfur, or nitrogen groups, in a straight-chain, branched, or cyclic form. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amide, substituted amide, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C 20 Cyclic groups, substituted C3-C 20 Cyclic groups, heterocyclic groups, substituted heterocyclic groups, amino acids, peptides, and polypeptide groups.

[0063] It should be understood that “substitution” or “substituted” includes the implicit condition that the substitution meets the permissible valence of the substituted atom and substituent, and that the substitution produces a stable compound, i.e. a compound that does not spontaneously undergo transformation (such as through rearrangement, cyclization, elimination, etc.).

[0064] As used herein, the term "hydrolyzable" refers to a group or portion capable of undergoing hydrolysis or solvent decomposition. For example, a hydrolyzable group can be hydrolyzed (i.e., converted into a hydrogen group) by exposure to water or a protic solvent at or near ambient temperature or high temperature and at or near atmospheric pressure or high pressure. In some cases, a hydrolyzable group can be hydrolyzed by exposure to acidic or basic water or acidic or basic protic solvents. Typical hydrolyzable groups include, but are not limited to, alkoxy, aryloxy, arylalkoxy, acyloxy, or halogen groups. As used herein, the term is generally used to refer to one or more groups bonded to a silicon atom in a silyl group.

[0065] As used herein, the terms “detect,” “detecting,” or “detection” can describe a specific observation of a composition of general behavior or detectable markers that is found or identified.

[0066] The term "nucleic acid" refers to a polymer of nucleotides and, unless otherwise limited, includes analogues of naturally occurring nucleotides that can function in a similar manner to naturally occurring nucleotides (e.g., hybridization). The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is the DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or viral RNA, or by amplification; synthetic or amplified DNA molecules; mRNA; and non-coding RNA. Nucleic acids can be of any origin, including mammals (e.g., humans), bacteria, viruses, or synthetic sources. The term nucleic acid encompasses double-stranded or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acid complexes, the nucleic acid strands do not need to be co-extended (i.e., a double-stranded nucleic acid does not need to be double-stranded along its entire length).

[0067] The term nucleic acid also encompasses any modifications to it, such as those made by methylation and / or by capping. Nucleic acid modifications can include the addition of chemical groups that incorporate additional charge, polarization, hydrogen bonding, electrostatic interactions, and functionality into a single nucleic acid base or the nucleic acid as a whole. Such modifications can include base modifications such as 2' sugar modification, 5' pyrimidine modification, 8' purine modification, modification at the amine site outside the cytosine ring, substitution of 5-bromouracil, sugar-phosphate backbone modification, unusual base pairing combinations (such as isobasic cytosine and isoguanidine), etc. More specifically, in some embodiments, nucleic acids may include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid as an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing a non-nucleotide backbone, such as polyamides (e.g., peptide nucleic acids (PNA)) and polymorpholino polymers (see, for example, Summerton and Weller (1997) “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Dev. 7:1817-195; Okamoto et al. (2002) “Development of electrochemically gene-analyzing method using DNA-modified electrodes,” Nucleic Acids Res. Supplement 2:171-172), and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, such as those found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, the disclosures of which, in their entirety, are incorporated herein by reference. Nucleic acids may be derived from entirely chemically synthesized processes (such as solid-phase mediated chemical synthesis), biological sources (such as isolation from any species that produces nucleic acids), or processes involving manipulation of nucleic acids using molecular biology tools (such as DNA replication, PCR amplification, reverse transcription), or combinations of these processes.

[0068] As used herein, the terms “oligonucleotide”, “polynucleotide”, etc., refer to molecules containing nucleic acids, including but not limited to DNA or RNA. The term “oligonucleotide” is used to refer to relatively short nucleic acids, typically shorter than 500 nucleotides, particularly shorter than 200 nucleotides, even more particularly shorter than 100 nucleotides, and most particularly shorter than 50 nucleotides. Typically, oligonucleotides are single-stranded DNA molecules. This term encompasses sequences of any known base analogues, including but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6 -Methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl-Q nucleoside, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, methyl uracil-5-hydroxyacetate, uracil-5-hydroxyacetic acid, oxybutoxynucleoside, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl N-uracil-5-hydroxyacetate, uracil-5-hydroxyacetic acid, pseudouracil, Q nucleoside, 2-thiocytosine, and 2,6-diaminopurine.

[0069] In some implementations, the length of the oligonucleotide can range from about 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to 75, or 12 to 50 nucleotides. An oligonucleotide can be referred to by its length; for example, a 24-residue oligonucleotide can be called a “24-mer.”

[0070] The term "nucleic acid amplification" encompasses any means of replicating at least a portion of at least one target nucleic acid in a template-dependent manner, including but not limited to a wide range of techniques for linearly or exponentially amplifying nucleic acid sequences. Exemplary means of performing the amplification step include polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLPA), post-ligation Q-replicaase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid chain-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), recombinase polymerase amplification, etc., including multiple versions and combinations thereof, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction—CCR), digital amplification, etc. Descriptions of these techniques can be found in the following literature and other sources: Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, ed., Cold Spring Harbor Press (1995); U.S. Patent Nos. 5,830,711, 6,027,889 and 5,686,243, each of which is incorporated herein by reference in its entirety.

[0071] As used herein, “sample” or “biological sample” includes various samples containing nucleic acids (e.g., DNA and / or RNA) from tissue, cell, or fluid samples isolated from a subject, including but not limited to, whole blood, white membrane, plasma, serum, immune cells (e.g., monocytes or macrophages), blood cultures, swabs, saliva, and sputum. In some embodiments, the sample contains buffers, such as anticoagulants and / or preservatives. In some embodiments, whole blood is mixed with heparin in a lithium heparin collection tube. The sample can be derived from any bodily fluid, tissue, or cell containing expressed biomarkers. Biological samples can be obtained from a subject using conventional techniques. For example, blood can be obtained by venipuncture or finger-puncture of a capillary, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art. In some aspects, the blood sample is placed in a tube specifically designed for assay. In some aspects, the sample is a nasal swab. For example, the swab is freshly obtained or stored in a transport medium (such as UTM or VTM).

[0072] "Reagent" refers to any reagent used in a reaction other than the analyte (e.g., the nucleic acid being analyzed). Illustrated reagents used in nucleic acid amplification reactions include, but are not limited to, affinity reagents, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, dNTPs, etc. Reagents used in enzyme reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

[0073] As used in this article, the term “detection” refers to “determining” the “presence” of an item (such as a nucleic acid sequence, for example, a nucleic acid sequence indicating the presence of a virus).

[0074] As used herein, “Clinical Laboratory Improvement Amendments (CLIA)” refers to the 1988 Clinical Laboratory Improvement Amendments (CLIA) regulations in effect as of the original filing date of this application. CLIA regulations comprise federal standards applicable to all facilities or sites in the United States that test human specimens for health assessment or for the diagnosis, prevention, or treatment of disease. “CLIA-compliant” testing is testing that complies with these regulations. “CLIA-exempt” testing includes testing that does not fully comply with all of these regulations. For example, CLIA-exempt testing includes testing systems approved by the U.S. Food and Drug Administration for home use, as well as those tests for which exemptions have been approved under CLIA standards.

[0075] solid carrier

[0076] This article provides solid carrier materials for isolating and purifying nucleic acids from samples containing nucleic acids. Figure 1 Exemplary embodiments of nucleic acid extraction and separation via a modified solid support 10 in the presence of affinity reagent 20 are shown. In some embodiments, the solid support 10 may be modified with functional surface groups 12 that bind to or interact with the affinity reagent described herein, thereby making the surface selective for interaction with nucleic acids. In some instances, the functional surface groups are chemically bonded to the surface of the solid support. Figure 1 A solid support 10 modified with a hydrophobic surface group 12 is shown, which can interact with at least a portion of an affinity reagent. For example, the functional surface group can be covalently bonded (e.g., via a triazole linker, such as...). Figure 4 (As shown) onto a solid support. In other examples, functional surface groups are electrostatically bonded to the solid support or interact with the solid support via polar, van der Waals, and / or hydrophobic interactions. In other embodiments, the solid support is bound to or interacts with an affinity reagent to make the surface pair selective for interaction with nucleic acids. In the disclosure provided herein, the solid support typically includes surface functional groups that can interact with and / or react with affinity reagents.

[0077] The solid support can be any substrate suitable for interaction with biomolecules, and may include, but is not limited to, paramagnetic particles, gels, fibers, controlled-pore glass, magnetic beads, microspheres, nanospheres, capillaries, filter membranes, columns, cloths, wipes, paper, planar supports, porous plates, porous membranes, porous monoliths, wafers, combs, or any combination thereof. Preferably, the solid support is a porous material and may include any suitable material, including but not limited to glass, silica, titanium dioxide, alumina, iron oxide, vinyl backbone polymers, polypropylene, polyethylene, polystyrene, ceramics, cellulose, nitrocellulose, magnetic silica particles (such as MagneSil™ particles obtained from Promega Corporation), and divinylbenzene. In some embodiments, the solid support includes materials selected from: polystyrene, glass, ceramics, polypropylene, polyethylene, silica, mica, titanium dioxide, polycarbonate, latex, PMMA, zeolite, polyethersulfone, carboxymethyl cellulose, cellulose, and combinations thereof. Examples of solid carriers include magnetic beads, glass beads, polystyrene beads, cellulose filters, polystyrene filters, polycarbonate filters, polyethersulfone filters, polytetrafluoroethylene filters, polyvinylpyrrolidone filters, or glass fiber filters. Solid carriers may also contain a polymer binder for binding particles or fibers within the solid carrier. Exemplary polymer binders include acrylic polymers.

[0078] In some embodiments, the solid carrier is a fibrous material, preferably a glass fiber filter (GFF). Fiber filters, such as glass fiber filters, offer several advantages over other porous carriers, such as glass beads. Porous glass fiber filters have a much larger surface area than a flat glass surface, but smaller than a porous glass bead. Unlike glass beads, the thin, paper-like sheets of glass fiber filters are easily processed in aqueous or organic solvents. The mechanical stability of glass fiber filters allows for the use of tapes and sheets in high-throughput manufacturing. Unlike beads, glass fiber filters simplify flow-through filtration without requiring a glass floc. This characteristic of GFFs allows for the construction of multi-layer devices, where several modified glass filters can be stacked on top of each other within a cylindrical flow-through housing. If greater DNA binding capacity is required, the effective thickness of the filter can be adjusted by stacking multiple discs.

[0079] Solid carriers preferably have a relatively high surface area to achieve high nucleic acid binding capacity. When the solid carrier is a fibrous material, the fibrous material can be characterized by fiber diameter, pore diameter, basis weight, thickness, and / or specific surface area. Fibers in the fibrous material may have the following average diameters: 1 μm or greater, 1.5 μm or greater, 2 μm or greater, 2.5 μm or greater, 3 μm or greater, 3.5 μm or greater, 4 μm or greater, 4.5 μm or greater, 5 μm or greater, 5.5 μm or greater, 6 μm or greater, 6.5 μm or greater, 7 μm or greater, 7.5 μm or greater, 8 μm or greater, 9 μm or greater, 10 μm or greater, 12 μm or greater, 15 μm or greater, 16 μm or greater, 18 μm or greater, 19 μm or greater, or 20 μm or greater, or any value between these values ​​(e.g., 11.4 μm). In some embodiments, the fibers in the fibrous material may have the following average diameters: 25 micrometers or less, 24 micrometers or less, 22 micrometers or less, 20 micrometers or less, 19 micrometers or less, 18 micrometers or less, 16 micrometers or less, 15 micrometers or less, 14 micrometers or less, 13 micrometers or less, 12 micrometers or less, 10 micrometers or less, 9 micrometers or less, 8 micrometers or less, 7.5 micrometers or less, 7 micrometers or less, 6 micrometers or less, 5.5 micrometers or less, or 5 micrometers or less, or any value between these (e.g., 17.1 micrometers). In some embodiments, the fibers in the fibrous material may have the following average diameters: 1 micrometer to 20 micrometers, 2 micrometers to 20 micrometers, 2 micrometers to 18 micrometers, 2.5 micrometers to 15 micrometers, 2.5 micrometers to 12 micrometers, 2.5 micrometers to 10 micrometers, 3 micrometers to 20 micrometers, 3 micrometers to 18 micrometers, 3 micrometers to 15 micrometers, 3 micrometers to 12 micrometers, 4 micrometers to 20 micrometers, 4 micrometers to 15 micrometers, or 5 micrometers to 20 micrometers.

[0080] The fibrous material may have the following effective pore sizes (or average pore diameters): 0.20 micrometers or larger, 0.30 micrometers or larger, 0.40 micrometers or larger, 0.50 micrometers or larger, 0.55 micrometers or larger, 0.60 micrometers or larger, 0.65 micrometers or larger, 0.70 micrometers or larger, 0.75 micrometers or larger, 0.80 micrometers or larger, 0.85 micrometers or larger, 0.90 micrometers or larger, 0.95 micrometers or larger, 1.0 micrometer or larger, or 1.1 micrometers or larger, or any value in between (e.g., 0.511 micrometers). In some embodiments, the fibrous material may have the following average pore sizes: 3.0 micrometers or less, 2.0 micrometers or less, 1.9 micrometers or less, 1.8 micrometers or less, 1.7 micrometers or less, 1.6 micrometers or less, 1.5 micrometers or less, 1.4 micrometers or less, 1.3 micrometers or less, 1.2 micrometers or less, 1.1 micrometers or less, 1.05 micrometers or less, 1.0 micrometers or less, 0.95 micrometers or less, 0.90 micrometers or less, 0.85 micrometers or less, 0.80 micrometers or less, 0.75 micrometers or less, or 0.70 micrometers or less, or any value in between (e.g., 1.67 micrometers). In some embodiments, the fibrous material may have the following average pore sizes: 0.2 µm to 3 µm, 0.20 µm to 2.0 µm, 0.20 µm to 1.5 µm, 0.40 µm to 1.5 µm, 0.40 µm to 1.3 µm, 0.40 µm to 1.2 µm, 0.50 µm to 1.5 µm, 0.50 µm to 1.3 µm, 0.50 µm to 1.2 µm, 0.60 µm to 1.5 µm, 0.60 µm to 1.3 µm, 0.60 µm to 1.2 µm, 0.70 µm to 1.5 µm, 0.70 µm to 1.3 µm, 0.70 µm to 1.2 µm, or 0.70 µm to 1.0 µm.

[0081] Fiber materials (such as glass fiber filters) may have pore sizes selected to accommodate beads of a corresponding size to facilitate mechanical cleavage. The beads may include glass beads, silica beads, or combinations thereof.

[0082] The thickness of the fiber material can be 100 micrometers or greater, 150 micrometers or greater, 200 micrometers or greater, 250 micrometers or greater, 300 micrometers or greater, 350 micrometers or greater, 400 micrometers or greater, 450 micrometers or greater, 500 micrometers or greater, 550 micrometers or greater, 600 micrometers or greater, 650 micrometers or greater, 700 micrometers or greater, 750 micrometers or greater, 800 micrometers or greater, 900 micrometers or greater, 1,000 micrometers or greater, 1,200 micrometers or greater, 1,500 micrometers or greater, 1,600 micrometers or greater, 1,800 micrometers or greater, 1,900 micrometers or greater, or 2,000 micrometers or greater, or any value in between (e.g., 1,709 micrometers). In some embodiments, the fibrous material may have the following thicknesses: 2,500 micrometers or less, 2,400 micrometers or less, 2,200 micrometers or less, 2,000 micrometers or less, 1,900 micrometers or less, 1,800 micrometers or less, 1,600 micrometers or less, 1,500 micrometers or less, 1,400 micrometers or less, 1,300 micrometers or less, 1,200 micrometers or less, 1,000 micrometers or less, 900 micrometers or less, 800 micrometers or less, 750 micrometers or less, 700 micrometers or less, 600 micrometers or less, 550 micrometers or less, or 500 micrometers or less, or any value in between (e.g., 867.1 micrometers). In some embodiments, the fibrous material may have the following thicknesses: 100 micrometers to 2,000 micrometers, 200 micrometers to 1,500 micrometers, 200 micrometers to 1,200 micrometers, 250 micrometers to 1,200 micrometers, 250 micrometers to 1,000 micrometers, 250 micrometers to 800 micrometers, 300 micrometers to 2,000 micrometers, 300 micrometers to 1,800 micrometers, 300 micrometers to 1,500 micrometers, 300 micrometers to 1,200 micrometers, 400 micrometers to 2,000 micrometers, 400 micrometers to 1,500 micrometers, or 500 micrometers to 2,000 micrometers.

[0083] The basis weight of the fiber material can be 10 g / m³ 2 or larger, 15 g / m 2 or larger, 20 g / m 2 Or larger, 25g / m 2 Or larger, 30 g / m 2 Or larger, 35 g / m 2 Or larger, 40 g / m 2 Or larger, 45 g / m 2 Or larger, 50 g / m 2 Or larger, 55g / m 2 Or larger, 60 g / m 2 Or larger, 65 g / m 2Or larger, 70 g / m 2 Or larger, 75 g / m 2 Or larger, 80 g / m 2 Or larger, 90 g / m 2 Or larger, 100 g / m 2 Or larger, 120 g / m 2 Or larger, 150 g / m 2 Or larger, 160 g / m 2 Or larger, 180g / m 2 Or larger, 190 g / m 2 Or larger, or 200 g / m 2 Or greater, or any value in between (e.g., 58 g / m³) 2 In some embodiments, the fiber material may have the following basis weight: 250 g / m². 2 or smaller, 240 g / m 2 or smaller, 220 g / m 2 or smaller, 200 g / m 2 or smaller, 190 g / m 2 or smaller, 180 g / m 2 or smaller, 160 g / m 2 or smaller, 150 g / m 2 or smaller, 140 g / m 2 or smaller, 130 g / m 2 or smaller, 120 g / m 2 or smaller, 100 g / m 2 or smaller, 90 g / m 2 or smaller, 80 g / m 2 or smaller, 75 g / m 2 or smaller, 70 g / m 2 or smaller, 60 g / m 2 or smaller, 55 g / m 2 Or smaller, or 50 g / m 2 Or less, or any value in between (e.g., 182 g / m³) 2 In some embodiments, the fiber material may have the following basis weight: 10 g / m³ 2 Up to 200 g / m 2 20 g / m 2 Up to 150 g / m 2 20 g / m 2 Up to 120 g / m 2 25 g / m 2 Up to 120 g / m 2 25 g / m2 Up to 100 g / m 2 25 g / m 2 Up to 90 g / m 2 30 g / m 2 Up to 200 g / m 2 30 g / m 2 Up to 180 g / m 2 30 g / m 2 Up to 150 g / m 2 30 g / m 2 Up to 100 g / m 2 40 g / m 2 Up to 150 g / m 2 40 g / m 2 Up to 100 g / m 2 or 50 g / m 2 Up to 90 g / m 2 .

[0084] In some instances, unmodified solid supports (which may be modified to include functional surface groups) are available from Pall Corporation with varying pore sizes and thicknesses: for example, glass fiber filters are available from Pall Corporation in types A / E, A / B, and A / C, all with a nominal pore size of 1 µm and thicknesses of 0.33 mm, 0.66 mm, and 0.25 mm, respectively. Other filter types are described below. Pall TCLP (Toxicity Characteristic Leaching Process) products have the same dimensions as Whatman (Cytiva) filters (0.7 µm pore size, 0.4 mm thickness). Exemplary glass fiber filter specifications include binder-free borosilicate glass with a nominal pore size of 3 µm and a thickness of 660 µm (26 mils); glass fibers with an acrylic binder having a nominal pore size of 1 µm and a thickness of 1270 µm (50 mils); ultrafine glass fibers with an acrylic binder having a nominal pore size of 0.5 µm and a thickness of 330 µm (13 mils); and binder-free borosilicate glass with a nominal pore size of 0.7 µm and a thickness of 432 µm (17 mils). In some embodiments, the solid carrier is a glass fiber filter with a thickness of 400 micrometers to 2000 micrometers and a pore size of 0.5 micrometers to 1 micrometer.

[0085] In other embodiments, the solid carrier is a particulate material, such as silica gel or silicon dioxide, with a pore diameter of about 30 to about 1000 angstroms, a particle size of about 2 to about 300 micrometers, and a specific surface area of ​​about 35 m². 2 / g to approximately 1000 m 2 / g. In some embodiments, the particulate material may have pore diameters of about 40 Å to about 500 Å, about 60 Å to about 500 Å, about 100 Å to about 300 Å, and about 150 Å to about 500 Å. In some embodiments, the particulate material may have particle sizes of about 2 to about 25 μm, about 5 to about 25 μm, about 15 μm, about 63 to about 200 μm, and about 75 to about 200 μm; and about 100 μm. 2 / g to approximately 350 m 2 / g, approximately 100 m 2 / g to approximately 500 m 2 / g, approximately 65 m 2 / g to approximately 550 m 2 / g, approximately 100 m 2 / g to approximately 675 m 2 / g and about 35 to about 750 m 2 Specific surface area per g.

[0086] As described herein, solid supports may contain functional surface groups that interact with and / or react with affinity reagents. These functional surface groups may include functional groups, such as silanized groups or other components that can facilitate binding to the solid support. For example, functional surface groups may include ethers, silyl ethers, siloxanes, carboxylic esters, sulfonates, aminosulfonates, sulfates, phosphonates, hypophosphonates, phosphates, silyl carboxylate esters, silyl sulfonate esters, silyl sulfinate esters, silyl sulfate esters, silyl phosphonate esters, silyl hypophosphonate esters, silyl phosphate esters, oxides, sulfides, carbocyclic rings, heterocyclic rings having at least one oxygen atom, heterocyclic rings having at least one nitrogen atom, heterocyclic rings having at least one sulfur atom, and heterocyclic rings having at least one silicon atom. Rings, carbodiimides (such as DCC and EDCI), phosphorus or ammonium (such as BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH), heterocycles derived from "click" reactions, carbocyclic rings derived from Diels-Alder reactions, heterocycles derived from Diels-Alder reactions, amides, imides, sulfides, thiolates, metal thiolates, carbamates, oximes, hydrazides, hydrazones, physico-adsorbed or chemi-adsorbed or otherwise non-covalently linked portions, or combinations thereof. In some embodiments, the functional surface groups include functional groups selected from: maleimide, acrylate, acrylamide, epoxide, aziridine, thiacyclopropane, aldehyde, ketone, azide, alkyne, disulfide, acid anhydride, carboxylic acid ester, phosphate ester, phosphonate, sulfate ester, sulfonate ester, nitrate ester, amidine, silane, siloxane (e.g., cyclic siloxane), triazine, heteroazosilane, silsesquioxane, silazane, cyanate ester, acetylene, cyanide, halogen, acetal, ketal, amino, carbonyl, carboxyl, biotin, cyclodextrin, adamantane, or vinyl groups that can be attached to a solid carrier.

[0087] Functional surface groups may also include functional binding groups that promote binding with affinity reagents. For example, functional surface groups may include hydrophobic binding moieties that bind with affinity reagents, negatively charged binding groups (e.g., sulfonic acid, sulfate, phosphate, phosphonic acid, or carboxyl groups), positively charged binding groups (e.g., primary amines, secondary amines, tertiary amines, and quaternary ammonium, heterocyclic amines, such as pyridine, pyrimidine, pyridinium, piperazine), polar binding moieties (e.g., chemical moieties containing polarized chemical bonds, such as C—O, C═O, C—N, C═N, C═N, N—H, O—H, C—F, C—Cl, C—Br, C—S, S—H, S—O, S═O, C—P, P—O, P═O, P—H, more specifically carboxyl groups, alcohols, thiols, amides, halides, amines, esters, ethers, or thioesters) or combinations thereof.

[0088] In some instances, the functional surface group is a silane. A silane contains at least one silicon atom having four bonds with other atoms, such as carbon, hydrogen, oxygen, etc. In specific instances, the functional surface group can be a silane containing one or more alkyl, heteroalkyl, cycloalkyl, cyclohexaalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, haloalkyl, or derivatives thereof. In other specific instances, the functional surface group may include silanes having one or more alkoxy groups, such as methoxysilane, dimethoxysilane, trimethoxysilane, ethoxysilane, diethoxysilane, triethoxysilane, chlorosilane, or derivatives thereof. Silanes containing alkoxy groups as described herein can be crosslinked with other silanes on a solid support. In further instances, the silane may contain hydrophobic groups, such as straight-chain alkyl groups. Straight-chain alkyl groups include, for example, C4-C… 20 Alkyl groups, C4-C 12 alkyl groups or C4-C 10 Alkyl groups. Silanes may include bipod silanes. Alternatively or otherwise, the functional surface may include branched alkyl groups, such as C6-C. 20 Alkyl groups, C8-C 12 alkyl groups or C8-C 10 Alkyl groups. In some embodiments, the functional surface groups are compounds having the following formula:

[0089]

[0090] Formula (IIa),

[0091] Where R' is the portion that interacts with and / or reacts with the affinity reagent, and R is the portion that interacts with and / or reacts with the solid support; or where R is the portion that interacts with and / or reacts with the affinity reagent, and R' is the portion that interacts with and / or reacts with the solid support. In some instances of R or R', each may be defined as the group -L-NM, where L is the linker and NM is the portion that binds to the affinity reagent or nucleic acid. The linker may be selected from an alkeneoxy (e.g., C2-C4 alkeneoxy) group, an alkylene (e.g., C2-C4 alkylene or C2-C3 alkylene) group, or a heteroalkylene (e.g., C4-C6 heteroalkylene). In some instances, the linker is a bond.

[0092] In some embodiments of Formula IIa, R' is a group -L-NM, where L is a linker and NM is a nucleic acid binding moiety. For example, NM can be an amino group or an intercalating agent, such as pyrene or a pyrene derivative. In some embodiments, the functional surface group can be selected from (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, N-(6-aminohexyl)aminomethyltriethoxysilane, or N-(2-aminoethyl)-3-aminopropyltrimethoxysilane.

[0093] In some instances of formula IIa, R' is an alkyl or fluoroalkyl group. For example, the functional surface group can be a compound having the following formula:

[0094]

[0095] Equation (IIb),

[0096] Where R is an alkyl group. In some examples of Formula IIb, the functional surface group is a trimethoxyalkylsilane or a triethoxyalkylsilane. For example, the functional surface group may be selected from trimethoxyhexylsilane, trimethoxyheptylsilane, trimethoxyoctylsilane, trimethoxynonylsilane, trimethoxydecylsilane, trimethoxyundecylsilane, trimethoxydodecylsilane, trimethoxytridecylsilane, trimethoxytetradecylsilane, trimethoxypentadecanylsilane, trimethoxyhexadecylsilane, trimethoxyheptadecylsilane, or trimethoxyoctadecylsilane. In other examples, the functional surface groups are triethoxyhexylsilane, triethoxyheptylsilane, triethoxyoctylsilane, triethoxynonylsilane, triethoxydecylsilane, triethoxyundecylsilane, triethoxydodecylsilane, triethoxytetrazylsilane, triethoxytetradecylsilane, triethoxypentadecanylsilane, triethoxyhexadecylsilane, triethoxyheptadecylsilane, or triethoxyoctadecylsilane.

[0097] In some embodiments, the functional surface groups may be selected from fluorinated silanes, such as heptadecafluorodecyltrimethoxysilane, heptadecafluorodecyltriethoxysilane, heptadecafluorononadecaalkyltrimethoxysilane, heptadecafluorononadecaalkyltriethoxysilane, tridecafluoroheptadecaalkyltrimethoxysilane, tridecafluoroheptadecaalkyltriethoxysilane, nonafluoropentadecaalkyltrimethoxysilane, nonafluoropentadecaalkyltriethoxysilane, nonafluorohexyltrimethoxysilane, nonafluorohexyltriethoxysilane, tridecafluorooctyltrimethoxysilane, tridecafluorooctyltriethoxysilane, or pentafluoroaryl, such as pentafluorophenoxyundecyltrimethoxysilane.

[0098] In some aspects, the surface of the solid support contains azide groups. Functionalization of the azide groups can be achieved by reacting with alkyne-containing functionalized surface groups. For example, the functionalized surface groups can react with alkyne-containing groups having the formula AL-NM, where "A" is an alkyne, "L" is a linker, and "NM" is an affinity-binding or nucleic acid-binding moiety. In some instances, the alkyne is a constrained alkyne, such as diazabicyclooctyne (DBCO). In some embodiments, the alkyne is a DBCO group, and NM is a hydrophobic group, such as an alkyl group.

[0099] In some embodiments, the solid support is a glass containing silanol and siloxane groups. Such silanol groups on the glass surface can react with silane- and siloxane-containing functional surface groups to provide a surface with compounds chemically bonded via siloxane bridges.

[0100] Functionalization of solid supports can be performed by any method known in the art. For example, functionalization can be performed by silanization. Silanization can be performed, for example, in a liquid solution or in the gas phase (e.g., physical vapor deposition (PVD), chemical vapor deposition (CVD), atomic layer deposition (ALD), etc.).

[0101] In some instances, functionalization of the solid support is performed via deposition, which may include one or more CVD steps. CVD may include loading the substrate into a vacuum oven, loading the silane to be deposited, evacuating the oven, allowing the substrate to stand, and optionally performing a water contact angle (WCA) test to measure the hydrophobicity of the surface. Deposition may be repeated more than once. For example, the deposition step may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. The substrate may be allowed to stand in the oven for at least about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes or longer. Conditions in the oven may include a reduced pressure atmosphere. For example, the pressure in the oven can be about 1, 2, 3, 4, 5, 10, 15, 20, 50, 100, 200, 500 or lower. Alternatively or additionally, the conditions in the oven may include a certain temperature. The temperature in the oven may be at least about 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, 140°C, 150°C or higher.

[0102] The density of functional surface groups can be controlled by deposition via liquid-phase or gas-phase methods. In some embodiments, the density of functional surface groups (and affinity reagents) is 1 nmol / cm³. 2 Or greater (e.g., 5 nmol / cm) 2 Or larger, 10 nmol / cm 2 Or larger, 15 nmol / cm 2 Or larger, 20 nmol / cm 2 Or larger, 25 nmol / cm 2 Or larger, 35 nmol / cm 2 Or larger, 40 nmol / cm 2 Or larger, 45 nmol / cm 2 Or larger, 50 nmol / cm 2 Or larger, 55 nmol / cm 2 Or larger, 60 nmol / cm 2 Or larger, 65 nmol / cm 2 Or larger, 70 nmol / cm 2 Or larger, 75 nmol / cm 2 Or larger, 80 nmol / cm 2 Or larger, 85 nmol / cm2 Or larger, 90 nmol / cm 2 Or larger, 95 nmol / cm 2 Or larger, 100 nmol / cm 2 Or larger, 200 nmol / cm 2 Or larger, 300 nmol / cm 2 Or larger, 400 nmol / cm 2 Or larger, 500 nmol / cm 2 Or larger, 1,000 nmol / cm 2 Or larger, 1,500 nmol / cm 2 Or larger, 2,000 nmol / cm 2 Or larger, 2,500 nmol / cm 2 Or larger, 3,000 nmol / cm 2 Or larger, 1-3,000 nmol / cm 2 1-1,500 nmol / cm 2 1-500 nmol / cm 2 10-100 nmol / cm 2 10-200 nmol / cm 2 10-500 nmol / cm 2 15-80 nmol / cm 2 30-100 nmol / cm 2 or 30-90 nmol / cm 2 30-200 nmol / cm 2 or 30-500 nmol / cm 2 and any values ​​in between, such as 452 nmol / cm 2 In some embodiments, the density of functional surface groups (and affinity reagents) is 3,000 nmol / cm³. 2 Or smaller (e.g., 2,000 nmol / cm) 2 Or smaller, 1,500 nmol / cm 2 Or smaller, 1,000 nmol / cm 2 Or smaller, 900 nmol / cm 2 or smaller, 800 nmol / cm 2 or smaller, 600 nmol / cm 2 or smaller, 500 nmol / cm 2 or smaller, 400 nmol / cm 2or smaller, 300 nmol / cm 2 or smaller, 200 nmol / cm 2 Or smaller, or 100 nmol / cm 2 Or smaller, and any value in between, such as 1,278 nmol / cm 2 ).

[0103] The density control of functional surface groups (and affinity reagents) can be achieved by varying the concentration of the reagents used to functionalize the solid surface. For example, in the liquid phase, the concentration of the reagent (such as a silane or alkyne-containing reagent) can be varied to provide the desired density. In gas-phase methods such as CVD, the amount of silane and deposition conditions (temperature, pressure, and exposure duration) can be varied to provide the desired density. Alternatively, the density of the solid support can be controlled by co-depositing a second compound on the surface. For example, a second silane group is deposited simultaneously or sequentially with a first silane on the solid support. The relative density of the first and second functional surface groups can vary depending on the characteristics of the functional groups, solution conditions, etc. A well-controlled density of the first and second functional surface groups and / or affinity reagents on the surface of the solid support provides high nucleic acid binding capacity.

[0104] In one embodiment, the present invention provides a solid support comprising a first functional surface group and a second functional surface group, the first functional surface group being an alkylsilane and the second functional surface group being an azide silane. For example, an alkylsilane is first deposited on the solid support, followed by the deposition of an azide silane. Alternatively, an azide silane is first deposited on the solid support, followed by the deposition of an alkylsilane. In yet another alternative, the alkylsilane and the azide silane are deposited simultaneously. Deposition can be performed, for example, by CVD. In some embodiments, the ratio of the first functional surface group to the second functional surface group can be less than 1:1, 1:2, 1:3, 1:5, or 1:10. In some embodiments, the ratio of the first functional surface group to the second functional surface group can be greater than 1:1, 1:2, 1:3, 1:5, or 1:10. In some embodiments, the ratio of the first functional surface group to the second functional surface group may be between 1:10 and 10:1, or between 1:10 and 5:1, or between 1:5 and 5:1, or between 1:5 and 1:2, or between 1:5 and 1:1.

[0105] Solid supports, together with affinity reagents, can have a strength of at least 10 µg / cm³. 2 DNA binding capacity (e.g., 15 µg / cm) 2 or larger, 20 µg / cm 2 or larger, 25 µg / cm 2or larger, 35 µg / cm 2 or larger, 40 µg / cm 2 or larger, 45 µg / cm 2 or larger, 50 µg / cm 2 or larger, 55 µg / cm 2 or larger, 60 µg / cm 2 or larger, 65 µg / cm 2 or larger, 70 µg / cm 2 or larger, 75 µg / cm 2 or larger, 80 µg / cm 2 or larger, 85 µg / cm 2 or larger, 90 µg / cm 2 or larger, 95µg / cm 2 Or larger, 100 µg / cm 2 Or larger, 30-100 µg / cm 2 Or 30-90 µg / cm 2 , or any value in between, such as 79 µg / cm 2 In some embodiments, the solid support has a density of at least 30 µg / cm³. 2 DNA binding ability.

[0106] Affinity reagents

[0107] As described in this article, this method utilizes affinity reagents capable of interacting with both the solid carrier and the nucleic acids from the sample. Back to Figure 1 The affinity reagent 20 comprises a first portion 22 that interacts and / or reacts with the solid support 10 and a second portion 24 that interacts and / or reacts with the nucleic acid 32. The affinity reagent may interact and / or react directly with the surface of the solid support, or indirectly with the solid support via functional surface groups on the solid support. For example, the affinity reagent may be directly covalently or nonvalently bonded to the surface of the solid support (e.g., via ionic bonding, hydrophobic or van der Waals interactions, polar interactions). In other instances, the affinity reagent may be indirectly covalently or nonvalently bonded to the solid support via functional surface groups on the surface of the solid support. When referring to the interaction and / or reaction of the affinity reagent with the solid support, both direct and indirect interactions and / or reactions are considered.

[0108] Figure 2Affinity reagent 20 is shown, which also contains a photolytically cleavable group 26, denoted as X-X'. Once the nucleic acid is extracted and isolated from the sample, it is released from the solid support by photolysis in the presence of an elution reagent. In some embodiments, the nucleic acid is released from the solid support by heating, and the nucleic acid is extracted and isolated from the sample, such as... Figure 3 As shown. Figure 3 A solid support 10 modified with a hydrophobic surface group 12 and an affinity reagent 20 is shown, the affinity reagent having a first portion 22 that binds to the hydrophobic surface group 12 and a second portion 24 that binds to nucleic acid 32.

[0109] As described herein, the affinity reagent comprises a first part that interacts with and / or reacts with the solid support. The first part of the affinity reagent may contain functional groups such as silanization groups, ethers, silyl ethers, siloxanes, carboxylic esters, sulfonates, aminosulfonates, sulfates, phosphonates, hypophosphonates, phosphates, silyl carboxylic acids, silyl sulfonates, silyl sulfinates, silyl sulfates, silyl phosphonates, silyl hypophosphonates, silyl phosphates, oxides, sulfides, carbocyclic rings, heterocycles having at least one oxygen atom, heterocycles having at least one nitrogen atom, heterocycles having at least one sulfur atom, heterocycles having at least one silicon atom, carbodiimides (such as DCC and EDCI), phosphorus or ammonium (such as BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH), and "point". Heterocycles derived from the "knock" reaction, carbocyclic rings derived from the Diels-Alder reaction, heterocycles derived from the Diels-Alder reaction, amides, imides, sulfides, thiols, metal thiols, carbamates, oximes, hydrazides, hydrazones, maleimides, acrylates, acrylamides, epoxides, aziridines, thiapropylcyclic rings, aldehydes, ketones, azides, alkynes, disulfides, acid anhydrides, carboxylic acid esters, phosphate esters, phosphonates, sulfate esters, sulfonates, nitrate esters, amidines, silanes, siloxanes (e.g., cyclic siloxanes), triazines, heteroazines, silsesquioxanes, silazanes, cyanates, acetylene, cyanides, halogens, acetals, ketals, amino groups, carbonyl groups, carboxyl groups, biotin, cyclodextrins, adamantane, physico- or chemi-adsorbed or otherwise non-covalently linked portions or combinations thereof. In some embodiments, the first portion of the affinity reagent may comprise a functional group that interacts with the solid support, such as a hydrophobic group, a negatively charged binding group (e.g., sulfonic acid group, sulfate group, phosphate group, phosphonate group, or carboxyl group), a positively charged binding group (e.g., primary amine, secondary amine, tertiary amine and quaternary ammonium, heterocyclic amine, such as pyridine, pyrimidine, pyridinium, piperazine), a polar group (e.g., a chemical moiety containing a polarized chemical bond, such as C—O, C═O, C—N, C═N, C═N, N—H, O—H, C—F, C—Cl, C—Br, C—S, S—H, S—O, S═O, C—P, P—O, P═O, P—H, more specifically carboxyl, alcohol, thiol, amide, halide, amine, ester, ether, or thioester) or a combination thereof. Hydrophobic groups, negatively charged binding groups, positively charged binding groups, and polar groups are as described herein. Preferably, the first portion of the affinity reagent comprises a functional group that interacts with and / or reacts with functional surface groups on the solid support. For example, the interaction between the affinity reagent and the solid support can be achieved via hydrophobic interaction. In this case, the affinity reagent comprises a first hydrophobic group that interacts with the solid support comprising a second hydrophobic group. In some embodiments, the first and second hydrophobic groups are independently alkyl groups.

[0110] In some instances, the interaction with the solid support and the affinity reagent is based on fluorine affinity. In such cases, both the affinity reagent and the solid support each contain at least one fluorine atom substituted. For example, the affinity reagent and / or the solid support each independently contain a perfluoroalkyl group.

[0111] In some instances, the interaction with the solid support and affinity reagent is achieved via the triazine moiety. The azide group reacts rapidly and quantitatively with strained cyclic alkynes to form stable cycloadducts. Furthermore, such chemical groups exhibit minimal reactivity to oligonucleotide functional groups (primary amines, secondary amines, primary alcohols, secondary alcohols, phosphate esters, ketones, aldehydes, etc.). Strained cyclic alkynes and azides can be used to generate bioorthogonal reactions for the functionalization of the solid support and affinity reagent. Therefore, modification with azides and / or strained cyclic alkynes on the solid support or affinity reagent can be used as a suitable way to functionalize the solid support. In some instances, the interaction with the solid support and affinity reagent is achieved via the imidazole or indole moiety.

[0112] The affinity reagent also includes a second part that interacts with and / or reacts with nucleic acids in the sample. This second part may be described herein as a “DNA-binding ligand,” a “nucleic acid-binding ligand,” or a “nucleic acid-binding moiety.” Nucleic acids are typically any oligonucleotide and may include RNA or DNA, double-stranded or single-stranded, or have a secondary (single-stranded DNA or RNA may form an internal double-stranded region, i.e., a secondary structure) or tertiary structure.

[0113] Nucleic acid binding moieties include any molecule capable of binding to or associating with nucleic acids. Such binding or association can be achieved via covalent bonding, ionic bonding, hydrogen bonding, van der Waals bonding, or any other type of reversible or irreversible association. The term "molecular part" is used herein to refer to any atom, ion, molecule, macromolecule (e.g., polypeptide), or combination of such entities. The term "binding moieties" is used interchangeably with the term "ligand."

[0114] The nucleic acid binding moiety can be selected from primary amines, secondary amines, triazoles, imidazoles, indole-containing compounds (such as alkylamines, cycloalkylamines, alkoxyamines, polyamines, or arylamines) or other heterocyclic moieties (such as aziridine, azacyclopropene, pyrrolidine, pyrrololine, pyrrole, pyrazolidine, imidazoline, pyrazole, tetrazolium, oxazole, thiadiazole, piperidine, piperazine, pyrimidine, pyrazine, triazine, oxazine, morpholine, isoyindole, indazole, azaindole, azaindazole, purine, adenine). (e.g., guanine, quinoline, quinazoline, cyclophosphamide, pteridine), intercalating agents (e.g., furanocoumarins, coumarins, anthracyclines, phenanthridines, psoralen derivatives, acridines, rose alkaloids, actinomycins, anthraquinones, and Tris compounds), groove binding agents (e.g., pyrrolo(1,4)benzodiazepines (PBD), anddavancin, pyrimethamine, fusiformin, tertimobin, chachomycin, CC-1065, and Hoechst 33258), polypeptides, amino acids (histidine), proteins (such as zinc finger proteins, homeodomain proteins, leucine zipper proteins, helical-circular-helical proteins, or β-sheet motifs), or combinations thereof.

[0115] In some aspects of the affinity reagents disclosed herein, the nucleic acid binding moiety may contain multiple amine groups (or polyamines), multiple amide groups (or polyamides), or combinations thereof (polyamine-amides). For example, the nucleic acid binding moiety may contain at least two, at least three, at least four, at least five, at least six amine or amide groups, or combinations thereof. In other instances, the nucleic acid binding moiety contains a single amine or amide group. The amine or amine group may be a primary, secondary, or tertiary amine. In some embodiments, the nucleic acid binding moiety contains a trialkylammonium group, such as triethylammonium acetate or a quaternary ammonium group.

[0116] In some aspects of affinity reagents, the nucleic acid binding moiety contains C1-C. 16 Alkylamines (e.g., C1-C) 12 Alkylamines, C1-C 10 Alkylamines, C1-C8 alkylamines, C2-C8 alkylamines or C2-C6 alkylamines), C3-C 12 Cycloalkylamines (e.g., C3-C) 10 Cycloalkylamines, C3-C8 cycloalkylamines, C3-C6 cycloalkylamines, C4-C8 cycloalkylamines or C4-C6 cycloalkylamines), C1-C 16 Alkoxyamines (e.g., C1-C) 12 Alkoxyamines, C1-C 10 Alkoxyamines, C1-C8 alkoxyamines, C2-C8 alkoxyamines or C2-C6 alkoxyamines), C6-C 12 Arylamines (e.g., C6-C) 10 Arylamines, C6-C8 arylamines), C6-C 12 Imidazole group, C3-C 14Heterocyclic alkylamines (e.g., C3-C) 10 Heterocyclic alkylamines, C3-C8 heterocyclic alkylamines, C3-C6 heterocyclic alkylamines, C4-C8 heterocyclic alkylamines or C4-C6 heterocyclic alkylamines) and C2-C 20 Heteroalkylamines (e.g., C1-C) 12 Heteroalkylamines, C1-C 10 (Heteroalkylamines, C1-C8 heteroalkylamines, C2-C8 heteroalkylamines, or C2-C6 heteroalkylamines) or combinations thereof. In some embodiments, the nucleic acid binding moiety comprises an alkylamine group, an imidazole group, or a combination thereof.

[0117] In some embodiments, the affinity reagent comprises a nucleic acid-binding moiety that interacts with the nucleic acid via electrostatic interactions. This nucleic acid-binding moiety can be an amine or other nitrogen-containing group that can bind the nucleic acid with high affinity when positively charged (at a relatively low pH) and release the nucleic acid when neutral (at a relatively high pH). In some instances, the nucleic acid-binding moiety is an imidazole. In other instances, the nucleic acid-binding moiety is selected from spermidine, spermine, methylamine, ethylamine, propylamine, cadaverine, putrescine, ethylenediamine, diethylenetriamine, 1,3-dimethyldipropylenediamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium chloride, tris(2-aminoethyl)amine, or combinations thereof.

[0118] The nucleic acid binding moiety can be an intercalator or a groove-binding agent (major groove or minor groove). Intercalators include, but are not limited to, doxorubicin, detecarboxylic acid, cyproterone acetate, doxorubicin, and pyrene. Minor groove-binding moieties include, for example, polyamides, such as heterocyclic polyamides (containing, for example, pyrrole and / or imidazole groups). Other minor groove-binding moieties include small molecules, such as fusiformin, tertamicin, pentamidine, benir, and doxorubicin.

[0119] The nucleic acid binding site can be a fluorescent dye. Suitable fluorescent dyes include PicoGreen, OliGreen, EvaGreen, GelGreen, GelRed, ethidium bromide, propidium iodide, acridine orange, 7-aminoactinomycin D, anthocyanin dye, bis(benzyl)imine, benzoxanthracene yellow, fusiformin, crystal violet, any SYTO dye, SYBR Green I, SYBR Green II, SYBR DX, OliGreen, CyQuant GR, SYTOX, POPO-1, BOBO-1, YOYO-1, TOTO-1, JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3, PO-PRO-1, YO-PRO-1, TO-PRO-1, JO-PRO-1, PO-PRO-3, YO-PRO-3, TO-PRO-3, TO-PRO-5, DAPI, and Hoechst 33258.

[0120] The first part (binding to the solid carrier) and the second part (nucleic acid binding portion) of the affinity reagent can be conjugated via a linker. In some embodiments, the linker is a branched group conjugated to either the first or second part. The linker may be selected from an alkeneoxy (e.g., C2-C4 alkeneoxy) group, an alkylene (e.g., C2-C4 or C2-C3 alkylene) group, or a heteroalkylene (e.g., C4-C6 heteroalkylene). In some instances, the linker is a bond. In other instances, the linker comprises polyethylene glycol, an alkyl group, an ester, an amide, a disulfide, a carbamate, or a thiol group. In other instances, the linker may contain a photolyzable group that allows convenient release of the nucleic acid from the solid carrier. Suitable photolyzable groups include nitrobenzyl linkers, coumarin linkers, anthracene linkers, and quinone linkers.

[0121] In some implementations, the linker contains one or more functional groups that interact with the solvent in the sample to improve the solubility of the affinity reagent. For example, when the linker contains polyethylene glycol (PEG) or similar polymer groups, the linker alters the physical and chemical properties of the affinity reagent, such as its conformation, electrostatic binding, and hydrophobicity. In some instances, affinity reagents having one or more PEG linkers alter the solubility of the affinity reagent in water or other solvents.

[0122] In some embodiments, the linker comprises an ester-based linker (such as a branched ester-based linker), an amide-based linker (such as a branched amide-based linker, including an N,N-dihexyl-3-imidazol-2-oxoamine linker), an amine-based linker (such as a 3-dibutylaminopropylamine linker, an N,N-dihexyl-3-aminopropylamine linker), an organophosphorus-based linker (such as an organophosphorus-based linker, an organophosphorus oxide-based linker, an organophosphonate-based linker, an organophosphoramide-based linker, an organophosphate-based linker, an organophosphoramide-based linker, or an organophosphonate-based linker), a glucuronic acid-based linker, a disulfide linker, a cathepsin B linker, or a combination thereof.

[0123] In some instances, the affinity reagent comprising the first and second parts is an alkylamine. The alkyl group can be straight-chain or branched. In some embodiments, the alkylamine is methylamine, ethylamine, diethylamine, triethylamine, propylamine, isopropylamine, diisopropylamine, butylamine, isobutylamine, sec-butylamine, tert-butylamine, pentylamine, isopentylamine, hexylamine, isohexylamine, cyclohexylamine, heptylamine, octylamine, nonylamine, decylamine, undecylamine, dodecylamine, tridecylamine, tetradecylamine, pentadecylamine, hexadecylamine, heptadecanylamine, octadecylamine, nonadecanylamine, or 3-dibutylaminopropylamine.

[0124] box

[0125] In some embodiments, solid carriers and affinity reagents are incorporated into automated cartridges (such as GeneXpert® cartridges). In one aspect, the present invention relates to a sample cartridge utilizing a valve body platform that allows for the detection of enveloped and free nucleic acid targets. In some embodiments, the valve body includes a sample processing area or lysis chamber that provides one or both of mechanical and chemical lysis. This allows a single cartridge to provide lysis for multiple different types of targets and can therefore be considered an "assay kit".

[0126] A sample cartridge can be any device configured to perform one or more processing steps related to the preparation and / or analysis of a biological fluid sample according to any of the methods described herein. In some embodiments, the sample cartridge is configured to perform at least sample preparation. The sample cartridge can also be configured to perform additional processes, such as the detection of target nucleic acids in a nucleic acid amplification assay (NAAT) (e.g., polymerase chain reaction (PCR) assay), by using a reaction vessel attached to the cartridge. In some embodiments, the reaction vessel extends from the body of the sample cartridge. The preparation of fluid samples typically involves a series of processing steps, which may include chemical, electrical, mechanical, thermal, optical, or acoustic processing steps depending on the specific protocol. Such steps can be used to perform a variety of sample preparation functions, such as cell capture, cell lysis, analyte binding, and binding of unwanted materials.

[0127] The cartridge applicable to this invention includes one or more transfer ports through which a prepared fluid sample can be transported to an attached reaction vessel for analysis. Figure 5 An exemplary assay kit 100, according to some embodiments, is shown that, when received in an instrument module, is suitable for sample preparation and analytical testing via PCR. The sample kit is configured to perform various sample procedures, including the chemical lysis of a target, and is configured for optional integrated nucleic acid analysis of the immunoPCR and target assay kit according to some embodiments of the invention.

[0128] The sample cartridge is attached to a reaction container 103 (also referred to as a "reaction tube" or "PCR tube") suitable for analyzing fluid samples processed within the sample cartridge 100. In some embodiments, the reaction container extends from the cartridge body. Such a sample cartridge 100 includes various components, including a main housing 102 having one or more chambers 108 for processing fluid samples, which typically includes sample preparation prior to analysis. The instrument module facilitates the processing steps required for sample preparation, and the prepared sample is delivered via one of a pair of transfer ports to a fluid conduit in the reaction container 103 attached to the housing of the sample cartridge 100. The prepared biofluid sample is then transferred to the reaction chamber of the reaction container, where nucleic acid amplification is performed. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, the excitation device and optical detection device of the module are used to detect optical emission indicating the presence or absence of a target nucleic acid analyte of interest (e.g., bacteria, viruses, pathogens, toxins, tumors, or other target analytes) while the biofluid sample is amplified. It should be understood that such a reaction container may include a variety of different chambers, conduits, or micropore arrays for detecting target analytes. The cartridge is equipped with means for preparing a biofluid sample prior to delivery into a reactor dish. Any chemical reagents required for cell lysis, or means for binding or detecting an analyte of interest (e.g., reagent beads), may be contained within one or more chambers of the cartridge and thus may be used for sample preparation.

[0129] Exemplary uses of a reaction vessel for analyzing biological fluid samples are described in commonly assigned U.S. Patent No. 6,818,185, filed May 30, 2000, entitled “Cartridge for Conducting a Chemical Reaction,” the entire contents of which are incorporated herein by reference for all purposes. Examples of sample boxes and related modules are shown and described in U.S. Patent No. 6,374,684, filed August 25, 2000, entitled “Fluid Control and Processing System”; U.S. Patent No. 8,048,386, filed February 25, 2002, entitled “Fluid Processing and Control”; U.S. Patent Application No. 63 / 218,672, filed July 1, 2021, entitled “Universal Assay Cartridge and Methods of Use”; U.S. Provisional Application No. 63 / 319,993, filed March 15, 2022, entitled “Unitary Cartridge Body and Associated Components and Methods of Manufacture”; and U.S. Patent No. 10,562,030, filed July 22, 2016, entitled “Molecular Diagnostic Assay System”; the entire contents of these documents are incorporated herein by reference for all purposes.

[0130] In some instances, the reaction vessel includes integrated optical sensor circuitry. This integrated optical sensor circuitry may include a CMOS substrate. The CMOS substrate can be a solid and planar surface of a semiconductor device fabricated using complementary metal-oxide-semiconductor (CMOS) microfabrication processes. Such a device may have dimensions (width and length) between 0.5 mm and 25 mm and a thickness between 0.05 mm and 1.5 mm. The surface of the CMOS substrate can be microfabricated using planar very large-scale integration (VLSI) processes to have an electrically insulating material, commonly referred to as a passivation layer. In some cases, the passivation layer on the CMOS substrate is optically transparent to allow the CMOS device to perform optical detection (e.g., optical detection of fluorescence signals). Example materials used for passivation layers are thin oxides, such as SiO2 and Si3N4.

[0131] The compositions, methods, and systems described herein may be part of an optical biochip system. An optical biochip system may include a CMOS biosensor array that uses optical detection methods, such as reverse fluorescence transduction (IFT) (see, for example, U.S. Patent No. 9,133,504, and Hassibi, A et al., Nat. Biotechnol. 2018, 36(8), 738-745). Such an optical biochip system may be part of a system or used in molecular detection assays in life science research and diagnostics, such as applications using nucleic acid molecules as their detection targets or molecular recognition elements (e.g., probes). Some examples of such applications are nucleic acid amplification assays using polymerase chain reaction processes, affinity-based detection systems utilizing two-dimensional and addressable DNA microarrays, and DNA sequencing arrays incorporating solid-phase synthesis sequencing (SBS) methods.

[0132] like Figure 5 As shown, the assay kit 100 includes a housing 102 containing multiple chambers 108 for reagent or buffer solutions and sample processing. The chambers are arranged around a central syringe barrel 106, which is in fluid communication with and sealed to a valve assembly 110 including a valve body 111 by a gasket 104. The valve body 111 may include a cap 112, and the entire housing can be supported on a base 101. Figures 6A to 6C As shown in more detail below, the valve body 111 typically includes one or more channels or cavities (chambers 114), which may contain ports 115 on the front surface 111a of the valve body. Figure 6B ) and the screen 116 on the rear surface 111b of the valve body ( Figure 6C ).

[0133] Figures 7A to 7E Various examples of valve assemblies 110a-110e within the measurement kit 100 are shown. For example, Figure 7A The valve assembly includes a valve body 111a, a cap 112a, a filter membrane 117, a gasket 104, and a central syringe barrel 106. The valve body 111a contains a channel 114a that supports the filter membrane 116. Figures 7B to 7E This illustrates how valve assemblies 110b-110e can be modified to accommodate other shapes, dimensions, and orientations of valve bodies 111b-111e, which include channel 114b, filter membrane 117, and filter column 118. Filter membrane 117 and filter column 118 can be suitable separation materials as described herein, which can be used for binding and eluting nucleic acids. In some embodiments, such as Figure 7D As shown, the filter membrane 117 may include filter particles or beads 119. The filter particles or beads 119 have a larger surface area than the filter membrane 117 and can be designed to bind and elute high concentrations of nucleic acids.

[0134] Figures 7A to 7CThe valve assembly demonstrates how each cartridge can be adapted for one or more types of target lysis, any of which can be used in a corresponding sample cartridge. According to the embodiments described herein, each cartridge includes a filter material with surface and reagent chemistry properties. Figure 7A The illustrated valve assembly housing performs mechanical lysis of more difficult-to-handle targets and includes a filter membrane modified with DNA-binding ligands. Figure 7B and Figure 7C The illustrated valve assembly housing performs chemical lysis of viruses, free neural networks (NAs), or more vulnerable targets, and includes a filter column. In some embodiments, the valve assembly includes... Figure 7D and Figure 7E The modified filter shown preferably has glass particles or a modified glass fiber filter 119, and its size is designed to be fixed between the valve cover and the valve body. The modified glass fiber filter 119 utilizes glass beads on a modified glass fiber filter, which is suitable for the mechanical lysis of certain types of targets. In this embodiment, the modified filter 119 is formed of glass fiber and has a pore size of 0.7 μm. In contrast, Figure 7D The filter in this design utilizes a filter formed as a polymer membrane (i.e., PCTE) disk, suitable for mechanical lysis but not for chemical lysis. By employing a filter with a pore size of 0.7 µm, the filter is suitable for receiving glass beads of appropriate size for mechanical lysis. The use of glass fibers to form the filter facilitates affinity binding with free nucleic acids released through chemical lysis. Therefore, this filter is suitable for both mechanical and chemical lysis.

[0135] In some embodiments, the cartridge also includes one or more temperature-controlled channels or chambers, which in some embodiments may function as thermal circulation chambers. A plunger (not shown) can be operated to draw fluid into the syringe barrel 106, and rotation of the valve body 111 provides selective fluid communication between various reagent chambers 108 and channels, reaction chambers, mixing chambers, and optionally any temperature-controlled zones. Thus, the various reagent chambers 108, reaction chambers, matrix materials, and temperature-controlled chambers or channels are selectively fluidly communicated via the rotation of the plunger, and reagent movement (e.g., chamber loading or unloading) is operated by the "syringe" action of the plunger within the valve assembly.

[0136] Back Figures 7A to 7E , can Figure 5 Different valve assemblies 110 are used in the sample boxes. For example, Figure 7A The valve assembly is configured to perform mechanical lysis and is suitable for lysis of difficult-to-handle targets (e.g., certain bacteria, spores). Figure 7B and Figure 7CThe valve assembly shown is configured to perform chemical lysis and is suitable for lysis of less difficult-to-handle targets (e.g., viruses, free NA, some spores, some bacteria, and yeast). A universal valve assembly can perform mechanical and chemical lysis on all types of targets. In all such cartridges, the valve assembly includes syringe tubing 106, valve bodies 111a-111c, and valve caps 112a-112c. The capability of the valve assembly in a sample cartridge depends on the filter or separation material described herein, and the specific workflow sequence performed by the module's instrument interface. For example, valve assembly 110a ( Figure 7A The valve body is shaped to have a circular cavity to support a filter disc for filtering samples, and the cover has an ultrasonic processing dome feature that engages with the ultrasonic processing amplitude rod of the cassette receiving module for ultrasonic disintegration of the target. In contrast, valve assemblies 110b and 110c ( Figure 7B and Figure 7C The valve assembly has a valve body with an elliptical filter recess in which a filter (e.g., a glass fiber filter column) is received. This filter is configured to bind to nucleic acids optionally released from the target via chemical cleavage. A general-purpose valve assembly has a more similar design. Figure 7A The valve assembly 110a is designed with a cap featuring an ultrasonically treated dome and a valve body with a circular cavity for supporting a disc filter; however, this design utilizes a glass fiber filter as described herein. Utilizing DNA-binding ligands to form a filter with a filter material (such as modified glass fiber) facilitates affinity binding to free nucleic acids released from biological samples, optionally via chemical lysis.

[0137] Although the compositions and methods described herein are primarily referenced to the GENEXPERT® kit from Cepheid Inc. (Sunnyvale, Calif.) (see, for example, Figure 5This method is described herein, but it should be recognized that, in light of the teachings provided herein, it can be implemented on other box / microfluidic systems, including alternative box designs with valve assemblies involving multiple interface components, and box bodies with multiple chambers defined by multiple interface components to form a box, such as those described in Korean Application No. 102293717B1; boxes that utilize ultrasound to lyse cells in biological samples, such as those described in International Application No. WO2021 / 245390A1; boxes and systems that utilize electrowetting meshes for microdroplet manipulation and are configured to detect analytes of interest, such as those described in International Application No. WO2016 / 077341A2; and boxes and systems that facilitate the movement of nucleic acids from one chamber by opening a vent bag. The methods described herein can be applied to cartridges that move to the next chamber, such as those described in International Application WO2012 / 145730A2; assay systems including addressable nucleic acid probe arrays attached to a solid surface, such as those described in U.S. Patent No. 9,708,647; multiplex assay systems including multiple thermal cycling units such that individual chambers can be heated, cooled, and / or compressed to mix fluids within the chambers or to propel fluids from one chamber to another, such as those described in International Application WO2015 / 138343A1; and systems for rapid nucleic acid amplification facilitated by temperature cycling of flexible portions of the sample cartridge to enable nucleic acid amplification, such as those described in International Application WO2017 / 147085A1. Furthermore, it should be understood that the methods described herein can also be implemented in entirely different systems, including: isothermal nucleic acid amplification systems, digital RT-PCR, electrochemical PCR, lateral flow assay cartridges, electrochemical sensors, nucleic acid sequencing, CRISPR / Cas-based technologies, chemiluminescence, and nanoparticle-based colorimetric detection.

[0138] In some embodiments, the sample cartridge may include: a) a cartridge having a plurality of chambers defined therein, wherein the plurality of chambers are in fluid communication via a fluid path of the cartridge, and wherein at least one chamber is configured to receive a biological sample; b) a reaction vessel configured to amplify nucleic acids by thermal cycling; and c) a filter disposed in the fluid path between the plurality of chambers and the reaction vessel, wherein the filter comprises a solid support material as disclosed herein, and wherein the plurality of chambers and the reaction vessel independently contain affinity reagents and other reagents for releasing nucleic acids from the biological sample, and primers and probes for detecting the nucleic acids.

[0139] In other embodiments, the sample cartridge may include: a) a housing having a plurality of chambers, wherein the plurality of chambers includes a sample chamber configured to receive a biological sample; b) a reaction vessel fluidly connected to the plurality of chambers and configured for amplifying nucleic acids and detecting a variety of amplification products; c) a filter disposed in a fluid path between the plurality of chambers and the reaction vessel, wherein the filter comprises a solid carrier having a surface capable of binding an affinity reagent; d) an affinity reagent disposed in one of the plurality of chambers, the affinity reagent comprising a first portion interacting with the filter and a second portion interacting with nucleic acids from the biological sample; and e) primers and / or probes disposed in one or more of the plurality of chambers or the reaction vessel for detecting nucleic acids. In some embodiments, the sample cartridge is a biosensor cartridge, such as those described in U.S. Patent No. 11,833,503B2, wherein primers and / or probes are fixed to a surface (e.g., a biosensor surface) in one or more of the plurality of chambers or the reaction vessel for detecting nucleic acids. The solid carriers and affinity reagents used in the sample kits are as described herein.

[0140] The pyrolysis chamber may optionally contain a pyrolysis reagent selected from liquid-dissolving agents, chelating agents, buffers, and detergents. The pyrolysis chamber may also include a valve body and a valve cover, wherein the valve body and valve cover engage to define the pyrolysis chamber therebetween, and wherein a filter is held within the pyrolysis chamber and fixed between the valve body and valve cover.

[0141] The lysis chamber has a fluid flow path between an inlet in the cap and an outlet in the valve body, the outlet being fluidly coupled to a fluid displacement region of the valve body. This fluid displacement region can be depressurized by movement of the syringe to draw fluid into the fluid displacement region and pressurized by movement of the syringe to expel fluid from the fluid displacement region. The sample cartridge, together with the reagents, allows flow rates up to approximately 100 µL per second, such as approximately 10 µL to approximately 100 µL. The sample cartridge, together with the reagents, allows pressures below 100 psi, below 80 psi, or below 60 psi. The sample cartridge allows sample volumes up to 1000 µL, such as 300 µL to 1000 µL.

[0142] The cassette may also include, for example, ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducers within the lysis chamber to facilitate mechanical lysis. The sample cassette may also include a syringe movable to facilitate fluid inflow and outflow from the lysis chamber via pressure fluctuations.

[0143] The box can be a single-use, disposable box. In some implementations, the box is an automated box.

[0144] To increase detection sensitivity, large sample volumes can be prepared. However, as described herein, large-volume preparation contradicts the requirements of microfluidic systems for automated lysis, processing, and / or analysis of biological samples. Therefore, a solution is needed that allows for the preparation of large sample volumes via, for example, filtration, and enables the isolated nucleic acids to be used in small volumes within the microfluidic system via a microfluidic interface. Sample cartridges comprising filters with surfaces capable of binding to affinity reagents and the methods described herein are used to address this need. This cartridge and method allow for the detection of target nucleic acids (e.g., DNA) from a wide variety of sample types, including whole blood, plasma, serum, semen, cerebrospinal fluid, tissue, tears, urine, feces, saliva, respiratory samples, nasopharyngeal samples, vaginal swabs, vaginal mucus samples, vaginal tissue samples, vaginal cell samples, bacterial cultures, mammalian cell cultures, viral cultures, human cells, bacteria, extracellular fluid, pancreatic juice, cell lysates, PCR reaction mixtures, or in vitro nucleic acid modification reaction mixtures, without requiring excessive sample processing steps from the user.

[0145] Methods for processing large-volume samples include introducing the biological sample into a sample cassette. In some cases, the sample may be mixed with reagents before being introduced into the cassette to break down particles present within the sample. However, samples introduced into the cassette may also be broken down within the cassette only during processing.

[0146] To handle large volumes, the volume of the sample chamber and / or lysis chamber within the sample cassette can be, in particular, at least 300 µL, at least 500 µL, at least 1,000 µL, at least 1,500 µL, at least 2,000 µL, at least 2,500 µL, at least 3,000 µL, at least 3,500 µL, at least 4,000 µL, at least 4,500 µL, at least 5,000 µL, at least 5,500 µL, at least 6,000 µL, at least 6,500 µL, at least 7,000 µL, at least 7,500 µL, at least 8,000 µL, at least 8,500 µL, at least 9,000 µL, at least 9,500 µL, or at least 10,000 µL. Preferably, the sample cassette can purify and process nucleic acids from liquid samples with volumes up to 10,000 µL, such as volumes of 300 µL to 5,000 µL, 300 µL to 3,000 µL, 300 µL to 2,000 µL, or 300 µL to 1,000 µL.

[0147] In some cases, biological samples in a sample cartridge may be disrupted using a lysis buffer (i.e., a solution). In other cases, biological samples are disrupted before being introduced into the sample cartridge. Typically, it is desirable to treat the samples with enzymes (such as lysozyme, proteinase K) and / or leukopenic agents before mixing them with a chemical lysis buffer. Furthermore, it may be desirable to have more than one lysis buffer and more than one wash buffer. Similarly, aspects of the instrument that can be used to improve the efficiency of extraction and purification of large sample volumes are not shown. For example: 1) a pre-filter for capturing particulate matter in the sample before or after lysis, but before sending the lysate to a modified nucleic acid-binding filter; 2) elements involving heating the sample during / before lysis; 3) elements involving sonicating or shearing the sample during lysis; 4) elements involving delivering heated or dehumidified air to the nucleic acid-binding matrix to improve drying; and similar features not shown but assumed to be included to improve the overall performance of the instrument. In some embodiments, multiple rounds of sample aspiration may be performed, followed by directing the “filtered” sample to waste until all samples or sample containers are empty.

[0148] method

[0149] Nucleic acid isolation

[0150] This document also provides methods for isolating and purifying nucleic acids from samples containing nucleic acids using the compositions disclosed herein. Samples containing nucleic acids can be selected from blood, plasma, serum, semen, vaginal swabs, vaginal mucus samples, vaginal tissue samples, vaginal cell samples, cerebrospinal fluid, tissue, tears, urine, feces, saliva, smear preparations, bacterial cultures, mammalian cell cultures, viral cultures, human cells, bacteria, extracellular fluid, PCR reaction mixtures, paraffin-embedded tissue samples, cell lysates, or in vitro nucleic acid modification reaction mixtures. In specific embodiments, the sample containing nucleic acids (biological sample) can be a fixed paraffin-embedded sample (e.g., from an FFPET sample), which can be used to identify the presence and / or expression level of a gene, and / or the mutational state of a gene. In some embodiments, the sample containing nucleic acids can be a liquid biopsy sample for detecting cancers such as prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, esophageal cancer, ovarian cancer, bile duct cancer, gastric cancer, and liver cancer. In some embodiments, the sample containing nucleic acids can be a respiratory sample for detecting infectious diseases. Samples containing nucleic acids may include human, bacterial, fungal, animal, or plant material. In other embodiments, samples containing nucleic acids may be obtained from nucleic acid modification reactions or nucleic acid synthesis reactions.

[0151] Nucleic acids encompass any synthetic or naturally occurring nucleic acid, such as DNA or RNA, in any possible conformation, i.e., double-stranded, single-stranded, aptamer, or any combination thereof. Nucleic acids can be DNA, including dsDNA, ssDNA, and their hybrids. Nucleic acids can also be RNA, such as mRNA, non-coding RNA, total RNA, etc. Nucleic acids can be synthetic nucleic acids. In some embodiments, the method described herein for isolating nucleic acids is well-suited for use in diagnostic methods, prognostic methods, methods for monitoring treatment (e.g., cancer treatment), etc. Therefore, target nucleic acids may include genomic DNA, total RNA, short DNA, small DNA, tumor-derived nucleic acids (including circulating tumor DNA), methylated DNA, microbial nucleic acids, bacterial nucleic acids, viral nucleic acids, cell-free nucleic acids, or combinations thereof. In some embodiments, nucleic acids isolated using the method described herein are used to detect the presence and / or copy number and / or expression level and / or mutation status of one or more cancer biomarkers.

[0152] Methods for isolating nucleic acids from a sample containing nucleic acids may include (a) contacting a solid carrier with the sample and an affinity reagent, wherein the affinity reagent comprises a first portion that interacts with the solid carrier and a second portion that interacts with the nucleic acids from the sample, and (b) concentrating the nucleic acids onto the solid carrier. In some embodiments, the sample is a biological sample that is contacted with a lysis solution prior to contact with the solid carrier, thereby lysing the cells contained in the biological sample and releasing the nucleic acids into the solution.

[0153] When used, the lysis solution may contain a dissociative agent, such as guanidine thiocyanate, guanidine hydrochloride, alkali metal perchlorate, alkali metal iodide, urea, formamide, and combinations thereof. In some embodiments, the lysis solution may contain a salt, such as sodium chloride or calcium chloride. In some instances, the lysis buffer contains one or more of a dissociative agent, salt, buffer, surfactant, defoamer, or combinations thereof. A sample can be lysed by contacting the sample with the lysis buffer before contacting it with a solid carrier and subsequently precipitating the nucleic acid. In some cases, the lysis solution contains one or more proteases. Suitable proteases include, but are not limited to, serine proteases, threonine proteases, cysteine ​​proteases, aspartic proteases, metalloproteinases, glutamate proteases, metalloproteinases, and combinations thereof. Illustrative suitable proteases include, but are not limited to, proteinase K (a broad-spectrum serine protease), subtilisin, trypsin, chymotrypsin, pepsin, papain, etc. Other proteases will be readily available to those skilled in the art using the teachings and examples provided herein.

[0154] In some embodiments, the methods disclosed herein do not require the use of a dissociation agent or high salt concentrations to lyse nucleic acid-containing samples. In some embodiments, the methods disclosed herein may require lower concentrations of dissociation agents or salts for lysing nucleic acid-containing samples compared to conventional lysis assays. For example, the dissociation agent may be used at concentrations less than 4.5 M, less than 2 M, or less than 1 M. In some embodiments, the methods disclosed herein do not require the use of a lysis buffer containing a dissociation agent.

[0155] Methods for isolating nucleic acids may also include filtering, centrifuging, precipitating, and / or washing the nucleic acids prior to elution to concentrate them. Conventionally, after nucleic acid lysis, the lysate is filtered onto a solid support in the presence of a binding agent (such as PEG) to bind the nucleic acids to the solid support. The binding agent may include one or more of the following: alcohols (e.g., methanol, ethanol, propanol, isopropanol); alkyldiols or alkyltriols having 2 to 6 carbon atoms; monocarboxylic acid esters or dicarboxylic acid diesters having 2 to 6 carbon atoms in the acidic component and 1 to 4 carbon atoms in the alcoholic component; (poly)ethylene glycol and its ether and ester derivatives; and poly(4-styrenesulfonic acid-co-maleic acid). For example, the binder may include one or more of the following: 1,2-butanediol, 1,2-propanediol, 1,3-butanediol, 1-methoxy-2-propanol acetate, 3-methyl-1,3,5-pentanetriol, DBE-2, DBE-3, DBE-4, DBE-5, DBE-6, diethylene glycol monoethyl ether (DGME), triethylene glycol monoethyl ether (TGME), diethylene glycol monoethyl ether acetate (DGMEA), ethyl lactate, ethylene glycol, poly(2-ethyl-2-oxazoline), sodium poly(4-styrenesulfonic acid-co-maleic acid), tetraethylene glycol (TEG), tetraethylene glycol, tetrahydrofurfuryl polyethylene glycol 200, tri(ethylene glycol) divinyl ether, anhydrous triethylene glycol, and triethylene glycol monoethyl ether. In some embodiments of the methods disclosed herein, no binder is required, or a lower concentration of binder can be used compared to conventional assays. For example, binding agents (such as PEG) can be used at concentrations of less than 40% v / v, less than 30% v / v, less than 20% v / v, or less than 10% v / v for filtration and / or washing. Solid carriers, together with the affinity reagents described herein, allow for the selective capture of nucleic acids (RNA and DNA) from biological matrices. In practice, solid carriers, together with affinity reagents, can capture free circulating nucleic acids as well as nucleic acids from cells without the use of lysis buffers, salts, or binding agents, or with very low concentrations of lysis buffers, salts, or binding agents. It is important to note that the inventions described herein cover the capture of complex genomic DNA or RNA from a variety of organisms in biological samples. For example, modified glass microscope slides are commonly used to fix DNA or RNA for microarray imaging. Typically, these flat, modified surfaces have a very low surface area and are not suitable for isolating DNA or RNA from large, complex samples.

[0156] Concentrated nucleic acids can optionally be washed on a solid carrier, for example, to remove components of the lysis buffer or unwanted components from the biological sample. Concentrated (bonded) nucleic acids can be washed in a buffer compatible with PCR reactions.

[0157] Nucleic acids are then eluted from the solid support using an elution buffer. Elution of nucleic acids from the solid support can be achieved by gradually or in a gradient increasing of the pH of the mobile phase or eluent. In some embodiments, the bonded nucleic acids can be eluted from the solid support by contact with an alkaline solution. The alkaline solution may contain ammonia or an alkali metal hydroxide, ammonium hydroxide, NaOH, or KOH at a concentration sufficient to disrupt the binding of the nucleic acids to compounds on the solid support. In some embodiments, the eluent has an alkaline pH. In some embodiments, the eluent has a pH greater than about 9, greater than about 10, greater than about 11, 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 higher than 10. Exemplary eluents contain 1% or more ammonia, 15 mM or more KOH (e.g., 25 mM KOH, 35 mM KOH, 40 mM KOH, or 50 mM KOH), or 15 mM or more NaOH (e.g., 25 mM NaOH, 35 mM NaOH, 40 mM NaOH, or 50 mM NaOH). As described herein, the use of high pH to elute nucleic acids (such as DNA) is particularly unique for the cassette described herein and provides improved speed and performance of the disclosed method. Speed ​​is provided by the rapid neutralization of acidic ammonium ions by a high concentration of hydroxide ions. Another advantage of high pH is the denaturing effect of KOH on captured DNA or RNA. Double-stranded structures and other secondary structures are disrupted but can be renatured upon neutralization, for example, with Tris HCl. The cassette described herein allows for the rapid neutralization of DNA or RNA eluted in KOH / NaOH. Tris reagents can be provided separately (e.g., in bead form) to react immediately with KOH / NaOH elution buffer to produce a final pH of approximately 8.5 for downstream PCR or other nucleic acid assays. In some embodiments, the elution buffer has a pH less than approximately 9, less than approximately 8.5, or less than approximately 8.

[0158] In some embodiments, the eluent comprises a polyanion. A polyanion is typically a polymer containing multiple anionic groups. In some embodiments, the anionic groups are phosphate, phosphonate, sulfate, or sulfonate groups, or combinations thereof. In some embodiments, the polyanion is a negatively charged polymer at a pH above about 7. Both synthetic and naturally occurring polyanions can 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, a carrier nucleic acid is a nucleic acid that does not interfere with subsequent detection of the concentrated nucleic acid (e.g., by PCR). Exemplary carrier nucleic acids include polyrA, polydA, herring sperm DNA, salmon sperm DNA, and other carrier nucleic acids well known to those skilled in the art. In some embodiments, the eluent comprises carrageenan and an alkali metal hydroxide, such as NaOH or KOH.

[0159] In general, nucleic acid chains, including DNA and RNA, are readily captured on the surface of solid supports by affinity reagents and impurities are washed away at pH 5 or higher. For affinity reagents containing alkylamines, nucleic acids can be effectively eluted with high pH buffers (8.5–12.5) or with at least 50 mM KOH, as demonstrated by the PCR assays described herein.

[0160] For affinity reagents with photolyzable groups, nucleic acids can be released optionally via photochemical or thermal cleavage of the adduct. In some cases, methods for isolating nucleic acids include eluting them from a solid carrier in the presence of an eluent, including heating concentrated (bonded) nucleic acids to 100°C. °C Or lower, 95 °C Or lower, 85 °C Or lower, 75 °C Or lower, 65 °C Or lower, 55 °C Or at lower temperatures; sonication of nucleic acids; photochemical cleavage of compounds; or combinations thereof.

[0161] The nucleic acids isolated using the methods and compositions described herein possess suitable quality for amplification to detect and / or quantify one or more target nucleic acid sequences in a sample. In fact, the nucleic acid isolation methods and compositions described herein are suitable for basic research aimed at discovering gene expression profiles relevant to disease diagnosis and prognosis. This method is also applicable to disease diagnosis and / or prognosis, the determination of specific treatment regimens, and / or the monitoring of treatment effectiveness.

[0162] Nucleic acid testing

[0163] The methods described herein simplify the isolation of nucleic acids from biological samples and efficiently produce nucleic acids highly suitable for isolation using RT-PCR systems. In some embodiments, nucleic acids isolated from samples containing nucleic acids using the methods described herein can be detected by any suitable known nucleic acid detection method. Therefore, methods for detecting nucleic acids in biological samples are disclosed. Detection methods may include nucleic acid amplification. In some embodiments, methods for detecting nucleic acids may include combining the eluent with PCR reagents, which may be present in a lyophilized granule in a cassette, after releasing and eluting the nucleic acids from a solid carrier with an eluent. In some embodiments, PCR uses a Taq polymerase with hot-start capability, such as AptaTaq (Roche, Switzerland). The polymerase chain reaction may be nested PCR, isothermal PCR, gradient PCR, qPCR, reverse transcriptase PCR, real-time PCR, multiplex PCR, nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), rolling circle amplification (RCA), or strand displacement amplification (SDA).

[0164] In some embodiments, a method for detecting nucleic acids in a biological sample obtained from a subject may include placing the biological sample in a housing as described herein, wherein the housing includes a plurality of fluidly connected chambers, a reaction tube configured for amplifying nucleic acids by thermal cycling, and a filter in a fluid path between the plurality of chambers and the reaction tube; lysing cells with a lysis reagent present in at least one of the plurality of chambers and capturing DNA released therefrom; and amplifying the DNA with primers and probes to detect the presence of nucleic acids.

[0165] In some implementations, target nucleic acids such as coronaviruses, such as alpha-coronavirus, beta-coronavirus, or SARS-CoV-2, adenovirus, Chlamydia pneumoniae, influenza A virus, influenza B virus, metapneumovirus, rhinovirus / enterovirus, mycoplasma, Bordetella spp., parainfluenza virus and respiratory syncytial virus (RSV), Hantavirus, cytomegalovirus, Coxsackievirus, herpes simplex virus, echovirus, influenza C virus, Streptococcus pneumoniae, Chlamydia pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus parainfluenzae, Group A Streptococcus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas spp., Neisseria spp., Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Candida spp., Aspergillus spp., Mucor spp., Cryptococcus neoformans, or Pneumocystis carinii biomarkers and / or optional controls can be detected. The target nucleic acid can be detected by: (a) contacting a nucleic acid from a sample with a set of primers and optional probes to detect the presence of the desired target nucleic acid; (b) subjecting the nucleic acid, primers, and optional probes to amplification conditions; (c) optionally detecting the presence of any amplification product via real-time PCR, melting curve analysis, or a combination thereof; and (d) optionally identifying the presence of the target nucleic acid in the sample based on the detection of or absence of the amplification product.

[0166] In some embodiments, the amplification method includes initial denaturation at about 90°C to about 100°C for about 1 to about 10 minutes, followed by cycling, which includes 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, the cyclic denaturation step is omitted for the first cycle after initial denaturation. The specific time and temperature will depend on the specific nucleic acid sequence being amplified and can be readily determined by those skilled in the art.

[0167] In some embodiments, nucleic acid isolation and detection are performed in an automated sample processing and / or analysis platform. In some embodiments, commercially available automated analysis platforms are utilized. For example, in some embodiments, the GeneXpert system (Cepheid, Sunnyvale, Calif.) is used. However, the present invention is not limited to a specific detection method or analysis platform. Those skilled in the art will recognize that any number of platforms and methods can be used. The GeneXpert system utilizes an integrated, single-use kit. Nucleic acid sample extraction, amplification, and detection can all be performed within this integrated "lab-in-a-kit."

[0168] Examples of other methods that can be used with the methods described herein include bead-based flow cytometry assays. See Lu J. et al. (2005) Nature 435:834-838, which is incorporated herein by reference and used in this description. An example of bead-based flow cytometry assays is the xMAP® technology of Luminex, Inc. See www.luminexcorp.com / technology / index.html. Another method uses microfluidic devices and single-molecule detection. See U.S. Patents 7,402,422 and 7,351,538 to Fuchs et al., US Genomics, Inc., each of which is incorporated herein by reference in its entirety. Yet another method is simple gel electrophoresis and detection with labeled probes (e.g., probes labeled with radioactive or chemiluminescent markers), such as via northern blotting.

[0169] While in some implementations the extracted nucleic acids are used for amplification reactions, other uses are also considered. Therefore, for example, the isolated nucleic acids or their amplification products can be used in a variety of sequencing or hybridization protocols, including but not limited to nucleic acid-based microarrays and next-generation sequencing.

[0170] Easy-to-automate methods are of interest. The methods described herein can be performed in a substantially automated manner using commercially available nucleic acid amplification systems. Exemplary non-limiting nucleic acid amplification systems that can be used to implement the methods of the present invention include the GENEXPERT® system, the GENEXPERT® Infinity system, and the GENEXPERT® Xpress system (Cepheid, Sunnyvale, Calif.). In some embodiments, the amplification system may be located in the same location as the individual being tested, such as the office, clinic, or community hospital of a healthcare provider, so that processing is not delayed by transporting the sample to another facility. Assays according to the methods described herein can be completed in 3 hours using automated systems (e.g., the GENEXPERT® system), in some embodiments in 2 hours, in some embodiments in 1 hour, in some embodiments in 45 minutes, in some embodiments in 35 minutes, and in some embodiments in 30 minutes. GENEXPERT® utilizes an integrated, single-use cartridge. Sample extraction, amplification, and detection can all be performed within this integrated cartridge as described herein.

[0171] Before amplification, the sample undergoes one or more sample preparation operations. These operations typically include processes such as extracting intracellular material (e.g., nucleic acids from whole-cell samples, viruses, etc.) to form a crude extract, and additional treatments to prepare the sample for subsequent operations (e.g., denaturation, purification, filtration, desalting, etc.) of contaminating (e.g., DNA-binding) proteins. The release of nucleic acids from sample cells or viruses and the denaturation of DNA-binding proteins can generally be performed using chemical, physical, or electrolytic lysis methods. For example, chemical methods typically employ a lysis agent to disrupt cells and extract nucleic acids from them, followed by treatment of the extract with a dissociative salt (such as guanidine isothiocyanate or urea) to denature any contaminating and potentially interfering proteins. Typically, when using chemical extraction and / or denaturation methods, appropriate reagents can be incorporated into the sample preparation chamber, a separate accessible chamber, or introduced externally. Preferably, sample preparation is performed in only one or no more than two steps. As described herein, this method simplifies the isolation of nucleic acids from biological samples and efficiently produces nucleic acids highly suitable for isolation using RT-PCR systems.

[0172] The method for detecting nucleic acids described herein can be implemented without transporting samples from the collection site. For example, the method can be performed at a P°C diagnostic site. P°C diagnostic sites include patient care environments, preferably hospitals, emergency care centers, emergency rooms, doctors' offices, health clinics, or homes. In some cases, the presence or absence of nucleic acids in a biological sample can be detected within 75 minutes or 60 minutes of sample collection from the subject.

[0173] Distinguishing between infectious and non-infectious (or live and dead) nucleic acids

[0174] Certain aspects of this disclosure generally relate to compositions, apparatuses, and methods for identifying viruses, such as coronaviruses. For example, some aspects relate to using the compositions, apparatuses, and methods disclosed herein to identify viruses. In the compositions and apparatus disclosed herein (comprising a solid carrier having an affinity reagent, wherein the affinity reagent comprises a first portion interacting with the solid carrier and a second portion interacting with nucleic acids), free RNA or other nucleic acids may be preferentially dispensed onto the solid carrier, while intact viruses may be present in solution (such as in a filtrate or wash buffer) or simultaneously in solution and on the solid carrier. Therefore, for example, free RNA or other nucleic acids may be preferentially removed compared to intact RNA or other nucleic acids present within the virus. In some cases, the solution phase containing intact viruses can be analyzed to determine, for example, the infectivity of a sample from a subject. This can be useful, for example, to distinguish between subjects capable of transmitting infection and those that are not infectious.

[0175] One aspect of this disclosure relates to the detection of whole viral particles in a biological sample containing the whole viral particles, other viral components (such as RNA (or other nucleic acids)) identified as belonging to the virus by a molecular detection system (e.g., RT-PCR), and other biological or non-biological components not identified as belonging to the virus by the molecular detection system. Some embodiments include techniques for positive molecular detection that distinguishes between whole viral particles and viral components using the same molecular detection system. This can provide the identification of samples that are capable of further infection and those that are not, which can, for example, be used in clinical isolation of patients who can transmit the infection and those who are ill but not infectious.

[0176] Some implementations rely on the spatial separation of whole viral particles from other components of the same virus, such as RNA. Specifically, free nucleic acids (such as RNA (e.g., not contained within the virus)) can be preferentially partitioned onto a solid carrier, for example, due to their net negative charge, along with an affinity reagent, while whole viral particles can be partitioned into a solution phase within the compositions and apparatus described herein, such as in the filtrate (or wash solution) of an aqueous buffer system used to bind free nucleic acids to a solid carrier. In some cases, if a sample contains both free RNA from dead virus and fully functional virions capable of further infecting, the sample can be detected by a simple molecular test on a phase that is substantially devoid of free RNA.

[0177] In one set of embodiments, the whole virus can be simultaneously distributed in solution and on a solid carrier. However, in other embodiments, the whole virus may be selectively or preferentially partitioned into the solution phase of, for example, the compositions and devices described herein. For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the whole virus may be found in the solution phase of the compositions and devices described herein. Additionally, in some embodiments, free RNA (or other nucleic acids) is preferentially partitioned onto the solid carrier of the compositions and devices described herein. For example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or all of the free RNA (or other nucleic acids) of the free nucleic acid molecule may be present on the solid carrier of the compositions and devices described herein.

[0178] In some embodiments, the determination of the solution phase (and / or nucleic acid on a solid carrier) can be used to determine the presence of nucleic acid. Examples of such determinations include, but are not limited to, RT / PCR or other detection techniques (e.g., sequencing) known to those skilled in the art. In some embodiments, aliquots of the solution phase of the compositions and devices described herein can be removed for analysis, although in other embodiments, the solution phase can be determined in situ within the device (such as a sample cartridge), i.e., it is not necessary to remove aliquots of the solution phase for analysis.

[0179] Examples of viruses that can be determined using the compositions, apparatus, and methods disclosed herein include, but are not limited to, coronaviruses, influenza viruses, or other viruses such as those described herein. In one set of embodiments, sample kits, such as those described herein, are used. Samples (e.g., biofluid samples collected from subjects) can be analyzed to determine the presence of a particular species of virus (e.g., SARS, MERS, COVID-19, etc.) and / or the presence of a particular type of virus (e.g., coronavirus). Biofluids can also be collected from subjects. In some cases, biofluids can be processed for further use. In some embodiments, a specific virus can be determined in the biofluid. Additionally, in some cases, different types of viruses can be distinguished from each other (e.g., coronavirus versus influenza virus).

[0180] Other non-limiting examples of viruses include infectious viruses such as coronaviruses or influenza viruses. Other non-limiting examples include adenoviruses, Coxsackieviruses, Epstein-Barr virus, hepatitis viruses (A, B, and C), herpes simplex virus (types 1 and 2), cytomegalovirus, herpesvirus (type 8), HIV, measles virus, mumps virus, papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, varicella-zoster virus, etc.

[0181] In some embodiments, coronaviruses can be identified. Examples of coronaviruses include, but are not limited to, HCoV-229E, HCoV-0C43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, or SARS-CoV-2. In some cases, one or more proteins of a coronavirus can be used to identify the virus, for example, by interaction with a binding moiety capable of binding to a protein or a target species. For example, in some cases, certain immunochemical assays, such as ELISA, can be used. Other assay methods can be used to determine the species within the solution phase or solid carrier of the device, typically depending on the identity and type of the present virus or other virus. Examples of other suitable assay techniques include, but are not limited to, spectroscopic, chemical, fluorescence, radiometric, and enzymatic assays. In some cases, a virus can be identified by identifying a virus-associated peptide or protein. Non-limiting examples include envelope particles, envelope proteins, membrane proteins, nucleocapsids, spike proteins, or hemagglutinins or neuraminidases. For example, suitable antibodies capable of interacting with such peptides or proteins can be used to identify such peptides or proteins. For example, in some cases, certain immunochemical assays, such as ELISA, can be used. Furthermore, other peptide or protein detection techniques can be used in some embodiments. These include, but are not limited to, direct spectrophotometry (e.g., monitoring absorbance at 280 nm) and dye-binding reactions with Coomassie Blue G-250 or fluorescein, phthalaldehyde, or other dyes and / or reagents. Free nucleic acids can also be determined using sequencing and other techniques, such as those described herein.

[0182] In some implementations, influenza viruses can be detected. Influenza viruses include genera such as influenza A virus, influenza B virus, influenza C virus, influenza D virus, salmon anemia virus, Togovirus, and Kwalanfair virus. Examples of influenza A viruses include, but are not limited to, H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, and H1ON7.

[0183] Examples of influenza B viruses include, but are not limited to, Victoria and Yamagata. In some cases, one or more proteins of the influenza virus can be used to identify the virus, for example, through interactions with binding sites or target species. Non-limiting examples of such viral proteins include hemagglutinin, neuraminidase, membrane proteins, glycoproteins, nucleocapsids, etc. Additionally, in some cases, the viral nucleus (e.g., RNA) can be identified, for example, through interactions with viral binding sites or target species.

[0184] Biological fluid samples may include fluids as disclosed herein, such as whole blood, serum, plasma, saliva, nasal secretions, sputum, urine, CNS fluid, nipple aspiration fluid, cerebrospinal fluid, semen, etc. In some cases, the subject is a suspected viral infection. For example, the subject may have been previously exposed to a person with the virus, or may be at least suspected of potentially having the virus. Furthermore, in some embodiments, the subject may not be suspected of potentially having the virus (e.g., fluids may be collected during routine screening).

[0185] In one respect, sample cassettes such as those discussed herein can be used to dispense free nucleic acids that may be present in a sample, such as those derived from dead or damaged virosomal particles. For example, sample cassettes can be used to isolate or concentrate free nucleic acids, such as DNA or RNA, for example, onto a solid carrier of the sample cassette. Additionally, in some cases, the amount or concentration of free nucleic acids in a sample can be determined and optionally compared with RNA (or other nucleic acids) present within a whole virus. The type, amount, and / or concentration of RNA (or other nucleic acids) within one or more phases (solid carrier or solution phase) can be determined using any suitable technique, such as via polymerase chain reaction (PCR), sequencing, and / or other assay techniques, including those described herein. The techniques used to determine whole virus and free nucleic acids can be the same or different. As a non-limiting example, as discussed herein, whole virus may be dispensed into a solution phase, while RNA (or other nucleic acids) not present within the whole virus may be preferentially dispensed onto a solid carrier.

[0186] In some implementations, the techniques used to determine intact viruses and cell-free nucleic acids (such as those described herein) can be correlated with disease severity, disease stage, infectivity of the subject, etc. As a non-limiting example, samples can be dispensed between a solid carrier and a solution phase in a sample cartridge such as those described herein. After equilibration, aliquots of the solution phase can be taken and processed in a manner such as to release any intact viruses present into the solution. However, in some cases, the virus can be determined in situ within the sample cartridge itself, i.e., it is not necessary to remove aliquots of one or more phases to be analyzed from the sample cartridge. Nucleic acids in the solution phase can be determined, for example, using PCR or other sequencing techniques. In some cases, the amount of a specific nucleic acid sequence (e.g., viral RNA) in the solution phase can be determined and used to determine the presence of intact viruses in the sample. This method can be particularly useful because the solid carrier of the sample cartridge has selective preference for cell-free nucleic acids, e.g., such that at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or all of the cell-free nucleic acids are present on the solid carrier. In some implementations, the differences can be quantified and used to determine the relative amount or concentration of the complete virus present in the sample.

[0187] Such systems can be used, for example, to distinguish between positive cases (e.g., determined by the presence of RNA or other free nucleic acids) and cases of positive infection (e.g., determined using free nucleic acids plus intact virus). This is possible, for example, because one phase (solution phase) may contain nucleic acids from the intact virus, while another phase (solid carrier) may also contain free RNA or other nucleic acids (e.g., DNA). Therefore, in some embodiments, measuring the intact virus in the solution phase and the nucleic acids on the solid carrier can advantageously indicate positivity and infectivity.

[0188] As discussed herein, solid carriers, together with affinity reagents, can be used in various aspects of this disclosure to identify one or more viruses (or other species). Generally, the properties of solid carriers, together with affinity reagents, that facilitate solute partitioning include parameters as described in more detail herein. However, since nucleic acids (such as RNA) are typically charged molecules, one parameter affecting their partitioning behavior between two phases is the difference in the ionic composition of the phases in the partitioning system, for example, via salts as additives and their distribution.

[0189] As mentioned herein, certain aspects of this disclosure generally relate to the study of viral states, although this disclosure is not limited to viruses. Other embodiments can be applied to substantially any molecular species and / or interactions, whether biological, biochemical, chemical, or other, and those skilled in the art will understand how this disclosure can be used in the context of other molecules. Therefore, it should be understood that whenever the term "virus" is used in the description herein, any other biological or non-biological molecule may also be used or studied in other embodiments. For example, a biological sample may comprise nucleic acid material from humans, bacteria, fungi, animals, or plants.

[0190] In some cases, viral distribution within the sample cassette can be surprisingly rapid. For example, in some cases, large-scale distribution can occur in less than 1 hour, less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute. In some cases, within these timeframes, sufficient distribution may have already occurred to allow for the identification of the virus and / or free nucleic acid, as discussed herein, for example, in one or more phases of the sample cassette.

[0191] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0192] Example

[0193] Example 1: CVD using alkylsilanes

[0194] Instrumentation: In this embodiment, a Lindberg / Blue M vacuum oven (Thermo Scientific) was used. The vacuum port of the vacuum oven was connected to the Hi-Vac trap assembly (Ace glass 8775-16), the vacuum gauge (Ace glass 14034-36), and then to the vacuum pump (Edwards RV12). The vacuum oven was installed inside a fume hood.

[0195] Materials: In this embodiment, a 1x3” silicon substrate with a 300 nm SiO2 layer on the top surface was used. High-purity chemical reagents from n-decyltriethoxysilane (Gelest SID2665.0), n-octyltrimethoxysilane (Gelest SIIO6715.5), or hexyltriethoxysilane (Gelest SIH6167.5) were used.

[0196] CVD Process: The vacuum oven was heated to a stable 120°C. A 1x3” Si substrate was then loaded onto a perforated rack in the oven, and 0.6 ml of chemical reagent was dispensed into an Al dish and placed at the bottom of the oven chamber. The oven chamber was immediately evacuated from atmospheric pressure. When the vacuum gauge showed a pressure less than 15 Torr, the vacuum valve (between the trap and the oven chamber) was closed. The process was allowed to stand for 90 min, then the chamber was evacuated to 1 Torr and subsequently degassed to atmospheric pressure. The treated Si wafer was removed from the chamber and cooled to room temperature. Due to surface modification with alkyl functional groups, a hydrophobic surface was expected on the treated wafer.

[0197] Example 2: Co-CVD using azidosilanes and alkylsilanes

[0198] Instrumentation: In this embodiment, a Lindberg / Blue M vacuum oven (Thermo Scientific) was used. The vacuum port of the vacuum oven was connected to the Hi-Vac trap assembly (Ace glass 8775-16), the vacuum gauge (Ace glass 14034-36), and then to the vacuum pump (Edwards RV12). The vacuum oven was installed inside a fume hood.

[0199] Materials: In this embodiment, a 1x3” silicon substrate with a 300 nm SiO2 layer on the top surface was used. High-purity chemical reagents from n-decyltriethoxysilane (Gelest SID2665.0) and 3-azidopropyltriethoxysilane (Gelest SIA0777.0) were used.

[0200] CVD Process: The vacuum oven was heated to a stable 120°C. A 1x3” Si substrate was then loaded onto a perforated rack within the oven. 0.6 ml of 3-azidopropyltriethoxysilane was dispensed into an Al dish and placed at the bottom of the oven chamber. The oven chamber was immediately evacuated from atmospheric pressure. When the vacuum gauge showed a pressure less than 2.7 Torr, the vacuum valve (between the trap and the oven chamber) was closed. This process was allowed to stand for 90 min, then the chamber was evacuated to 1 Torr and exhausted to atmospheric pressure. Next, another 0.6 ml of fresh Si substrate was added… 3-Azide-propyltriethoxysilane was dispensed into an Al dish and placed at the bottom of the oven chamber. The oven chamber was immediately evacuated from atmospheric pressure. When the vacuum pressure displayed on the vacuum gauge was less than 2.7 Torr, the vacuum valve (between the trap and the oven chamber) was closed. This process was allowed to stand for 90 min, then the chamber was evacuated to 1 Torr and exhausted to atmospheric pressure. Finally, 0.6 ml of n-decyltriethoxysilane was dispensed into an Al dish and placed at the bottom of the oven chamber. The oven chamber was immediately evacuated from atmospheric pressure. When the vacuum pressure displayed on the vacuum gauge was less than 2.7 Torr, the vacuum valve (between the trap and the oven chamber) was closed. This process was allowed to stand for 90 min, then the chamber was evacuated to 1 Torr and exhausted to atmospheric pressure. The treated Si wafer was removed from the chamber and cooled to room temperature. The treated surface was modified with both azide and alkyl functional groups.

[0201] Example 3: Continuous CVD using azidosilanes and alkylsilanes

[0202] Instrumentation: In this embodiment, a Lindberg / Blue M vacuum oven (Thermo Scientific) was used. The vacuum port of the vacuum oven was connected to the Hi-Vac trap assembly (Ace glass 8775-16), the vacuum gauge (Ace glass 14034-36), and then to the vacuum pump (Edwards RV12). The vacuum oven was installed inside a fume hood.

[0203] Materials: In this embodiment, a 1x3” silicon substrate with a 300 nm SiO2 layer on the top surface was used. High-purity chemical reagents from n-decyltriethoxysilane (Gelest SID2665.0) and 3-azidopropyltriethoxysilane (Gelest SIA0777.0) were used.

[0204] CVD Process: The vacuum oven was heated to a stable 120°C. A 1x3” Si substrate was then loaded onto a perforated rack in the oven, and 0.6 ml of a 1:1 mixture of 3-azidopropyltriethoxysilane and n-decyltriethoxysilane was dispensed into an Al dish and placed at the bottom of the oven chamber. The oven chamber was immediately evacuated from atmospheric pressure. When the vacuum gauge showed a pressure less than 2.7 Torr, the vacuum valve (between the trap and the oven chamber) was closed. This process was allowed to stand for 90 min, then the chamber was evacuated to 1 Torr and exhausted to atmospheric pressure. Next, another 0.6 ml of freshly mixed... A 1:1 ratio of 3-azidopropyltriethoxysilane and n-decyltriethoxysilane was dispensed into an Al dish and loaded at the bottom of the oven chamber. The oven chamber was immediately evacuated from atmospheric pressure. When the vacuum gauge showed a pressure less than 2.7 Torr, the vacuum valve (between the trap and the oven chamber) was closed. This process was allowed to stand for 90 min, then the chamber was evacuated to 1 Torr and exhausted to atmospheric pressure. The treated Si wafer was removed from the chamber and cooled to room temperature. The treated surface was modified with both azide and alkyl functional groups.

[0205] Example 4: Measurement of amine loading

[0206] The determination was performed essentially as described by Noel et al., Bioconjugate Chemistry, 2007. Orange II dye was adsorbed / desorbed onto the amine-functionalized surface at pH 3 and eluted at pH 10. The amount of dye bound to the filter was measured by obtaining absorbance at 484 nm on a Molecular Devices iD3 microplate reader. The amount of bound dye was proportional to the number of amine groups immobilized on the surface being measured.

[0207] Example 5: Retention of target analytes using hydrophobic carriers and soluble affinity reagents

[0208] A C8 filter (SPE C8-47mm, Restek) was punched into a smaller disc with a diameter of 5 / 16” and placed inside a centrifuge column (EconoSpin, Epoch Life Science). Aqueous solutions of butylamine (Sigma Aldrich) were prepared at concentrations of 1.5 mM and 15 mM. 500 µL of this solution was loaded onto the centrifuge column containing the C8 filter, and the column was centrifuged at 1000 rcf for 5 min in a microcentrifuge. Finally, Orange II binding was measured using the assay described in Example 4.

[0209] As shown below, using butylamine as an affinity reagent, the negatively charged orange II dye (used here as a substitute for DNA) was successfully captured and eluted from the hydrophobic C8 alkyl substrate.

[0210] 10-fold dilution of C8 eluent using 1.5 mM butylamine 0.272 0.0029 10-fold dilution of C8 eluent using 15 mM butylamine 0.289 0.0027

[0211] Example 6: Retention of DNA analytes using hydrophobic carriers and soluble affinity reagents

[0212] Filters (Restek SPE C8 #24048 and homemade C8-functionalized Whatman GF / F, Cytiva #1825-915) were cut to size and installed into empty centrifuge column housings. GF008 filters were plasma-cleaned before functionalization, while GF007 filters were not. Filters were then treated with 500 µL of pure water or 100% methanol by centrifuging the solution through the filter (2000 x g for 1 min for this and all subsequent centrifugations), and the flow-through was discarded. Three subsequent 500 µL washes with pure water were then performed. The filters were then treated with 500 µL of 15 mM butylamine pH 6.0 solution. 500 µL of λDNA (Thermo SD0011, 30 ng / µL in 15 mM butylamine pH 6.0) was then added and centrifuged through the filter, collecting the flow-through for analysis. The filter membrane was then washed three times with 500 µL of pure water or 15 mM butylamine at pH 6.0. The bound DNA was eluted by treating with 500 µL of water adjusted to pH 12.0 with NaOH. The eluent was collected for analysis. The DNA content of all flow-through and eluent solutions was determined using the Qubit 1x Broad Range DNA Kit (Thermo Q33266) according to the kit instructions. 10 µL of each sample was added to a final analysis volume of 200 µL.

[0213] The table below summarizes DNA binding and elution as measured by fluorescence assay. All values ​​are reported in ng / µL. SPE = Restek C8 filter, GF007 = C8-functionalized GF / F, GF008 = plasma-cleaned, C8-functionalized GF / F. An asterisk (*) indicates filter damage / breakage during assay.

[0214] water water SPE 28.4 0.579 water water GF007 < 0.2 2.48 water water GF008 < 0.2 8.29 methanol water SPE 8.43 0.627 methanol water GF007 < 0.2 3.72 methanol water GF008 < 0.2 6.96 water Butylamine SPE 25.5 1.55 water Butylamine GF007 3.17 9.13 water Butylamine GF008 < 0.2 18.2 methanol Butylamine SPE 26.7* < 0.2 methanol Butylamine GF007 < 0.2 13.3 methanol Butylamine GF008 < 0.2 18.2

[0215] Example 7: A modified glass fiber filter for selective detection of active viral infections with background nucleic acid removal.

[0216] Glass fiber filters (or other surfaces) can be modified with aminosilanes that have been shown to have an affinity for nucleic acids. Passing a swab sample through a glass filter with this amine modification (such as in a cartridge pre-filter) can remove any free nucleic acids from the sample. Viral particles have no affinity for the filter and will pass through unaffected. Therefore, the test will detect only viral particles and not residual RNA / DNA.

[0217] Figures 8A to 8E The glass fiber filter modified with diethylenetriaminosilane (GF / D from Cytiva; 5-micron pore size) can capture approximately 90% of COVID RNA (UTM containing CoV2 RNA from Exact Sciences) without capturing inactivated virus (Zepto Natrol from Zeptometrix).

[0218] Glass fiber filters modified with aminopropyltriethoxysilane (APTES) (GF / D from Cytiva; 5-micron pore size) were cut into discs for assembly into the pre-filters of the Cepheid GX box. Figures 8B to 8E The kit demonstrates that a 3-layer GFF material pre-filter can remove viral RNA from a sample (Copan UTM medium) without affecting the intact viral particles.

[0219] Example 8: Retention of target analytes using hydrophobic supports and branched-chain soluble affinity reagents

[0220] A C8 filter (SPE C8-47mm, Restek) was punched into a smaller disc with a diameter of 5 / 16” and placed inside a centrifuge column (EconoSpin, Epoch Life Science). Methanol solutions of 3-dibutylaminopropylamine (Sigma Aldrich) were prepared at concentrations of 1.5 mM and 15 mM. 500 µL of this solution was loaded onto the centrifuge column containing the C8 filter, and the column was centrifuged at 1000 rcf for 5 min in a microcentrifuge. Finally, OrangeII binding was measured using the assay described in Example 4.

[0221] As shown below, using butylamine as an affinity reagent, the negatively charged orange II dye (used here as a substitute for DNA) was successfully captured and eluted from the hydrophobic C8 alkyl substrate.

[0222] 10-fold dilution of C8 eluent using 1.5 mM butylamine 0.272 0.0029 10-fold dilution of C8 eluent using 15 mM butylamine 0.289 0.0027

[0223] Example 9: Synthesis of a linker for synthesizing branched soluble affinity reagents

[0224] N,N-Dihexyl-3-aminopropylamine: 10 mL of acrylonitrile was added to a suspension containing 1 g of dihexylamine (2 mmol) in 10 mL of methanol and 10 mL of DCM. The reaction was continued at room temperature for 24 h. The solvent was removed under vacuum, and the product was purified by silica gel using ethyl acetate and hexane as solvents. The product (0.8 g) was then dissolved in 10 mL of diethyl ether, and 1 g of LiAlH4 was added. Reduction was allowed to proceed overnight at room temperature. The reaction was terminated at 0°C with a dilute aqueous solution of NaOH. The reaction mixture was filtered and extracted with ethyl acetate. The organic solvent was dried over sodium sulfate and evaporated to give 0.2 g of white powder. Mass detected by ESI-MS (+ve): 243 m / z. Overall yield: 15%.

[0225] N,N-Dihexyl-3-imidazol-2-oxoamine: HATU (0.89 mmol) and DIPEA (1 mmol) were added to a solution of imidazolium propionic acid (50 mg, 0.4 mmol) in DCM (10 mL). Dihexylamine (0.48 mmol) was then added, and the mixture was stirred at room temperature for 1 day. The reaction was quenched with H2O and extracted with DCM. The organic phase was washed with H2O and brine, dried over Na2SO4, and evaporated under reduced pressure to give an oil. The oil was purified by alumina column chromatography using DCM:methanol as a solvent.

[0226] Example 10: Measurement of amine loading in polar solvents

[0227] The affinity reagent containing 3-dibutylaminopropylamine was dissolved in water using a polar solvent (such as methanol or DCM). Once dissolved, the affinity reagent was applied to the column. Orange II dye adsorbed / desorbed onto the amine-functionalized surface at pH 3 and eluted at pH 12. The amount of dye bound to the filter was measured by obtaining absorbance at 484 nm on a Molecular Devices iD3 microplate reader. The amount of dye bound was proportional to the number of amine groups immobilized on the surface being measured.

[0228] Samples containing 20% ​​and 10% w / v affinity reagents of 3-dibutylaminopropylamine showed similar binding in 20% v / v methanol to pure 3-dibutylaminopropylamine and 3-dibutylaminopropylamine in water. The 10% (w / v) affinity reagent containing 3-dibutylaminopropylamine was dissolved in 20% methanol.

[0229] Example 11: Measurement of DNA binding to 3-dibutylaminopropylamine

[0230] An affinity reagent consisting of approximately 10% w / v 3-dibutylaminopropylamine in 20% v / v methanol was applied to a C8 column. The column was washed with buffer at pH 6, 7, or 8. DNA in the buffer was adsorbed / desorbed onto the amine-functionalized surface, washed three times with buffer, and eluted at pH 12. The flow-through and elution buffers for DNA binding were measured using a Quibit assay. Figure 9A and Figure 9B The graph shows DNA binding and elution as measured by fluorescence assay. As shown, tris pH 8 provides optimal capture and release, achieving approximately 71% load and approximately 22% capture.

[0231] Example 12: Effect of salt on the binding of DNA to 3-dibutylaminopropylamine

[0232] An affinity reagent of approximately 10% w / v 3-dibutylaminopropylamine in 20% v / v methanol was applied to a C8 column. The column was washed with a buffer at pH 8 and 100 mM salt (e.g., KCl, NaCl, LiCl, NaPi, and GuHCl). DNA in the buffer was adsorbed / desorbed onto the amine-functionalized surface, washed three times with buffer, and eluted at pH 12. The flow-through and elution buffers for DNA binding were measured using a Quibit assay. Figure 10A and Figure 10B The effects of salts on DNA binding and elution, as measured by fluorescence assay, are shown. As illustrated, the addition of KCl and similar salts improves DNA capture and recovery. Figure 11A and Figure 11B The effects of washing with KCl, NaCl, LiCl, NaPi, and GuHCl are demonstrated. For C8, the binding rate is close to 100% under any 100 mM salt condition (30 ng / uL in 500 uL λDNA). Approximately 50% of the loading is eluted with various salts at 100 mM concentrations.

[0233] Example 13: Effect of 3-Dihexyl-3-aminopropylamine (DAP) on DNA binding

[0234] An affinity reagent consisting of approximately 10% w / v 3-dibutylaminopropylamine or 5% w / v 3-dihexyl-3-aminopropylamine (DAP) in 20% v / v methanol was applied to the filter. The filter was washed with a buffer at pH 8 and 100 mM salt (e.g., KCl, NaCl, LiCl, NaPi, and GuHCl). DNA in the buffer was adsorbed / desorbed onto the amine-functionalized surface, washed three times with buffer, and eluted at pH 12. The flow-through and eluent for DNA binding were measured using a Quant-iT assay. Figure 12DNA binding and elution were compared using affinity reagents 3DP and DAP.

[0235] All publications, patents, patent applications and other documents cited in this application are hereby incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, patent application or other document were individually indicated as being incorporated herein by reference for all purposes.

[0236] Although various specific implementation schemes have been described and illustrated, it should be understood that changes may be made without departing from the spirit and scope of the invention.

Claims

1. A method for isolating nucleic acids from a sample, the method comprising: The solid carrier is brought into contact with the affinity reagent and the sample, wherein the affinity reagent comprises: The first part interacts with the solid carrier. The second part of the interaction with nucleic acids in the sample, and The nucleic acid is concentrated onto the solid carrier.

2. The method of claim 1, wherein the affinity reagent further comprises a connector that interacts with the solvent and increases the solubility of the affinity reagent.

3. The method of claim 1 or 2, wherein the solid support comprises functional surface groups that interact with the first portion of the affinity reagent.

4. The method of any one of claims 1 to 3, wherein the functional surface groups comprise hydrophobic binding groups, negatively charged binding groups, positively charged binding groups, polar binding groups, or combinations thereof, and Optionally, the functional surface groups include hydrophobic binding groups selected from alkyl groups, cycloalkyl groups, haloalkyl groups, aryl groups, or combinations thereof.

5. The method of any one of claims 1 to 4, wherein the functional surface group comprises an azide group, and the first portion of the affinity reagent comprises a cycloalkyne, and wherein the azide group reacts with the cycloalkyne to form a nitrogen-containing heterocycle.

6. The method of any one of claims 1 to 5, wherein the functional surface group is bonded to the solid support via a triazole group, a triazine group, an imidazole group, an indole group, a silane group, a heteroazosilane tricyclic group, a siloxane group, a cyclic siloxane group, a sesquioxane group, a silazane group, or a combination thereof.

7. The method of any one of claims 1 to 6, wherein the solid carrier is a porous material.

8. The method of claim 7, wherein the porous material is derived from silica, glass, cellulose, vinyl backbone polymers, mica, polycarbonate, zeolite, titanium dioxide, magnetic materials, polyethersulfone, polytetrafluoroethylene, polyvinylpyrrolidone, or combinations thereof.

9. The method of any one of claims 1 to 8, wherein the first portion of the affinity reagent that interacts with the solid support comprises a hydrophobic group, a negatively charged binding group, a positively charged binding group, a polar group, or a combination thereof.

10. The method of claim 9, wherein the first portion of the affinity reagent comprises a hydrophobic group, wherein the hydrophobic group is selected from alkyl groups, cycloalkyl groups, haloalkyl groups, aryl groups, or combinations thereof.

11. The method of claim 9 or 10, wherein the first portion of the affinity reagent comprises a straight-chain alkyl group or a branched-chain alkyl group, optionally wherein the straight-chain alkyl group is selected from C4-C6. 20 Alkyl groups, C4-C 12 alkyl groups or C4-C 10 Alkyl groups, optionally wherein the branched alkyl groups are selected from C6-C6. 20 Alkyl groups, C8-C 12 alkyl groups or C8-C 10 Alkyl groups.

12. The method of any one of claims 1 to 11, wherein the second portion of the affinity reagent that interacts with the nucleic acid comprises an amine, a heterocyclic compound (such as a triazole, imidazole, or indole), an intercalating agent, a DNA groove binding agent, a peptide, an amino acid, a protein, or a combination thereof.

13. The method of claim 12, wherein the second portion of the affinity reagent comprises an amine, optionally wherein the amine is selected from alkylamines, cycloalkylamines, branched alkylamines, alkoxyamines, polyamine moieties, arylamines, or combinations thereof.

14. The method of claim 12 or 13, wherein the second portion of the affinity reagent comprises an alkylamine, an imidazole, a bisbenzylimine small groove binder, a polycyclic intercalator, or a combination thereof.

15. The method of any one of claims 12 to 14, wherein the second portion of the affinity reagent comprises spermine, methylamine, ethylamine, propylamine, ethylenediamine, diethylenetriamine, 1,3-dimethyldipropylenediamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium chloride, tris(2-aminoethyl)amine, 3-dibutylaminopropylamine, or combinations thereof.

16. The method of any one of claims 1 to 15, wherein the solvent is an aqueous solvent.

17. The method of any one of claims 2 to 16, wherein the connector of the affinity reagent further comprises one or more functional groups that interact with the solvent in the sample to increase the solubility of the affinity reagent.

18. The method of claim 17, wherein the connector comprises a hydrophilic group selected from polyethylene glycol connectors, ester-based connectors (such as branched ester-based connectors), amide-based connectors (such as branched amide-based connectors, including N,N-dihexyl-3-imidazol-2-oxoamine connectors), amine-based connectors (such as 3-dibutylaminopropylamine connectors, N,N-dihexyl-3-aminopropylamine connectors), organophosphorus-based connectors (such as organophosphorus-based connectors, organophosphorus oxide connectors, organophosphonates, organophosphoramide connectors, organophosphate connectors, organophosphonamide connectors, or organophosphonate connectors), glucuronic acid-based connectors, disulfide connectors, cathepsin B connectors, or combinations thereof.

19. The method of any one of claims 1 to 18, wherein contacting the solid carrier with the sample and the affinity reagent is performed under conditions that effectively allow interactions between a) the nucleic acid and the affinity reagent and b) the solid carrier and the affinity reagent.

20. The method of any one of claims 1 to 19, further comprising releasing the nucleic acid concentrated on the solid carrier.

21. The method of claim 20, wherein releasing the nucleic acid concentrated on the solid carrier comprises exposing at least one of the solid carrier, the affinity reagent, or the nucleic acid to an eluent, heat, ultrasonic treatment, photochemical lysis conditions, or a combination thereof.

22. The method of claim 21, wherein releasing the nucleic acid concentrated on the solid carrier comprises exposing at least one of the solid carrier, the affinity reagent, or the nucleic acid to an eluent, optionally wherein the eluent has a pH greater than about 7, greater than about 8, greater than about 9, and / or a salt concentration higher than that of the sample.

23. The method of claim 22, wherein the functional surface groups interact covalently or non-covalently with the first portion of the affinity reagent or a combination thereof.

24. The method of any one of claims 1 to 23, wherein the solid carrier is a glass fiber filter modified with functional surface groups, and optionally wherein the functional surface groups include hydrophobic silane compounds.

25. The method of any one of claims 1 to 24, wherein the affinity reagent further comprises a photodegradable group for releasing the nucleic acid concentrated on the solid carrier.

26. The method of any one of claims 1 to 25, wherein contacting the solid carrier with the sample and the affinity reagent is performed under conditions that effectively allow interactions between a) the nucleic acid and the affinity reagent and b) the solid carrier and the affinity reagent.

27. The method of claim 26, wherein the conditions include a pH less than 7.

28. The method of claim 26 or 27, further comprising contacting the sample with a lysis buffer before or simultaneously with contacting the solid carrier with the sample.

29. The method of claim 28, wherein the lysis buffer comprises one or more of a dissociation agent, a salt, a buffer, a surfactant, an antifoaming agent, and a binding agent.

30. The method of any one of claims 20 to 29, wherein releasing the nucleic acid concentrated on the solid carrier comprises exposing at least one of the solid carrier, the affinity reagent, or the nucleic acid to heat, optionally wherein the nucleic acid, the affinity reagent, and the solid carrier are heated to 35°C. °C or higher, 45 °C Or higher, 55 °C Or higher, 65 °C Or higher, 75 °C Or higher, 85 °C Or higher, 95 °C Or higher or up to 100 °C Or higher temperatures.

31. The method of any one of claims 20 to 30, wherein releasing the nucleic acid concentrated on the solid carrier comprises exposing at least one of the solid carrier, the affinity reagent, or the nucleic acid to ultrasonic treatment.

32. The method of any one of claims 20 to 31, wherein releasing the nucleic acid concentrated on the solid carrier comprises exposing at least one of the solid carrier, the affinity reagent, or the nucleic acid to photochemical lysis conditions of the affinity reagent, optionally wherein the photochemical lysis conditions comprise exposing the affinity reagent to UV light.

33. The method of any one of claims 1 to 32, further comprising washing the solid carrier with a washing solution.

34. The method of claim 33, wherein the washing solution has a pH of less than 7.

35. The method of any one of claims 1 to 34, wherein the method is performed in a box, optionally in an automated box.

36. A method for detecting nucleic acids in a biological sample, comprising: (a) Isolating the nucleic acid from the sample using the method defined in any one of claims 1 to 35; (b) Releasing the nucleic acid from the solid carrier using an elution agent; and (c) Detect the nucleic acid.

37. A sample kit for isolating and detecting nucleic acids from biological samples, the sample kit comprising: A housing having multiple chambers, wherein the multiple chambers include a sample chamber configured to receive the biological sample; A reaction vessel, which is fluidly connected to the plurality of chambers and is configured for amplifying nucleic acids and detecting a variety of amplification products; A filter, disposed in the fluid path between the plurality of chambers and the reaction vessel, wherein the filter comprises a solid carrier having a surface capable of binding affinity reagents. The affinity reagent is disposed in one of the plurality of chambers and comprises a first portion that interacts with the solid carrier and a second portion that interacts with nucleic acids from the biological sample; and Primers and / or probes, wherein the primers and / or probes are disposed in one or more of the plurality of chambers or reaction vessels for detecting the nucleic acid.

38. The sample cassette of claim 37, wherein the affinity reagent further comprises a connector that interacts with the solvent and increases the solubility of the affinity reagent.

39. The sample cassette of claim 37 or 38, wherein the primers and / or probes are fixed on the surface of one or more of the plurality of chambers or the reaction vessel for detecting the nucleic acid.

40. The sample box of any one of claims 37 to 39, wherein the plurality of chambers includes a lysis chamber in fluid communication with the sample chamber, the lysis chamber including a lysis reagent for releasing nucleic acids, optionally wherein the sample chamber and the lysis chamber are identical.

41. The sample box of any one of claims 37 to 39, wherein the plurality of chambers further comprises a binding reagent, a filtration buffer, a washing reagent, an elution buffer, or a combination thereof.

42. The sample cartridge of any one of claims 37 to 41, wherein the solid carrier comprises functional surface groups that interact with the first portion of the affinity reagent. Optionally, the functional surface groups interact with the first portion of the affinity reagent covalently (e.g., click / SPAAC reaction) or non-covalently (e.g., charged interactions such as ionic bonding or polar interactions, hydrophobic or van der Waals interactions, biotin-streptavidin interactions) or combinations thereof.

43. The sample box according to any one of claims 37 to 42, wherein the functional surface groups comprise hydrophobic binding groups, negatively charged binding groups (e.g., sulfonic acid, sulfate, phosphate, phosphonic acid, or carboxyl groups), positively charged binding groups (e.g., primary amines, secondary amines, tertiary amines, and quaternary ammonium, heterocyclic amines, such as pyridine, pyrimidine, pyridinium, piperazine), polar binding groups (e.g., chemical moieties containing polarized chemical bonds, such as C—O, C═O, C—N, C═N, C═N, N—H, O—H, C—F, C—Cl, C—Br, C—S, S—H, S—O, S═O, C—P, P—O, P═O, P—H, carboxyl, alcohol, thiol, amide, halide, amine, ester, ether, or thioester) or combinations thereof, and Optionally, the functional surface groups include hydrophobic binding groups selected from alkyl groups, cycloalkyl groups, haloalkyl groups (e.g., fluoroalkyl groups), aryl groups, or combinations thereof.

44. The sample box of claim 43, wherein the hydrophobic binding group comprises a straight-chain alkyl group, optionally wherein the straight-chain alkyl group is selected from C4-C6. 20 Alkyl groups, C4-C 12 alkyl groups or C4-C 10 Alkyl groups.

45. The sample box according to any one of claims 37 to 44, wherein the functional surface groups are bonded to the solid carrier via a triazole group, a triazine group, an imidazole group, an indole group, a silane group, a heteroazosilane tricyclic group, a siloxane group, a cyclic siloxane group, a sesquioxane group, a silazane group, or a combination thereof.

46. ​​The sample box according to any one of claims 41 to 45, wherein the functional surface groups are bonded to the solid carrier via silane groups.

47. The sample box according to any one of claims 37 to 46, wherein the solid carrier is a porous material, and optionally wherein the solid carrier is a fibrous porous material.

48. The sample box of claim 47, wherein the porous material is derived from silica, glass (e.g., glass beads or glass filters), cellulose (e.g., cellulose filters), vinyl skeleton polymers, mica, polycarbonate (e.g., polycarbonate filters), zeolite, titanium dioxide, magnetic materials (e.g., magnetic beads), polyethersulfone (e.g., polyethersulfone filters), polytetrafluoroethylene (e.g., polytetrafluoroethylene filters), polyvinylpyrrolidone (e.g., polyvinylpyrrolidone filters), or combinations thereof.

49. The sample box according to any one of claims 46 to 48, wherein the porous material is a glass fiber filter.

50. The sample box of any one of claims 37 to 49, wherein the solid carrier is a glass fiber filter modified with functional surface groups, optionally wherein the functional surface groups include hydrophobic silane compounds.

51. The sample kit of any one of claims 37 to 50, wherein the first portion of the affinity reagent that interacts with the solid carrier comprises a hydrophobic group, a negatively charged binding group, a positively charged binding group, a polar group, or a combination thereof.

52. The sample kit of any one of claims 37 to 51, wherein the first portion of the affinity reagent comprises a hydrophobic group, wherein the hydrophobic group is selected from alkyl groups, cycloalkyl groups, haloalkyl groups (e.g., fluoroalkyl groups), aryl groups, or combinations thereof.

53. The sample kit of any one of claims 37 to 52, wherein the first portion of the affinity reagent comprises a straight-chain alkyl group or a branched-chain alkyl group, optionally wherein the straight-chain alkyl group is selected from C4-C6. 20 Alkyl groups, C4-C 12 alkyl groups or C4-C 10 Alkyl groups, optionally wherein the branched alkyl groups are selected from C6-C6. 20 Alkyl groups, C8-C 12 alkyl groups or C8-C 10 Alkyl groups.

54. The sample kit of any one of claims 37 to 53, wherein the second portion of the affinity reagent that interacts with the nucleic acid comprises an amine, a nitrogen-containing heterocycle, an intercalating agent, a DNA groove binding agent, a peptide, an amino acid, a protein, or a combination thereof.

55. The sample kit of any one of claims 37 to 54, wherein the second portion of the affinity reagent comprises an amine, the amine being optionally selected from alkylamines, cycloalkylamines, branched alkylamines, alkoxyamines, polyamine moieties, arylamines, or combinations thereof.

56. The sample kit of any one of claims 37 to 55, wherein the second portion of the affinity reagent comprises an alkylamine, an imidazole, a bis(benzyl)imine small groove binder, a polycyclic intercalator, or a combination thereof.

57. The sample kit of any one of claims 37 to 56, wherein the second portion of the affinity reagent comprises spermine, methylamine, ethylamine, propylamine, ethylenediamine, diethylenetriamine, 1,3-dimethyldipropylenediamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium chloride, tris(2-aminoethyl)amine, 3-dibutylaminopropylamine, or combinations thereof.

58. The sample cassette of any one of claims 37 to 57, wherein the connector comprises a hydrophilic group selected from polyethylene glycol connectors, ester-based connectors (such as branched ester-based connectors), amide-based connectors (such as branched amide-based connectors, including N,N-dihexyl-3-imidazol-2-oxoamine connectors), amine-based connectors (such as 3-dibutylaminopropylamine connectors, N,N-dihexyl-3-aminopropylamine connectors), organophosphorus-based connectors (such as organophosphorus-based connectors, organophosphorus oxide connectors, organophosphonates, organophosphoramide connectors, organophosphate connectors, organophosphonamide connectors, or organophosphonate connectors), glucuronic acid-based connectors, disulfide connectors, cathepsin B connectors, or combinations thereof.

59. The sample cassette of any one of claims 37 to 58, wherein the affinity reagent further comprises a photodegradable group for releasing the nucleic acid concentrated on the solid carrier.

60. The sample cartridge of any one of claims 37 to 59, wherein the sample cartridge, together with the reagent, allows a flow rate of up to about 100 µL per second, such as about 10 µL to about 100 µL.

61. The sample cassette of any one of claims 37 to 60, wherein the sample cassette, together with the reagent, allows pressures below 100 psi, below 80 psi, or below 60 psi.

62. The sample box as claimed in any one of claims 37 to 61, wherein the box is an automated box.

63. The method or sample kit of any one of claims 1 to 62, wherein the affinity reagent is administered at 10 nmol / cm² based on the solid carrier. 2 Or larger, 20 nmol / cm 2 Or larger, 35 nmol / cm 2 Or larger or 30-100 nmol / cm 2 The surface density exists.

64. The method or sample kit of any one of claims 1 to 63, wherein the affinity reagent is administered at 3,000 nmol / cm² based on the solid carrier. 2 Or smaller, 2,500 nmol / cm 2 Or smaller, 2,000 nmol / cm 2 Or smaller, 1,000 nmol / cm 2 or smaller, 500 nmol / cm 2 or smaller, 400 nmol / cm 2 or smaller, 300 nmol / cm 2 or smaller, 200 nmol / cm 2 Or smaller, or 100 nmol / cm 2 Or it may exist with a smaller surface density.

65. The method or sample kit of any one of claims 1 to 64, wherein the solid carrier together with the affinity reagent has a concentration of at least 10 µg / cm³. 2 20 µg / cm 2 or larger, 35 µg / cm 2 Or larger or 30-100 µg / cm 2 DNA binding ability.

66. The method or sample box according to any one of claims 1 to 65, wherein the solid carrier has a pore size of 0.2 µm to 3 µm, 0.2 µm to 2 µm, 0.5 µm to 1.0 µm, or 0.6 µm to 0.8 µm.

67. The method or sample cassette of any one of claims 1 to 66, wherein the solid carrier (e.g., a glass fiber filter) comprises beads to facilitate mechanical lysis, wherein the beads are selected from glass beads, silica beads, or combinations thereof.

68. The method or sample cassette of any one of claims 1 to 67, wherein the solid carrier (e.g., a glass fiber filter) has a density of 35 g / m³. 2 Up to 100 g / m 2 Preferably 50 g / m 2 Up to 85 g / m 2 or 70 g / m 2 Up to 85 g / m 2 The base weight.

69. The method or sample cassette of any one of claims 1 to 68, wherein the solid carrier (e.g., a glass fiber filter) has a fiber diameter of 1 µm to 100 µm, preferably 1 µm to 50 µm or 1 µm to 25 µm.

70. The method or sample cassette of any one of claims 1 to 69, wherein the solid carrier (e.g., a glass fiber filter) has a thickness of 250 µm to 2,000 µm, 300 µm to 1,500 µm, 300 µm to 1,000 µm, 300 µm to 750 µm, or 350 µm to 500 µm.

71. A method for detecting nucleic acids in a biological sample obtained from a subject, the method comprising: a) Contact the nucleic acid from the biological sample with a set of primers and optional probes in the sample cassette according to any one of claims 37 to 70; b) subject the nucleic acid, primer pair, and optional probe to amplification conditions; c) Optionally, the presence of amplification products can be detected by real-time PCR, melting curve analysis, or a combination thereof; as well as d) Detect the presence of the nucleic acid in the biological sample based on the detection of the amplification product.

72. The method of claim 71, wherein the method does not involve the use of a dissociation agent, a lysis buffer, or a binding agent.

73. The method or sample kit of any one of claims 1 to 72, wherein the biological sample is blood, blood culture, plasma, serum, semen, cerebrospinal fluid, tissue, tears, urine, feces, saliva, smear preparation, respiratory sample, nasopharyngeal swab sample, anterior nasal sample, middle turbinate nasal sample, oropharyngeal sample, vaginal swab, vaginal mucus sample, vaginal tissue sample, vaginal cell sample, bacterial culture, mammalian cell culture, viral culture, human cells, bacteria, extracellular fluid, pancreatic juice, cell lysate, PCR reaction mixture or in vitro nucleic acid modification reaction mixture, preferably wherein the biological sample is blood, plasma, respiratory sample or vaginal swab.

74. The method or sample kit of any one of claims 1 to 73, wherein the biological sample comprises nucleic acids selected from: genomic DNA, total RNA, short DNA, small DNA, tumor-derived nucleic acids, methylated DNA, microbial nucleic acids, bacterial nucleic acids, viral nucleic acids, cell-free nucleic acids, or combinations thereof.

75. The method or sample kit of any one of claims 1 to 74, wherein amplification is performed by nested PCR, gradient PCR, isothermal PCR, qPCR or RT-PCR.

76. The method or sample kit of any one of claims 1 to 75, wherein amplification is performed by real-time PCR multiplex assay.

77. A solid support comprising a first functional surface group and a second functional surface group, wherein the first functional surface group is an alkylsilane and the second functional surface group is an azide silane.

78. The solid support of claim 77, wherein the alkylsilane is first deposited on the solid support, and then the azide silane is deposited.

79. The solid support of claim 77, wherein the azide silane is first deposited on the solid support, and then the alkyl silane is deposited.

80. The solid support of claim 77, wherein the alkylsilane and azide silane are deposited simultaneously.

81. A method for immobilizing nucleic acids onto a solid support, wherein the solid support comprises a first functional surface group and a second functional surface group, the first functional surface group being an alkylsilane and the second functional surface group being an azide silane.

82. The method of claim 81, wherein the nucleic acid comprises a reactive group capable of reacting with an azide group.

83. The method of claim 82, wherein the reactive group is an alkynyl group.

84. A method comprising: Biological samples containing viruses and cell-free nucleic acids are dispensed into sample cartridges containing a solid carrier having a modified or affinity-binding surface, wherein the affinity reagent comprises a first portion that interacts with the solid carrier and a second portion that interacts with the cell-free nucleic acids from the biological sample. At least 70% of the free nucleic acid, together with the affinity reagent, is dispensed onto the solid carrier; and Identify the virus within the sample box.

85. The method of claim 84, wherein the affinity reagent further comprises a connector that interacts with the solvent and increases the solubility of the affinity reagent.

86. The method of claim 84, wherein the affinity reagent is according to any one of claims 1 to 18.

87. The method of claim 84, further comprising: Contact the solid carrier with the affinity reagent and the biological sample, and The free nucleic acid is concentrated onto the solid carrier.

88. The method of any one of claims 84 to 87, comprising: Optionally, the virus can be isolated from the biological sample. Lysing the virus to release nucleic acid from it, and The nucleic acid released from the virus is detected to identify the virus.

89. The method of any one of claims 84 to 88, wherein detecting the nucleic acid released from the virus comprises: The nucleic acid from the virus is brought into contact with a set of primers and optional probes in the sample cassette; The nucleic acid, primer pair, and optional probe are subjected to amplification conditions; The presence of amplification products can be detected, optionally via real-time PCR, melting curve analysis, or a combination thereof; as well as The presence of the nucleic acid is detected based on the detection of the amplification product.

90. The method of any one of claims 84 to 89, wherein at least 95% of the free nucleic acid is dispensed onto the solid carrier together with the affinity reagent of the sample cassette.

91. The method of any one of claims 84 to 90, wherein the free nucleic acid is derived from a non-viable virus.

92. The method of any one of claims 84 to 81, further comprising determining the free nucleic acid within the sample cassette.

93. The method of any one of claims 84 to 82, wherein the dispensing occurs in less than 15 minutes.

94. The method of any one of claims 84 to 93, wherein the biological sample is derived from a subject suspected of being infected with a virus.

95. The method of any one of claims 84 to 94, wherein the biological sample is selected from whole blood, serum, plasma, saliva, nasal discharge, sputum, urine, CNS fluid, nipple aspiration fluid, cerebrospinal fluid, or semen.

96. The method of any one of claims 84 to 95, wherein the subject is a human being.

97. The method of any one of claims 84 to 96, wherein the virus is a coronavirus.

98. The method of claim 97, wherein the coronavirus is COVID-19.

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