HK1196433A

HK1196433A - Use of porous polymer materials for storage of biological samples

Info

Publication number: HK1196433A
Application number: HK14109848.9A
Authority: HK
Inventors: 艾米丽．弗朗西丝．希尔德; 洪韦博
Original assignee: 塔斯马尼亚大学
Priority date: 2011-07-12
Filing date: 2012-07-11
Publication date: 2014-12-12

Description

Use of porous polymer materials for storing biological samples

Technical Field

The present invention relates generally to the use of porous polymeric materials as a medium for storing biological samples. The invention also relates to a method for drying and storing a biological sample on a porous polymeric material. The biological samples include blood and plasma samples.

Background

A sampling technique known as Dried Blood Spotting (DBS) was developed by the microbiologist Robert Guthrie in 1963. The sample collection procedure is extremely simple, involving the collection of very small amounts of blood from small incisions in the heel or fingers. A drop of blood was then applied directly to the sampling paper and dried for future analyte extraction. DBS sampling is now a common and established practice for quantitative and qualitative screening of metabolic diseases in neonates (Edelbrook, P.M., J.van der Heijden and L.M.L.Stolk, Dried Blood Spot Methods in Therapeutic Drug Monitoring: Methods, Assays, and Pitfalls. Therapeutic Drug Monitoring, 2009.31 (3): page 327-.

Conventional sampling techniques employ plasma or serum as the biological matrix of choice for analysis (biological matrix). These techniques require large volumes of blood to be collected directly from the veins of the test subject. In contrast, DBS sampling requires much smaller sample volumes (microliters, rather than milliliters), which allows sample collection in situations where collection in a traditional manner is difficult, and DBS sampling is now routinely used in epidemiological studies, e.g., it has been successfully implemented for the determination of many biological markers, such as amino acids (Corso, G, et al, Rapid Communications in Mass Spectrometry,2007.21 (23): 3777-.

The DBS method is particularly suitable for analyzing infectious agents (infectious agents) such as HIV and HCV, since the reduced sample volume minimizes the risk of infection and the blood is no longer considered biologically harmful once it is dry, which greatly simplifies storage and transport of the sample (alanson, a.l. et al, journal Pharmaceutical and biological Analysis, 2007, 44 (4): page 963-. The sample can be easily and cost-effectively transported around the world without special storage requirements. Another advantage provided by this technique is that no equipment such as centrifuges and freezers are required for sample processing or storage.

DBS technology is also applied to pharmacokinetic analysis to analyze components in blood.

Media used in current DBS processes, which include drying and storing blood and plasma samples prior to future extraction and analysis, include paper-based cellulosic materials. For example, modified paper-based materials have been developed for simplifying the isolation of nucleic acids; where paper is chemically treated with compounds to promote long-term storage of DNA. However, paper-based cellulose materials are not particularly suitable for accelerating the drying process (especially for plasma) and are not suitable for introducing a specific functionality for selective extraction of components from blood.

There is therefore a need to identify alternative materials that provide properties that facilitate drying and storage of biological samples (including body fluids such as blood and plasma samples) for future extraction and analysis; or to allow specific functionalities to be incorporated into the storage medium.

Disclosure of Invention

In a first aspect, there is provided the use of a porous polymeric material as a medium for drying and storing a biological fluid sample, wherein the porous polymeric material is selected from a porous polymeric matrix material or a porous polymeric monolith material, wherein the porous polymeric monolith material is formed by a step-growth polymerization process.

The biological fluid sample may be a body fluid selected from blood, urine, mucus (mucous), synovial fluid (synovalous), cerebrospinal fluid, tears, or other body secretions. In one embodiment, the use of the porous polymeric material as a medium is for storing whole blood. In a preferred embodiment, the use is for dry blood spot sampling (DBS). In another embodiment, the use of the porous polymeric material as a medium is for storing blood plasma. In a preferred embodiment, the use is for dry plasma spot sampling (DPS).

In one embodiment, there is provided the use of a porous polymeric matrix material as a medium for drying and storing a biological fluid sample. In another embodiment, the present invention provides the use of a porous polymeric monolithic material as a medium for drying and storing a biological fluid sample.

The porous polymeric material medium has an integral body having a pore size and/or a specific surface area adapted to facilitate drying and storage of body fluids.

In one embodiment, the porous polymeric material has a pore size of 5 to 10,000nm, 50 to 5,000nm, 100 to 2,000nm, 200 to 1000 nm. Smaller pore sizes correspond to larger surface areas that promote adsorption of biological fluids (e.g., blood and plasma). In another embodiment, the porous polymeric material has a specific surface area of 0.5 to 1000m when measured by nitrogen adsorption (nitrogenadsorption) using a BET isotherm (BET isotherm)²G, 1 to 500m²G, 5 to 200m²10 to 100 m/g²G, 20 to 60m²G, 30 to 50m²/g。

The porous polymeric material medium as described above is capable of receiving a biological fluid sample in liquid form and subsequently drying to facilitate storage, transport and/or future analysis of the sample. The porous polymeric material medium may be adapted to promote the adsorption or adhesion of body fluids such as blood and plasma. In a particular embodiment, the medium is adapted for storing blood and/or plasma. For example, the porous polymeric material may provide chemical functionality (e.g., hydrophilic groups). Chemical functionality can be incorporated into the polymeric material based on the polymerization of the polymeric material. The chemical functionality may be incorporated after polymerization, for example during preparation of the media or during functionalization after preparation of the media. Chemical functionality may include covalent bonding of functional groups into the polymer chain. The chemical functionality may be adapted to facilitate pre-analysis on the medium or purification of the biological sample in situ, e.g., extraction of one or more specific components in the sample.

In another embodiment, functionality may be incorporated into the porous polymeric material for the in situ elimination of undesired components in the blood that hinder the detection of other specific components (e.g., analytes, such as drugs or New Chemical Entities (NCEs)). In a particular embodiment, at least the surface of the porous polymeric material is modified to provide ion exchange properties to facilitate analysis of any analyte present in the sample after storage. In another particular embodiment, the surface region of the porous polymeric material may be provided with ion exchange properties such that selected pharmaceutical agents adhere thereto or selected contaminants present in the body fluid do not adhere thereto. Thus, the porous polymeric material can be used to analyze bodily fluids dried thereon without the need for chemical-based pretreatment. In another particular embodiment, the ion exchange properties may be provided by functional groups present on the monomer from which the porous polymeric material is formed and/or post-polymerization surface modifications or other chemical modifications including post-polymerization grafting (grafting). In a preferred embodiment, the post-polymerization surface modification is photografting.

In one embodiment, there is provided the use of a porous polymeric matrix material as a medium for drying and storing a biological fluid sample.

In one embodiment, the porous polymeric matrix material is selected from at least one of the following: polyolefins, polyethers, polyesters, polyamides, polycarbonates, polyurethanes, polyanhydrides, polythiophenes, polyethylenes, and epoxy resins, preferably at least one of the following: polyolefin, polyester or polyamide. Suitable polyolefins include polyethylene, polypropylene and polystyrene.

The porous polymeric matrix material may optionally be functionalized with at least one group selected from: hydroxyl, alkyl, sulfonyl, phosphonyl, carboxyl, amino, nitro, acrylate (acrylate) and methacrylate (methacrylate).

The porous polymeric matrix material may be a porous polymeric particulate material or a porous polymeric fibrous material. The porous polymeric matrix material may be provided in a variety of forms selected from or including foam (foam), sponge (sponge), woven or non-woven fabric (fabric), agglomerated particles (agglomerated particles) or fiber-based materials, or composites thereof. The porous polymer matrix material may provide an open cell (open cell) interconnected network structure.

In one embodiment, the porous polymeric matrix material is a porous polymeric particulate material formed by sintering (sinter) agglomerates of polymeric particles and optionally one or more additives. In one embodiment, the polymeric particles are selected from at least one of the following: a polyester; polyethylene, including high density polyethylene, polyethylene terephthalate (polyethylene terephthalate), polyvinylidene fluoride (PVDF), and Polytetrafluoroethylene (PTFE); and polypropylene, such as high density polypropylene.

In one embodiment, the porous polymeric matrix material is a porous polymeric fiber material comprising agglomerates of polymeric fibers and optionally one or more additives. In one embodiment, the polymeric fiber is selected from at least one of the following: a polyester; polyethylene, including polyethylene terephthalate, polyvinylidene fluoride (PVDF), and Polytetrafluoroethylene (PTFE); and polypropylene, such as high density polypropylene.

In one embodiment, there is provided the use of a porous polymeric monolith as a medium for drying and storing a biological fluid sample, wherein the porous polymeric monolith is formed by a step growth polymerization process.

The step growth polymerization process may comprise polymerizing one or more monomers having one or more functional groups selected from the group consisting of: hydroxyl, carboxylic acid, anhydride, acid halide, alkyl halide, anhydride, acrylate, methacrylate, aldehyde, amide, amine, guanidine, malimide (malimide), thiol, sulfonate, sulfonic acid, sulfonyl ester, carbodiimide, ester, cyano, epoxide, proline, disulfide, imidazole, imide (imide), imine, isocyanate, isothiocyanate, nitro or azide functional groups. The monomer may have one or more functional groups selected from hydroxyl, ester, amine, aldehyde, and carboxylic acid.

In one embodiment, the monomer is an acrylic monomer, such as a methacrylate monomer, such as hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EDMA).

In one embodiment, the porous polymeric monolith may be prepared by polymerizing a polymerization mixture comprising one or more monomers in the presence of a crosslinking monomer (crosslinking monomer), an initiator (initiator), and a porogen (porogen). The polymerization mixture can be disposed on and/or in a support material that can comprise the porous polymeric matrix material described herein, and polymerization can be initiated thereon to form a porous polymeric monolith, which can then be washed with a suitable solvent to remove the porogen. The polymeric mixture may also be first prepared and polymerized and then disposed on the support material.

The porous polymeric monolith may be derived from a polymerization mixture comprising from 10 to 90 volume percent, more typically from 20 to 80 volume percent of the monomer, from 10 to 90 volume percent, more typically from 20 to 80 volume percent of the porogen and from 0.5 to 5 volume percent, more typically about 1 volume percent of the initiator.

In a second aspect, there is provided a method of storing a bodily fluid for future analysis comprising applying a biological fluid sample to a porous polymeric material as described herein, and drying the biological fluid sample such that the sample at least partially solidifies and adsorbs or adheres to the porous polymeric material.

In a third aspect, there is provided a method of storing a body fluid for future analysis, comprising:

applying one or more biological fluid samples to one or more regions of a porous polymeric material medium as described herein;

partially drying the one or more samples applied to the medium;

optionally separating any one or more regions of the medium to which the sample is applied from regions to which the sample is not applied;

optionally further drying the one or more samples applied to the one or more regions of the medium; and

one or more samples applied to one or more regions of the medium are stored.

In one embodiment, the method comprises the step of separating any one or more regions of the medium to which the sample is applied from regions to which no sample is applied. In another embodiment, the method comprises the step of further drying the one or more samples applied to the one or more regions of the medium prior to storing the one or more samples applied to the one or more regions of the medium.

In one embodiment, separating any one or more regions of porous polymeric material media to which sample has been applied from regions to which sample has not been applied may comprise substantially removing any media to which bodily fluid has not been applied from around the sample, for example trimming or cutting away media located at or near the periphery of the sample. The medium can be trimmed or cut away from around the sample so that the sample substantially covers the surface of the area to which the sample is applied. In a particular embodiment, a hole-punch (hole-punch) is used to separate and obtain one or more areas of the porous polymeric material medium to which the sample is applied.

The method may further comprise identifying and detecting an analyte from a stored sample applied to the medium. In one embodiment, stored bodily fluid samples can be analyzed without pretreatment and/or removal from the porous polymeric material medium. In another embodiment, the method may comprise pre-treating the sample stored on the medium prior to analyzing the sample.

In one embodiment, drying of the biofluid sample (e.g., blood or plasma) is enhanced by applying at least one of elevated temperature, forced convection (forced convection), or reduced pressure. The elevated temperature may be in a temperature range above ambient temperature and below a temperature at which the integrity of the storage medium or sample is compromised. In a particular embodiment, the elevated temperature is from 30 to 150 ℃, from 40 to 120 ℃, and more particularly from about 60 to 100 ℃, or above 30 ℃, above 50 ℃, above 70 ℃, above 90 ℃, above 110 ℃ or above 130 ℃. In a particular embodiment, the elevated temperature is greater than about 90 ℃. In another particular embodiment, the reduced pressure is from 5 to 760 mmHg.

In a fourth aspect, there is provided an assay method comprising identifying and detecting an analyte from a stored biological fluid sample adsorbed or adhered to a porous polymeric material medium as described herein.

In one embodiment, stored biological fluid samples are analyzed without pretreatment and/or removal from the porous polymeric material medium. The analysis is typically for an analyte. Analytes may include small molecules and low molecular weight compounds present in blood or plasma samples, such as pharmaceutical agents, including Novel Chemical Entities (NCE) and any metabolites, peptides, proteins, oligonucleotides, oligosaccharides, lipids, or other labile compounds thereof. In another embodiment, the analysis involves simultaneous analysis of at least two analytes. In a particular embodiment, the at least two analytes comprise NCE and its metabolites.

In a fifth aspect, there is provided a method for storing and subsequent analysis of a biological fluid sample comprising genetic material, the method comprising:

applying a biological fluid sample comprising one or more analytes to a porous polymeric material medium as described herein;

drying the sample applied to the medium;

storing the sample;

recovering the sample;

optionally pretreating the sample; and

the sample is analyzed for one or more analytes.

Drawings

FIG. 1 is a photograph showing a container used to prepare the porous polymeric monolith on a support membrane of example 2;

FIG. 2 is a graph showing the hematocrit of human versus the area of a dried blood spot (in example 2, Whatman FTADMPK-C)^TMCard and Agilent Bond Elut DMS^TMOn-card) a map of the effects of;

FIG. 3 is a graph showing the hematocrit in sheep responding to gabapentin versus the area of the dried blood spot (Whatman FTA DMPK-C in example 2^TMCard and Agilent Bond Elut DMS^TMOn-card) a map of the effects of;

FIG. 4 shows a graph of the effect of sheep hematocrit on dry blood spot area in response to fluconazole;

figure 5 shows a graph of the effect of sheep hematocrit on dry plaque area in response to ibuprofen;

FIG. 6 shows a graph of consistency in the recovery of gabapentin from different locations (2, 3, 4 and 5) in a dried blood spot normalized to location 1;

figure 7 shows a graph of the consistency of recovery of fluconazole from different positions (2, 3, 4 and 5) in dried blood spots normalized to position 1;

figure 8 shows a graph of the consistency of recovery of ibuprofen from different positions (2, 3, 4 and 5) in dried blood spots normalized to position 1.

Detailed description of the abbreviations

In the examples, the following abbreviations will be referred to, wherein:

AFM atomic force microscope

APP applications

C degree centigrade

Class Cl

[] Concentration of

EMAA polyethylene methacrylic acid

F degree of Fahrenheit

FTIR Fourier transform infrared

h hours

HDPE high density polyethylene

Mn number average molecular weight

Mw weight average molecular weight

MW molecular weight

Relative humidity of RH

SEM scanning electron microscope

SENB single edge notched bar

TDCB taper double cantilever beam (tapered double cantilever beam)

TETA triethyltetramine

Weight percent of specific components in Wt% composition

XPS X-ray photoelectron spectroscopy

DEGDMA diethylene glycol dimethacrylate

DMPAP 2, 2-dimethoxy-2-phenyl-acetophenone

EDMA ethylene glycol dimethacrylate

GMA glycidyl methacrylate

HEMA 2-hydroxyethyl methacrylate

MAA methacrylic acid

3- (trimethoxysilyl) propyl gamma-MAPS methacrylate

META methacryloyloxyethyl trimethyl ammonium chloride

SPMA 3-sulfopropyl methacrylate

UHMWPE ultrahigh molecular weight polyethylene

Relative area of RE

Coefficient of variation of CV

Detailed Description

In order to identify alternative materials that provide properties that facilitate drying and storage of biological fluid samples (such as blood and plasma samples) for future extraction and analysis, or to identify materials that may allow incorporation of specific functionalities therein, it has now been found that biological fluid sample storage media can be formed from some porous polymeric materials. Some non-limiting specific embodiments of the invention are described below.

The present invention relates generally to the use of porous polymeric materials as a medium for storing dried biological fluids, particularly blood and plasma. Thus, the porous polymeric materials described herein may provide a suitable medium for DBS processes as an alternative to the paper-based cellulosic materials currently in use. In some particular embodiments, the porous polymeric material provides a modified medium for storing biological substances for later analytical testing, such as storing blood and plasma samples for future detection and identification of analytes, including small molecules (such as pharmaceutical agents and related metabolites) and low molecular weight compounds (such as proteins and oligonucleotides). Porous polymeric materials have excellent properties that have proven to be capable of efficiently drying and storing biological fluid samples (including blood and plasma) over long periods of time.

Another advantage of using the porous polymeric materials as DBS sorbents is that these materials allow a degree of control over the morphology and surface chemistry of the material.

Typically, the porous polymeric material is a highly crosslinked synthetic polymer. For example, the porous polymeric material is not a cellulose or paper-based material.

Term(s) for

"porous polymeric matrix material" generally refers to a continuous porous polymeric matrix having a monolithic body in which the porosity of the material is formed in a post-polymerization process.

"porous polymeric particulate material" generally refers to a continuous porous polymeric matrix having an integral body comprising agglomerates of polymeric particles, wherein the porosity of the material is formed in a post-polymerization process.

"porous polymeric fibrous material" generally refers to a continuous porous polymeric matrix having an integral body comprising agglomerates of polymeric fibers, wherein the porosity of the material is formed in a post-polymerization process.

"porous polymeric monolithic material" generally refers to a continuous porous polymeric matrix having a monolithic body comprising a fused array of microspheres (microrogobule) separated by pores, wherein the porosity of the material is formed during in situ polymerization.

"step-growth polymerization" refers to a type of polymerization mechanism in which a bi-or multi-functional monomer reacts to form polymer chains and a crosslinked network.

"biological fluid sample" or "body fluid" refers to any fluid that can be considered a sample from the body of an organism, and which may contain a detectable analyte or genetic material, for example, blood or plasma from a human or animal subject.

"analytes" include, but are not limited to, small molecules and low molecular weight compounds that can be detected in body fluids, such as pharmaceutical agents present in blood or plasma samples obtained from human or animal subjects. For example, an "analyte" may include an agent that includes an NCE, a peptide, a protein, an oligonucleotide, an oligosaccharide, a lipid, or other labile compound.

The term "media" when used in conjunction with another term (e.g., "porous polymeric material media") generally refers to a material by itself or further in conjunction with a support material, such as one or more additional layers, including a backing layer or protective layer. The medium may provide a stationary support for the biological fluid sample.

A "support material" or similar term is a support layer or structure that may be bonded to the polymeric monolith by attachment, detachable attachment, or non-attachment, e.g., the polymeric material may be polymerized on the support material or may be simply placed on the support material with or without other spacer layers, which may also be bonded to the polymeric material and the support material by attachment, detachable attachment, or non-attachment. The support material may be flexible, semi-rigid, or rigid, and may be in any desired form (e.g., a film or membrane), and may be formed from any suitable material, including glass, polymers, metals, ceramics, or combinations thereof.

The term "alkyl" refers to any saturated or unsaturated, branched or unbranched, cyclic, or combination thereof, typically having 1 to 10 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, which may be optionally substituted with methyl.

The term "alkylene" refers to any branched or unbranched, cyclic, or combination thereof, typically having 1 to 10 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, which may be optionally substituted with methyl.

The term "polymer" includes copolymers and the term "monomer" includes comonomers (co-monomers).

The term "porogen", "porogen solvent" or similar term refers to a solvent capable of forming pores in a polymer matrix during polymerization of the polymer matrix, including but not limited to aliphatic hydrocarbons, aromatic hydrocarbons, esters, amides, alcohols, ketones, ethers, solutions of soluble polymers, and mixtures thereof.

The term "initiator" refers to any free radical generator capable of initiating polymerization by thermal initiation, photoinitiation, or redox initiation.

Porous polymeric matrix materials

The porous polymeric matrix material comprises a continuous porous polymeric matrix having a unitary body, wherein the porosity of the material is formed in a post-polymerization process.

The porous polymeric matrix material may be a porous polymeric particulate material or a porous polymeric fibrous material.

The porous polymeric matrix material may be provided in a variety of sizes, configurations, shapes or forms depending on the particular intended purpose. The material may be formed by at least one method selected from the group consisting of: sintering, extrusion (extrusion), emulsification, interfacial polymerization, and woven fiber preparation.

Porous polymer matrix materials involve post-polymerization processes to introduce porosity. For example, a polymeric material that may include functionality and that includes one or more additives is first prepared. The prepared polymeric material can then be machine processed or processed (e.g., milled, ground, or extruded) into extrudates, units, strands, fibers, or particles of a certain size to facilitate handling and incorporation of additional components or materials. Thereafter, the extrudates, units, strands, fibers or particles, among other additives, may be combined or agglomerated together (e.g., by sintering into a solid material) to form a medium containing a particular porosity. The media or material can be processed to introduce porosity (e.g., by washing and removing additives present in the polymeric material).

In one embodiment, the porous polymeric matrix material is selected from at least one of the following: polyolefins, polyethers, polyesters, polyamides, polycarbonates, polyurethanes, polyanhydrides, polythiophenes, polyethylenes, and epoxy resins, preferably at least one of the following: polyolefin, polyester or polyamide.

Suitable polyolefins include polyethylene, polypropylene and polystyrene. The polyethylene polymer (polyethylene copolymer) may be selected from at least one of the following or mixtures thereof: ultra-high molecular weight polyethylene, high density polyethylene, polytetrafluoroethylene, ethylene vinyl acetate, ethylene methyl acrylate, ethylene-propylene rubber (ethylene-propylene rubber), ethylene-propylene-diene rubber, poly (1-butene), poly (2-butene), poly (1-pentene), poly (2-pentene), poly (3-methyl-1-pentene), poly (4-methyl-1-pentene), 1, 2-poly-1, 3-butadiene, 1, 4-poly-1, 3-butadiene, polyisoprene, polychloroprene, poly (vinyl acetate), poly (vinylidene chloride), poly (tetrafluoroethylene) (PTFE), poly (vinylidene fluoride) (PVDF), polyacrylates, polymethacrylates, and the like, PET or PTFE. The polystyrene may be acrylonitrile-butadiene-styrene (ABS). The polyether may be selected from at least one of the following: ether Ketone (PEEK) (poly (oxy-1, 4-phenylene-carbonyl-1, 4-phenylene)) and Polyethersulfone (PES). The polyamide may be selected from nylons, such as nylon-6.

The porous polymeric matrix material may optionally be functionalized with at least one group selected from: hydroxyl, alkyl, sulfonyl, phosphonyl, carboxyl, amino, nitro, acrylate and methacrylate.

It will be appreciated that the porous nature of the polymer matrix material provides one or more channels through which gas or liquid molecules may pass. The average pore size may be about 0.1 μm to 1000 μm. Particularly suitable average pore sizes may be from about 1 μm to about 500 μm, for example from 1 to 150 μm, from 5 μm to 100 μm or from 10 μm to 50 μm. It will be appreciated that average pore size and pore density can be readily determined using mercury porosimetry or scanning electron microscopy.

Various methods known to those skilled in the art may be used to fabricate porous media of polymeric materials, such as by sintering, use of blowing agents and/or leaching agents (leaching agents), minicell formation methods, drilling, reverse phase precipitation, or hydro-acupuncture (hydrojet). The porous material may comprise a regular arrangement of channels of random or well-defined diameter and/or randomly positioned pores of different shapes and sizes. Pore size generally refers to the average diameter, even though the pores themselves are not necessarily spherical.

In one embodiment, the porous polymeric particulate material may be formed by sintering polymeric particles (optionally with one or more additives).

The particular method used to form the pores or channels of the porous polymeric material and the resulting porosity (i.e., average pore size and pore density) may vary depending on the desired application. The desired porosity, which may be affected by the porous polymeric material, may alter the physical properties (e.g., tensile strength and durability) of the material.

The relative amounts of polymer and optional additives used to provide the porous polymeric material can vary depending on the particular material used, the desired functionality of the surface of the material, and the strength and flexibility of the material itself.

The polymer, functionality additive, or optional other materials, which may be in particulate form, may be mixed to provide a homogeneous mixture, which may then be sintered. This can be done using a die, a beltline (beltline), or other techniques known to those skilled in the art, depending on the desired size and shape of the final product (e.g., block, tube, cone, cylinder, sheet, or film). Suitable molds are commercially available and are well known to those skilled in the art. Specific examples of molds include, but are not limited to, flat sheets (sheetings) and cylinders of varying heights and diameters. Suitable mold materials include, but are not limited to, metals and alloys (e.g., aluminum and stainless steel), high temperature thermoplastics, and other materials known in the art and disclosed herein.

In one embodiment, a compression mold (compression mould) is used to provide the sintered material. The mold is heated to the sintering temperature of the polymer, allowed to equilibrate, and then pressurized. This pressure typically ranges between about 1psi and about 10psi depending on the composition of the mixture being sintered and the desired porosity of the final product. Generally, the greater the pressure applied to the mold, the smaller the average pore size and the greater the mechanical strength of the final product. The duration of the applied pressure also varies depending on the desired porosity of the final product, and is typically from about 2 minutes to about 10 minutes.

Once the porous material is formed, the mold is allowed to cool. If pressure has been applied to the mold, cooling can be performed while pressure is still being applied or after the pressure is removed. The material is then removed from the mold and optionally processed. Examples of optional processing include, but are not limited to, sterilization, cutting, milling, polishing, encapsulation, and coating.

A variety of materials of different sizes and shapes may be used to provide suitable porous materials. The narrow particle size distribution allows for the production of materials with uniform porosity (i.e., substrates comprising pores that are uniformly distributed throughout the substrate and/or have about the same size), which allows for more uniform flow of solutions and gases through the material and provides the material with fewer structural weaknesses.

Porous polymeric fiber materials are continuous porous polymeric matrices having a specific pore size range with a unitary body formed from polymeric fibers. A typical method of producing porous polymeric fibrous materials involves initially forming polymeric fibers which are combined together in a subsequent step to form the porous polymeric fibrous material. The pore characteristics of porous polymeric fibrous materials are not determined during the initial polymerization process, but rather during the process of joining previously produced fibers together as the material is formed, during the reformation of the material, or during the modification of the material after formation.

The polymer fibers may agglomerate to form an interconnected porous polymer network. The interconnected porous polymer network may be open-celled. The polymer fibers may be oriented or randomly agglomerated. The polymeric fibers may be woven or non-woven fabrics. The porous polymeric fibrous material may comprise one or more types of continuous polymeric fibers. The porous polymeric fibre material may comprise one or more types of discontinuous fibres, such as cut fibre (cut fibre) or mixed fibre. The fiber may be comprised of a core and a shell. Different types of fibers may be mixed together. The porous polymeric fibrous material may comprise a fibrous structure. A rigid open cell structure can be formed. The material may be provided in various shapes and sizes, which may include sheets, tubes, rods, or other three-dimensional geometries.

The polymeric fibers of the porous polymeric fiber material may be selected from at least one of the following: a polyester; polyethylene, including polyethylene terephthalate, polyvinylidene fluoride (PVDF), and Polytetrafluoroethylene (PTFE); and polypropylene, such as high density polypropylene. The polymer fibers or materials may be further modified to increase hydrophilicity. The polymers may be mixed or different types of polymer fibers may be combined.

A variety of structural fibers may be added to the material to provide strength and rigidity.

Particularly suitable pore sizes for the polymeric material may range from about 10 μm to about 250 μm. Particularly suitable pore volumes may range from 25% to 95%. The porous polymeric fibrous material may have a density in the range of, for example, 12 g/cc to 0.6 g/cc.

Porous polymeric monolithic materials

The porous polymer monolith typically has a highly crosslinked structure that can serve as a stationary support. The internal structure of the porous polymeric monolithic material consists of a fused array of microspheres separated by pores and whose structural rigidity is ensured by a large number of crosslinks. The porosity of the monolithic material is formed in an in situ polymerization process that forms the monolithic material.

The porous polymeric monolith may be fabricated from a mixture comprising an initiator and monomers, including crosslinking monomers, dissolved in a pore-forming solvent, referred to as a porogen. Formation of the monolith is triggered by the decomposition of an initiator by an external source (e.g., photoinitiation), wherein the initiator decomposes to produce free radicals that induce the formation of polymer chains that precipitate out of the polymerization mixture, eventually clumping together to form a continuous solid structure. The morphology of the monolith can be controlled by a number of variables: the crosslinking monomers employed, the composition and percentage of the porogenic solvent (porogen), the concentration of the free radical initiator, and the method used to initiate the polymerization.

Since polymer monoliths generally have a continuous, rigid structure, they can be easily manufactured in situ in a variety of forms, shapes or sizes. Monoliths have been commonly manufactured within the context of chromatography columns or capillary columns for a variety of chromatographic applications. However, the monolith can also be manufactured in the form of a flat sheet if there is a suitable mold. The flat monolith sheet provides a particularly suitable medium for storing whole blood, which allows for easy storage and transport of blood samples.

Another advantage of using porous polymeric monolithic materials for DBS is the ability to control both pore properties and specific surface chemistry. The ability to introduce specific functionalities to the surface of the monolith allows for specific extraction of analytes, such as pharmaceutical agents or New Chemical Entities (NCEs), as well as facilitates matrix elimination that can reduce future analysis. Future assays may include Solid Phase Extraction (SPE), which is based on the physical adsorption of the analyte on a suitable medium, and therefore the medium should have a large surface area for maximum analyte recovery. The pore properties of the media can also be used to control the specific surface chemistry (e.g., surface area) to some extent, and thus the ion exchange capacity of the media is dependent on the pore properties. Detection and identification of analytes may include small molecules and low molecular weight compounds present in blood or plasma samples, such as pharmaceutical agents, including NCEs, peptides, proteins, oligonucleotides, oligosaccharides, lipids, or other labile compounds.

The porous polymeric monolith is formed by a step growth polymerization process. Step-growth polymerization generally refers to the type of polymerization mechanism in which a di-or multi-functional monomer reacts into a polymer chain that can have a high degree of crosslinking.

The step-growth polymerization process may include polymerizing one or more monomers having a functional group selected from at least one of: hydroxyl, carboxylic acid, anhydride, acid halide, alkyl halide, anhydride, acrylate, methacrylate, aldehyde, amide, amine, guanidine, malimide, thiol, sulfonate, sulfonic acid, sulfonyl ester, carbodiimide, ester, cyano, epoxide, proline, disulfide, imidazole, imide, imine, isocyanate, isothiocyanate, nitro or azide functional groups. The monomer may have at least one functional group selected from: hydroxyl, ester, amine, aldehyde, and carboxylic acid. In another embodiment, the functional group may comprise a zwitterionic group, such as a sulfoalkyl betaine-based zwitterionic compound, for example N, N-dimethyl-N-methacryloyloxyethyl N- (3-sulfopropyl) betaine ammonium (SPE).

In one embodiment, the monomer is an acrylic monomer, such as a methacrylate monomer, for example, hydroxy methacrylate [ HEMA ] and ethylene glycol dimethacrylate (EDMA).

In one embodiment, the porous polymeric monolith may be prepared by polymerizing a polymerization mixture comprising one or more constituent monomers of a polymer in the presence of an initiator and a porogen. The polymerization mixture can be disposed on and/or in a support material that can comprise the porous polymeric matrix material described herein, and polymerization can be initiated thereon to form a porous polymeric monolith, which can then be washed with a suitable solvent to remove the porogen. The polymeric mixture may also be prepared and polymerized prior to being disposed on the support material.

The polymerization mixture can include monomers in an amount of about 10 to 60 volume percent, and more particularly about 15 to 40 volume percent, about 45 to 85 volume percent porogen, and about 1 volume percent initiator. In one embodiment, the polymerization mixture comprises about 20% to 80% monomers (including crosslinking monomers), about 20 to 80% by volume of porogen, and about 1% by volume of initiator. The respective ranges of the monomer, crosslinking monomer, and porogen may vary depending on the intended use.

Flat sheets of porous polymeric monolithic materials can be successfully fabricated, for example, by anchoring the monolithic sheet to a rigid glass plate by imparting methacryl functionality to the glass surface. The methacryloyl functionality participates in the polymerization process, resulting in covalent attachment of the entire species to the slide during the polymerization process.

In one embodiment, the porous polymeric media thereof is a sheet or membrane having a thickness of up to about 1mm, specifically a thickness of about 300 μm to 900 μm, more specifically a thickness of about 500 μm to 700 μm. The thickness of the polymer monolith may be up to 500 μm, in particular about 200 to 400 μm. Other forms and thicknesses of monolithic or monolithic media are contemplated and may be formed depending on the particular application (e.g., the type of post-storage analysis contemplated).

Other preferred polymers include polymers incorporating functional groups along the backbone of the polymer to facilitate further modification or interaction with blood or plasma. For example, the porous polymeric monolith may be configured to be capable of providing a plurality of blood spot samples thereon, and optionally configured to facilitate removal of excess monolith from around each blood spot sample.

Changing the porogen in the process of preparing the porous polymeric monolithic material only affects the porous structure of the material, while changing other parameters modifies the composition and stiffness of the material. Increasing the concentration of the non-solvent porogen induces early precipitation during the polymerization process, which typically results in a larger material pore size. Thus, the porogenic solvent and its relative composition are selected to design a material with a desired pore structure.

The composition and percentage of the porogenic solvent may be used to control pore properties by varying or adjusting the percentage of the porogenic solvent mixture and the porogen (e.g., water or an organic solvent such as cyclohexanol, methanol, hexane, propanol, or butanediol). This affects both the median pore size and the median pore volume of the monoliths produced. A wide range of pore sizes can be readily achieved by simply adjusting the composition of the porogenic solvent.

In one embodiment, the porogen used to prepare the porous polymer monolith may be selected from a variety of different types of materials. For example, suitable liquid porogens include solutions of organic solvents, aliphatic hydrocarbons, aromatic hydrocarbons, esters, amides, alcohols, ketones, ethers, soluble polymers, and mixtures thereof. The porogen is generally present in the polymerization mixture in an amount of about 40 to 90 volume percent, more preferably about 50 to 80 volume percent. In a particular embodiment, the porogen or porogen solvent comprises dodecanol, cyclohexanol, methanol, hexane, or a mixture thereof. In a preferred embodiment, the porogen is 1-decanol, cyclohexanol, methanol, or hexane. In another particular embodiment, the porogenic solvent comprises at least 35% dodecanol in combination with cyclohexanol or methanol in combination with hexane.

Percent porosity is the percentage of pore volume based on the total volume of the monolith substrate. The term "pore volume" as used herein refers to the volume of pores in a 1 gram monolith. In one embodiment, the porous polymeric monolithic material has a macroporous structure with a percentage of porosity of between about 45% and 85%, more particularly between about 60% and 75%. In another embodiment, the porous polymer monolith can have a pore size of 5Bm to 10,000um, 50um to 5,000nm, 100um to 2,000nm, 200um to 1,000 nm. Smaller pore sizes are associated with larger surface areas that increase the loading capacity of bodily fluids (e.g., blood and plasma). In another embodiment, the specific surface area of the porous polymer matrix is from 0.5 to 1000m, as measured by nitrogen adsorption using BET isotherms (Atkins P, Physical Chemistry, oxford university press)²G, 1 to 500m²G, 5 to 200m²10 to 100 m/g²G, 20 to 60m²G, 30 to 50m²/g。

Polymerization can be carried out by a variety of methods of free radical initiation mechanisms including, but not limited to, gamma irradiation, thermal initiation, photoinitiation, redox initiation. In one embodiment, about 0.1 to 5 wt% (relative to the monomer) of a free radical or hydrogen abstraction photoinitiator (hydrogen abstraction photoinitiator) may be used to produce the porous polymer monolith matrix. For example, 1 wt% (relative to the monomer) of a hydrogen abstraction photoinitiator can be used to initiate the polymerization process. The hydrogen abstraction photoinitiator may include benzophenone, 2-dimethoxy-2-phenylacetophenone (DMPAP), dimethoxyacetophenone, xanthone, and thioxanthone. If the photoinitiator selected is poorly soluble, the desired initiator concentration can be achieved by homogenizing the initiator in the emulsion and having a higher concentration by adding a surfactant.

In another embodiment, wherein the polymerization is carried out by thermal initiation, the thermal initiator is typically a peroxide, hydroperoxide (hydroperoxide), peroxy-or azo compound selected from benzoyl peroxide (benzoperoxide), potassium persulfate (potassium peroxidise), ammonium persulfate (ammonium peroxidise), t-butyl hydroperoxide, 2' -Azobisisobutyronitrile (AIBN), and azobisisobutyric acid (azodicarbonobutyric acid), and the thermally induced polymerization is carried out by heating the polymerization mixture to a temperature of from 30 ℃ to 120 ℃.

In another embodiment, wherein the polymerization is initiated by a redox initiator, the redox initiator may be selected from a mixture of benzoyl peroxide-dimethylaniline peroxide and a mixture of ammonium persulfate-N, N, N ', N' -tetramethylene-1, 2-ethylenediamine.

Incorporating functional groups into the porous polymeric monolith increases the polarity of the surface and thus increases wettability. Since blood is mainly composed of water, incorporation of a polar monomer into the whole body is advantageous for adsorption of blood.

Varying the type and amount of porogen solvent can provide control over the pore size distribution of the monolith, which can be verified by Mercury Intrusion Porosimetry (MIP). Polar monomers increase the concentration of less polar porogens (such as 1-dodecanol), generally providing a monolith with larger pores.

It has been found that increasing the percentage of dodecanol to 38% to 100% of the porogenic solvent in the mixture of dodecanol and cyclohexanol maintains a pore size distribution of about 600 nm. The same ratio of methanol and hexane in a binary porogenic solvent was used to obtain large pores in the bulk. The pore size distribution may be about 7000 nm. Smaller pore size monoliths, such as monoliths comprising 40% dodecanol and 20% cyclohexanol, are more reproducible.

The visual appearance of the monolith is considered a reliable indicator of pore size due to light scattering. The studied monolith appeared chalky (chalky), indicating a macroporous material (i.e., pore size greater than about 50 nanometers). This was confirmed by analysis of the MIP with a median pore diameter measuring about 600nm and an overall porosity of 68%. The specific surface area of the monolith was determined by BET analysis.

Various types of step-growth polymers can be used, including groups capable of multiple branching types, such as star, comb, brush, ladder, and tree monomers, comonomers, or polymer groups.

Support material

The support material for the porous polymeric monolith may be a flexible, semi-rigid, or rigid film, membrane, or backing layer. This bonding between the support material and the polymer matrix may be adhesive, removably attached, or non-attached. The support material may comprise a porous polymeric matrix material as described herein.

Optional additives

The porous polymeric material according to any one of the above embodiments may further comprise other additives such as rheology modifiers (rheology modifiers), fillers, toughening agents, heat or UV stabilizers, flame retardants, lubricants, surfactants. The additives are generally present in an amount less than about 10 percent (based on the total weight of the activation treatment or combination of solvent, agent, and additive). Examples include:

(a) rheology modifiers such as hydroxypropyl methylcellulose (e.g., Methocell311, Dow), modified urea (e.g., Byk411, 410), and polyhydroxycarboxylic acid amides (e.g., Byk 405);

(b) film formers, for example, esters of dicarboxylic acids (e.g., Lusolvan FBH, BASF) and glycol ethers (e.g., Dowanol, Dow);

(c) wetting agents such as fluorochemical surfactants (e.g., 3M Fluorad) and polyether modified polydimethylsiloxanes (e.g., Byk307, 333);

(d) surfactants such as fatty acid derivatives (e.g., Bermadol SPS2543, Akzo) and quaternary ammonium salts;

(e) dispersants, such as primary alcohol-based nonionic surfactants (e.g., Merpol4481, Dupont) and alkylphenol-formaldehyde-disulfide condensates (e.g., Clariants 1494);

(f) defoaming agents;

(g) preservatives, such as phosphate esters (e.g., ADD APT, AnticorC6), alkylammonium salts of (2-benzothiazolylthio) succinic acid (e.g., Irgacor153CIBA), and triazine dithiol;

(h) stabilizers such as benzimidazole derivatives (e.g., Bayer, Preventol BCM, bactericidal membrane protection);

(i) leveling agents such as fluorocarbon modified polymers (e.g., EFKA 3777);

(j) pigments or dyes, such as fluorescers (royal Pigment and chemicals);

(k) organic and inorganic dyes, such as fluorescein (fluorescein);

(l) Lewis acids such as lithium chloride, zinc chloride, strontium chloride, calcium chloride and aluminum chloride.

(m) suitable flame retardants that retard flame propagation, heat release and/or smoke generation, which may be added separately or optionally comprise:

phosphorus derivatives, such as molecules containing phosphate, polyphosphate, phosphite, phosphazene and phosphine functional groups, for example, melamine phosphate, di (melamine) phosphate, melamine polyphosphate, ammonium phosphate, ammonium polyphosphate, pentaerythritol phosphate, melamine phosphite and triphenylphosphine.

Nitrogenous derivatives, such as melamine, melamine cyanurate, melamine phthalate, phthalimide melamine, melam, melem, melon, cyclohexane tetramine, imidazole, adenine, guanine, cytosine and thymine.

Molecules containing borate/ester functions, such as ammonium borate and zinc borate.

Molecules containing two or more alcohol groups, such as pentaerythritol, polyvinyl alcohol (polyethylene alcohol), polyethylene glycol and carbohydrates (e.g. glucose, sucrose and starch).

Molecules that endothermically release non-flammable decomposition gases, such as metal hydroxides (e.g., magnesium hydroxide and aluminum hydroxide).

Expandable graphite.

The additive may be selected from one or more of the following: silicon powder, silica gel, chopped glass fiber (CPG), Controlled Pore Glass (CPG), glass beads (glass beads), ground glass fiber (glass fiber), glass bubbles (glass bubbles), kaolin, alumina, nano-sintered diamond. The additive may be fiberglass.

In one embodiment of the porous polymeric matrix material, the other additives may include lubricants, fibers, colorants, fillers, functional additives, active agents (e.g., antimicrobial agents), or antistatic agents. The functional additive may comprise a compound having one or more functionalities selected from the group consisting of: hydroxyl, carboxylic acid, anhydride, acid halide, alkyl halide, aldehyde, olefin, amide, amine, guanidine, malimide, thiol, sulfonate, sulfonic acid, sulfonyl ester, carbodiimide, ester, cyano, epoxide, proline, disulfide, imidazole, imide, imine, isocyanate, isothiocyanate, nitro or azide functional groups. The functional additive may include a compound having hydroxyl, amine, aldehyde, or carboxylic acid functionality. The active agent can be a drug, hydrophilic moiety, catalyst, antibiotic, antibody, antifungal, carbohydrate, cytokine, enzyme, glycoprotein, lipid, nucleic acid, nucleotide, oligonucleotide, peptide, protein, ligand, cell, ribozyme, or a combination thereof.

Preparation, storage and analysis of body fluids

The porous polymeric materials described herein are used to store biological or bodily fluid samples (particularly blood and plasma) for future analysis (e.g., analytes including pharmaceutical agents or metabolites thereof). The blood or plasma sample may be applied directly to the porous polymeric material. The combination of the sample and the porous polymeric material is then dried to form a cured sample that adsorbs or adheres to the storage medium.

Body fluid samples typically comprise genetic material (e.g., DNA and RNA) and can be obtained from any source, for example, physiological/pathological body fluids (e.g., blood, urine, secretions, excretions, exudates, and exudates (transudates)) or cell suspensions (e.g., blood, lymph, synovial fluid, semen, saliva including buccal cells).

A porous polymeric material is provided for storage or subsequent analysis of a stored sample. The porous polymeric material may be comprised of a solid matrix comprising a functionality and/or a composition or one or more active agents that may prevent the degradation of genetic material stored on the porous polymeric material or facilitate the inactivation of microorganisms (e.g., microorganisms associated with the sample that may degrade the sample or be potentially pathogenic to a human handler), facilitate the extraction of a particular analyte, or facilitate the removal of the matrix to aid in the identification and analysis of analytes.

The dried body fluid sample on the porous polymeric material may be analysed at a later stage, for example for the pharmacokinetic analysis of agents present in blood and plasma samples. After drying of the body fluid sample on the porous polymeric material, the porous polymeric material is particularly suitable for storing and transporting such samples, in particular whole blood and plasma samples, since at this stage they are considered to be relatively safe to handle and not infectious (e.g. for infectious diseases that may be carried in blood, such as HIV).

The porous polymeric material may be configured or adapted to be capable of storing bodily fluids for a number of years, including any one of the following periods of time: at least one day, one week, one month, 6 months, one year, two years, 5 years, 10 years, 20 years, or up to 50 years or more.

In one embodiment, long term storage of bodily fluids on porous polymeric materials may be facilitated by encapsulating the porous polymeric material (particularly porous polymeric monolithic material) in a protective material, such as a plastic material (e.g., polystyrene), which may be removed when subsequent contact with a stored sample is required.

In the storage of blood, a blood sample may be applied to the porous polymeric material as a blood spot. The functionality, components, or one or more reagents may be added to or incorporated into the porous polymeric material to provide specific optional properties suitable for a variety of purposes (e.g., for denaturing proteins, eliminating matrices, or reducing or removing any pathogenic organisms in a sample). At the same time, the blood (and genetic material and/or analytes therein) can be protected from degradation factors and methods so that a relatively stable dry blood sample can then be stored and transported to a diagnostic laboratory. The analyte or genetic material may be extracted, analyzed or used in situ on the porous polymeric material.

The composition or active agent used with the porous polymeric material may comprise, for example, a monovalent weak base (e.g., "Tris", trimethylolmethane, whether as the free base or as a carbonate), a chelating agent (e.g., EDTA, ethylenediaminetetraacetic acid), an anionic detergent (e.g., SDS, sodium dodecyl sulfate), guanidine, or uric acid or a urate salt. Other reagents may include a retaining agent to reduce the loss of analyte in subsequent analyses that may occur during storage or pre-analysis processing.

Monomers with specific functionalities can be incorporated to help eliminate the biological matrix from the sample. While the ability to functionalize the surface of paper-based media is limited, a simple scheme for modifying polymeric material media to incorporate functionality is well established.

In another embodiment, functionality may be incorporated into the porous polymeric material for the in situ elimination of undesirable components in the blood that hinder the detection of a particular analyte (e.g., a pharmaceutical agent or other low or small molecular weight compound). In a particular embodiment, the surface region of the porous polymeric material may be provided with ion exchange properties such that selected agents present in the body fluid adhere thereto or selected contaminants do not adhere thereto. Thus, the porous polymeric material can be used to analyze bodily fluids dried thereon without the need for chemical-based post-or pre-treatments. In another particular embodiment, ion exchange properties may be provided by functional groups present on the monomers or comonomers forming the porous polymeric material and/or post-polymerization surface modifications including co-grafting or other chemical modifications. The chemical modification may be photografting, for example as described in U.S. patent No.7,431,888, which is incorporated herein by reference. The photografting may be by UV or gamma irradiation. The chemical modification may be chemical C-H activation, such as may be mediated by a transition metal complex.

Grafting is the way in which the surface chemistry is tailored. Several methods have been used to graft polymers onto thermoplastic polymer surfaces, including a number of different methods such as flame treatment, corona discharge treatment (corona discharge treatment), plasma treatment (plasma treatment), use of monomeric surfactants, acid treatment, free radical polymerization, and high energy radiation. See, e.g., Uyama, Y et al, adv.Polym.Sci.1998, 137, 1.

Attachment of polymer chains to sites at the pore surfaces within a typical monolithic or porous polymeric material provides a variety of functionalities from each surface site and significantly increases the density of surface functionalities. Examples of grafting and functionalizing porous polymeric materials, including porous polymeric monolithic materials, using free radical polymerization initiation can be found in the art. Viklund, C et al incorporated zwitterionic sulfobetaine groups into porous polymer monoliths in Macromolecules2000, 33, 2539. Peters et al, previously shown in U.S. Pat. No.5,929,214, can graft a thermally responsive polymer to the surface of pores within a polymer monolith by a two-step grafting process that requires (i) vinylation of the pores, followed by (ii) in situ free radical polymerization of a selected vinyl monomer or mixture of selected monomers. The thermally responsive polymer changes flow properties through the pores in response to the temperature difference.

Surface photografting with vinyl monomers has been used to functionalize polymer fibers, films, and sheets, such as described by Ranby B et al in nuclear. Photografting can be used to modify flat two-dimensional surfaces or for three-dimensional highly crosslinked porous polymer monoliths.

In one embodiment, the chemical modification of the surface of the porous polymeric material is performed by UV-initiated photografting. For example, UV-initiated photografting, mediated by a hydrogen abstraction photoinitiator, can be used to modify the surface of channels, to create porous monoliths or materials, and to modify monoliths or materials in selected areas. Modification and surface functionalization of porous polymer materials can be accomplished by photo-initiated grafting within specific spaces (i.e., microfluidic channels or portions thereof), which allows layering and patterning of different functionalities on the polymer surface.

The blood sample may be lysed to promote adhesion of the swatch to the medium prior to adsorption or adhesion of the blood sample to the medium. The pore size of the porous polymeric material medium may be provided at or above the diameter of the red blood cells (typically about 6,000nm to 8,000nm) to promote adhesion of the blood sample to the medium.

In one embodiment, a method of storing a bodily fluid for future analysis is provided that includes applying a bodily fluid sample to a porous polymeric material medium and drying the bodily fluid such that the sample at least partially solidifies and adsorbs or adheres to the porous polymeric material medium.

In another embodiment, a method of storing a bodily fluid for future analysis may comprise:

applying one or more bodily fluid samples to one or more regions of a porous polymeric material medium;

partially drying the one or more samples applied to the medium;

one or more samples applied to one or more regions of the medium are stored.

applying one or more bodily fluid samples to one or more regions of a porous polymeric material medium as described herein;

partially drying the one or more samples applied to the medium;

separating any one or more regions of the medium to which the sample is applied from regions to which the sample is not applied;

one or more samples applied to one or more regions of the medium are stored.

partially drying the one or more samples applied to the medium;

further drying the one or more samples applied to the one or more regions of the medium; and

one or more samples applied to one or more regions of the medium are stored.

Separating any one or more regions of the porous polymeric material to which the sample is applied from regions to which the sample is not applied may comprise substantially removing any medium to which bodily fluid is not applied from around the sample, for example trimming or cutting away medium located at or near the periphery of the sample. The medium can be trimmed or cut away from around the sample so that the sample substantially covers the surface of the area to which the sample is applied (e.g., by using a punch having a smaller diameter than the plaque sample). In other words, the blood spot sample may extend to or near the outer edge of the region of the porous polymeric material medium to which the sample is applied. One advantage of this embodiment is that cracking of the sample during drying of the sample can be reduced or prevented. Removing any media not in contact with the sample can promote adhesion and non-cracking of the sample after drying. The sample is typically cut or perforated to separate from the excess media.

The sample applied to the media is typically about 1mm to 20mm in diameter, and may be about 2mm to 15mm or 5mm to 10mm in diameter, for example typically of size l0 to 100mm²Is spherical. For example, the one or more samples may be selected from the following sizes (mm)²) Any one of: 1. 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100. In another embodiment, the one or more regions may be selected from the following dimensions (mm)²) Any one of: 1. 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100. It is understood that depending on the process, application, or device used, the application of the sample to the medium may vary, and ranges above, below, or between these dimensions are also within the scope of the present invention. The medium may also be sized or shaped to substantially cover its surface with the bodily fluid sample, for example by providing one or more individual areas of the medium on a support material (e.g. an array) of a size to which a sample can be applied that covers its surface. Various arrangements and patterns of one or more samples to one or more regions also fall within the scope of these embodiments. For example, an array of bodily fluid samples may be applied to the medium, for example by providing about 20mm²A separate 5 x 5 array of samples. In another embodiment, the sample array may be applied to and/or excised from a single medium, or applied to an array of one or more separate regions of a medium.

Drying of a body fluid (such as blood or plasma) is enhanced by applying at least one of elevated temperature, forced convection, or reduced pressure. The elevated temperature may be in a temperature range above ambient temperature but below a temperature at which the integrity of the storage medium or sample is compromised. In a particular embodiment, the elevated temperature is from 30 to 150 ℃, from 40 to 120 ℃, more particularly about 60 to 100 ℃, or above 30 ℃, above 50 ℃, above 70 ℃, above 90 ℃, above 110 ℃ or above 130 ℃. In a particular embodiment, the elevated temperature is greater than about 90 ℃, which may enhance future analysis of the sample or prevent cracking of the sample after drying, for certain types of monolithic media and samples. The sample may be dried at elevated temperature for about 10 to 20 minutes. In a particular embodiment, the reduced pressure is from 5 to 760 mmHg. The reduced pressure may be applied by a vacuum device.

Also provided herein are analytical methods involving the identification and detection of analytes from stored bodily fluid samples adsorbed or adhered to a porous polymeric material medium.

In one embodiment, stored bodily fluid samples can be analyzed without pretreatment and/or removal from the porous polymeric material medium. In other words, the sample stored on the medium can be used directly for analysis without further modification. Analytes may include small molecules and low molecular weight compounds present in blood or plasma samples, such as agents, including Novel Chemical Entities (NCE) and any metabolites, peptides, proteins, oligonucleotides, oligosaccharides, lipids, or other labile compounds thereof. In another embodiment, the analysis involves simultaneous analysis of at least two analytes. In a particular embodiment, the at least two analytes comprise NCE and its metabolites.

Porous polymeric materials for selective extraction and matrix elimination

Ion exchange functionality is incorporated into the porous polymeric material to facilitate selective extraction of a particular analyte (e.g., a drug or NCE) and to facilitate matrix elimination. Both copolymerization and surface modification techniques can be employed to incorporate functionality into the polymeric material.

Typically the porous polymeric material has a hydrophilic surface to facilitate absorption of body fluids. Functionality may be incorporated into the porous polymeric material to facilitate in situ sample purification or matrix elimination, to facilitate specific extraction (e.g., for analytes), or to facilitate biological analysis. For example, strong cation exchange (SCX) functionality may be provided by the incorporation of sulfonic acid type surface groups (e.g., HEMA-co-SPMA), weak cation exchange (WCX) functionality may be provided by carboxylic acid surface groups, strong anion exchange (SAX) functionality may be provided by quaternary amine surface groups, and weak anion exchange (WAX) may be provided by tertiary amine surface groups.

Solid Phase Extraction (SPE) methods involve sample preparation, purification and concentration of analytes from a matrix by adsorption to a medium followed by elution with an appropriate solvent. The analytes partition between the solid phase and the solvent and only those analytes with high affinity for the solid phase remain. Following matrix elimination, the analyte can be eluted from the solid phase and analyzed.

Polymeric materials (e.g., monoliths) having acidic functional groups can be made for selective extraction of NCEs containing basic functional groups, while polymeric monoliths having basic functionality allow for selective extraction of NCEs having a degree of acidity. The incorporation of functionality into porous polymeric materials is generally well established and can be achieved using several different strategies.

Two possible methods of incorporating specific functionalities into the porous polymeric monolithic media are: by direct incorporation of the functional monomer into the polymerization mixture, or by post-polymerization of the monolithic scaffold. The direct incorporation of the functional monomers together with the structural monomers into the polymerization mixture is by far the simplest approach, since no subsequent modification is required. However, since the functional monomer is part of the polymerization mixture, it is likely that a large proportion of the ionizable groups will be trapped in the bulk medium and not available at the bulk surface and thus not interact with the NCE.

The second method is a post-polymerization reaction, which directly imparts functional groups to the surface of the material through covalent attachment. The materials can be optimized separately, which means that a variety of functionalities can be imparted to them. The advantage of using post-polymerization is that the functionality is imparted directly to the material surface, which means that it is easier to synthesize higher capacity materials for high sample loading. Two very different methods can be used to impart surface functionality; the first is to change the surface chemistry by a chemical reaction. This approach requires that the structural monomers contain reactive groups. The second option is to complete a second polymerization reaction on the previously formed material; this technique is called surface grafting.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described, the embodiments herein are to be considered in all respects illustrative and not restrictive.

It will be understood that, if any prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the art in australia or in any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The invention will now be described with reference to the following non-limiting examples.

Examples

Example 1 preparation and use of porous Polymer matrix media

The macroporous structure of all polymeric materials was measured by mercury intrusion porosimetry using a Micromeritics AutoPore IV9505(Norcross, GA, USA) porosimeter. Specific surface area the specific surface area was measured by a Brunauer-Emmet-Teller (BET) [ Brunauer S et al, Journal of the American chemical Society, 1938.60: page 309-319 ].

An OAI LS30/5Deep UV irradiation system (San Jos é, CA, USA) with 500W of HgXelam was used for all UV exposures. The lamp was calibrated to 20.0mW/cm using an OAI Model306 intensity meter with a 260nm probe²。

Porous high density polyethylene films (X-4913, median pore diameter of 90 to 130 μm) were obtained from Porex (GA, USA).

Preparation of the modified Medium

A porous high density polyethylene film was immersed in a degassed solution consisting of 15 wt% 2-acrylamido-2-methyl-1-propanesulfonic acid, 0.22 wt% benzophenone, 63.6 wt% t-butanol, and 21.1 wt% water. The substrate was allowed to stand in this solution for at least 10 minutes, with air excluded. The substrate was covered with a microscope slide and grafting was achieved by UV irradiation for an irradiation time of 15 minutes. The substrate was then washed with water by continuous stirring in a rocking bath (rocking bath) for at least 2 hours, after which it was allowed to dry at room temperature.

Using media for DBS

To demonstrate the potential of the modified porous polymer matrix as a medium or adsorbent for storage of whole blood, 15 μ L aliquots of whole human blood were spotted directly onto both the unmodified and modified matrices. Blood did not penetrate the unmodified matrix and dried to irregular sized spots. On the modified matrix, blood penetrated the entire thickness of the matrix (. about.2 mm) and showed excellent uniformity for both spot size and shape. The blood spots were dried by contact (touch dry) on the substrate at room temperature within 1 hour.

Example 2-preparation of porous polymeric monolithic Material on a supporting Membrane

The macroporous structure of all polymeric materials was measured by mercury intrusion porosimetry using a Micromeritics AutoPore IV9505(Norcross, GA, USA) porosimeter. Specific surface area the specific surface area was measured by a Brunauer-Emmet-Teller (BET) [ Brunauer S et al, Journal of the American chemical Society, 1938.60: page 309-319 ]. All monoliths were degassed in Micromeritics vacprep at 50 ℃ for 24 hours.

A flat sheet monolith on a support film was prepared using a rectangular sandwich container (sandwich container) as shown in fig. 1. The jacketed vessel was made of stainless steel and had dimensions (W.times.Ltimes.H) of 11.3X 24.5X 2.3 cm. It consists of two parts (two haircolors): a base (base) of thickness 1.4cm and an upper rectangular border of thickness 0.45 cm. The empty space of 8.1 x 21.5cm of the bezel allows UV exposure in the middle. The central part of the substrate is a shallow cavity (shallow cavity) with dimensions (W × L) of 8 × 21.5cm and a depth of 600L μm. An 8.8 x 22.0cm Viton O-ring was used to form a barrier along the edge of the shallow chamber to prevent solution leakage. A piece of 9.5 x 22.8cm glass plate with a thickness of 0.4cm was placed in the container between the two parts to seal the cavity, forming an integral body on the inside.

Preparation of the polymerization mixture

A polymerization mixture (17.58g) was prepared by weighing the appropriate initiator, monomer, crosslinking monomer and porogen in a vial. The polymerization mixture consisted of 19.3% (w/w) monomer (2-hydroxyethyl methacrylate, HEMA), 19.3% (w/w) crosslinking monomer (ethylene glycol dimethacrylate, EDMA), 30.7% (w/w) of each porogen (methanol and n-hexane) mixed with UV initiator (2, 2-dimethoxy-2-phenylacetophenone (DMAP)) to give a clear organic solvent mixture. The amount of initiator used corresponds to 1% (w/w) of the total amount of monomer and crosslinking monomer. The mixture was sonicated for 10 minutes to ensure the components were dissolved.

Preparation of Polymer monoliths on membranes

1. Placing a support film with a size of 7 × 20.5cmIs placed on the central part of the casting mould (cast). The support film was a non-woven polyester fiber (OTH 001 sold by BMPAmerica) having a thickness of 0.59mm and a weight of 130g/m²。

2. The polymerization mixture was injected into the shallow cavity with a Pasteur tube (Pasteur pipette) just enough to wet the entire membrane.

3. The mold was covered with a 9.5X 22.8cm thick glass plate in the middle of the two parts of the container.

4. The two parts are secured together with 8 screws at a distance of 7.5cm from each other.

5. The polymerization mixture was injected into the container through a syringe equipped with a 25 gauge (gauge) syringe needle until the entire space was occupied by the mixture.

6. After the solution was in place and the two parts of the jacketed vessel were fixed, Spectrolinker was used^TMXL-1500Series (Spectronics Corporation, Westbury, NY, USA) irradiated the vessel for 50 minutes under UV.

7. After polymerization, the support membrane containing the monolith was separated from the casting mold and transferred to a container containing methanol and washed overnight on a shaker (rock) (Gyro-rock STR9, sturt instruments, Bibby Scientific Limited, UK).

8. The washed support membrane containing the monolith flat was allowed to dry in a vacuum oven at ambient temperature overnight.

Polymer monoliths were used for Dry Blood Spot (DBS) sampling techniques for drug development (3mm spots, nominal concentration 2500ng/ml)

The purpose of this example was to test the diffusion properties and variation of hematocrit levels of DBS using a polymeric monolithic material and a support membrane prepared as described above.

Human hematocrit versus example 2, Whatman FTA DMPK-C^TMCard and Agilent BondElut DMS^TMEffect of dried blood spot area on card

The maximum difference between hematocrit levels of example 2, Whatman and Agilent were 9%, 26% and 10%, respectively. The spot area was measured by integration using the program lmageJ. Convert Pixel count to mm². The differences at each extreme of example 2, Whatman and Agilent cards were 9%, 14% and 9%, respectively. This measurement is more accurate because we use ImageJ to measure the total blood spot area rather than using the blood spot diameter to calculate the area (the blood spot may not be circular). The results are listed in table 1 below and graphically illustrated in fig. 2.

TABLE 1

Example 2

The effect of human hematocrit in response to gabapentin, fluconazole and ibuprofen is shown in figures 3 to 5. On example 2, the percent difference between gabapentin and ibuprofen with respect to HCT 45% was over 15%. In addition, greater percent error was observed when using HCT20 and HCT80 on Whatman. For gabapentin and ibuprofen, the Agilent card was sensitive to low hematocrit levels of HTC20 and HTC 30. In summary, lower percent error was observed with fluconazole on all three card types.

Polymer monoliths for Dried Blood Spot (DBS) sampling technology for drug development

The purpose of this example is to demonstrate the consistency (or lack thereof) of the recovery of analytes from different locations within a dried blood spot, i.e. to demonstrate the homogeneity of DBS.

Example 2A is a porous polymer monolith on the support membrane of example 2, 800 microns thick, with a membrane thickness of 400 microns and a monolith thickness of 400 microns.

Example 2B is a porous polymer monolith on the support membrane of example 2, having a thickness of 640 to 700 microns, with a membrane thickness of 400 microns and a monolith thickness of 240 to 300 microns.

Process for producing a metal oxide

20 μ L of 2500ng/mL blood samples containing gabapentin, fluconazole and ibuprofen (7500ng/mL) were spotted onto different card types.

Spots were allowed to dry for 1 hour on examples 2A and 2B and 2 hours on other card types.

Punch a 1.50mm disk from each dried spot and place in an Eppendorf tube.

Add 300 μ L of 0.1% formic acid in 80% methanol (containing 5nm/mL deuterated internal standard mixture) to the sample, then vortex (vortex) and soak (soak) for about 2 hours (or sonicate if possible).

The sample was centrifuged (14000 rpm. times.5 min), and 250. mu.L of the supernatant was collected and transferred to a 0.5mL tube.

The sample was evaporated to dryness overnight in a vacuum oven at 35 ℃.

Reconstitute the sample in 200 μ L water: methanol (9: 1) or (60ng/mL sample and 7.5ng/mL i.s.), centrifuge (14000rpm × 5 min), and then transfer 100 μ L to 250 μ L sample vials for analysis.

These results are shown in table 2 below.

TABLE 2

The peak area ratios for the individual positions are mostly reproducible except for the spots on the Agilent card. The peak area ratios on the Agilent cards are particularly inconsistent with the deviations of the center hole chip.

The results are graphically shown in fig. 6 to 8.

Claims

1. Use of a porous polymeric material as a medium for drying and storing a biological fluid sample, wherein the porous polymeric material is selected from a porous polymeric matrix material or a porous polymeric monolithic material, wherein the porous polymeric monolithic material is formed by a step growth polymerization process.

2. The use of claim 1, wherein the biological fluid sample is whole blood or plasma.

3. Use according to claim 1 or claim 2 for dry blood spot sampling or dry blood plasma spot sampling.

4. Use according to any one of claims 1 to 3, wherein the porous polymeric material medium has a unitary body with a pore size and a specific surface area suitable for facilitating drying and storing body fluids, and wherein the medium is optionally combined with one or more support layers.

5. Use according to claim 4, wherein the porous polymeric material has a pore size of from 5nm to 10,000nm and a specific surface area of 0.5m when measured by nitrogen adsorption using the BET isotherm²G to 1000m²/g。

6. Use according to any one of claims 1 to 5, wherein the porous polymeric material incorporates chemical functionality to facilitate pre-analysis or in situ purification of a biological sample on the medium.

7. Use according to any one of claims 1 to 6, wherein the porous polymeric material is a porous polymeric matrix material.

8. Use according to claim 7, wherein the porous polymeric matrix material is selected from at least one of: polyolefins, polyethers, polyesters, polyamides, polycarbonates, polyurethanes, polyanhydrides, polythiophenes, polyethylenes, and epoxy resins.

9. Use according to claim 8, wherein the polyolefin is selected from at least one of the following: polyethylene, polypropylene and polystyrene.

10. Use according to claim 9, wherein the polyethylene is selected from at least one of the following: high density polyethylene, polyethylene terephthalate, polyvinylidene fluoride (PVDF), and Polytetrafluoroethylene (PTFE).

11. Use according to any one of claims 7 to 10, wherein the porous polymeric matrix material is in the form of: foam, sponge, woven or non-woven fabric, agglomerated particles or fiber-based materials or composites thereof.

12. Use according to claim 11, wherein the porous polymer matrix material has an open-celled interconnected network structure.

13. Use according to any one of claims 7 to 12, wherein the porous polymeric matrix material is a porous polymeric particulate material formed by sintering agglomerates of polymeric particles with optionally present one or more additives.

14. Use according to any one of claims 7 to 12, wherein the porous polymeric matrix material is a porous polymeric fibre material comprising agglomerates of polymeric fibres with optionally present one or more additives.

15. Use according to any one of claims 1 to 6, wherein the porous polymeric material is a porous polymeric monolithic material formed by a step growth polymerisation process.

16. The use of claim 15, wherein the step-growth polymerization process for the porous polymeric monolithic material comprises polymerizing one or more monomers having functional groups selected from one or more of the following: hydroxyl, carboxylic acid, anhydride, acid halide, alkyl halide, anhydride, acrylate, methacrylate, aldehyde, amide, amine, guanidine, malimide, thiol, sulfonate, sulfonic acid, sulfonyl ester, carbodiimide, ester, cyano, epoxide, proline, disulfide, imidazole, imide, imine, isocyanate, isothiocyanate, nitro or azide functional groups.

17. The use of claim 16, wherein the monomer is an acrylic monomer.

18. The use of claim 17, wherein the acrylic monomer is a methacrylate monomer.

19. The use according to claim 18, wherein the methacrylate monomer is selected from at least one of hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EDMA).

20. The use according to any one of claims 15 to 19, wherein the porous polymeric monolithic material is prepared as follows: polymerizing a polymerization mixture comprising one or more monomers in the presence of a crosslinking monomer, an initiator, and a porogen to provide a material comprising: 10 to 90 volume percent monomer, 10 to 90 volume percent porogen, and 0.5 to 5 volume percent initiator.

21. Use according to claim 20, wherein the polymeric mixture is disposed on and/or in a support material.

22. Use according to claim 21, wherein the support material is a polymer matrix material as defined in any one of claims 7 to 14.

23. A method of storing a body fluid for future analysis comprising applying a biological fluid sample to a porous polymeric material as defined in any one of claims 1 to 20 and drying the biological fluid sample such that the sample at least partially solidifies and adsorbs or adheres to the porous polymeric material.

24. A method of storing a body fluid for future analysis, comprising:

applying one or more samples of biological fluid to one or more regions of a porous polymeric material medium as defined in any one of claims 1 to 20;

partially drying the one or more samples applied to the medium;

optionally separating any one or more regions of the medium to which the sample is applied from regions to which no sample is applied;

storing the one or more samples applied to the one or more regions of the medium.

25. An assay method comprising identifying and detecting an analyte from a stored biological fluid sample adsorbed or adhered to a porous polymeric material medium as defined in any one of claims 1 to 20.

26. A method for storing and subsequently analyzing a biological fluid sample comprising genetic material, the method comprising:

applying a sample of biological fluid comprising one or more analytes to a porous polymeric material medium as defined in any one of claims 1 to 20;

drying the sample applied to the medium;

storing the sample;

recovering the sample;

optionally pre-treating the sample; and

analyzing the sample for the one or more analytes.