NUCLEIC ACID EXTRACTION AND ISOLATION WITH HEAT LABILE SILANES AND CHEMICALLY MODIFIED SOLID SUPPORTS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to, and the benefit of, U.S. Provisional Application No. 63/337,014, filed on April 29, 2022, which is incorporated by reference herein for all purposes.

FIELD OF THE DISCLOSURE

[0002] The invention relates generally to the field of molecular biology. In certain embodiments the invention provides devices, kits, and methods relating to the isolation and detection of nucleic acids.

BACKGROUND

[0003] Isolating nucleic acids is typically the first step of most molecular biological inquiries including polymerase chain reaction (PCR), DNA hybridization, restriction enzyme digestion, DNA sequencing, and array-based experiments. As such, there is a need for simple and reliable methods for isolating nucleic acid, and in particular, for isolating high quality nucleic acid. A variety of techniques for isolating nucleic acids from a sample have been described, one of the most common involving lysing the nucleic acid source in a chaotropic substance (for example, guanidinium salt, urea, and sodium iodide), in the presence of a DNA binding solid phase (for example, glass beads or fibers). The released nucleic acid is bound to the solid phase in a one-step reaction, where the solid phase is washed to remove any residual contaminants. As an example, glass fiber disc having a 30 mm diameter, 0.7 pm pore size has been shown to capture 150 pg of plasmid DNA (binding capacity about 30 pg/cm2 ) with 2 M guanidine hydrochloride (GuHCl) lysates. Kim, Y-C and Morrison, S.L. PLoS ONE (2009) 4, 11, e7750. Although these methods have proven to be fast, they have resulted in a moderate level of DNA shearing and some level of contamination. Residual GuHCl or GuSCN can poison downstream PCR reactions and must be removed with extensive washing. There is a need for methods of isolating a nucleic acid from a sample that are fast, economical, and produce high yields.

[0004] In order to increase sensitivity of nucleic acid detection, large sample volumes can be prepared. The preparation of large volumes, however, is contradictory to fluidic systems for automatic lysis, processing and/or analysis of biological samples. Particularly, current

automative on-market products for processing samples, such as whole blood for the detection of pathogenic targets are limited to tolerating only a few hundred microliters of blood per test. Existing technologies capable of processing multiple milliliters of blood samples for pathogen detection involves a laborious process with multiple manual steps that must be followed correctly by the user, including centrifugation, decantation, vortexing, and glass column-based DNA precipitation and purification. After using the existing sample processing kit, the user is then still responsible for preparing a PCR set-up that can accurately analyze what is produced by the sample kit. There is a market need for automated sample processing of multiple milliliters of blood within a single device. It would also be useful if the method minimized the required manipulation of the sample and could be performed using a single device. Certain embodiments of the invention described herein provide for such methods. Other embodiments of the invention described herein provide for devices and kits which may be used for isolating nucleic acids from a sample. Still other embodiments of the invention provide for the detection of a nucleic acid in a sample.

Summary

[0005] Described herein are compositions, systems, and methods for isolating and purifying nucleic acid from a biological sample. The compositions, systems, and methods utilize a modified solid support comprising a DNA binding ligand as a separating material, thereby reducing and/or eliminating the amount of lysis reagents and nucleic acid binding agents conventionally used in PCR processes.

[0006] In some aspects, the compositions and systems for isolating a nucleic acid from a biological sample comprise: a compound bonded to a solid support, the compound being derived from a structure represented by the formula:

Y— (L)y— SiX3

wherein, Y is a DNA binding ligand selected from an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an intercalating agent, a minor groove binder (e.g., a bisbenzimide minor groove binder), a peptide, an amino acid, an arylamine, or a combination thereof; L is a linker selected from an alkyl group, a heteroalkyl group, an alkene group, a heteroalkene group, a polyacrylic acid, a Diels-Alder adduct, or a combination thereof; each X, independently for each occurrence, is selected from a hydrolyzable group, an alkyl group, a heteroalkyl group, an alkenyl group, or two or three Xs combine to form one or more cyclic groups, or one X combines with Y to form a cyclic azasilane,; and y is 0 or 1.

[0007] The DNA binding ligand or the substituent Y can comprise a plurality of amine groups; a plurality of amide groups; or a combination thereof. For example, the DNA binding ligand or Y can comprise at least two, at least three, at least four, at least five, at least six amine or amide groups, or a combination thereof. In some embodiments, the DNA binding ligand or Y comprises an alkylamine group, an imidazole group, or a combination thereof. Representative examples of the amine group include spermine, methylamine, ethylamine, propylamine, ethylenediamine, di ethylene triamine, 1,3 -dimethyldipropylenediamine, 3-(2-aminoethyl)aminopropyl, (2-aminoethyl)trimethylammonium hydrochloride, tris(2-aminoethyl)amine, or a combination thereof.

[0008] The linker, L, is present (or y is 1) in some aspects of the compound disclosed herein. The linker can be selected from an alkyleneoxy group, an alkylene group, or a Diels-Alder adduct.

[0009] The group X facilitates attachment of the compounds disclosed herein with the solid support. Figure 4, for example, shows how individual silane molecules can be incorporated into a solid support material by bonding to the solid support material and/or with neighboring silane molecules. In some embodiments, each X, independently for each occurrence, can be selected from a moiety that facilitates hydrogen bonding, electrostatic attraction, covalent bonding, horizontal and/or vertical polymerization, with functionalities present at the interface or the solid support material and/or with neighboring silanes. In some aspects of the compositions and systems, at least two Xs can be independently selected from a halogen, an alkoxy, a dialkylamino, a trifluoromethanesulfonate, or combine together with the Si atom to which they are attached to form an oligomeric or polymeric silane, a silatrane, a cyclic siloxane, a polysilsesquioxane, or a silazane. For example, at least two Xs can be independently selected from an alkoxy group (such as ethoxy or methoxy).

[0010] In other aspects, the compositions and systems for isolation of a nucleic acid from a biological sample comprises a Diels-Alder adduct, the Diels-Alder adduct including a DNA binding ligand, wherein the Diels-Alder adduct is optionally bonded to the solid support. As defined herein, the DNA binding ligand can comprise an amine group, an intercalating agent, a minor groove binder, a peptide, an amino acid, or a combination thereof. In some examples, the DNA binding ligand is selected from an alkylamine, a cycloalkylamine, an alkyloxy amine, an arylamine, a polyamine moiety, or a combination thereof. The Diels-Alder adduct described herein can be derived from an unsaturated cyclic imido group.

[0011] In some embodiments, the compound or the Diels-Alder adduct bonded to the solid support can be derived from a structure represented by the general Formula,

their isomers, salts, tautomers, or combinations thereof, wherein Y' is the DNA binding ligand, and L, X, and y are as defined herein. For example, Y' can comprise an alkylamine group, an imidazole group, or a combination thereof. In some examples, Y' can further comprise more than one DNA binding ligand, such as in FIG. 8, which shows an alkylamine group further linked to a minor groove DNA binding ligands. L is optionally present and can be selected from an alkyleneoxy group, an alkylene group, cyanuric chloride, an alkylamine, or a combination thereof. At least two Xs can be independently selected from a halogen, an alkoxy, a dialkylamino, a trifluoromethanesulfonate, or combine together with the Si atom to which they are attached to form a silatrane, a cyclic siloxane, a polysilsesquioxane, or a silazane.

[0012] In some examples, the compound or the Diels- Alder adduct can be derived from one of the following structures:

, 3 -aminopropyltrimethoxy silane, 3 -aminopropyltri ethoxy silane, an aminoalkylsilatrane, 3-(2-aminoethyl)aminopropyltri ethoxy silane, 3 -(2-aminoethyl)aminopropyltrimethoxy silane, or a combination thereof, and wherein n is an integer from 0 to 10, from 1 to 10, or from 1 to 5.

[0013] The solid support described herein can comprise a material selected from silica, glass, ethylenic backbone polymer, mica, polycarbonate, zeolite, titanium dioxide, magnetic bead, glass bead, cellulose filter, polycarbonate filter, polytetrafluoroethylene filter, polyvinylpyrrolidone filter, polyethersulfone filter, glass fiber filter or a combination thereof. In some examples, the solid support is a glass fiber filter. The glass fiber filter can have a pore size selected to accommodate correspondingly sized beads to facilitate mechanical lysis. For example, the glass fiber filter can have a pore size from 0.2 pm to 3 pm, from 0.2 pm to 2 pm, from 0.5 pm to 1.0 pm, or from 0.6 pm to 0.8 pm. Further, the glass fiber filter can have a basis weight from 35 g/m2 to 100 g/m2, preferably from 50 g/m2 to 85 g/m2, or from 70 g/m2 to 80 g/m2. The glass fiber filter can have a fiber diameter from 1 pm to 100 pm, preferably from 1 pm to 50 pm, or from 1 pm to 25 pm. The glass fiber filter can have a thickness from 250 pm to 2,000 pm, from 300 pm to 1,500 pm, from 300 pm to 1,000 pm, from 300 pm to 750 pm, or from 350 pm to 500 pm. As described herein, the glass fiber filter can accommodate beads to facilitate mechanical lysis The beads can include glass beads, silica beads, or a combination thereof. The compound or the Diels-Alder adduct can be bonded to the solid support via a siloxane bridge, a carboxylate bridge, as ester bridge, an ether bridge, or a combination thereof.

[0014] Separating materials for nucleic acid isolation and purification are also disclosed herein. The separating material can comprise a compound or compositions disclosed herein, comprising a DNA binding ligand. For example, the separating material can comprise a glass fiber solid support and a compound bonded to the glass fiber solid support. As described herein, the compound can be derived from a structure represented by the formula: Y — (L)y — SiXs, and wherein Y, L, X, and y are as defined herein. In other examples, the separating material can comprise a glass fiber solid support comprising a Diels-Alder adduct having a DNA binding ligand, wherein the adduct is chemically bonded to a glass fiber solid support. The glass fiber solid support may further comprise a polymeric binder. In some examples, the glass fiber solid support can be in the form of a 500 microns to 2000 microns thick glass fiber disk having an effective pore size of 0.5 microns to 1 micron.

[0015] Systems comprising the compounds, compositions, and separating materials disclosed herein are also provided. The systems can be a sample cartridge, preferably an automated sample cartridge. Accordingly, disclosed herein are systems comprising a sample cartridge for isolation and detection of nucleic acid from a biological sample. The sample cartridge can comprise: a) a cartridge body having a plurality of chambers defined therein, wherein the plurality of chambers are in in fluidic communication through a fluidic path of the cartridge, and wherein at least one chamber is configured to receive the biological sample, b) a reaction vessel configured for amplification of the nucleic acid by thermal cycling, and c) a filter disposed in the fluidic path between the plurality of chambers and the reaction vessel, wherein the filter comprises a compound, separating material, or composition as disclosed herein, wherein the plurality of chambers and the reaction vessel independently comprise reagents for releasing nucleic acid from the biological sample, and primers and probes for detection of the nucleic acid. In other aspects, the sample cartridge can comprise a) a cartridge body having a plurality of chambers therein, wherein the plurality of chambers include: a sample chamber having at least a fluid outlet in fluid communication with another chamber of the plurality; and a lysis chamber in fluidic communication with the sample chamber, the lysis chamber comprising reagents for releasing nucleic acid, optionally wherein the sample chamber and lysis chamber are the same; b) a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for amplification of nucleic acid and ii) detection of a plurality of amplification products; c) a filter disposed in the fluidic path between the lysis chamber and the reaction vessel, wherein the filter comprises a solid support modified with a DNA binding ligand selected from an alkylamine, a cycloalkylamine, an alkyloxy amine, a polyamine moiety, an intercalating agent, a minor groove binder (e.g., a bisbenzimide minor groove binder), a peptide, an amino acid, a protein, an arylamine, or a combination thereof, and d) a plurality of primers and/or probes disposed in one or more chambers of the plurality of chambers or reaction vessel for detection of the nucleic acid. The sample cartridge is configured to carry out non-isothermal amplification such as by thermal cycling, gradient /temperature differential), or temperature oscillation, and isothermal amplification.

[0016] The sample cartridge may further comprise a syringe that is movable to facilitate fluid flow into and from the lysis chamber by fluctuation of pressure.

[0017] In some aspects of the sample cartridge, the lysis chamber may further comprise a valve body and a valve cap, wherein the valve body interfaces with the valve cap to define the lysis chamber therebetween, and wherein the filter is held within the lysis chamber secured between the valve body and the valve cap. The lysis chamber can have a fluid flow path between an inlet in the cap and an outlet in the valve body that is fluidically coupled to a fluid displacement region of the valve body, wherein the fluid displacement region is depressurizable by movement of the syringe to draw fluid into the fluid displacement region and pressurizable by movement of the syringe to expel fluid from the fluid displacement region.

[0018] The lysis chamber optionally comprises lysis reagents, the lysis reagents selected from a chaotropic agent, a chelating agent, a buffer, a detergent, or combinations thereof, to facilitate chemical lysis. The cartridge body can further comprise an ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducer, for example in the lysis chamber to facilitate mechanical lysing. The cartridge body in one or more of the plurality of chambers may further comprise a binding agent.

[0019] The disclosed compositions and systems provide improvements in performance of the sample cartridge disclosed herein by facilitating reduction and/or eliminating the amount of lysis reagent and binding reagent conventionally used in cartridge design. Particularly, the sample cartridges disclosed herein have reduction in the amount of chaotropic agent and/or PEG contained in lysis buffer, wash buffer, elution buffer, and binding reagent. Furthermore, the volume of lysis buffer, wash buffer, elution buffer, and binding reagent stored within the cartridge can be reduced.

[0020] In some prior experiments, the maximum fluid volume that could be processed with conventional cartridges was 300 pL at which volume pressure aborts (at 100 psi or greater) occurred approximately 50% of the time, thus volumes in conventional cartridges were reduced and limited to 125 pL to avoid pressure aborts. The current cartridges allow for higher sample volumes (e.g., 300 pL, 700 pL, 1,000 pL, up to 5,000 pL) without reaching or exceeding the maximum pressure allowable (100 psi). The attribute of the cartridge that allowed for the processing of higher volumes was related to the reduction in chaotropic agent and binding agent as well as the filter material. Flow rates for these experiments were 10 pL per second for conventional cartridges. Flow rates are limited by pressure in sample cartridges, but the compounds and compositions in the disclosed sample cartridges allow for flow rates up to at least 100 pL per second. Accordingly, more total sample are able to be processed with the disclosed cartridges as compared to conventional cartridges in less time while maintaining viable pressures below 100 psi. overall, the disclose sample cartridge together with the reagents can allow for higher flow rates up to about 100 pL per second, such as from about 10 pL to about 100 pL, compared to conventional cartridges. The disclosed sample cartridge together with the reagents can allow for pressure below 100 psi, below 80 psi, or below 60 psi. The disclosed sample cartridge can allow for sample volumes up to 5000 pL, such as from 300 pL to 5,000 pL, from 300 pL to 3,000 pL, from 300 pL to 2,000 pL, or from 300 pL to 1,000 pL.

[0021] The cartridge can be a single-use disposable cartridge. In some embodiments, the cartridge is an automated cartridge.

[0022] Methods for isolating a nucleic acid from a biological sample are also provided. The method can comprise (a) causing the nucleic acid to contact a compound bonded to a solid

support as provided in the compositions and systems disclosed herein, and (b) eluting the nucleic acid from the solid support. In other aspects, the methods for isolation of a nucleic acid from a biological sample comprises (a) causing the nucleic acid to contact a composition comprising a Diels-Alder adduct, the Diels-Alder adduct including a DNA binding ligand as disclosed herein, and (b) concentrating the nucleic acid onto a solid support. In some aspects, the method for detecting nucleic acid in a biological sample obtained from a subject can comprise a) contacting nucleic acid from the biological sample with a set of primers and optional probes in a sample cartridge as described herein; b) subjecting the nucleic acid, primer pairs, and optional probes to amplification conditions; c) detecting the presence of amplification product(s), optionally via real-time PCR, melt curve analysis, or a combination thereof, and d) detecting the presence of the nucleic acid in the biological sample based on detection of the amplification products. Said contacting nucleic acid from the sample with the set of primers and optional probes in a sample cartridge can comprise placing the biological sample in the cartridge comprising a cartridge body having a plurality of chambers in fluidic communication, a reaction vessel having one or more reaction chambers and configured for amplification of the nucleic acid, a fluidic path between the plurality of chambers and the reaction vessel, and a filter in the fluidic path, and if the biological sample comprises cells, lysing cells in the biological sample with one or more lysis reagents present within at least one of the plurality of chambers. Said subjecting the nucleic acid, primer pairs, and optional probes to amplification conditions can comprise amplifying the nucleic acid with primers and probes present in solution within at least one of the plurality of chambers. Said subjecting the nucleic acid, primer pairs, and optional probes to amplification conditions can comprise amplifying the nucleic acid with primers and probes present in solution within at least one of the plurality of chambers.

[0023] The methods are used for isolating and purifying nucleic acid from a biological sample. The biological sample can be selected from blood, plasma, serum, semen, spinal fluid, tissue, tear, urine, stool, saliva, smear preparation, respiratory sample, nasopharyngeal sample, vaginal swab, vaginal mucus sample, vaginal tissue sample, vaginal cell sample, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, pancreatic fluid, cell lysate, PCR reaction mixture, or in vitro nucleic acid modification reaction mixture. In some embodiments, the biological sample is blood, plasma, respiratory sample, or vaginal swab. In some examples, the biological sample comprises nucleic acid selected from genomic DNA, total RNA, short-DNA, small DNA, tumor-derived nucleic acid, methylated

DNA, microbial nucleic acid, bacterial nucleic acid, viral nucleic acid, cell free nucleic acid, or combinations thereof. In some embodiments, the biological sample comprises cell free nucleic acid.

[0024] In the methods disclosed herein, the biological sample may be contacted with a buffer prior to or simultaneously with step a) causing the nucleic acid to contact the composition or compound bonded to a solid support. The buffer can be a lysis buffer and include one or more of a chaotropic agent, a salt, a buffering agent, a surfactant, a defoaming agent, a binding agent, a precipitating agent, or a combination thereof. The chaotropic agent can be selected from guanidinium thiocyanate, guanidinium hydrochloride, alkali perchlorate, alkali iodide, urea, formamide, or combinations thereof. The chaotropic agent can be utilized at a lower concentration compared to conventional lysis assay. For example, the chaotropic agent can be used at a concentration of less than 4.5 M, less than 2 M, or less than 1 M. of the lysis buffer. In some embodiments, the methods disclosed herein do not utilize a chaotropic agent or a lysis buffer.

[0025] When a chaotropic agent or lysis buffer is not used in the methods disclosed herein, the biological sample can be contacted with a buffer comprising saline (inorganic salts such as CaCh, MgSC , KC1, NaHCCh, NaCl, etc.), phosphate buffer, Tris buffer, 2-amino-2-hydroxymethyl-l,3-propanediol, HEPES, PBS, citrate buffer, TES, MOPS, PIPES, Cacodylate, SSC, MES, saccharide or disaccharide, or combinations thereof. For example, the buffer can be a commercially available buffer such as Hanks’ Balanced Salt Solution available from Sigma Aldrich or TE Buffer available from Fisher BioReagents.

[0026] In some embodiments, the methods further comprise contacting the nucleic acid with a binding agent, a filtering reagent, a washing reagent, or a combination thereof, simultaneously with concentrating or prior to eluting the nucleic acid. The binding reagent (such as PEG or a salt) can promote binding of nucleic acids to the filter while removing non-target material. The filtering agent and/or the washing agent may comprise the binding agent. The binding agent can be utilized at a lower concentration compared to conventional lysis assay. For example, the binding agent can be used at a concentration of less than 40% v/v, less than 30% v/v, less than 20% v/v, or less than 10% v/v, of the filtering agent and/or the washing agent. In some embodiments, the compositions, systems, and methods disclosed herein do not utilize a binding agent. In some instances, neither lysis buffer (or a chaotropic agent) nor binding reagents are used in the methods disclosed herein. In such cases, proteinase K or a mechanical treatment

such as sonication can be used to release nucleic acids from the sample which subsequently bind to the modified filter.

[0027] Eluting may comprise heating the nucleic acid on the filter to a temperature of 100°C or less, 95°C or less, 85°C or less, 75°C or less, 65°C or less, 55°C or less; sonicating the nucleic acid; photochemically cleaving the compound/composition; or a combination thereof, in the presence of an eluting agent. In some embodiments, the methods for isolating and purifying a nucleic acid can comprise eluting the nucleic acid with an eluting agent. The eluting agent can have a pH greater than about 9, greater than about 10, greater than about 11, or greater than about 12. The eluting agent can have a pH greater than about 10. The eluting agent can have a pH of about 10 to about 13. In some Examples provided herein, 50 mM KOH (pH 12.7) was used for eluting the nucleic acid. The use of high pH to elute nucleic acid such as DNA is unique especially to the cartridges described herein and provides improved speed and performance of the disclosed methods. Speed is provided by the rapid neutralization of acidic ammonium ions by the high concentration of hydroxide ions. Alkylamines have a pKa -10-11 and are immediately deprotonated at pH 12.7, to form the neutral free base on the solid surface, and release the cationic DNA. A further advantage of the high pH is the denaturing effect of KOH on captured DNA or RNA. Acidic functional groups in the heterocyclic bases of DNA or RNA are immediately deprotonated and cannot form Watson-Crick bonds. Double stranded structures and other secondary structures are disrupted, but can re-nature when neutralized for example, with Tris HC1. This chemical denaturing of captured genomic DNA can be an advantage for isothermal assays that do not undergo the usual heat denaturing of PCR. The cartridges provided herein allows for rapid neutralization of eluted DNA or RNA in KOH followed by reaction with Tris to produce a final pH of about 8.5 for downstream PCR or other nucleic acid assays. In some embodiments, the eluting agent can have a pH less than about 9, less than about 8.5, or less than about 8. This lower pH elution of bound DNA or RNA can be an advantage, especially for devices that don’t facilitate rapid neutralization of the KOH solution. It is known that RNA is hydrolyzed by high pH, so short exposure times to KOH are important for good quality RNA. In some examples, the eluting agent comprises a polyanion, a polycation, ammonia or an alkali metal hydroxide. For example, the eluting agent may comprise a polyanion such as a carrageenan, a carrier nucleic acid, or a combination thereof.

[0028] Methods for detecting a nucleic acid in a biological sample are also disclosed. The methods can include (a) isolating the nucleic acid from the biological sample using a method as defined herein; (b) eluting the nucleic acid from the solid support with an eluting agent; and (c) detecting the nucleic acid. Detecting the nucleic acid can comprise amplifying the nucleic acid by polymerase chain reaction. The polymerase chain reaction can be selected from a nested PCR, an isothermal PCR, qPCR, or RT-PCR.

[0029] In other embodiments, the methods for detecting a nucleic acid in a biological sample can include placing the biological sample in a cartridge body as disclosed herein, lysing cells optionally with one or more lysis reagents present within at least one of the plurality of chambers and capturing nucleic acid released therefrom; and amplifying the nucleic acid with primers and probes for detecting the presence of the nucleic acid. The nucleic acid can be detected within the biological sample within 75 minutes or within 60 minutes of collecting the sample from the subject.

Brief Description of the figures

[0030] The application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0031] FIG. 1A shows attachment of DNA binders to glass. The image on the left shows reactive groups such as amines, carboxylic acids and epoxides are attached to glass surfaces using silane reagents. Aliphatic amines can bind DNA directly. The silane density, thickness, crosslinking, reactive groups, surface charge or surface density can be varied to promote faster extraction, exclude/reduce amounts of a binding agent (such as PEG) or chaotropic agent (such as guanidine thiocyanate). The types of rea may also be varied. The image on the right shows other DNA binding ligands conjugated to glass via a linker. The DNA binder type, linker type, loading density, surface charge or surface density can be varied to promote faster extraction, exclude/reduce amounts of a binding agent (such as PEG) or chaotropic agent (such as guanidine thiocyanate).

[0032] FIG. IB shows a scanning electron micrograph (SEM) of glass fiber filter (GFF). GFF of specified pore size (0.7 um) and thickness (0.43 mm) is standard for EPA compliant leaching assays. The filter materials are supplied as round discs of various diameter and are used as received from the manufacturer for the silanization methods described here.

[0033] FIG. 2 shows a scheme for synthesis of PFP ester reagent, and DMT assay for measuring surface alkylamine groups. Accessible surface amino groups on the glass surface react with DMT (dimethoxytrityl) containing PFP ester. DMT cation releases with acid treatment and absorbance is measured.

[0034] FIG. 3 shows measurement of surface amine density in aminopropyl (AP) coated glass fiber filters (AP-GFF). AP-GFF were prepared using Method B as detailed in the examples. For kinetics experiment, 0.33 mm thick, 1 pm pore size AP-GFF filters were treated with 0.1 M PFP ester.

[0035] FIG. 4 shows an APTES-derived solid support layer: individual silane molecules can be incorporated into the layer via (a) hydrogen bonding, (b) electrostatic attraction, (c) covalent bonding with the substrate, (d) horizontal and (e) vertical polymerization with neighboring silanes, and (f) oligomeric/polymeric silanes can also react/interact with functionalities present at the interface. The figure shows multilayer coating, crosslinking, and non-covalent bonding occurs at the surface.

[0036] FIG. 5 shows rigid linker in CL silanes increases (such as double) amine density vs. aliphatic amines. The structure of flexible aliphatic DETA and rigid CL54 linker structures are also shown in Fig. 5. Both linkers can form 3 ionizable alkylammonium groups to ionically bind the polyanionic DNA. Structure of an ionically “captured” DNA dinucleotide illustrates relative dimensions of the silanized glass surface.

[0037] FIG. 6 shows a comparison of EDA and APTES coated GFF (Method A). These data show DNA extraction efficiency with the 2 amine coating types. The 2% and 4% APTES showed slightly lower capture efficiency. In this example, EDA retained DNA better at all concentrations. Amine loading values (nmole/cm2) are as follows: APTES: 2% is at 32.5, 4% is at 33.3, 6% is at 31, 8% is at 35. EDA: 0.5% is at 48, 1% is at 50, 2% is at 45, and 4% is at 47.

[0038] FIG. 7 shows EDC coupling of BisTris to succ-AP-GFF. Aminopropyl GFF is first succcinylated to provide carboxylic acid coated surface. EDC activation and coupling to BisTris gives ester coating. At pH 6, unprotonated amine of Bis-Tris can also react.

[0039] FIG. 8 shows AP-GFF coated with a minor groove binding DNA ligand. Bis-benzimide with attached hexylamine linker (BB-NH2) was prepared and conjugated to AP-GFF using a novel cyanuric chloride (CC) activated surface (CC-GFF). BB-CC-AP-GFF binds DNA efficiently, but it does not elute at high pH.

[0040] FIG. 9 shows heat release (top) and alkaline release (bottom) of DNA. Glass Fiber Filters are first silanized with cleavable linker silane (CL53) to give CL53-GFF (top). The amine coating is released from GFF by heating at 95°C for a few minutes. CL53-GFF was further coated with bis-benzimide ligands. DNA capture/heat release was demonstrated.

Alternately, GFF are first silanized with APTES (bottom). This coating binds DNA at pH < 10 and releases at pH > 12.

[0041] FIGS. 10A-10C show an overview of a sample cartridge with a valve assembly configured for performing differing sample processes, including chemical lysing of targets, which is configured for immuno-PCR and optional integrated nucleic acid analysis of the target assay panel in accordance with some embodiments of the invention. FIG. 10A shows the sample cartridge body with reaction vessel, FIG. 10B shows an exploded view of the sample cartridge, and FIG. 10C shows components of the valve assembly, in accordance with some embodiments.

[0042] FIG. HA illustrates various valve assemblies A, B, C, and a universal cartridge each suited for one or more types of target lysing, any of which may be used in a respective sample cartridge. Each cartridge includes a filter material having a surface, in accordance with embodiments, is modified with a DNA binding ligand. Cartridge A performs only mechanical lysing for more hardy targets and includes a filter membrane modified with a DNA binding ligand. Cartridges B and C perform only chemical lysing for viruses, free NA or more fragile targets and include a filter column modified with a DNA binding ligand. The universal cartridge includes a modified filter (40), preferably a modified glass fiber filter and is sized to be secure between the valve cap and valve body.

[0043] FIG. 11B illustrates a universal valve assembly, in accordance with some embodiments, which utilizes glass beads son the modified glass fiber filter suited for mechanical lysis of certain types of targets, as compared to conventional assemblies in conventional sample cartridges. In this embodiment, the modified filter 40 is formed of glass fibers and has a 0.7 um pore size. In contrast, Cartridge A utilizes a filter formed as a disk of a polymer film (i.e., PCTE), which while suitable for mechanical lysing, but not suited for chemical lysing. By utilizing a filter having a pore size of 0.7 um, the filter is suitable for receiving suitably sized glass beads for mechanical lysing. Utilizing glass fibers to form the filter facilitates affinity bonding with the free nucleic acid released by chemical lysing. Thus, this filter is suited for both mechanical and chemical lysing.

[0044] FIG. 12 shows a comparison of glass fiber filters modified with 2% APTES by chemical vapor deposition (CVD1-4) compared to unmodified glass fiber filter (85 A and RCC).

DETAILED DESCRIPTION

Definitions

[0045] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:

[0046] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of compounds, reference to “an amine group” includes mixtures of two or more such amine groups, and the like.

[0047] Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0048] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0049] The term “alkyl” includes both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

[0050] Unless the number of carbons is otherwise specified, “alkyl” as used herein means an alkyl group, as defined above, but having from one to twenty carbons, more preferably from one to ten carbon atoms in its backbone structure. Likewise, “alkenyl” and “alkynyl” have similar chain lengths.

[0051] The alkyl groups can also contain one or more heteroatoms within the carbon backbone. Examples include oxygen, nitrogen, sulfur, and combinations thereof. In certain embodiments, the alkyl group contains between one and four heteroatoms.

[0052] The term “heteroalkyl”, as used herein, refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quatemized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0053] “Alkenyl” and “Alkynyl”, as used herein, refer to unsaturated aliphatic groups containing one or more double or triple bonds analogous in length (e.g., C2-C30) and possible substitution to the alkyl groups described above.

[0054] “Aryl”, as used herein, refers to 5-, 6- and 7-membered aromatic rings. The ring can be a carbocyclic, heterocyclic, fused carbocyclic, fused heterocyclic, bicarbocyclic, or biheterocyclic ring system, optionally substituted as described above for alkyl. Broadly defined, “Ar”, as used herein, includes 5-, 6- and 7-membered single-ring aromatic groups that can include from 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 the ring structure can also be referred to as “heteroaryl”, “aryl heterocycles”, or “heteroaromatics”. The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, — CF3, and — CN. The term “Ar” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles, or both rings are aromatic.

[0055] “Alkylaryl” or “aryl-alkyl”, as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or hetero aromatic group).

[0056] “Heterocycle” or “heterocyclic”, as used herein, refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ring atoms, containing carbon and one to four heteroatoms each selected from non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (C1-4) alkyl, phenyl or benzyl, and optionally containing one or more double or triple bonds, and optionally substituted with one or more substituents. The term “heterocycle” also encompasses substituted and unsubstituted heteroaryl rings. Examples of heterocyclic ring include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4a7/-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2J/,6J/-l,5,2-dithiazinyl, dihydrofuro[2,3-Z>]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, UT-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothi azole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4/7-quinolizinyl, quinoxalinyl, quinuclidinyl, 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, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.

[0057] “Heteroaryl”, as used herein, refers to a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms each selected from non-peroxide oxygen, sulfur, and N(Y) where Y is absent or is H, O, (Ci-Cs) alkyl, phenyl or benzyl. Nonlimiting examples of heteroaryl groups include furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide) and the like. The term "heteroaryl" can include radicals of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto. Examples of heteroaryl include, but are not limited to, furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide), thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide), and the like.

[0058] The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined below. The alkoxy and aroxy groups can be substituted as described above for alkyl.

[0059] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: -NR9R10 or NR9R10 10, wherein R9, Rio, and R'10 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)m-R's or R9 and Rio taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R's represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In some embodiments, only one of R9 or Rio can be a carbonyl, e.g., R9, Rio and the nitrogen together do not form an imide. In some embodiments, the term “amine” does not encompass amides, e.g., wherein one of R9 and Rio represents a carbonyl. In some embodiments, R9 and Rio (and optionally R’ 10) each independently represent a hydrogen, an alkyl or cycloakly, an alkenyl or cycloalkenyl, or alkynyl. Thus, the term "alkylamine" as used herein means an amine group, as defined above, having a substituted (as described above for alkyl) or unsubstituted alkyl attached thereto, /.