Patent Application
20160349156
The present invention relates to an apparatus and method for moiety specific sample extraction from a complex sample matrix in a flow through process. An integrated membrane is used for separation and preservation of biomolecules. Classes of biomolecules, such as those in blood, can be separated and preserved.
The present invention relates to separation and analysis of biomolecules, especially those of bodily fluids such as blood. It provides for moiety-specific sample extraction from a complex sample matrix in a flow through process. More particularly, the present invention relates to an integrated membrane for separation and preservation of classes of biomolecules, such as those in blood and other bodily fluids. These separated components are then available to be used for the diagnosis and testing for diseases and other medical conditions.
Many techniques and devices are available to separate and analyze biomolecules, particularly those biomolecules found in the human body. Present day medical care and treatment focuses on the diagnosis of disease, disfunctions and abnormalities based on the laboratory analysis of bodily fluids, such as blood, using the tools of modern science. Biomolecule sample work-up prior to profiling often requires preservation and the separation into components. Evaluation may be of the various individual components, or groups of components.
Medical testing of a wide variety of different body fluid components is desirable based on developed testing and diagnostic protocols. Such testing and analysis is used to indicate and evaluate various diseases, or other medical conditions. Having the ability to separate and preserve the biomolecules into components for sampling reliably, cost effectively and without contamination enhances the ability to discover and develop new knowledge about diseases, treatments and cures.
Methods for medical and scientific evaluation of components significantly depend on the ability to adequately separate and preserve the components intact, providing pure and uncompromised samples for evaluation based on comparison of healthy and diseased biomolecules. Many techniques and devices are available to make these analyses. The particular ones used will vary with the biomolecule components of concern. These procedures typically require considerable care and attention. The ability to easily collect, separate and analyze biomolecules without risk of contamination is important.
To acquire biomolecules for sampling and analysis is not trivial. For example, the storage of highly labile biomolecules, such as proteins and RNA, requires the use of low (4° C. and −20° C.) and ultra-low (−80° C.) freezers and/or a host of specialized and expensive chemicals to preserve the structural and chemical integrity of the biomolecule. Because of these storage requirements, storing millions of samples to use for early diagnosis is cost prohibitive and impractical. Presently, alternatives to low and ultra-low temperature storage have significant drawbacks.
Evaluation of blood and its components is a particular focus for diagnosis and treatment of many different medical conditions. Human blood comprises a plasma component, and a blood corpuscle component. The blood corpuscle component comprises erythrocytes (i.e., red blood cells), leucocytes (i.e., white blood cells), and blood platelets. The blood plasma component comprises water (90% by volume), dissolved proteins, nucleic acids, glucose, clotting factors, mineral ions, hormones and gases (e.g., carbon dioxide).
Information derived from the analysis of proteins and nucleic acids derived from blood and other fluids and tissues has provided early detection of and diagnosis capability for many medical conditions. For example, analysis of blood proteins and nucleic acids can detect illnesses such as post-traumatic stress disorder (PTSD), traumatic brain injury (TBI) as well as other “hidden” illnesses. The ability to provide early detection and diagnosis facilitates the successful evaluation and treatment of these and other illnesses.
To avoid the stringent demands for low and ultra-low temperature storage required preserve structural and chemical integrity for blood and other fluids, there are preservation methodologies such as those for ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) storage that provide for stabilizing the RNA during collection, processing and storage; however, these technologies are subjectively dependent on the selective ability to extract and purify RNA with minimal exposure to contaminating ribonucleases (RNases). Because RNases are ubiquitous in the clinic and laboratory, clinicians are unable to institutionalize reliable procedures for avoiding contamination.
In an attempt to overcome the inherent problems with RNases contamination, several commercially reagents, such as RNAlater® (Ambion, Austin, Tex.), have been developed to avoid RNases degradation during sample collection, processing and storage. To avoid contamination, these reagents provide for the in situ precipitation of degenerative RNases in cells/living tissues via use of ammonium sulfate. While these reagents theoretically provide for the storage of RNA samples at near room temperature for up to seven (7) days, actual use shows that there is significant mRNA degradation that may occur after three (3) days of near-room temperature storage.
More recently, a new RNA storage reagent trading under the name of RNAstable® (Biomatrica, San Diego, Calif.) was introduced using anhydrobiosis as a method to stabilize and protect biomolecules from degradation at near room temperature. RNAstable® has been shown to store total RNA at near room temperature, and is claimed to do so for up to twelve (12) years according to manufacturer's specifications.
While RNAstable® and other reagents may ultimately provide for longer RNA storage, the use of a reagent generally is highly dependent on the ability to collect, process and, if necessary, purify samples while minimizing any incidence of RNases contamination.
With regard to protein storage, there is currently no commercially available means of storage other than the low and ultra-low temperature methodology. Alternative methodologies exist but are deficient. For example, for blood analysis, alternatives include drying out the sample and adhering it to dried blood spot (DBS) papers or cards (Shleicher & Scheull 903). Current DBS technology is limited and an inferior substitute for low and ultra-low temperature storage of samples. Special training of personnel handling the placement of the blood on the papers/cards is required or large variations and errors can result. For example, DBS papers in use are subject to operator inefficiencies and lack of skill. Operator errors in the distribution of the samples on the matrix is often non-uniform, leading to assay variations (upon recovery). And, samples are recovered using a punch device, which can lead to cross-contamination among samples.
Accordingly, there is a need for an improved method and apparatus for preserving and storing biomolecules. There is an additional need for an improved method and apparatus for long-term storage of biomolecules. There is also a need for an improved method and apparatus for storage of biomolecules at ambient conditions. There is a further need for a method and apparatus that reduces the possibility of sample cross-contamination. There is an additional need for a practical and cost-effective method and apparatus for storing samples. The present invention satisfies these needs and provides other related advantages.
Targeted extraction is possible with the present invention. It provides moiety-specific sample extraction from a complex sample matrix (such as blood, food, soil, etc.) using a flow through process.
One or more membranes with conjugate parts may be integrated in the apparatus to extract just targeted components. Variations will occur in the sequence of the sorting, which will cause the apparatus to be modified by one of ordinary skill in the art for the apparatus to provide for the best sequence and separation.
It is possible to provide moiety-specific extraction for dry materials also through the use of a buffer. With the present invention, it is possible separate of classes of biomolecules such as cells, proteins and nucleic acids (DNA/RNA) fully preserved from whole blood.
Sample extraction can be done for materials within the cells as well, by using, for example, lysing buffers. The process is performed rapidly. With blood separation, this avoids proteins and RNA degradation.
An integrated membrane illustrated herein provides for preservation of biomolecules, such as proteins and nucleic acids. While the apparatus and method of the present invention are described further for the use of blood-derived biomolecules, the invention is not so restricted and can be used for the preservation generally of biomolecules.
An integrated membrane illustrated herein provides preservation of biomolecules for long-term storage. An integrated membrane illustrated herein provides for storage of biomolecules at ambient conditions.
An integrated membrane illustrated herein provides for rapid separation and processing of samples to avoid degradation.
An integrated membrane illustrated herein provides for separation and preservation of biomolecules without the use of solvents or additives.
An integrated membrane illustrated herein provides one or more membranes with conjugate parts to extract just targeted components.
An integrated membrane illustrated herein provides optimized sequencing and separation of components. By way of example, and not limitation, for nucleic acids, it is necessary to sequence the filtering process to first filter cells, and then proteins to avoid degradation of the nucleic acids.
In an embodiment, an apparatus for preserving biomolecules is provided. The apparatus includes a housing having a matrix portion, and a fluid collection portion disposed within an inner cavity thereof. The housing also includes an aperture permitting access to the inner cavity. A matrix disposed within the matrix portion includes a first layer for collecting substantially all cells from the fluid, a second layer for protein adsorption, and a third layer for nucleic acid adsorption. Fluid enters the housing through the aperture, passes through the matrix, and into the fluid collection portion.
In another embodiment, the apparatus includes a desiccant disposed within the fluid collection portion.
In a further embodiment, the housing of the apparatus includes a barrier disposed between the matrix portion and the fluid collection portion.
In an additional embodiment, the barrier includes openings through which fluids passes from the matrix portion to the fluid collection portion.
In yet another embodiment, the housing of the apparatus includes a locking mechanism for engaging a syringe.
In yet a further embodiment, the aperture is disposed on a distal end of a neck extending away from the housing.
In an embodiment, each layer comprises a fluid permeable membrane.
In another embodiment, each layer comprises a membrane held within a holder.
In a further embodiment, each membrane is disposed between upper and lower portions of the holder.
In an additional embodiment, each holder removably engages at least one adjacent holder.
In yet another embodiment, at least the nucleic acid adsorption layer is separable from the matrix.
In yet a further embodiment, at least the nucleic acid adsorption layer includes at least one line of weakness defining a plurality of removable sections.
In an embodiment, a method for preserving biomolecules is provided. The method includes passing fluid through a matrix; collecting substantially all cells from the fluid in a layer of the matrix; adsorbing protein from the fluid in a layer of the matrix; and adsorbing nucleic acid from the fluid in a layer of the matrix.
In another embodiment, the method includes drying the fluid that has passed through the matrix.
In a further embodiment, the method includes drying at least the nucleic acid adsorbing layer.
In an additional embodiment, the method includes separating the matrix from the fluid that has passed through the matrix.
In yet a further embodiment, the method includes containing the matrix within a housing.
In yet another embodiment, the method includes injecting the fluid into the housing using a syringe matingly engaged with the housing.
In an embodiment, the method includes containing each layer within a holder.
In another embodiment, the method includes separating at least the nucleic acid adsorbing layer.
In an additional embodiment, the method includes defining at least the nucleic acid adsorbing layer into separate removable sections.
In an embodiment, a system for preserving biomolecules is provide. The system includes a housing containing a matrix therein. The matrix includes a first layer for collecting substantially all cells from the fluid, a second layer for protein adsorption, and a third layer for nucleic acid adsorption. The system also includes a storage case for containing at least the third layer after separation from the matrix.
In a further embodiment, an apparatus for preserving biomolecules is provided. The apparatus includes a housing comprising a matrix portion, and a fluid collection portion disposed within an inner cavity thereof; wherein the housing includes an aperture permitting access to the inner cavity. The apparatus also includes a matrix disposed within the matrix portion, comprising an asymmetric layer for filtering material from the fluid. Fluid enters the housing through the aperture, passes through the matrix, and into the fluid collection portion.
In another embodiment of the apparatus, the matrix further includes a layer for adsorption of a first component of the material.
In an additional embodiment of the apparatus, the matrix further includes a layer for adsorption of a second component of the material.
This brief summary has been provided so that the nature of the invention may be understood quickly. A more complete understanding of the invention can be obtained by reference to the following detailed description of the preferred embodiments thereof concerning the attached drawings.
The various present embodiments now will be discussed in detail with an emphasis on highlighting the advantageous features with reference to the drawings of various embodiments. The illustrated embodiments are intended to illustrate, but not to limit the invention. These drawings include the following figures, in which like numerals indicate like parts:
The following detailed description describes the present embodiments with reference to the drawings. In the drawings, reference numbers label elements of the present embodiments. These reference numbers are reproduced below in connection with the discussion of the corresponding drawing features.
The term “fluid” used in the specification refers to, but is not limited to, bodily fluids, such as blood in its original composition, partially fractionated blood, blood plasma, and the like.
The terms “corpuscle” or “cell” used in the specification refer to, but are not limited to erythrocytes, leucocytes, blood platelets and the like.
The term “biomolecule” used in the specification refers to, but is not limited to, a molecule produced by living cells (e.g., protein, carbohydrate, lipid, or nucleic acid).
The term “nucleic acid” used in the specification refers to, but is not limited to, ribonucleic acid, deoxyribonucleic acid and the like.
As shown in
The integrated membrane avoids the possibility of rapid degeneration of biomolecules such as highly labile macromolecules. The technology allows the user to apply a sample (e.g., blood) directly on a multi-layer membrane wherein nucleic material is extracted, purified and preserved in one rapid step. Samples are passed through the integrated membrane in a flow-through process. For example, blood is drawn with the syringe or other blood collection device and, after blood draw (e.g., ˜50-500 μl), the blood is dispensed across the integrated membrane and the processing occurs automatically. A preferred maximum volume for whole blood draw is 500 μl to reduce the possibility the integrated membrane being clogged with cells.
The integrated membrane provides: (1) rapid, on-site collection of samples, such as nucleic material (e.g., RNA, DNA); and (2) autonomous collection of samples, precluding any variations due to operator error. A benefit of rapid, on-site collection is, that prolonged exposure to contaminants is reduced. For example, with blood, for collection of nucleic material, prolonged exposure to contaminants such as RNases/DNases is minimized. A benefit to autonomous collection of RNA/DNA is that the potential for variations due to operator error is eliminated. The integrated membrane obviates the need for the use of protein denaturants, which are typically used during the sampling process, to inactivate RNases/DNases, which provides for the use of unaltered use of the RNA/DNA. This provides for recovery of the RNA/DNA from the integrated membrane using a standard RNA/DNA suspension buffer (e.g., a nuclease free TE buffer) without the need to remove protein denaturants (necessary for inactivation of the RNases/DNases) prior to analysis. Avoiding the use of protein denaturants is desirable, as the removal of protein denaturants typically requires the use of toxic reagents (e.g., phenol/chloroform) that can cause significant loss of RNA/DNA. Thus, eliminating the use of protein denaturants saves time, reduces expense, and increases RNA/DNA yield.
An integrated membrane embodying the present invention overcomes limitations of current DBS technology and other technologies by enabling autonomous collection. For example, collection of proteins from whole blood using the present invention avoids inaccuracies and variations in results. The integrated membrane also can incorporate a rapid drying process. For proteins in blood samples, this enables the proteins to be uniformly distributed in the matrix thus reducing any sources of assay variations. The membrane or membranes selected are dependent upon the sample and the particular separation and preservation sought. For example, if the membrane selected has a high binding capacity, the integrated membrane can be sufficiently small enough to make the use of a punch device upon recovery unnecessary. To elute/recover proteins, the integrated membrane can be designed to provide for incubation and/or washing in an organic solvent (e.g., a physiological buffer with hydrophobic elution solvents and low salt (e.g., lucine)). The integrated membrane may also be used in conjunction with a solvent that extracts proteins from cells without denaturing the proteins; as such, the proteins are captured in their native states within the protein adsorption matrix.
As shown in
The assembly 10 comprises a sample collection layer 12 (alternatively referred to as a cell collection layer), a protein adsorption layer 14, and a highly absorbent nucleic material (e.g., RNA/DNA) preservation layer 16. In the sample collection layer 12, cells from whole blood or tissues are captured and subsequently lysed in the presence of a nucleic acid/protein extraction buffer such e.g., 0.8 M guanidine hydrochloride, 0.1% triton X-100. The sample collection layer 12 comprises an asymmetric membrane that has varying pore size (large to small) across the flow direction of the sample. An example of an asymmetric membrane is a VIVID Plasma Separation Membrane (Pall Corporation, Port Washington, N.Y.). The protein adsorption layer 14 selectively captures and stabilizes proteins from cell extracts immediately upon lysis. In this manner, the protein adsorption matrix protects the RNA/DNA from RNA/DNA-degrading enzymes (e.g., RNases/DNases). The protein adsorption layer 14 may be made from cellulose linked with C18 polymeric chains. Stearic acid and octadecanoic acid are the most common fatty acids found in nature and also constitute the functional part of the most common reverse phase resin, C18, used to as part of the protein adsorption layer 14. The RNA/DNA preservation layer 16 stabilizes the RNA/DNA using a bio-inspired reagent (e.g., trehalose) that protects bio-molecules in a dry state. The preservation layer 16 may be cellulose-based, and infused with trehalose to minimize oxidative stress and promote stability. Alternatives to trehalose include rapid freezing. Pore size in the preservation layer 16 ranges between one hundred (100) to four hundred (400) nanometers to one (1) angstrom.
The sample collection layer 12, protein adsorption layer 14 and the RNA/DNA preservation layer 16 are subsequently peeled away manually (e.g., by hand, with or without the use of tweezers), dried and stored. Alternatively, the layers 12, 14, 16 may be dried while still a single unit and subsequently separated from one another prior to storage. In another alternative, the assembly 10 may be stored intact after being dried. The layer(s) may be stored in various containers including a storage case having separate compartments/recesses for each layer, moisture-barrier pouches with desiccants (e.g., salts, swellable silica) or the like for long-term storage at ambient temperature. The integrated membrane assembly 10 can be used in conjunction with a solvent (e.g., 0.8 M guanidine hydrochloride, 0.1% triton X-100) that serves to extract, purify and preserve the proteins and nucleic acids in individual layers within the assembly 10.
In one preferred embodiment designed for use with blood separation and preservation, the assembly 10 eliminates the need to pre-process the blood sample, as whole blood is directly applied to the sample collection layer 12. Once applied to the sample collection layer 12, the solvent (e.g., 0.8 M guanidine hydrochloride, 0.1% triton X-100) is added to lyse the cells enabling the lysate (containing intracellular proteins/DNA/RNA) to flow through the matrix and into the protein adsorption matrix 14 where proteins (e.g., nucleases) are retained. The lysis buffer breaks open the blood cells and releases the cellular content (e.g., proteins and nucleic acids). The asymmetric membrane of the sample collection layer 12 traps and filters out cellular debris, followed by the protein adsorption layer 14 (i.e., the C18 conjugated cellulose membrane) which selectively adsorbs all proteinacious material as it is highly hydrophobic. Finally, the highly hydrophilic RNA/DNA preservation layer 16 (e.g., a cellulose membrane infused with trehalose) adsorbs and stabilizes all nucleic acids. The immediate removal of proteins (e.g., nucleases) from the lysate provides a mechanism for protecting the RNA/DNA from degradation. Moreover, it avoids the need for protein denaturation agents (e.g., Guanidinium HCl) to inactivate RNases/DNases.
Protein denaturation agents are likely to interfere with downstream applications/reactions, such as quantitative polymerase chain reaction (PCR). Therefore, the absence of these reagents will enable a user (e.g., a researcher, lab technician, physician or the like) to directly use the protein and/or RNA/DNA upon elution from the matrix. As the RNA/DNA can be stored in the matrix without any protein contaminants, the RNA/DNA is not likely to degrade upon elution of the sample from the matrix. The integrated membrane assembly 10 avoids the use of protein denaturants (e.g., Guanidine-HCl) to inactivate RNases/DNases, as proteins are immediately separated from the RNA/DNA upon cell lysis. Because protein denaturants are unnecessary, the user is able to (1) capture proteins in their native states within the protein adsorption matrix and (2) use the RNA/DNA directly, upon elution from the matrix, without the need to remove the protein denaturants. The avoidance of protein denaturants is advantageous as protein denaturants are known to interfere with most downstream applications, such as PCR.
The integrated membrane assembly 10 of the present invention provides a seamless process. In a preferred embodiment, a first stage is the sample collection layer 12 that enables the autonomous collection of whole cells (as well as damaged cells) from the blood sample within the integrated membrane assembly 10. The sample collection layer 12 (stage 1) is designed to capture intact cells from whole blood such that cells are lysed only upon addition of the cell lysis buffer. The buffer may be pre-loaded into the syringe prior to blood draw so that upon blood draw, the buffer mixes in with the blood sample and starts the lysis process. Alternatively, the buffer may be introduced to the integrated membrane assembly 10 after the blood sample is applied to the upstream side 20 of the integrated membrane assembly 10. In either case, blood cells are collected in the membrane intact thus preventing the release of intracellular RNases/DNases prior to the addition of cell lysis buffer. The sample collection layer 12 all cellular debris, including the membranes of ruptured/damaged cells. The sample collection layer 12 may be discarded if a user is only interested in nucleic acids. Some users may want to analyze the cellular debris and look for membranes—which is also possible by eluting out the cellular debris from the top membrane (i.e, the sample collection layer 12).
The sample collection layer 12 comprises an asymmetric membrane with a minimum pore size of 0.2 μm. Asymmetric membranes differ from symmetric micro-porous membranes in that an asymmetric membrane comprises larger pores on an upstream portion of the membrane and smaller pores on a downstream portion of the membrane. The larger upstream pores act as a pre-filter while the smaller downstream pores act as an absolute cut-off layer or exclusion zone. Accordingly, the asymmetric membrane is able to efficiently capture a larger number of cells, as compared to conventional micro-porous membranes, while also reducing the possibility of clogging on the upstream side of the membrane. Moreover, the asymmetric membrane captures erythrocytes, which are able to traverse the narrowest blood vessel.
Once the cells in the blood sample have been collected by sample collection layer 12, in a second stage, the protein adsorption layer 14 extracts proteins followed by a third stage where the RNA/DNA is extracted and purified (in preparation for long-term preservation at ambient temperature) from the blood sample. After the blood cells are collected in the sample collection layer 12, the captured cells are lysed using a cell lysis buffer. The cell lysates then enter the protein adsorption layer 14 immediately upon lysis. The purpose of the protein adsorption layer 14 is to selectively deplete proteins from the lysates such that the nucleic acids are preserved in a protein-free environment in the RNA/DNA adsorption layer 16. The protein adsorption layer 14 comprises a membrane conjugated to an 18-mer hydrocarbon chain (C18) wherein proteins are selectively adsorbed from the solution via high-affinity binding to non-polar residues. The protein adsorption layer 14 enables hydrophilic molecules, such as RNA/DNA, to flow through the layer 14 for subsequent preservation of protein-free nucleic acids in the RNA/DNA adsorption layer 16.
Once the cell lysates have passed through the protein adsorption layer 14, the protein-free RNA/DNA can be captured in the highly adsorbent nucleic acid capture layer (i.e., the RNA/DNA adsorption layer 16). The RNA/DNA adsorption layer 16 comprises a cellulose membrane infused with anyhydrobiotic and/or stabilizing agents (e.g., trehalose).
As outlined above, the layers 12, 14, 16 are stacked and pressed together as a single unit and sealed with the hydrophobic coating 18 to provide directional flow of the blood sample/solvent mixture through all the layers 12, 14, 16. Once the lysates have flowed through the integrated membrane assembly 10, the protein and nucleic acid adsorption layers 14, 16 are peeled away from the sample collection layer 12, air dried and stored in an opaque, moisture-barrier container (not shown) (e.g., a case, pouch, or the like) so that the layers 14, 16 are protected from light and moisture for long-term storage in ambient conditions. The term “peeled away” is used only in the sense that layers 12, 14, 16 are pressed together to form the integrated membrane assembly 10, and the layers 12, 14, 16 may stick to each other due to the presence of lingering moisture from the sample/buffer or from the layers 12, 14, 16 adhering to each after having dried. The container may also include desiccants for moisture control. If necessary, the adsorption layers 14, 16 can be encased in a plastic film 24 prior to storage in the container.
The cell lysis buffer/solvent can have a dual functionality in that it may acts as both a cell lysis buffer and a liquid medium that facilitates selective adsorption of protein contaminants. To achieve efficient cell lysis, the solvent comprises a non-ionic surfactant, such as Triton X-100, to solubilize proteins and lipids. Triton X-100 helps bind proteins to the protein adsorption layer 14. A concentration of 0.1% Triton X-100 in the membrane of the protein adsorption layer 14 enhances protein binding to the layer 14 without adversely affecting red/white blood cell lysis. The buffer further comprises 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA and protease inhibitors.
In another preferred embodiment, as shown in
The layers 32, 34, 36 may be in the form of fluid-permeable cellulose membranes such as those discussed above with respect to assembly 10. Silica membranes are an alternative membrane for nucleic acid capture due to their strong nucleic acid binding capacity, facile and high recovery, rapid drying, and buffer compatibility properties. Like a hydrophilic cellulose membrane infused with trehalose, Silica (SiO2) is positively charged and forms an electrostatics connection to the phosphate backbone of the nucleic acid. Silica matrices are the most popular solid phase support for binding nucleic acids due to their quick and efficient purification procedures. DNA and RNA bind to silica matrices with high affinity due to their negatively charged phosphate backbone and the positively charged silica particles (silicon oxide). Silica membranes used as an alternative to cellulose membranes, including, but not limited to, for any one or more of the layers 32, 34, 36 will have the porous structure described with respect to the layers 32, 34, 36. The basic scientific method is a well-established as it has been implemented in a number of blood DNA extraction techniques. The main limitation of this technique in traditional laboratory process is the relatively high protein contamination present in a sample. However, the integrated membrane assembly 30 can circumvent this limitation by having a protein pre-filter step prior to nucleic acid binding, thereby leading to a greater purity in the overall process.
Based on the silica matrix surface and binding buffers, silica gels can be selectively tailored for capturing and eluting specific nucleic acid species. Silica gels have a high binding capacity of up to 1 μg of nucleic acid per 10-3 cubic centimeters. Since blood has about 0.5 μg/μl of nucleic acids, a volume of 0.3 cc of silica matrix is sufficient to extract all nucleic acid species present in 500 μl of whole blood. All parameters of the silica membrane are being optimized for greater than 99% capture of all nucleic acid in a sample as well as species-specific elution.
A commercially available desiccant may be used (e.g., Ecopack Clay Desiccant, Inter Dry Moisture Control). This material combines the moisture absorption property of calcium chloride with the ruggedness of clay to render a single use desiccant capable of rapidly removing enough water for 70% of its own weight 21. The increased hygroscopy relative to silica gel (7× more) enable the rapid drying of the nucleic acid carrying membrane 36.
Each layer 32, 34, 36 comprises a membrane held within a holder 64 where the membrane is sandwiched between upper and lower portions 66, 68 of the holder 64. Each portion 66, 68 includes an upwardly extending annular ring capable of engaging an annular recess on the bottom side of an above adjacent portion 66, 68. This allows the membrane to be held firmly between the portions 66, 68. This also allows the upper portion 66 of the holder 64 for layer 34 to engage the bottom portion 68 of the adjacent holder 64 for layer 32. Likewise, this further also the lower portion 68 of the holder 64 for layer 34 to engage the upper portion 66 of the adjacent holder 64 for layer 36. In this manner, each holder 64 is capable of removably engaging at least one adjacent holder 64.
Each layer 32, 34, 36 may include at least one line of weakness 70 (e.g., perforations or slits in the membrane) defining a plurality of removable sections or segments (
A fluid-impermeable gasket (e.g., silicone, rubber) may be disposed between adjacent layers 32, 34, 36 to provide directional flow of the blood sample/solvent mixture through all the layers 32, 34, 36 of the integrated membrane assembly 30 and prevent the blood-sample/solvent mixture from moving between the layers 32, 34, 36 and the sidewall of the inner cavity of the housing 42.
The canister assembly 40 and the holders 64 are re-usable after being sterilized. Although the canister assembly 40 and holders 64 are illustrated as being circular in cross-section, they are not limited to that shape. The canister assembly 40 and holders 64 may be come in various polygonal cross-sectional shapes including, but not limited to, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal or the like. The desiccant may be discarded after each use.
A storage case 70 may be used to store the membranes of each layer 32, 34, 36 after they have been removed from their respective holders 64. Alternatively, the membranes for layers 32, 34 containing cellular debris and proteins may be discarded while only the membrane for layer 36 containing nucleic material is storage. The storage case 70 includes upper and lower portions 72, 74 that matingly engage using a press-fit or other known conventional methods. One or more recesses (not shown) may be formed in an upper surface of the lower portion 72 in which the membranes to be stored may be disposed and kept separate from one another. For example, the upper surface of the lower portion 72 may include three recesses (i.e., one recess per membrane so that the membrane of each layer 32, 34, 36 from a particular sample may be stored.
As shown in
In order to ensure both patient safety and optimal function, all the components of the canister assembly 40 are sterilized. A labile molecule such as RNA requires the utmost care in handling, which includes ensuring that there is no RNases contamination. Starting with fabrication, all elements and materials acquired for the canister assembly 40 are made under good manufacturing practice (GMP) with proper sterilizing techniques used for each element (autoclave, gamma irradiation, e-beam). Ensuring that the sample remains as sterile as possible, all canister assemblies 40 are individually wrapped when shipped from the factory. The principals applied in the instant specification allow extracted sterile nucleic acids stored at room temperature under dry conditions to remain intact for at least 5 years if not much more.
A system (not shown) may comprise a canister assembly, and a storage case. The canister assembly and storage case are same as/similar to those described above.
The present invention is directed to rapidly extracting components, such as nucleic acid species from complex body tissue (e.g., whole blood) through a moiety specific sorting process that separates tissue constituents by type of biological molecule. A device embodying the present invention comprises an integrated membrane composed of distinct matrices designed to autonomously extract, purify and preserve the intended sample, such as RNA/DNA. Preferably it may also contain a low-cost, sterile syringe/capillary sample collector pre-loaded with solvent. As shown in
The initial design for the cell collection layer 82 comprised a cellulose membrane impregnated with a compound (e.g., trehalose) that stabilizes RNA/DNA in the dry state. The membrane for capturing cells from whole blood without inducing cell lysis during the collection process in layer 82 was adapted from a BTS Asymmetric Membrane (Pall Corporation). The membrane for separating nucleases and other degradative enzymes from the extracted RNA/DNA in layer 84 was adapted from an Empore™ Disk, C18 (3M Corporation). The membrane for Stores RNA/DNA in a dry format in layer 86 was adapted from Whatman® Cellulose Chromatography Paper Grade 3 MM (GE Healthcare).
It should be noted that a user may not prefer to lyse the cells. This can occur in a situation where the user wants to capture only the freely circulating RNA/DNA—such as from some cancers, viruses, etc. In this case, because the user does not want RNA/DNA from inside the cells, the user has to be extremely careful not to rupture the cells. So, the user captures it (without crushing the cells) with the asymmetric membrane 12. Therefore, a lysing buffer is used only in those situations when a user wishes to capture RNA/DNA present inside the cells. The choice is up to the user/application.
As discussed previously, the integrated membrane performs a seamless process; in a preferred embodiment it may be a three stage process, but more or less stages may be optimum depending on the sample to be gathered, separated and/or preserved. For a three stage process, the first stage is typically a sample collection matrix that enables the autonomous collection of whole cells within the integrated membrane (
In a preferred embodiment, the membrane may be asymmetrical. Due to its unique design, an asymmetric membrane separates a large number of blood cells using a single 25 mm disk. Moreover, the asymmetric membrane is able to separate erythrocytes that are able to traverse the narrowest blood vessel—a feature that have made these cells difficult to capture using conventional symmetric microporous membranes. Asymmetric membranes differ from conventionally cast symmetric microporous membranes in that the larger pores upstream act as a pre-filter while the smaller pores downstream act as an absolute cut-off layer or exclusion zone. As such, the asymmetric membrane is able to efficiently capture a larger number of cells, as compared to conventional symmetric microporous membranes.
For blood, different types of filters and membranes were tested in order to determine the optimal material and method for ˜100% cell removal manually, without triggering cell lysis. A test was performed of the sample collection matrix using a prototype device seen in
Shown in
Referring to
The purpose of the protein adsorption matrix is to selectively deplete proteins from the lysates such that the nucleic acids are preserved in a protein-free environment in the RNA/DNA adsorption matrix. The protein adsorption matrix is a membrane conjugated to an 18-mer hydrocarbon chain (C18) wherein proteins are selectively adsorbed from the solution via high-affinity binding to non-polar residues. The C18 membrane enables hydrophilic molecules, such as RNA/DNA, to flow through the membrane for subsequent preservation of protein-free nucleic acids in the RNA/DNA adsorption matrix.
To determine total protein adsorption using the candidate reversed phase membranes, 5-50 μl of whole bovine blood was lysed in the solvent at a 1:200 dilution for a total load volume of 1 ml. The total protein concentration of the load was determined using the Pierce® 660 nm Protein Assay Reagent.
indicates data missing or illegible when filed
TABLE 1 shows RNA and DNA Concentrations of the lysed whole blood, pre- and post-loading. Shown in (A) and (B) of Table 1, data for RNA (A) and DNA (B) concentration of the lysed whole blood prior to loading (indicated as load) and post-loading (indicated as flow-through (FT)) using the indicated membranes is presented. The percentage yield (% yield) of the RNA/DNA in the flow-through is shown to indicate the efficacy of the membranes. The table shows that the C18 membrane was able to recover greater than 93% of the RNA and greater than 80% in whole blood enabling the collection of ˜24 μg RNA and ˜5 μg of protein-free DNA per 50 μl sample.
In order to demonstrate that the reversed phase membranes are capable of selectively depleting proteins from lysed whole blood, the amount of RNA and DNA in the load was quantified and the flow-through used the Quant-It™ Assay Reagents. As shown in Table 1, the DNA concentration in the load was equivalent to 119 ng of DNA per 1 μl of blood—a value that is consistent with those observed in healthy bovine blood and only slightly higher than those observed in normal human blood (˜90 ng/μl). The RNA concentration in the load was equivalent to 516 ng of RNA per 1 μl of blood. The RNA content of a typical mammalian cell is three (3) to five (5) times greater than the DNA content. As the RNA concentration (516 ng/μl) in the load is ˜4× greater than the DNA concentration (119 ng), the RNA concentration is consistent with those identified in healthy bovine blood. Numerous candidate membranes were evaluated and the results are summarize in Table 1. In sum, the C18 membrane is capable of achieving 100% protein adsorption while recovering 93% of the RNA and 80% of the DNA in 5 μl of whole blood. Additional studies using larger volumes of the C-18 membrane confirmed similar results starting with 50 μl of whole bovine blood—indicating that the integrated membrane assembly technology can be scaled to process larger volumes of blood (˜0.5 ml). These results demonstrate a major advantage for the integrated membrane assembly, as the system is now able to autonomously extract ˜25 μg of protein-free RNA as well as ˜5 μg of protein-free DNA from only 50 μl of whole blood.
Alternatives to the C18 membrane include the SDB-XC and SDB-RPS membranes. The SDB-XC membrane is a poly (StyreneDivinylBenzene (SDB)) copolymer membrane used as a reversed phase sorbent for solid phase extraction. The SDB-XC membrane is not bonded to silica but is a 100% copolymeric particle that is spherical, porous and cross-linked. It does not exhibit the secondary cationic (silanol) interactions and pH limitations common to bonded silica sorbents, so it offers more predictable and reproducible reversed phase interaction. The SDB-XC membrane may be substituted into methods that use C18 or C8 bonded silica and often demonstrates greater capacity. The structure of the SDB-XC membrane provides unique selectivity, especially in the retention of moderately polar, water-soluble analytes. However, since the SDB-XC membrane displays both aromatic and aliphatic interactions and does not display secondary cationic attraction, some existing methods may require adjustment to optimize analyte retention and elution.
The SDB-RPS (Reversed Phase Sulfonate) membrane is a poly(StyreneDivinylBenzene) copolymer membrane that has been modified with sulfonic acid groups to make it hydrophilic. SDB-RPS is not bonded to silica but is a 100% copolymeric particle that is spherical, porous and cross-linked. The sulfonation imparts unique selectivity for organic analytes that are polar in nature, such as drugs, drug metabolites, water-soluble pesticides and pesticide metabolites. SDB-RPS is also useful for comprehensive drug screening, in which a wide range of analytes are simultaneously extracted. Although SDB-RPS is sulfonated, it has much lower capacity than a typical strong cation exchanger. Because SDB-RPS displays reversed phase and cation exchange interactions, both functionalities can be considered for designing selective extractions. SDB-RPS can be used as a reversed phase sorbent and substituted into existing methods as an alternative to the C18 membrane. Organic analytes with polar character have been shown to extract better on SDB-RPS than on traditional C18 bonded silica. However, new dimensions in selectivity and elution can be achieved by considering the influence of the sulfonic acid groups to provide some cation exchange affinity for amine-containing analytes.
Development and optimization of the process for purifying RNA/DNA from whole blood prior to storage involves ensuring that buffers do not interfere with downstream processing. Guanidine hydrochloride (Gu-HCl) and 0.1% Triton X-100 selectively absorb proteins from whole cell lysates in blood and is an important component of RNA extraction buffers enabling the dissociation of nucleoprotein complexes. However, guanidine is a known contaminant that interferes with several downstream applications (e.g., gel electrophoresis, PCR). The challenge is to determine ideal concentrations of guanidine that would enable a user to rapidly and efficiently process/store RNA without needing to remove the excess guanidine prior to analysis. This was achieved by optimizing the reagents starting with commercial off-the-shelf (COTS) buffers and chemicals (see Table 2 below.
Table 2 lists all the custom optimized reagents and buffers used by the integrated membrane system based on the optimization of COTS components/chemicals shown above. By leveraging existing, readily available chemicals, the integrated membrane system is composed of cost-effective and established method that enhances its efficacy and acceptance within the user community.
A solvent consisting of 0.8M Gu HCl and 0.1% Triton X-100 was thus formulated and tested in order to ascertain whether the solvent is still able to recover intact RNA from blood cells (bovine blood samples) in a protein-free environment (greater than 99% protein depletion). To visualize the RNA on an analytical gel and thus measure the integrity of the RNA, the level of the isolated RNA was increased by isolating only leukocytes from whole blood using the 1× Red Blood Cell Lysis Buffer (BioLegend, San Diego, Calif.). By enriching for leukocytes, the integrated membrane system is challenged by introducing a sample that is likely to have a higher RNase/DNase content due to the presence of intracellular nucleases, as compared to whole blood. 2×106 blood cells were lysed with solvent containing 0.8M Guanidine HCl and 0.1% Triton X-100. Upon lysis, the blood cell lysates were applied to a pre-activated C18 membrane. Analysis of the flow-through for (1) RNA/DNA integrity occurred using analytical gels, and (2) total protein content using the Pierce® 660 nm Protein Assay. As shown in (C) of
While purified DNA may be stored for weeks at ambient temperatures, purified RNA may only be stored for hours at room temperature. The challenge therefore was to identify the proper storage conditions to store highly labile molecules, such as RNA, for periods longer than a few hours at room temperature. The integrated membrane system employs a method by which purified RNA/DNA is stored in a dry state—a mechanism that inhibits RNA/DNA biodegradation by hydrolysis thus enabling the storage of RNA/DNA for longer periods at ambient temperatures.
As previously described, the isolated RNA/DNA as extracted from whole blood using the integrated membrane is free from protein contaminants. Table 1-2 shows a representative data set wherein the integrated membrane was able to recover ˜93% RNA and ˜80% of the DNA while achieving 100% protein depletion. Given that relatively large amounts of purified RNA/DNA would be required to assess the stability of the RNA/DNA using standard agarose gel electrophoresis, ˜400 μg of RNA and ˜100 μg DNA was prepared from 2×107 leukocytes (as isolated from heparinized bovine blood) using a standard phenol:chloroform extraction method—a method that enables the large-scale preparation of protein-free RNA/DNA. This allowed for preparation of RNA/DNA stocks mimicking those prepared using the integrated membrane. The RNA was prepared using a standard phenol: chloroform extraction, as described previously. The dried RNA was prepared by pipetting 2 μg of purified RNA onto a matrix in the presence and absence of trehalose (a chemical stabilizer). Upon drying, the RNA was stored at −80° C. (as a positive control) or 50° C. for 8 days. It should be noted that storage for 8 days at 50° C. is equivalent to 56 days at 22° C. according to the accelerated aging theory. To assess the integrity of the RNA, samples were solubilized in nuclease-free water, electrophoresed in a 0.8% formaldehyde-treated agarose gel and visualized using SYBR Green II (Sigma, St. Louis, Mo.). As shown in
The ability of the integrated membrane system to enable the long-term storage of the purified DNA at ambient temperatures was determined. The stability of dried DNA (as compared to DNA stored in nuclease-free water) was tested after incubation for 24 hours at 37° C. and 50° C., which are temperatures that mimic 3 and 7 days respectively of storage at 22° C. The dried DNA was prepared by pipetting 0.5 μg of purified DNA onto a matrix in the presence of trehalose (a chemical stabilizer). Upon drying, the DNA was stored at −20° C. (as a positive control), 37° C. and 50° C. for 24 hours. DNA samples stored in nuclease-free water were prepared by pipetting 0.5 μg of purified DNA in 10 μl of nuclease-free water and storing the samples at −20° C. (as a positive control), 37° C. and 50° C. for 24 hours. To assess the integrity of the DNA, samples were solubilized (if dried) in nuclease-free water, electrophoresed in a 0.8% agarose gel and visualized using ethidium bromide. As shown in
Turning to
Successful recovery of ˜93% RNA and ˜80% DNA was obtained while achieving greater than 99% protein depletion from whole blood processed through the integrated membranes. Furthermore, the extracted DNA and RNA were shown to be well preserved for greater than 50 days (accelerated aging tests).
The integrated membrane assemblies of the present invention are shown to have numerous technical advantages including, but not limited to, the examples provided herein. For example, the integrated membrane assemblies allow proteins to be captured in their native states with the protein adsorption layer via use of a non-denaturing solvent that acts to simply/passively extract intracellular proteins. In another example, RNA/DNA is extracted, purified and preserved without the use of protein denaturants which enables a user to directly use the RNA/DNA upon elution from the matrix (use of protein denaturants (as are typically used in sample processing) requires the user to perform additional steps prior to analysis, as protein denaturants can interfere with many downstream applications). In an additional example, the integrated membrane facilitates one-step extraction, purification and long-term preservation of protein/DNA/RNA molecules at near room temperature for point-of-service (POS) use (the user is simply required to apply the sample on the membrane followed by the solvent and wait until the membrane is ready to be peeled and dried). In yet another example, the integrated membrane is compatible with Clinical Laboratory Improvement Amendments-waived (CLIA-waived) devices (e.g., the integrated membrane employs a hands-free methodology for protein/RNA/DNA collection that is so simple and accurate that it renders the likelihood of erroneous results negligible). In a further example, the integrated membrane works with a minimal sample volume (i.e., the integrated membrane captures adequate amounts of protein/RNA/DNA for subsequent analysis from blood volumes as low as 10 μl). In yet another example, the integrated membrane captures substantially all proteins and nucleic acids from minimal volumes (5-10 μl) of whole blood. In another example, the integrated membrane is able to process each blood sample in less than two minutes, and preferably within 1.5 minutes (i.e., the fluid sample passes through the integrated membrane and the layers are ready for subsequent use by a user within 1.5 minutes). In an additional example, the protein/nucleic acid adsorption layers are impregnated with anhydrobiotic agents that stabilize RNA/DNA molecules for long-term storage at ambient conditions, as described herein.
The integrated membrane assemblies of the present invention have numerous economic advantages including, but not limited to, the following examples. For example, the integrated membrane can be assembled from inexpensive commercial off-the-shelf (COTS) materials at an estimated cost of $2 per sample. In another example, the integrated membrane is stored at near-room temperature before and after sample application, which eliminates the need for laboratory freezers (allowing for ambient storage conditions with minimal logistic burden). In an additional example, minimal volume is needed to store the layers as each sample collection blot is approximately 25 mm in diameter (providing compact package volume).
Although the present invention has been discussed above in connection with blood, the present invention is not limited to that particular fluid and may also be used with any fluid that may contain nucleic material including, without limitation, bodily fluids (e.g., urine, semen, saliva, lymphatic fluid, etc.) or non-bodily fluids (e.g., water). A user may also choose not to use the lyse buffer/solvent if the user wishes to preserve cells such that foreign proteins and/or nucleic freely circulating in a subject's bloodstream or other bodily fluid (e.g., the proteins and/or nucleic material of bacteria or other organisms in a subject's bloodstream) may be collected.
Furthermore, the integrated membrane can be used to perform moeity specific sample extraction from a variety of complex sample matrix (e.g., blood, food, soil, etc.) in a flow through process. Even dry material can be used because the material can be buffer, as described above. The above described embodiments demonstrates separation of classes of biological material, such as cells, proteins and nucleic acids (DNA/RNA) fully preserved from whole blood. The subtlety here is that for many components, this has to be done rapidly—otherwise components such as proteins and RNA may degrade. As shown above, material from within cells can be extracted (by using lysing buffers). Other manifestations include targeted extraction, e.g., a specific protein or miRNA. In that example, a membrane with conjugate components can be integrated in the membrane assembly to extract just that targeted component. All this done in a single flow-through process after the best sequence of sorting has been determined. As described above, if a user desires to obtain nucleic acids, then the user determines that the user need to filter out the cells and then the proteins, or otherwise the nucleic acids can degrade.
Likewise, although the integrated membrane disclosed above has been discussed above in connection with a canister, the present invention is not limited to that specific construction and, in particular, the integrated membrane may be incorporated into various other constructs including, without limitation, a bandage, a swab, filter, or any other object used for, or capable of being used for, collecting a fluid in which proteins and/or nucleic material may be found.
In an alternative, an integrated membrane may be placed directly in a syringe or syringe attachment. The fluid sample (e.g., blood) is then pumped across the integrated membrane (either during uptake or dispense mode). In an example of an integrated membrane in a syringe attachment, the syringe attachment may be matingly engaged to a hypodermic needle on one end and a syringe on the other end, with the integrated membrane disposed within the syringe attachment. After blood is drawn, the syringe and needle are disengaged from the attachment and the apertures (where the attachment had engaged syringe and needle) sealed. As blood is drawn, the blood is dispensed across the layers of the integrated membrane, with the layers automatically processing the blood in the manner described above. The fluid remaining after passing through the integrated membrane exits the attachment and enters the syringe. The fluid within the syringe may be stored in a vial or other container for subsequent use or discarded with the syringe.
In addition, the claimed invention is not limited in size and may be constructed in various sizes in which the same or similar principles of operation as described above would apply. Furthermore, the figures (and various components shown therein) of the specification are not to be construed as drawn to scale.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results.
The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.
The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed.
When an element or layer is referred to as being “on”, “engaged to”, “connected to” or “coupled to” another element or layer, it may be directly on, engaged, connected or coupled to the other element or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to”, “directly connected to” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc.). As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
Spatially relative terms, such as “inner,” “outer,” “beneath”, “below”, “lower”, “above”, “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially relative terms may be intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as “below” or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, the example term “below” can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
The above description presents the best mode contemplated for carrying out the present invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains to make and use this invention. This invention is, however, susceptible to modifications and alternate constructions from that discussed above that are fully equivalent. Consequently, this invention is not limited to the particular embodiments disclosed. On the contrary, this invention covers all modifications and alternate constructions coming within the spirit and scope of the invention as generally expressed by the following claims, which particularly point out and distinctly claim the subject matter of the invention.
This application is a non-provisional patent application claiming priority to U.S. provisional patent application No. 61/685,330 filed on Mar. 14, 2012, entitled “An integrated membrane for the preservation of biomolecules utilizing membrane-adsorbent technology to autonomously fractionate and preserve proteins and nucleic acids for long-term storage at conditions” incorporated by reference herein.
This invention was made under a contract with an agency of the U.S. Government, Army Phase I Contract No: W81XWH-12-C-0002.
| Number | Date | Country | |
|---|---|---|---|
| 20140273058 A1 | Sep 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61685330 | Mar 2012 | US |
