Sample preparation device and method of use thereof

Abstract

A sample preparation device is disclosed. The sample preparation device includes a housing defining a passage way between a first opening and a second opening; and a sample filter occupying a section of said passage way. The housing is configured in the shape of a pipette tip. The sample filter contains a filter material having a porosity and thickness that reduce back pressure and allows effective binding of an analyte of interest to the sample filter.

Description

DOCKET NO.: 2001-032 PCTTITLE SAMPLE PREPARATION DEVICE AND METHOD OF USE THEREOF

[0001] This application claims priority from U.S. Provisional Patent App. No.63 / 762,834, filed February 25, 2025, and U.S. Patent App. No. 19 / 082,369, filed on March 18, 2025, both or which are hereby incorporated by reference.FIELD

[0002] The technical field is sample preparation devices in the biochemical art and, in particular, sample preparation devices using a porous filter for sample filtration, separation and purification.BACKGROUND

[0003] The purification of nucleotides from a bio-sample is a necessary first step. To molecular-based testing. Many devices have been developed for the purification of nucleic acids and proteins from bio-samples. However, there still exists a need for sample preparation devices that are easy to use and can be manufactured at low cost.SUMMARY

[0004] One aspect of the present application relates to a sample preparation device for isolating nucleic acids. The sample preparation device comprises: (1) a housing defining a passage way between a first opening and a second opening, wherein the housing is configured in the shape of a pipette tip, and (2) a sample filter occupying a section of the passage way, wherein the sample filter comprises a monolithic porous composite filter made from a silica material and one or more polymeric materials, wherein the monolithic porous composite filter has a porosity in the range of 1 micron to 100 micron and a thickness of 0.5 mm to 5.0 mm.

[0005] Another aspect of the present application relates to a sample preparation kit for isolating nucleic acids. The kit comprises one or more sample preparation devices of the present application and one or more sample preparation solutions.

[0006] Another aspect of the present application relates to a method for isolating nucleic acids from a sample using the sample preparation device of the present application. The method comprises the steps of passing the sample through the samplefilter of the sample preparation device of the present application under conditions that allow nucleic acids in the sample to bind to the sample filter, washing the sample filter to remove non-specifically bound materials and eluting the bound nucleic acids from the sample filter with a separate solution.

[0007] Another aspect of the present application relates to a method for normalizing output of a nucleic acid sample, comprising the steps of: passing the nucleic acid sample through a sample preparation device, wherein the sample preparation device comprises: a housing defining a passage way between a first opening and a second opening; and a monolithic porous composite filter occupying a section of the passage way, wherein the monolithic porous composite filter comprises a silica material and one or more polymeric materials, binds specifically to nucleic acids, and has a nucleic acid binding capacity that is lower than the amount of nucleic acid in the nucleic acid sample such that the monolithic porous composite filter is saturated with bound nucleic acids after the passing step; washing the monolithic porous composite filter; and eluting bound nucleic acids from the monolithic porous composite filter to normalize the nucleic acid sample, wherein the monolithic porous composite filter in each sample preparation device has the same nucleic acid binding capacity, and wherein the normalized nucleic samples comprise similar amounts of nucleic acids.DESCRIPTION OF THE DRAWINGS

[0008] The detailed description will refer to the following drawings, wherein like numerals refer to like elements, and wherein:

[0009] FIG. 1 shows a schematic of an embodiment of the sample preparation device of the present application.

[0010] FIG. 2 shows the result of tolerance testing of an embodiment of the sample preparation device of the present application.

[0011] FIG. 3 shows the extraction efficiency of genomic DNA with an embodiment of the sample preparation device of the present application and a comparative product.

[0012] FIG. 4 shows the comparison between a 1ml Akonni sample tip and a comparative product on viral RNA recoveries from pooled saliva without Influenza A H5N1 (Sample A) and with Influenza A (Sample B, Matrix gene). Replicate extractions of 140 pL pooled saliva samples were extracted using the Akonni sample tip (n=88 for Sample A and n=96 for Sample B) and the comparative product (n=24 for Sample A and n=24 for Sample B). RPP30 is the internal control.

[0013] FIG. 5 shows the extraction sensitivity comparison of 1ml Akonni sample tip with a comparative product. Blue boxes represent the Influenza A H5N1 Matrix gene and the red boxes represent the RPP30 internal control. The data show as good or better performance of the automated Akonni sample tip method (particularly at the lowest titer, 0.316 TCIDso / mL) compared to the comparative product. The sensitivity of these same two methods (1ml Akonni sample tip and the comparative product) were also compared across an Influenza A titer dilution series ranging from 31600 TCIDso / mL to 0.316 TCIDso / mL, with extractions of each dilution point run in triplicate for both methods. All dilutions and replicates for the Akonni sample tips were run simultaneously on a Hamilton STARlet pipetting station. While the two methods show comparable performance, at the lowest titer Akonni sample tip showed higher concentration (lower Ct) and greater precision than the comparative product.

[0014] FIG. 6 shows binding of PCR amplified DNA to the sample preparation device of the present application. The plateau illustrates that the sample filter is saturated to allow normalization of the DNA concentrations of the PCR-amplified libraries above this concentration.

[0015] FIG. 7 shows normalization of PCR amplified DNA with sample preparation devices having different surface binding areas.

[0016] FIG. 8 is a composite of bioanalyzer electropherograms. Top Panel: Prior to PCR amplification of M13 DNA; strong peaks from the primers can be seen. Middle Panel: After PCR, before Normalization / Cleanup; primer-dimer peak is present because of the absence of a cleanup step. Bottom Panel: After PCR, and after Normalization / Cleanup; no peaks other than the 140 base pair (bp) amplicon product is present. These results demonstrate the use of a single binding matrix in a pipette tip to simultaneously normalize (reduction in signal) and perform PCR cleanup (shows a single clean peak).DETAILED DESCRIPTION

[0017] Reference will be made in detail to certain aspects and exemplary embodiments of the application, illustrating examples in the accompanying structures and figures. The aspects of the application will be described in conjunction with the exemplary embodiments, including methods, materials and examples, such description is non-limiting and the scope of the application is intended to encompass all equivalents, alternatives, and modifications, either generally known, or incorporated here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this applicationbelongs. One of skill in the art will recognize many techniques and materials similar or equivalent to those described here, which could be used in the practice of the aspects and embodiments of the present application. The described aspects and embodiments of the application are not limited to the methods and materials described.

[0018] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0019] As used herein, “about,” “approximately,” “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and / or with e.g. a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. 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. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to "the value," greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed.

[0020] Where a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includesone or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0021] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0022] The drawing figures are not necessarily to scale and certain features of the invention may be shown exaggerated in scale or in somewhat schematic form in the interest of clarity and conciseness. In the description, relative terms such as "front," "back," "up," "down," "top" and "bottom," as well as derivatives thereof, should be construed to refer to the orientation as then described or as shown in the drawing figure under discussion. These relative terms are for convenience of description and normally are not intended to require a particular orientation. Terms concerning attachments, coupling and the like, such as "connected" and "attached," refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise.

[0023] In describing the invention, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0024] In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be evident, however, to one skilled in the art that the present invention may be practiced without these specific details.

[0025] In describing embodiments of the present invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. It is to be understood that each specific element includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.I. Sample preparation device

[0026] One aspect of the present invention relates to a sample preparation device. In one embodiment, the sample preparation device includes a housing that defines a sample passage way between two openings, and a sample filter embedded in a section of the passage way. The sample filter comprises a monolithic porous composite filter that specifically binds to an analyte. In some embodiments, the analyte comprises nucleic acids. In some embodiments, the sample preparation device further comprises an aerosol filter to prevent cross-contamination between a sample and a liquid delivery system, such as a manual pipettor, an electronic pipettor, or a robotic pipetting station.

[0027] A. The housing

[0028] The housing comprises an enclosure with two openings connected by an elongated passage way. In some embodiments, the housing has a volume in the range of about 0.1 pl to about 20 ml. In some embodiments, the housing has a volume in the range of about 10 pl to about 10 ml. In some embodiments, the housing has a volume in the range of about 5 pl to about 5 ml. Suitable materials for the housing are not particularly limited, and include plastics (such as polyethylene, polypropylene, and polystyrene), glass and stainless steel. The shape of the housing is not particularly limited. In some embodiments, the housing has a cylindrical shape, a conical cylindrical shape, or combinations thereof. In some embodiments, the housing has the shape of a pipette tip.

[0029] B. The sample filter

[0030] (1) Sample filter material

[0031] In some embodiments, the sample filter is made of a monolithic porous composite filter that binds specifically to nucleic acids. The term “monolithic porous composite filter” as used herein, refers to a porous, three-dimensional adsorbent material having a continuous interconnected pore structure in a single piece. The term"monolithic porous composite" or “monolithic porous composite filter” is meant to be distinguished from a collection of individual adsorbent particles packed into a bed formation or embedded into a porous matrix, in which the end product comprises individual adsorbent particles. The term "monolithic porous composite" or “monolithic porous composite filter” is also meant to be distinguished from a collection of adsorbent fibers or fibers coated with an adsorbent, such as filter papers or filter papers coated with an adsorbent.

[0032] In some embodiments, the monolithic porous composite filter comprises a mixture of a silica material, such as glass beads, glass fibers and / or woven glass fibers, and one or more polymeric materials, such as epoxy, vinylester, polyester thermosetting plastic, phenol formaldehyde resins, polyamide, acetal homopolymers and copolymers, polypropylene, polyethylene, low density polyethylene, high density polyethylene, ultra high molecular weight polyethylene, polyvinyl chloride, polyphenylene sulfide, polyethylene oxide, polycarbonate, polyethersulphone, acrylonitrile butadiene styrene, liquid crystal polymers, polyether ether ketone, polyethylenimine, and polyethylene terephthalate. In some embodiments, the monolithic porous composite filter of the present application comprises glass beads and one or more polymeric materials, wherein the glass beads and one or more polymeric materials form a monolithic porous composite structure.

[0033] In some embodiments, the amount of silica material in the monolithic porous composite filter is in the range of 1-5 wt%, 1-10 wt%, 1-15 wt%, 1-25 wt%, 1-50 wt%, 1-75 wt%, 5-10 wt%, 5-15 wt%, 5-25 wt%, 5-50 wt%, 5-75 wt%, 10-15 wt%, 10-15 wt%, 10-25 wt%, 10-50 wt%, 10-75 wt%, 15-25 wt%, 15-50 wt%, 15-75 wt%, 25-50 wt%, 25-75 wt%, or 50-75 wt% of the monolithic porous composite filter. In some embodiments, the amount of polymer materials in the monolithic porous composite filter is in the range of 25-99 wt%, 25-95 wt%, 25-90 wt%, 25-85 wt%, 25-75% wt, 25-50 wt%, 50-99 wt%, 50-95 wt%, 50-90 wt%, 50-85 wt%, 50-75% wt, 75-99 wt%, 75-95 wt%, 75-90 wt%, 75-85 wt%, 85-99 wt%, 85-95 wt%, 85-90 wt%, 90-99 wt%, 90-95 wt%, or 95-99 wt% of the monolithic porous composite filter.

[0034] In some embodiments, the monolithic porous composite filter in the sample preparation device of the present application is designed to intentionally saturate to normalize the nucleic acid concentration to a specific amount so that all nucleic acid concentrations across multiple samples are the same, which is common practice for library preparation in sequencing. In some embodiments, the monolithic porous composite filter or the sample filter in the sample preparation device has nucleic acid binding capacity in the range of 0. Ing to lOpg. (As used herein, the term “nucleic acid binding capacity” refers to themaximum amount of nucleic acids that may bind to, and be eluted from, the sample filter in a sample preparation device of the present application under the binding and eluting conditions described in the present application).

[0035] While the monolithic porous composite filter is a good adsorbent for nucleic acids, a skilled artisan would recognize that the monolithic porous composite filter may also be used to absorb other types of molecules. For example, the monolithic porous composite of the present application can be used as passive adsorption material for solid phase extraction. In other embodiments, the monolithic porous composite filter may be coated with antibodies to extract other ligand of interest from the sample. In one embodiment, the monolithic porous composite filter is derivatized in polymethylmethacrylate (PMMA) and cyclo- olefin-copolymer (COC) with antibodies as capture moieties for microbes and toxin. The term "antibody", as used herein, is used in the broadest possible sense and may include but is not limited to an antibody, a recombinant antibody, a genetically engineered antibody, a chimeric antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a heteroantibody, a monoclonal antibody, a polyclonal antibody, a camelized antibody, a deimmunized antibody, and an anti- idiotypic antibody. The term "antibody" may also include but is not limited to an antibody fragment such as at least a portion of an intact antibody, for instance, the antigen binding variable region. Examples of antibody fragments include Fv, Fab, Fab', F(ab'), F(ab')2, Fv fragment, diabody, linear antibody, single-chain antibody molecule, multispecific antibody, and / or other antigen binding sequences of an antibody. In another embodiment, the monolithic porous composite filter is coated with lectins, which bind to carbohydrates found in bacteria coats and can be used to capture bacteria in a sample.

[0036] (2) Sample filter porosity

[0037] The term “porosity,” refers to a measure of the void spaces in a material, and is a fraction of the volume of voids over the total volume, between 0 and 1 , or as a percentage between 0% and 100%. Strictly speaking, some tests measure the "accessible void", the total amount of void space accessible from the surface.

[0038] The porosity of the porous monolithic composite filter is application dependent. In general, the porous monolithic material should have a porosity that allows for a desired sample flow rate for a particular application. In some embodiments, the porosity of the porous monolithic composite filter is in the range of 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40%, 40-80%, 40-70%, 40-60%, 40-50%, 50-80%, 50-70%, 50-60%, 60-80%, 60-70% or 70-

[0039] (3) Sample filter pore size

[0040] The term “pore size,” as used herein, refers to the average pore size of pores in a porous material. The pore sizes are determined using methods well-known in the art. In some embodiments, the monolithic porous composite filter has a pore size in the range of 1-500, 1-400, 1-300, 1-200, 1-100, 1-50, 1-20, 1-10, 2-500, 2-400, 2-300, 2-200, 2-100, 2-50, 2-20, 2-10, 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 5-20, 5-10, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-20, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400 or 400-500 microns.

[0041] In some embodiments, the monolithic composite filter has a pore size in the range of between about 2 microns and about 220 microns. In some embodiments, monolithic porous composite filter has a pore size in the range of between about 2 microns and about 100 microns. In some embodiments, the monolithic porous composite filter has a pore size in the range of between about 40 microns and about 75 microns. In some embodiments, the monolithic porous composite filter has a pore size in the range of between about 150 microns and about 200 microns. In yet another embodiment, the monolithic porous composite filter has a pore size in the range of between about 2 microns and about 20 microns.

[0042] In some embodiments, the monolithic porous composite filter has a thickness in the range of 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-2, 2-30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25 or 25-30 mm. In some embodiments, the monolithic porous composite filter has a thickness in the range of between about 1 mm and about 20 mm. In some embodiments, the monolithic porous composite filter has a thickness in the range of between about 2 mm and about 5 mm. In some embodiments, the monolithic porous composite filter has a thickness in the range of between about 2 mm and about 3 mm. In some embodiments, the monolithic porous composite filter has a thickness of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mm.

[0043] In some embodiments, the sample filter is a filter made from composites of a silica material (e.g., glass beads) and one or more polymeric materials, such as epoxy, vinylester, polyester thermosetting plastic, phenol formaldehyde resins, polyamide, acetal homopolymers and copolymers, polypropylene, polyethylene, low density polyethylene, high density polyethylene, ultra high molecular weight polyethylene, polyvinyl chloride, polyphenylene sulfide, polyethylene oxide, polycarbonate,polyethersulphone, acrylonitrile butadiene styrene, liquid crystal polymers, polyether ether ketone, polyethylenimine, and polyethylene terephthalate.

[0044] (4) Sample filter tolerance

[0045] The sample filter of the present application has elastomeric properties that allow deformation of the filter during the manufacturing process of the sample preparation device of the present application. The elasticity may be expressed by the tolerance test as shown in FIG. 2. In some embodiments, the monolithic porous composite filter has a diameter tolerance in the range of ±10%, ±8%, ±7% ±6%, ±5%, ±4%, ±3%, ±2% or ±1%. This larger tolerance reduces the stringency (and cost) of the manufacturing process and allows for insertion of the filter into the pipette tips without requiring heat. The elastomeric nature of the composite filter also ensures a tight fit to the side walls of the housing to prevent liquid from going around the sides of the filter.

[0046] As used herein, the term “diameter tolerance” define the allowable diameter variance for assembling the sample preparation device, and in particular, for inserting the sample filter into the passage way of the housing and forming an interference fit or force fit.

[0047] (5) The analytes

[0048] The analyte can be any components or molecules of interest in a sample. In some embodiments, the analyte comprises nucleic acids. The term "nucleic acid," as used in the embodiments described hereinafter, refers to individual nucleic acids and polymeric chains of nucleic acids, including DNA and RNA, whether naturally occurring or artificially synthesized (including analogs thereof), or modifications thereof, especially those modifications known to occur in nature, having any length. Examples of nucleic acid lengths that are in accord with the present invention include, without limitation, lengths suitable for PCR products (e.g., about 50 to 700 base pairs (bp)) and human genomic DNA (e.g., on an order from about kilobase pairs (kb) to gigabase pairs (gb)). Thus, it will be appreciated that the term "nucleic acid" encompasses single nucleic acids as well as stretches of nucleotides, nucleosides, natural or artificial, and combinations thereof, in small fragments, e.g., expressed sequence tags or genetic fragments, as well as larger chains as exemplified by genomic material including individual genes and even whole chromosomes.

[0049] (6) Specific binding to analyte

[0050] The term "specifically bind to" or "specific binding," as used in the embodiments described hereinafter, refers to the binding of the adsorbent to an analyte (e.g., nucleic acids) with a specificity that is sufficient to differentiate the analyte fromother components or contaminants of a sample. In one embodiment, the dissociation constant of the adsorbent / ligand complex is less than about IxlO'6M. A person of ordinary skill in the art understands that stringency of the binding and elution of the analyte to the adsorbent can be controlled by binding and elution buffer formulations. For example, elution stringencies for nucleic acids can be controlled by salt concentrations using KC1 or NaCl. Nucleic acids, with their higher negative charge, are more resistant to elution than proteins. Temperature, pH, and mild detergent and other treatments that could be used for selective binding and elution. In some embodiments, The analytes are nucleic acids and the selective binding to the sample filter is carried out at a pH value in the range of pH 3-9, pH 3-8, pH 3-7, pH 3-6, pH 3-5, pH 3-4, pH 4-9, pH 4-8, pH 4-7, pH 4-6, pH 4-5, pH 5-9, pH 5-8, pH 5-7, pH 5-6, pH 6-9, pH 6-8, pH 6-7, pH 7-9, pH 7-8 or pH 8-9. Thermal consistency of the binding and elution may be maintained with a heat block or a water bath. The manipulation of the binding buffer is preferable since the impact of the modified elution buffer on the downstream analyzer would need to be evaluated.

[0051] C. The aerosol filter

[0052] The aerosol filter is configured to prevent cross contamination between the liquid sample and the tip of a pump device such as a manual pipettor, an automatic pipettor or a robotic pipetting station. The aerosol filter has a pore size that is larger than the pore size of the sample filter and does not bind specifically to the analyte. The aerosol filter is typically placed in the proximity of the first opening so as to avoid direct contact with the liquid sample during operation.

[0053] Exemplary device

[0054] Referring now to FIG. 1, an exemplary embodiment of the sample preparation device 100 includes a housing 110, a sample filter 150. The housing 100 defines a sample passage way 140 between a first opening 120 and a second opening 130. The shape and size of the housing 110 are not particularly limited. The preferred housing configuration is substantially cylindrical so that the flow vectors during operation are substantially straight, thereby minimizing or avoiding dilutional washing that might occur with non-cylindrical configurations. In the embodiments shown in FIG.1, the housing 110 has a pipette tip geometry, i.e., the first opening 120 has a diameter that is greater than the diameter of said second opening 130, and the first opening 120 is dimensioned to fit onto the tip of a pipettor. A sample filter 150 is placed in the close proximity of the second opening 130 so that samples are filtered immediately after being taken into the housing 110 through the second opening 130. In one embodiment, thesample filter 150 is contiguous with the second opening 130. In another embodiment, the sample filter 150 is separated from the second opening 130 by a distance of 1-20 mm. In another embodiment, the housing 110 has a column geometry.

[0055] The sample filter 150 is shaped to fit tightly into the passage way 140 to prevent the sample from channeling or bypassing the sample filter 150 during operation. In one embodiment, the filter 150 is fitted into the passage way 140 through mechanical means such as crimping and press fitting. In another embodiment, the filter 150 is attached to the interior of passage way 140 through an adhesive. In yet another embodiment, the side of the filter is tapered to have a frustoconical shape to the contour of the passage way 140. In the embodiments shown in FIG. 1 , the housing 110 has the shape of a frustoconical pipette tip with the first opening 120 dimensioned to fit on the end of a liquid delivery system, such as a manual pipettor or an electronic pipetting device. Samples are taken up through the second opening 130, passed through the sample filter 150 and then retained in the section of the housing 110 that is above the sample filter 150. In one embodiment, the liquid delivery system is an electronic pipetting device, such as an electronic pipettor or a robotic pipetting station.

[0056] In some embodiments, the housing 110 further contains an aerosol filter (not shown in FIG. 1) placed between the first opening 120 and the sample filter 150. The aerosol filter has a pore size that is larger than the pore size of the sample filter 150 and does not bind specifically to nucleic acids. The aerosol filter is placed in the proximity of the first opening 120 to avoid direct contact with liquid samples in the passage way 140.

[0057] Integrating the sample filter of the present application into a pipette tip has the benefit of bidirectional, multi-pass flow, which increases the nucleic acid residence time with the porous matrix without the need for small pores and hence allows the use of filters with larger porosities and thicker matrices. Larger porosities and thicker matrices offer a benefit of reduced backpressure, which is a function of the pore diameter to the fourth power verse the length to the first power, according the following Hagen-Poiseuille equation:

[0058] where p is the pressure drop across the pore, p is the viscosity, L is the length of a single pore, Q is the flow rate, and d is the diameter of the pore. Thus, larger diameter and shorter length pores have lower back pressure than smaller diameter andlonger length pores.

[0059] Lower backpressure becomes particularly important for implementations that utilize low pressures while trying to achieve high flow rates over short durations. One such implementation is incorporation of a filter in a pipette tip. The advantage of a high porosity, thick filter in a pipette tip is that bi-directional, multi-pass flow can be controlled with conventional pipetting motions while achieving sufficient binding capacities. Additionally, thicker matrices are more useful for pipette tip insertion to maintain its structural rigidity during insertion. To this end, the use of glass-reinforced, a glass-filled, or woven glass fiber filters or composites of glass and polymeric materials (composite glass filters) can serve to provide the necessary binding surface sites for nucleic acid adsorption. In some embodiments, the pore sizes of the sample filters are in the range of 1 pm to 100 pm, preferably in the range of 10 pm to 40 pm, 10 pm to 15 pm or 1 pm to 40 pm. The thickness of the sample filters are in the range of 0.5 mm to 10.0 mm. In some embodiments, the sample filters are constructed to bind specifically to nucleic acids, or nucleic acids of a desired size range under suitable ionic and pH conditions. In some embodiments, the suitable pH condition is in the range of pH 3-9, pH 3-8, pH 3-7, pH 3-6, pH 3-5, pH 3-4, pH 4-9, pH 4-8, pH 4-7, pH 4-6, pH 4-5, pH 5-9, pH 5-8, pH 5-7, pH 5-6, pH 6-9, pH 6-8, pH 6-7, pH 7-9, pH 7-8 or pH 8-9.

[0060] In some embodiments, the sample filter 150 consists of a single layer of the monolithic porous composite of the present application with an uniform distribution of porosity and pore size. In some embodiments, the sample filter 150 consists of a single monolith layer of the monolithic porous composite of the present application with two or more porous regions with different pore sizes. In some embodiments, the sample filter 150 consists of a single layer of the monolithic porous composite of the present application with a first porous region and a second porous region, with pore sizes in the range of 2-20 pm, 2-15 pm, 2-10 pm, 5-20 pm, 5-15 pm, 5-10 pm, 7-20 pm, 7-15 pm, and 7-10 pm in the first regions and pore sizes in the range of 25-50 pm, 25-40 pm, 25-35 pm, 25-30 pm, 30-50 pm, 30-40 pm, 30-35 pm, 35-50 pm, 35-40 pm, or 40-50 pm in the second region. In some embodiments, the sample filter 150 consists of a single layer of the monolithic porous composite of the present application with a first porous region and a second porous region, with pore sizes in the range of 1 - 10 pm in the first region and about 10-50 pm in the second region. In some embodiments, the sample filter is made from a blend of polymeric materials (such as polyethylene) and silica materials (such as glass beads or glass fibers). The polymer / silica blend is then heated or sintered to generate a single monolithic porous composite filter with two regions of poresizes, as described above.

[0061] In some embodiments, the sample filter 150 has a conical shape or disc shape that fits into the interior space of a pipette tip, wherein the second region has an average diameter that is smaller than the average diameter of the first region. In some embodiments, the sample filter 150 has a diameter in the range of 2-10 mm, 2-8 mm, 2-6 mm, 2-5 mm, 2-4 mm, 2-3 mm, 3-10 mm, 3-8 mm, 3-6 mm, 3-5 mm, 3-4 mm, 4-10 mm, 4-8 mm, 4-6 mm, 4-5 mm, 5-10 mm, 5-8 mm, 5-6 mm, 6-10 mm, 6-8 mm or 8-10 mm.

[0062] In some embodiments, the sample filter 150 has a thickness in the range of 0.5-10 mm. In some embodiment, the sample filter 150 has a thickness of 0.5-8.0 mm, 0.5-6.0 mm, 0.5-5.0 mm, 0.5-4.0 mm, 0.5-3.0 mm, 0.5-2.0 mm, 1.0-8.0 mm, 1.0-6.0 mm, I.0-5.0 mm, 1.0-4.0 mm, 1.0-3.0 mm, 1.0-2.0 mm, 2.0-8.0 mm, 2.0-6.0 mm, 2.0-5.0 mm, 2.0-4.0 mm, 2.0-3.0 mm, 3.0-8.0 mm, 3.0-6.0 mm, 3.0-5.0 mm, or 3.0-4.0 mm. In some embodiments, the sample filter 150 has a thickness of about 4 mm.

[0063] The sample filter 150 may be placed at any position within the housing of the pipette tip. In some embodiments, the sample filter 150 is placed in close proximity of the pipette tip so that samples are filtered immediately after being taken into the housing 140 through the second opening 130. In one embodiment, the sample filter 150 is contiguous with the second opening 130.

[0064] In another embodiment, the sample filter 150 is separated from the second opening 130 by a distance of 0-40 mm, preferably 0-20 mm. In other embodiments, the sample filter 150 is separated from the second opening 130 by a distance of 40-120 mm. In yet other embodiments, the sample filter is separated from the second opening 130 by a distance of 60-80 mm, e.g., 75 mmII. Kit

[0065] Another aspect of the present application relates to a kit for sample preparation. The kit comprises (1) one or more sample preparation device of the present application and (2) one or more solutions for sample preparation with the sample preparation device of the present application.III. Method of use

[0066] Another aspect of the present application relates to a method for isolating an analyte in a sample with the sample preparation device of the present application. In some embodiments, the analyte is nucleic acid. In some embodiments, the sample is a saliva sample. In some embodimnets, the method comprises the steps of (1) passing the nucleic acid sample through a sample preparation device of the present application, wherein the sample preparation device comprises a housing defining a passage waybetween a first opening and a second opening and a monolithic porous composite filter occupying a section of the passage way, wherein the monolithic porous composite filter comprises a silica material and one or more polymeric materials, binds specifically to nucleic acids, (2) washing the monolithic porous composite filter to remove non-specific binding, and (3) eluting bound nucleic acids from the monolithic porous composite filter.

[0067] Another aspect of the present application method for normalizing output from a plurality of nucleic acid samples, such as nucleic acid library samples. In some embodiments, the method comprises the steps of (1) passing the nucleic acid sample through a sample preparation device of the present application, wherein the sample preparation device comprises a housing defining a passage way between a first opening and a second opening and a monolithic porous composite filter occupying a section of the passage way, wherein the monolithic porous composite filter comprises a silica material and one or more polymeric materials, binds specifically to nucleic acids, and has a nucleic acid binding capacity that is lower than the amount of nucleic acids in the nucleic acid samples such that the monolithic porous composite filter is saturated with bound nucleic acids after the passing step; (2) washing the monolithic porous composite filter to remove non-specific binding; and (3) eluting bound nucleic acids from the monolithic porous composite filter to produce a normalized nucleic acid sample. In this method, the monolithic porous composite filter in each sample preparation device has the same (or similar) nucleic acid binding capacity. Since each nucleic acid sample contains more nucleic acids than the monolithic porous composite filter can absorb (or bind), only a fraction of the nucleic acids in each nucleic acid sample binds to the filter and is subsequently eluted from the filter. Accordingly, each normalized nucleic sample comprises similar amounts of nucleic acids to other nucleic samples processed the same way (as determined by the nucleic acid binding capacity of the monolithic porous composite filter in the sample preparation device).

[0068] In some embodiments, the plurality nucleic acid samples are nucleic acid library samples containing PCR primers and PCR amplification products. In some embodiments, the normalized nucleic samples are free from PCR primers.EXAMPLES

[0069] Example 1 : Methods

[0070] Extraction of genomic DNA

[0071] Saliva samples are lysed with commercially available lysing agents.Genomic DNA in the lysed samples are extracted with a sample preparation device of the present application (1 ml Akonni sample tip) using the nucleic acid extractionprocedure described below or with a comparative device using a corresponding procedure.

[0072] Extraction of saliva RNA

[0073] Saliva samples are lysed with commercially available lysing agents.Genomic DNA in the lysed samples are extracted with a sample preparation device of the present application (1 ml Akonni sample tip) using the nucleic acid extraction procedure described below described below or with a comparative device using a corresponding procedure.

[0074] Nucleic acid extraction procedure for lysed samples with 1 ml Akonni sample tip and buffers

[0075] - pipetting lysed samples with 1 ml Akonni sample tip by 15 pipetting cycles (aspiration + dispense) to allow nucleic acids binding to the sample filter in the Akonni sample tip,

[0076] -washing the sample filter by 5 pipetting cycles with Akonni Wash J buffer

[0077] - washing the filter by 5 pipetting cycles with Akonni Wash K buffer

[0078] - drying filter-bound nucleic acids by 100 pipetting cycles (aspirate + dispense air)

[0079] - eluting filter-bound nucleic acids by 5 cycles pipetting in Akonni Elution A2 buffer.

[0080] Example 2: Tolerance test of the monolithic porous composite filter of the present application

[0081] 1 -ml Akonni sample tips were assembled with three different diameters: nominal, 5% above the mean, and 5% below the mean. Saliva samples were spiked with hCoV-NL63 virus at 6.3x106 TCID50 / mL. Thirty (30) tips per filter diameter were used to process the saliva. The results in FIG. 2 show that both the internal RNA polymerase RPP30 control and the spiked NL63 virus had equivalent mean Ct values for each condition.

[0082] As shown in FIG. 2, effective viral RNA extraction was achieved with all the test sizes, suggesting that the monolithic porous composite filters of the present application are have elastomeric properties and allows for a greater tolerance as compared to the rigid nature of sintered glass filters. As a consequence, sintered glass filters require that the tips are heated when the filters are inserted. The monolithic porous composite filters of the present application do not require heat for insertion.

[0083] Example 3: Extraction efficiency of genomic DNA from saliva

[0084] Saliva samples were lysed and genomic DNA was extracted from each sample using either 1ml Akonni sample tip or a comparative device. As shown in FIG.3, the Akonni sample tip produced more genomic DNA than the comparative device.

[0085] The Akonni sample tips also isolate genomic DNA in a shorter period of time than comparative devices using glass fiber filters for nucleic acid isolation (e.g., 10 minutes using the Akonni sample tips vs 30 mintues using the comparative devices).

[0086] Example 4: Extraction efficiency of viral RNA from saliva

[0087] Saliva samples were lysed and RNA was extracted from each sample using either 1ml Akonni sample tip or a comparative device. Reproducibility using an automated Akonni sample tip nucleic acid purification procedure was compared directly to manual extractions using a comparative manual RNA extraction kit using a 140pL input for both methods. Samples used comprised of saliva collected in OMNIgene saliva collection tubes (OMR-610), then pooled and divided into two homogenous replicates. One replicate received no virus (Sample A) and one replicate was spiked with Influenza A H5N 1 (strain A / turkey / Ontario / 1966) to a titer of 316 TCID50 / mL (Sample B). Two rounds of automated and manual extractions were performed on individual aliquots of Sample A and Sample B (88 and 96 respective total for automated and 24 and 24 respective total for manual). The second round of automated extractions employed a checkboard pattern of sample plating to identify potential cross-contamination.Extraction methods were compared via detection of Influenza A Matrix gene and control human RNAse P (RPP30) RNA using real-time reverse-transcriptase PCR assay (Luna® Universal Probe One-Step RT-qPCR Kit, NEB). FIG. 4 shows the Influenza A H5N1 RNA recoveries (in blue) and RPP30 recoveries (in red) of the two extraction methods from pooled saliva with no false positives detected for any of the Influenza-negative samples. The automated processing time for Akonni sample tip for 96 samples was 70-75 minutes compared to approximately 2 hours for 24 samples for the manual QI Amp Viral RNA Extraction kit. The Akonni sample tip extraction process resulted in better precision and comparable recovery compared to the comparative device.

[0088] Example 5: Extraction sensitivity of viral RNA from saliva

[0089] Saliva samples were lysed and RNA was extracted from each sample using either 1ml Akonni sample tip or a comparative device. The sensitivity of these same two methods (Akonni sample tip and the comparative product) were compared across an Influenza A titer dilution series ranging from 31600 TCID50 / mL to 0.316 TCID50 / mL, with extractions of each dilution point run in triplicate for both methods. All dilutions and replicates for the 1ml Akonni sample tip were run simultaneously on aHamilton STARlet pipetting station. As shown in FIG. 5, while the two methods show comparable performance, at the lowest titer 1ml Akonni sample tip showed higher concentration (lower Ct) and greater precision than the comparative product.

[0090] Example 6: Normalization

[0091] Ml 3 bacteriophage DNA was PCR amplified, as an internal PCR control, using the KAPA Library Quantification Kit-Illumina / ABI Prism. A dilution series from stock (no dilution) to 400X of the amplified product were created to determine if the sample filter in a 1ml sample tip saturates and thus could be used to normalize a range of input sequencing library concentrations. Each dilution of the amplified product (simulating a range of library DNA concentrations) was combined with a binding buffer. The mixture (input) was processed through one sample tip for each dilution (sample), which were designed to have low binding capacity matrices so that it saturates below the expected concentrations of the PCR amplified libraries. As shown in FIG. 6, the output concentration of DNA plateaued at dilution 1:5 and lower, indicating that the sample filter in the sample tip had reached its nucleic acid binding capacity at these dilutions. As shown in FIG. 7, it appears that the nucleic acid binding capacity of a sample tip is proportional to the surface area of the sample filter in the sample tip.

[0092] The undiluted (“dirtiest”) product from this study was analyzed on a bioanalyzer to determine if binding matrix was also able to “cleanup” and isolate only the amplified product. As shown in FIG. 8, after normalization, there is only a single “clean” and narrow peak in the electropherogram at the expected molecular weight (140bp), with no evidence of primer-dimer peaks, demonstrating successful PCR-cleanup. These data show that the binding matrix offers the potential to normalize sequencing libraries and do PCR cleanup with a single tip.

[0093] While various embodiments have been described above, it should be understood that such disclosures have been presented by way of example only and are not limiting. Thus, the breadth and scope of the subject compositions and methods should not be limited by any of the above-described exemplary embodiments but should be defined only in accordance with the following claims and their equivalents.

[0094] The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present application, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present application, which is defined by the following claims. The claims are intended to coverthe components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

Claims

WHAT IS CLAIMED IS:

1. A sample preparation device, comprising:a housing defining a passage way between a first opening and a second opening, wherein said housing is configured in the shape of a pipette tip; anda sample filter occupying a section of said passage way, wherein said sample filter comprises monolithic porous composite filter comprising a silica material and one or more polymeric materials,wherein said monolithic porous composite filter has a pore size in the range of 1 micron to 100 micron and a thickness of 0.5 mm to 5.0 mm.

2. The sample preparation device of claim 1, wherein the monolithic porous composite filter binds specifically to nucleic acids.

3. The sample preparation device of claim 1 or 2, wherein the monolithic porous composite filter has a porosity in the range of 20-70%.

4. The sample preparation device of any one of claims 1-3, wherein the monolithic porous composite filter has a diameter tolerance of ±%5.

5. The sample preparation device of of any one of claims 1-4, wherein said first opening is configured for connecting to a pipetting device.

6. The sample preparation device of of any one of claims 1-5, further comprising an aerosol filter, placed in the proximity of the first opening.

7. The sample preparation device of of any one of claims 1-6, wherein the sample filter is placed in the proximity of the second opening.

8. A kit for isolating nucleic acid from a sample, comprising:one or more sample preparation device of of any one of claims 1-7; and one or more solutions for nucleic acid isolation.

9. A method for isolating nucleic acids from a liquid sample, comprising the steps of:passing the liquid sample through the sample filter of the sample preparation device of of any one of claims 1 -7 to allow nucleic acids in the liquid sample to specifically bind to the sample filter;washing the sample filter; andeluting the bound nucleic acids from the sample filter.

10. A method for normalizing output from a plurality of nucleic acid samples, comprising the steps ofpassing each of the plurality of nucleic acid samples to a sample preparation device, wherein the sample preparation device comprises: a housing defining a passage way between a first opening and a second opening; and a monolithic porous composite filter occupying a section of the passage way, wherein the monolithic porous composite filter comprises a silica material and one or more polymeric materials, binds specifically to nucleic acids, and has a nucleic acid binding capacity that is lower than the amount of nucleic acids in each of the plurality of nucleic acid samples such that the monolithic porous composite filter is saturated with bound nucleic acids after the passing step;washing the monolithic porous composite filter; andeluting bound nucleic acids from the monolithic porous composite filter to produce a normalized nucleic acid sample,wherein the monolithic porous composite filter in each sample preparation device has the same nucleic acid binding capacity, and wherein the normalized nucleic samples comprise similar amounts of nucleic acids.

11. The method of claim 10, wherein each of the plurality nucleic acid samples is a nucleic acid library sample containing PCR primers and PCR amplification products.

12. The method of claim 11, wherein the normalized nucleic samples are free from PCR primers.

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