Patent Application
20120100531
Various tests are available that can be used to assess the presence of biological analytes in a sample (e.g. surface, water, air, etc). Such tests include those based on the detection of ATP using the firefly luciferase reaction, tests based on the detection of protein using colorimetry, tests based on the detection of microorganisms using microbiological culture techniques, and tests based on detection of microorganisms using immunochemical techniques. Surfaces can be sampled using either a swab device or by direct contact with a culture device such as an agar plate. The sample can be analyzed for the presence of live cells and, in particular, live microorganisms.
Results from these tests are often used to make decisions about the cleanliness of a surface. For example, the test may be used to decide whether food-processing equipment has been cleaned well enough to use for production. Although the above tests are useful in the detection of a contaminated surface, they can require numerous steps to perform the test, they may not be able to distinguish quickly and/or easily the presence of live cells from dead cells and, in some cases, they can require long periods of time (e.g., hours or days) before the results can be determined.
The tests may be used to indicate the presence of live microorganisms. For such tests, a cell extractant is often used to release a biological analyte (e.g., ATP) associated with living cells. The presence of extracellular material (e.g., non-cellular ATP released into the environment from dead or stressed animal cells, plant cells, and/or microorganisms) can create a high “background” level of ATP that can complicate the detection of live cells.
In spite of the availability of a number of methods and devices to detect live cells, there remains a need for a simple, reliable test for detecting live cells and, in particular, live microbial cells.
In general, the present disclosure relates to articles and methods for detecting live cells in a sample. The articles and methods make possible the rapid detection (e.g., through fluorescence, chemiluminescence, or a color reaction) of the presence of cells such as bacteria on a surface. In some embodiments, the inventive articles are “sample-ready”, i.e., the articles contain all of the necessary features to detect living cells in a sample. The methods feature the use of a cell extractant to facilitate the release of biological analytes from biological cells. The inventive articles and methods include a release element, which controls the release of an effective amount of cell extractant into a liquid mixture comprising a sample. In some aspects, the inventive articles and methods provide a means to distinguish a biological analyte, such as ATP or an enzyme, that is associated with eukaryotic cells (e.g., plant or animal cells) from a similar or identical biological analyte associated with prokaryotic cells (e.g., bacterial cells). Furthermore, the inventive articles and methods provide a means to distinguish a biological analyte that is free in the environment (i.e., an acellular biological analyte) from a similar or identical biological analyte associated with a living cell.
In one aspect, the present disclosure provides an article for detecting cells in a sample. The article can comprise a housing with an opening, a sample acquisition device, a cell extractant, and a release element comprising the cell extractant. The housing can be configured to receive the sample acquisition device. In some embodiments, the release element can be disposed in the housing. In some embodiments, the release element can be disposed in the sample acquisition device.
In another aspect, the present disclosure provides an article for detecting cells in a sample. The article can comprise a housing with an opening configured to receive a sample, a sample acquisition device comprising a reagent chamber, a cell extractant, and a release element comprising the cell extractant. The release element can be disposed in the reagent chamber.
In any one of the above embodiments, the article can further comprise a frangible barrier that forms a compartment in the housing. In some embodiments, the frangible barrier can comprise the release element comprising the cell extractant. In some embodiments, the compartment can comprise the release element.
In another aspect, the present disclosure provides an article for detecting cells in a sample. The article can comprise a housing with an opening configured to receive a sample, a cell extractant, a release element comprising the cell extractant; a detection reagent, and a carrier comprising the detection reagent. In some embodiments, the release element and the carrier are disposed in the housing.
In any one of the above embodiments, the housing can further comprise a compartment. In any one of the above embodiments, the compartment can further comprise a detection reagent.
In any one of the above embodiments, the detection reagent is selected from the group consisting of an enzyme, an enzyme substrate, an indicator dye, a stain, an antibody, and a polynucleotide.
In another aspect, the present disclosure provides a sample acquisition device comprising a release element. The release element can comprise a cell extractant. The release element can be disposed on the sample acquisition device. In some embodiments, the cell extractant can comprise a microbial cell extractant. In some embodiments, the cell extractant can comprise a somatic cell extractant.
In another aspect, the present disclosure provides a kit. The kit can comprise a housing that includes an opening configured to receive a sample, a cell extractant, a release element comprising the cell extractant, and a detection system. Optionally, the kit can further comprise a sample acquisition device and the opening can be configured to receive the sample acquisition device. In some embodiments, the detection system can comprise a carrier comprising a detection reagent. In some embodiments, the detection reagent is selected from the group consisting of an enzyme, an enzyme substrate, an indicator dye, a stain, an antibody, and a polynucleotide.
In another aspect, the present disclosure provides a method of detecting cells in a sample. The method can comprise providing a cell extractant, a release element comprising the cell extractant, and a sample suspected of containing cells. The method further can comprise forming a liquid mixture comprising the sample and the release element. The method further can comprise detecting an analyte in the liquid mixture.
In another aspect, the present disclosure provides a method of detecting cells in a sample. The method can comprise providing a sample acquisition device and a housing. The housing can include an opening configured to receive the sample acquisition device, a cell extractant, and a release element comprising the cell extractant. The release element can be disposed in the housing. The method further can comprise obtaining sample material with the sample acquisition device, forming a liquid mixture comprising the sample material and the release element, and detecting an analyte in the liquid mixture.
In another aspect, the present disclosure provides a method of detecting cells in a sample. The method can comprise providing a sample acquisition device and a housing. The sample acquisition device can include a release element comprising a cell extractant. The housing can comprise an opening configured to receive the sample acquisition device. The method further can comprise obtaining sample material with the sample acquisition device, forming a liquid mixture comprising the sample material and the release element, and detecting an analyte in the liquid mixture.
In another aspect, the present disclosure provides a method of detecting cells in a sample. The method can comprise providing a sample acquisition device and a housing. The housing can include an opening configured to receive the sample acquisition device, a cell extractant, and a release element. The release element can comprise the cell extractant. The method further can comprise obtaining sample material with the sample acquisition device, forming a liquid mixture comprising the sample material and the release element, and detecting an analyte in the liquid mixture.
In any one of the above embodiments, the release element can comprise a coated substrate. In some embodiments, the substrate can comprise metal, plastic, or glass. In some embodiments, the substrate can comprise a film. In some embodiments, the substrate can comprise cavities. In any one of the above embodiments, the coated substrate can further comprise a barrier layer. In any one of the above embodiments, the coated substrate can further comprise a binder.
In any one of the above embodiments, the method further can comprise detecting the analyte using a detection system. In any one of the above embodiments, the method further can comprise quantifying an amount of the analyte. In any one of the above embodiments, the method further can comprise quantifying an amount of the analyte two or more times. In any one of the above embodiments, the method further can comprise compressing the release element.
“Biological analytes”, as used herein, refers to molecules, or derivatives thereof, that occur in or are formed by an organism. For example, a biological analyte can include, but is not limited to, at least one of an amino acid, a nucleic acid, a polypeptide, a protein, a polynucleotide, a lipid, a phospholipid, a saccharide, a polysaccharide, and combinations thereof. Specific examples of biological analytes can include, but are not limited to, a metabolite (e.g., staphylococcal enterotoxin), an allergen (e.g., peanut allergen(s), a hormone, a toxin (e.g., Bacillus diarrheal toxin, aflatoxin, etc.), RNA (e.g., mRNA, total RNA, tRNA, etc.), DNA (e.g., plasmid DNA, plant DNA, etc.), a tagged protein, an antibody, an antigen, and combinations thereof.
“Sample acquisition device” is used herein in the broadest sense and refers to an implement used to collect a liquid, semisolid, or solid sample material. Nonlimiting examples of sample acquisition devices include swabs, wipes, sponges, scoops, spatulas, pipettes, pipette tips, and siphon hoses.
As used herein, “chromonic materials” (or “chromonic compounds”) refers to large, multi-ring molecules typically characterized by the presence of a hydrophobic core surrounded by various hydrophilic groups (see, for example, Attwood, T. K., and Lydon, J. E., Molec. Crystals Liq. Crystals, 108, 349 (1984)). The hydrophobic core can contain aromatic and/or non-aromatic rings. When in solution, these chromonic materials tend to aggregate into a nematic ordering characterized by a long-range order.
As used herein, “release element” refers to a structure that is capable of containing a cell extractant. The release element includes physical and/or chemical components selected to limit the diffusion of a cell extractant from a region of relatively high concentration to a region of relatively low concentration.
“Encapsulating agent” refers to a type of release element. An encapsulating agent, as used herein, is a material that substantially surrounds the cell extractant.
As used herein, “shell structure” refers to a structure or framework forming a type of release element. Generally, the shell structure forms the exterior of the release element.
As used herein, the term “hydrogel” refers to a polymeric material that is hydrophilic and that is either swollen or capable of being swollen with a polar solvent. The polymeric material typically swells but does not dissolve when contacted with the polar solvent. That is, the hydrogel is insoluble in the polar solvent. The swollen hydrogel can be dried to remove at least some of the polar solvent.
“Cell extractant”, as used herein, refers to any compound or combination of compounds that alters cell membrane or cell wall permeability or disrupts the integrity of (i.e., lyses or causes the formation of pores in) the membrane and/or cell wall of a cell (e.g., a somatic cell or a microbial cell) to effect extraction or release of a biological analyte normally found in living cells.
“Detection system”, as used herein, refers to the components used to detect a biological analyte and includes enzymes, enzyme substrates, binding partners (e.g. antibodies or receptors), labels, dyes, and instruments for detecting light absorbance or reflectance, fluorescence, and/or luminescence (e.g. bioluminescence or chemiluminescence).
The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances.
Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
As used herein, “a,” “an,” “the,” “at least one,” and “one or more” are used interchangeably. Thus, for example, a housing that comprises “a” detection reagent can be interpreted to mean that the housing can include “one or more” detection reagents.
The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
The invention will be further explained with reference to the drawing figures listed below, where like structure is referenced by like numerals throughout the several views.
a is a top view of one embodiment of a release element comprising a substrate with cavities.
b is a cross-sectional view of the release element of
All patents, patent applications, government publications, government regulations, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, will control.
Biological analytes can be used to detect the presence of biological material, such as live cells in a sample. Biological analytes can be detected by various reactions (e.g., binding reactions, catalytic reactions, and the like) in which they can participate.
Chemiluminescent reactions can be used in various forms to detect cells, such as bacterial cells, in fluids and in processed materials. In some embodiments of the present disclosure, a chemiluminescent reaction based on the reaction of adenosine triphosphate (ATP) with luciferin in the presence of the enzyme luciferase to produce light provides the chemical basis for the generation of a signal to detect a biological analyte, ATP. Since ATP is present in all living cells, including all microbial cells, this method can provide a rapid assay to obtain a quantitative or semiquantitative estimate of the number of living cells in a sample. Early discourses on the nature of the underlying reaction, the history of its discovery, and its general area of applicability, are provided by E. N. Harvey (1957), A History of Luminescence: From the Earliest Times Until 1900, Amer. Phil. Soc., Philadelphia, Pa.; and W. D. McElroy and B. L. Strehler (1949), Arch. Biochem. Biophys. 22:420-433.
ATP detection is a reliable means to detect bacteria and other microbial species because all such species contain some ATP. Chemical bond energy from ATP is utilized in the bioluminescent reaction that occurs in the tails of the firefly Photinus pyralis. The biochemical components of this reaction can be isolated free of ATP and subsequently used to detect ATP in other sources. The mechanism of this firefly bioluminescence reaction has been well characterized (DeLuca, M., et al., 1979 Anal. Biochem. 95:194-198).
The inventive articles and methods of the present disclosure provide simple means for conveniently controlling the release of biological analytes from living cells in order to determine the presence, optionally the type (e.g., microbial or nonmicrobial), and optionally the quantity of living cells in an unknown sample. The articles and methods include a release element comprising a cell extractant.
Release elements according to the present disclosure include coated substrates. The coating, the substrate and/or the coated substrate is adapted to act as a physical barrier and/or a diffusion barrier to prevent the immediate dissolution, for a brief period of time, of an effective amount of cell extractant into an aqueous mixture.
Coated substrates include a coating and a substrate. The coating comprises a cell extractant. The coating can be applied to the substrate using coating processes that are known in the art such as, for example, dip coating, knife coating, curtain coating, spraying, kiss coating, gravure coating, offset gravure coating, and/or printing methods such as screen printing and inkjet printing. In some embodiments, the coating can be applied in a pre-determined pattern (e.g., stripes, grids, spots). The choice of the coating process will be influenced by the shape and dimensions of the solid substrate and it is within the grasp of a person of ordinary skill in the appropriate art to recognize the suitable process for coating any given solid substrate.
The substrate onto which the coating is applied includes a variety of substrate materials. Nonlimiting examples of suitable substrate materials onto which a coating of the present disclosure can be applied include plastic (e.g., polycarbonate, polyalkylenes such as polyethylene and polypropylene, polyesters, polyacrylates, and derivatives and blends thereof), metals (e.g., gold, graphite, platinum, palladium, and nickel), glass, cellulose and cellulose derivatives (e.g., filter papers), ceramic materials, open-cell foams (e.g., polyurethane foam), nonwoven materials (e.g., membranes, PTFE membranes), and combinations thereof (e.g., a plastic-coated metal foil). The substrate can be configured in a variety of forms including, for example, fibers, nonwoven materials, sheets, and films.
In some embodiments, the surface of the substrate can be relatively smooth. In some embodiments, at least a portion of the substrate can comprise cavities.
Substrates, as used herein, includes microreplicated substrates. “Microreplication” or “microreplicated” means the production of a microstructured surface (e.g., microchannels) through a process where the structured surface features retain an individual feature fidelity during manufacture, from product-to-product, that varies no more than about 50 micrometers. The microreplicated surfaces preferably are produced such that the structured surface features retain an individual feature fidelity during manufacture, from product-to-product, which varies no more than 25 micrometers. In accordance with the present invention, a microstructured surface comprises a surface with a topography (the surface features of an object, place or region thereof) that has individual feature fidelity that is maintained with a resolution of between about 50 micrometers and 0.05 micrometers, more preferably between 25 micrometers and 1 micrometer. Suitable microreplicated substrates are described in U.S. Patent Application Publication No. US 2007/0212266 A1, which is incorporated herein by reference in its entirety.
In some applications, it may be desirable that the release element containing a cell extractant is in a dry or partially-dried state. Certain release elements (e.g., water-swollen hydrogels) can be dried, for example, by methods known to those skilled in the art, including evaporative processes, drying in convection ovens, microwave ovens, and vacuum ovens as well as freeze-drying. When the dried or partially-dried release element is exposed to a liquid or aqueous solution, the cell extractant can diffuse from the release element. The cell extractant can remain essentially dormant in the release element until exposed to a liquid or aqueous solution. That is, the cell extractant can be stored within the dry or partially-dried release element until the release element is exposed to a liquid. This can prevent the waste or loss of the cell extractant when not needed and can improve the stability of many moisture sensitive cell extractants that may degrade by hydrolysis, oxidation, or other mechanisms.
In some embodiments, certain release elements (e.g., water-swollen hydrogels) that do not contain a cell extractant can be dried. Optionally, the dried material can be packaged (e.g., in a vacuum package). The dried material subsequently can be rehydrated in a solution comprising a cell extractant, thereby loading the rehydrated hydrogel with the cell extractant. Advantageously, this process allows a hydrogel to be produced and dried at one location and transported in a dry state to a second location, where the dried hydrogel can be loaded with a cell extractant by rehydrating the dried hydrogel in a solution (e.g., an aqueous solution) comprising a cell extractant. After The hydrogel may be coated onto a substrate before or after it is loaded with the cell extractant. Optionally, after rehydrating the hydrogel with the cell-extractant solution, the swollen hydrogel can be dried with the cell extractant therein and/or thereon, as described above.
The covered channels 1105 can present a relatively small opening (i.e., one or both ends of the channel) to contact a liquid in which the release element 1140 is suspended, thereby limiting and/or delaying the diffusion of an effective amount of a cell extractant out of the covered channels 1105 and into the liquid.
Release elements with microreplicated surfaces and/or cavities can be coated to fill the microchannels or cavities with a cell extractant composition. The cell extractant composition may be liquid, solid, semi-solid, or a combination of any two or more of the foregoing.
In some embodiments, the substrate can be a filter, such as Grade 4, 20-25 μm Qualitative Filter Paper, Grade 30, Glass-Fiber Filter Paper, Grade GB005, a thick (1.5 mm) highly absorbent blotting paper (all obtained from Whatman, Inc, Florham Park, N.J.), Zeta Plus Virosorb 1MDS discs (CUNO, Inc, Meriden, Conn.) and 0.45 μm MF-Millipore membrane (Millipore, Billerica, Mass.). Any one of the above substrates can be loaded a cell extractant solution containing polyvinyl alcohol. Any one of the above substrates can be loaded a cell extractant solution containing VANTOCIL (Arch Chemicals, Norwalk, Conn.). Any one of the above substrates can be loaded a cell extractant solution containing CARBOSHIELD (Lonza, Walkersville, Md.). Any one of the above substrates can be loaded a cell extractant solution containing 5% benzalkonium chloride solution.
In some embodiments, the substrate can be coated with a matrix material comprising a cell extractant. Suitable matrix materials comprising cell extractants are described in U.S. Patent Application No. 61/175,980, filed May 6, 2009 and entitled ARTICLES WITH MATRIX COMPRISING A CELL EXTRACTANT AND BIODETECTION METHODS THEREOF, which is incorporated herein by reference in its entirety. In some embodiments, the matrix material (e.g., a polymeric material or a nonpolymer material such as a ceramic) can be substantially insoluble in a liquid (for example, an aqueous liquid comprising a sample). Additionally, or alternatively, the matrix material can be substantially insoluble in an organic solvent. In some embodiments, the matrix material can comprise an excipient that is substantially soluble and/or dispersible at ambient temperature in a liquid mixture (e.g., an aqueous solution) comprising a sample. In some embodiments, the matrix material can comprise an excipient that is substantially insoluble and nondispersible at ambient temperature in an aqueous solution (i.e., the dissolution or dispersion of the excipient can be triggered by a temperature shift and/or the addition of a chemical trigger).
In some embodiments, the matrix material used to coat the substrate can be a pre-formed matrix (e.g., a polymer matrix) comprising a cell extractant. In some embodiments, a mixture comprising matrix precursors and cell extractant are coated onto the substrate and the matrix can be formed subsequently on the substrate using, for example, polymerization processes known in the art and/or described herein. In some embodiments, a pre-formed matrix is coated onto the substrate or a matrix is formed on the substrate and, subsequently, the cell extractant is loaded into the substrate using processes known in the art and/or described herein.
Matrices, according to the present disclosure, can comprise a cell extractant admixed with an excipient. “Excipient” is used broadly to include, for example, binders, glidants (e.g., flow aids), lubricants, disintegrants, and any two or more of the foregoing. In some embodiments, coated substrate can comprise an outer coating, which may influence the release of an active substance (e.g., a cell extractant) when the coated substrate is contacted with a liquid (e.g., an aqueous liquid comprising a sample). In some embodiments, matrices can comprise fillers (e.g., a sugar such as lactose or sorbitol) as a bulking agent for the matrix. Disintegrants (e.g., a polysaccharide such as starch or cellulose) may promote wetting and/or swelling of the matrix and thereby facilitate release of the active substance when the matrix is contacted with a liquid. Sorbitol and mannitol are excipients that can promote the stability of certain cell extractants (e.g., enzymes). Mannitol can be used to delay the release of the cell extractant. In some embodiments, polyethylene glycol (PEG) is a preferred excipient to control the release of active substances from a matrix. In some embodiments, PEG compounds with molecular weights of 3300 and 8000 daltons can be used to delay the release of an active substance from a matrix.
In some embodiments, the coating mixture comprises an additive (e.g., a binder or viscosifier) to facilitate the coating process and/or to facilitate the adherence of the coating to the substrate. Non-limiting examples of additives include gums (e.g., guar gum, xanthan gum, alginates, carrageenan, pectin, agar, gellan), polysaccharides (e.g., starch, methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, agarose), and polypeptides (e.g., gelatin,).
Matrix materials, cell extractants, substrates, and coating additives should be selected for their compatibility with the detection system used to detect cells in a sample. This compatibility can be tested by 1) detecting an amount of analyte in a detection system (e.g., a combining ATP with luciferin and luciferase and measuring the amount of luminescence with a luminometer, as described in Example 6); 2) repeating the detection step with the matrix material, cell extractant, substrate or coating additive; and 3) comparing the results of step 1 with the results of step 2 to determine whether the additive substantially inhibits the detection and/or measurement of the analyte in the reaction.
In some embodiments, the material used to coat the substrate may comprise a shell structure comprising a cell extractant. Suitable shell structures are described in U.S. Patent Application No. 61/175,996, filed May 6, 2009 and entitled “ARTICLES WITH SHELL STRUCTURES INCLUDING A CELL EXTRACTANT AND BIODETECTION METHODS THEREOF” and in U.S. Pat. No. 7,485,609, each of which is incorporated herein by reference in its entirety. The compatibility of a particular shell structure with a particular detection system can be determined as described herein.
In some embodiments, the coated substrate may further comprise a barrier layer. The barrier layer may serve as a means to delay the release of a cell extractant from the coated substrate. Barrier layers include, for example, erodible coatings such as those known in the art to control and/or delay the release of active ingredients from tablet or capsule medications. Barrier layers also include layers that can be disintegrated by physical or mechanical manipulation (e.g., a wax layer that can disintegrate at increased temperatures).
In some embodiments, the encapsulating materials may be activated to release an effective amount of cell extractant after the encapsulant is exposed to an activating stimulus such as pressure, shear, heat, light, pH change, exposure to another chemical, ionic strength change and the like. Activation may result in, for example, dissolution or partial dissolution of the encapsulating material, permeabilization of the encapsulating material, and/or disintegration or partial disintegration of the encapsulating material (e.g., by fracturing or melting a solid material such as, for example microcrystalline wax).
In some embodiments, chemical cell extractants include biochemicals, such as proteins (e.g., cytolytic peptides and enzymes). In some embodiments, the cell extractant increases the permeability of the cell, causing the release of biological analytes from the interior of the cell. In some embodiments, the cell extractant can cause or facilitate the lysis (e.g., rupture or partial rupture) of a cell.
In some embodiments, cell extractants include chemicals and mixtures of chemicals that are known in the art and include, for example, surfactants and quaternary amines, biguanides, surfactants, phenolics, cytolytic peptides, and enzymes. Typically, the cell extractant is not avidly bound (either covalently or noncovalently) to the means for delaying the release of a cell extractant and can be released from the means when the means is contacted with an aqueous liquid.
Surfactants generally contain both a hydrophilic group and a hydrophobic group. The means for delaying the release of a cell extractant may contain one or more surfactants selected from anionic, nonionic, cationic, ampholytic, amphoteric and zwitterionic surfactants and mixtures thereof. A surfactant that dissociates in water and releases cation and anion is termed ionic. When present, ampholytic, amphoteric and zwitterionic surfactants are generally used in combination with one or more anionic and/or nonionic surfactants. Nonlimiting examples of suitable surfactants and quaternary amines include TRITON X-100, Nonidet P-40 (NP-40), Tergitol, Sarkosyl, Tween, SDS, Igepal, Saponin, CHAPSO, benzalkonium chloride, benzethonium chloride, ‘cetrimide’ (a mixture of dodecyl-, tetradecyl- and hexadecyl-trimethylammoium bromide), cetylpyridium chloride, (meth)acrylamidoalkyltrimethylammonium salts (e.g., 3-methacrylamidopropyltrimethylammonium chloride and 3-acrylamidopropyltrimethylammonium chloride) and (meth)acryloxyalkyltrimethylammonium salts (e.g., 2-acryloxyethyltrimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride, 3-acryloxy-2-hydroxypropyltrimethylammonium chloride, and 2-acryloxyethyltrimethylammonium methyl sulfate). Other suitable monomeric quaternary amino salts include a dimethylalkylammonium group with the alkyl group having 2 to 22 carbon atoms or 2 to 20 carbon atoms. That is, the monomer includes a group of formula —N(CH3)2(CnH2n+1)+ where n is an integer having a value of 2 to 22. Exemplary monomers include, but are not limited to monomers of the following formula
where n is an integer in the range of 2 to 22.
Non-limiting examples of suitable biguanides, which include bis-biguanides, include polyhexamethylene biguanide hydrochloride, p-chlorophenyl biguanide, 4-chloro-benzhydryl biguanide, alexidine, halogenated hexidine such as, but not limited to, chlorhexidine (1,1′-hexamethylene-bis-5-(4-chlorophenyl biguanide), and salts thereof.
Non-limiting examples of suitable phenolics include phenol, salicylic acid, 2-phenylphenol, 4-t-amylphenol, Chloroxylenol, Hexachlorophene, 4-chloro-3,5-dimethylphenol (PCMX), 2-benzyl-4-chlorophenol, triclosan, butylated hydroxytoluene, 2-Isopropyl-5-methyl phenol, 4-Nonylphenol, xylenol, bisphenol A, Orthophenyl phenol, and Phenothiazines, such as chlorpromazine, prochlorperazine and thioridizine.
Non-limiting examples of suitable cytolytic peptides include A-23187 (Calcium ionophore), Dermaseptin, Listerolysin, Ranalexin, Aerolysin, Dermatoxin, Maculatin, Ranateurin, Amphotericin B, Direct lytic factors from animal venoms, Magainin, Rugosin, Ascaphin, Diptheria toxin, Maxymin, Saponin, Aspergillus haemolysin, Distinctin, Melittin, Staphylococcus aureus toxins, (α, β, χ, ), Alamethicin, Esculetin, Metridiolysin, Streptolysin O, Apolipoproteins, Filipin, Nigericin, Streptolysin S, ATP Translocase, Gaegurin, Nystatin, Synexin, Bombinin, GALA, Ocellatin, Surfactin, Brevinin, Gramicidin, P25, Tubulin, Buforin, Helical erythrocyte lysing peptide, Palustrin, Valinomycin, Caerin, Hemolysins, Phospholipases, Vibriolysin, Cereolysin, Ionomycin, Phylloxin, Colicins, KALA, Polyene Antibiotics, Dermadistinctin, LAGA, Polymyxin B.
Non-limiting examples of suitable enzymes include lysozyme, lysostaphin, bacteriophage lysins, achromopeptidase, labiase, mutanolysin, streptolysin, tetanolysin, a-hemolysin, lyticase, lysing enzymes from fungi, cellulase, pectinase, Driselase®, Viscozyme® L, pectolyase.
Any other known cell extractants that are compatible with the precursor compositions or the resulting hydrogels can be used. These include, but are not limited to, chlorhexidine salts such as chlorhexidine gluconate (CHG), parachlorometaxylenol (PCMX), triclosan, hexachlorophene, fatty acid monoesters and monoethers of glycerin and propylene glycol such as glycerol monolaurate, Cetyl Trimethylammonium Bromide (CTAB), glycerol monocaprylate, glycerol monocaprate, propylene glycol monolaurate, propylene glycol monocaprylate, propylene glycol moncaprate, phenols, surfactants and polymers that include a (C12-C22) hydrophobe and a quaternary ammonium group or a protonated tertiary amino group, quaternary amino-containing compounds such as quaternary silanes and polyquaternary amines such as polyhexamethylene biguanide, transition metal ions such as copper containing compounds, zinc containing compounds, and silver containing compounds such as silver metal, silver salts such as silver chloride, silver oxide and silver sulfadiazine, methyl parabens, ethyl parabens, propyl parabens, butyl parabens, octenidene, 2-bromo-2-nitropropane-1,3 diol, or mixtures of any two or more of the foregoing.
Suitable cell extractants also include dialkyl ammonium salts, including N-(n-dodecyl)-diethanolamine; cationic ethoxylated amines, including ‘Genaminox K-10’, Genaminox K-12, ‘Genamin TCL030’, and ‘Genamin C100’; amidines, including propamidine and dibromopropamidine; peptide antibiotics, including polymyxin B and nisin; polyene antibiotics, including nystatin, amphotericin B, and natamycin; imidazoles, including econazole, clotramizole and miconazole; oxidizing agents, including stabilized forms of chlorine and iodine; and the cell extractants described in U.S. Pat. No. 7,422,868, which is incorporated herein by reference in its entirety.
Cell extractants are preferably chosen not to inactivate the detection system (e.g., a detection reagent such as luciferase enzyme) of the present invention. For microbes requiring harsher cell extractants (e.g., ionic detergents etc.), modified detection systems (such as luciferases exhibiting enhanced stability in the presence of these agents, such as those disclosed in U.S. Patent Application Publication No. 2003/0104507, which is hereby incorporated by reference in its entirety) are particularly preferred.
Methods of the present invention provide for the release of an effective amount of cell extractant from a release element to cause the release of biological analytes from a live cell. The present disclosure includes a variety of cell extractants known in the art and each of which may be released from the release element at a different rate and may exert its effect on living cells at a different concentration than the others. The following will provide guidance concerning the factors to be considered in selecting the cell extractant and the in determining an effective amount to include in the release element.
It is known in the art that the efficacy of any cell extractant is determined primarily by two factors—concentration and exposure time. That is, in general, the higher the concentration of a cell extractant, the greater the effect (e.g., permeabilization of the cell membrane and/or release of biological analytes from the cell) it will have on a living cell. Also, at any given concentration of cell extractant, in general, the longer you expose a living cell to the cell extractant, the greater the effect of the cell extractant. Other extrinsic factors such as, for example, pH, co-solvents, ionic strength, and temperature are known in the art to affect the efficacy of certain cell extractant. It is known that these extrinsic factors can be controlled by, for example, temperature controllers, buffers, sample preparation, and the like. These factors, as well as the cell extractant, can also have effects on the detection systems used to detect biological analytes. It is well within the grasp of a person of ordinary skill to perform a few simple experiments to determine an effective amount of cell extractant to produce the articles and perform the methods of the present disclosure. Further guidance is provided in the Examples described herein.
Initial experiments to determine the effect of various concentrations of the cell extractant on the cells and/or the detection system can be performed using an enzyme assay (e.g., an ATP assay as described in Example 6). Initially, a putative release element comprising a cell extractant can be screened for its effect on the biological analyte detection system. For example, the release element comprising a cell extractant can be placed into an ATP assay (without bacterial cells). The assay can be run with solutions of reagent-grade ATP (e.g. from about 0.1 to about 100 picomoles of ATP) and the amount of bioluminescence emitted by the luciferase reaction in the sample with the release element comprising a cell extractant can be compared to the amount of bioluminescence emitted by a sample without the release element comprising a cell extractant. Preferably, the amount of bioluminescence in the sample with the release element comprising a cell extractant is greater than 50% of the amount of bioluminescence in the sample without the release element comprising a cell extractant. More preferably, the amount of bioluminescence in the sample with the release element comprising a cell extractant is greater than 90% of the amount of bioluminescence in the sample without the release element comprising a cell extractant. Most preferably, the amount of bioluminescence in the sample with the release element comprising a cell extractant is greater than 95% of the amount bioluminescence in the sample without the release element comprising a cell extractant.
Additionally, the effect of the cell extractant on the release of the biological analyte from the cells can be determined experimentally, as described in Example 6. For example, liquid suspensions of cells (e.g., microbial cells such as Staphylococcus aureus) are exposed to relatively broad range of concentrations of a cell extractant (e.g., BARDAC 205M) for a period of time (e.g. up to several minutes) in the presence of a detection system to detect biological analytes from a cell (e.g., an ATP detection system comprising luciferin, luciferase, and a buffer at about pH 7.6 to 7.8). The biological analyte is measured periodically, with the first measurement usually performed immediately after the cell extractant is added to the mixture, to determine whether the release of the biological analyte (in this example, ATP) from the cells can be detected. The results can indicate the optimal conditions (i.e., liquid concentration of cell extractant and exposure time) to detect the biological analyte released from the cells. The results may also indicate that, at higher concentrations of cell extractant, the cell extractant may be less effective in releasing the biological analyte (e.g., ATP) and/or may interfere with the detection system (i.e., may absorb the light or color generated by the detection reagents).
After the effective amount of cell extractant in liquid mixtures is determined, consideration should be given to the amount of cell extractant to incorporate into the release element by the methods described herein. When the release element comprising a cell extractant forms a liquid mixture (e.g., a sample suspected of containing live cells in an aqueous suspension) the cell extractant diffuses out of the release element until a concentration equilibrium of the cell extractant, between the release element and the liquid, is reached. Without being bound by theory, it can be assumed that, until the equilibrium is reached, a concentration gradient of cell extractant will exist in the liquid, with a higher concentration of extractant present in the portion of the liquid proximal the release element. When the concentration of the cell extractant reaches an effective concentration in a portion of the liquid containing a cell, the cell releases biological analytes. The released biological analytes are thereby available for detection by a detection system.
Achieving an effective concentration of cell extractant in the liquid containing the sample can be controlled by several factors. For example, the amount of cell extractant loaded into the release element can affect final concentration of cell extractant in the liquid at equilibrium. Additionally, the amount of release element and, in some embodiments, the amount of surface area of the release element in the liquid mixture can affect the rate of release of the cell extractant from the release element and the final concentration of cell extractant in the liquid at equilibrium. Furthermore, the temperature of the aqueous medium can affect the rate at which the release element releases the cell extractant. Other factors, such as the ionic properties and or hydrophobic properties of the cell extractant and the release element may affect the amount of cell extractant released from the release element and the rate at which the cell extractant is released from the release element. All of these factors can be optimized with routine experimentation by a person of ordinary skill to achieve the desired parameters (e.g., manufacturing considerations for the articles and the time-to-result for the methods) for detection of cells in a sample. In general, it is desirable to incorporate at least enough cell extractant into the release element to achieve the effective amount (determined by the experimentation using the cell extractant without a release element) when the cell extractant reaches equilibrium between the release element and the volume of liquid comprising the sample material. It may be desirable to add a larger amount of cell extractant to the release element (than the amount determined by experimentation using the cell extractant without a release element) to reduce the amount of time it take for the release element to release an effective amount of cell extractant.
The release element can be contacted with the liquid sample material either statically, dynamically (i.e., with mixing by vibration, stirring, or aeration, for example), or a combination thereof. In certain embodiments wherein the release element comprises a hydrogel coating that includes the cell extractant, compressing the release element (e.g., pressing the composition against a surface and/or crushing the composition) can cause a faster release of an effective amount of cell extractant. Thus, in some embodiments, mixing can advantageously provide a faster release of cell extractant and thereby a faster detection of biological analytes (e.g., from live cells) in a sample. In some embodiments, compressing the release element (e.g., by exerting pressure against the composition using a sample acquisition device such as a swab or a spatula, a conveyor (described below) or some other suitable implement) can advantageously provide a faster release of cell extractant and thereby a faster detection of biological analytes in a sample. Additionally, the step of compressing the release element can be performed to accelerate the release of the cell extractant at a time that is convenient for the operator. In some embodiments, static contact can delay the release of an effective amount of cell extractant and thereby provide additional time for the operator to carry out other procedures (e.g., reagent additions, instrument calibration, and/or specimen transport) before detecting the biological analytes. In some embodiments, it may be advantageous to hold the mixture statically until a first biological analyte measurement is taken and then dynamically mix the sample to reduce the time necessary to release an effective amount of cell extractant.
It is fully anticipated that the most preferred concentration(s) or concentration range(s) functional in the methods of the invention will vary for different microbes and for different cell extractants and may be empirically determined using the methods described herein or commonly known to those skilled in the art.
Articles and methods of the present disclosure provide for the detection of biological analytes in a sample. In some embodiments, the articles and methods provide for the detection of biological analytes from live cells in a sample. In certain preferred embodiments, the articles and methods provide for the detection of live microbial cells in a sample. In certain preferred embodiments, the articles and methods provide for the detection of live bacterial cells in a sample.
The term “sample” as used herein, is used in its broadest sense. A sample is a composition suspected of containing a biological analyte (e.g., ATP) that is analyzed using the invention. While often a sample is known to contain or suspected of containing a cell or a population of cells, optionally in a growth media, or a cell lysate, a sample may also be a solid surface, (e.g., a swab, membrane, filter, particle), suspected of containing an attached cell or population of cells. It is contemplated that for such a solid sample, an aqueous sample is made by contacting the solid with a liquid (e.g., an aqueous solution) which can be mixed with hydrogels of the present disclosure. Filtration of the sample is desirable in some cases to generate a sample, e.g., in testing a liquid or gaseous sample by a process of the invention. Filtration is preferred when a sample is taken from a large volume of a dilute gas or liquid. The filtrate can be contacted with hydrogels of the present disclosure, for example after the filtrate has been suspended in a liquid.
Suitable samples include samples of solid materials (e.g., particulates, filters), semisolid materials (e.g., a gel, a liquid suspension of solids, or a slurry), a liquid, or combinations thereof. Suitable samples further include surface residues comprising solids, liquids, or combinations thereof. Non-limiting examples of surface residues include residues from environmental surfaces (e.g., floors, walls, ceilings, fomites, equipment, water, and water containers, air filters), food surfaces (e.g., vegetable, fruit, and meat surfaces), food processing surfaces (e.g., food processing equipment and cutting boards), and clinical surfaces (e.g., tissue samples, skin and mucous membranes).
The collection of sample materials, including surface residues, for the detection of biological analytes is known in the art. Various sample acquisition devices, including spatulas, sponges, swabs and the like have been described. The present disclosure provides sample acquisition devices with unique features and utility, as described herein.
Turning now to the Figures,
The sample acquisition device 130 further comprises an elongated shaft 134 and a tip 139. In some embodiments, the shaft 134 can be hollow. The shaft 134 comprises a tip 139, positioned near the end of the shaft 134 opposite the handle 131. The tip 139 can be used to collect sample material and can be constructed from porous materials, such as fibers (e.g., rayon or Dacron fibers) or foams (e.g., polyurethane foam) which can be affixed to the shaft 134. In some embodiments, the tip 139 can be a molded tip as described in U.S. Patent Application No. 61/029,063, filed Dec. 5, 2007 and entitled, “SAMPLE ACQUISITION DEVICE”, which is incorporated herein by reference in its entirety. The construction of sample acquisition devices 130 is known in the art and can be found, for example, in U.S. Pat. No. 5,266,266, which is incorporated herein by reference in their entirety.
Optionally, the sample acquisition device 130 can further comprise a release element 140 comprising a cell extractant. In some embodiments, the release element 140 is positioned in or on the sample acquisition device 130 at a location other than the tip 139 that is used to collect the sample (e.g., on the shaft 134, as shown in
In use, the tip 139 of a sample acquisition device 130 is contacted with a sample material (e.g., a solid, a semisolid, a liquid suspension, a slurry, a liquid, a surface, and the like) to obtain a sample suspected of containing cells. The sample acquisition device 130 can be used to transfer the sample to a detection system as described herein.
In use, sample acquisition device 230 can be used to contact surfaces, preferably dry surfaces, to obtain sample material. After the sample is obtained, the tip 239 of the sample acquisition device 230 is moistened with a liquid (e.g. water or a buffer; optionally, including a detection reagent such as an enzyme and/or an enzyme substrate), thereby permitting an effective amount of the cell extractant to be released from the release element 240 and to contact the sample material. The release of an effective amount of cell extractant from release element 240 permits the sample acquisition device 230 to be used in methods to detect biological analytes from live cells as described herein.
Another embodiment (not shown) of a sample acquisition device including a release element comprising a cell extractant can be derived from the “Specimen Test Unit” disclosed by Nason in U.S. Pat. No. 5,266,266 (hereinafter, referred to as the “Nason patent”). In particular, referring to FIGS. 7-9 of the Nason patent, the handle of the sample acquisition devices described herein can be modified to embody Nason's functional elements of the housing base 14 (which forms reagent chamber 36) and the seal fitting 48, which includes central dispense passage 50 (optional, with housing cap 30) connected to the hollow swab shaft 22. The central passage 50 of the seal fitting 48 can be closed by a break-off nib 52 in the form of an extended rod segment 54 connected to the seal fitting 48 at the inboard end of the passage 50 via a reduced diameter score 56. Thus, in one embodiment of the present disclosure, the sample acquisition device handle comprises a reagent chamber, as described by Nason. The reagent chamber located in the handle of the sample acquisition device of this embodiment includes release elements (e.g., coated substrates) comprising a cell extractant. Thus, the sample acquisition device of this embodiment provides an enclosure (reagent chamber 36) containing the release element. In this embodiment, the release element particles are not suspended in a liquid medium that causes the release of the cell extractant from the composition. The release element particles are proportioned and shaped to allow free passage of the individual particles into and through the central passage 50 and the hollow shaft 22.
In use, the sample acquisition device comprising a handle including a reagent chamber can be used to obtain a sample as described herein. If the sample is a liquid, the break-off nib 52 can be actuated, as described in the Nason patent, enabling the passage of the release element through the shaft to contact the liquid sample in the swab tip, thereby forming a liquid mixture comprising the sample and the composition. The liquid mixture comprising the sample and the release element can be used for the detection of a biological analyte associated with a live cell, as described herein. If the sample is a solid or semi-solid, the tip of the sample acquisition device can be contacted or submersed in a liquid solution and the break-off nib 52 can be actuated, as described in the Nason patent, enabling the passage of the release element through the shaft to contact the liquid sample in the swab tip, thereby forming a liquid mixture comprising the sample and the composition. The liquid mixture comprising the sample and the release element can be used for the detection of a biological analyte associated with a live cell, as described herein.
In
It should be recognized that in this and all other embodiments (for example, the illustrated embodiments of
The wall 324 of the housing 320 can be cylindrical, for example. It will be appreciated that other useful geometries, some including a plurality of walls 324, are possible and within the grasp of one of ordinary skill in the appropriate art. The housing 320 can be constructed from a variety of materials such as plastic (e.g., polypropylene, polyethylene, polycarbonate) or glass. Preferably, at least a portion of the housing 320 is constructed from materials that have optical properties that allow the transmission of light (e.g., visible light). Suitable materials are well known in devices used for biochemical assays such as ATP tests, for example.
Optionally, housing 320 can comprise a cap (not shown) that can be shaped and dimensioned to cover the opening 322 of the housing 320. It should be recognized that other housings (for example, housings 420 and 520 as shown in
In some embodiments, the housing 320 can be used in conjunction with a sample acquisition device (not shown). Optionally, the sample acquisition device may comprise a release element, such as, for example, sample acquisition devices 130 or 230 shown in
The housing 320 can be used in methods to detect live cells in a sample. During use, the operator can form a liquid (e.g., an aqueous liquid or aqueous solutions containing glycols and/or alcohols) mixture in the housing 320, the mixture comprising a liquid sample and the release element 340 comprising a cell extractant. In some embodiments, the mixture can further comprise a detection reagent. The liquid mixture comprising the sample and the release element 440 comprising a hydrogel can be used for the detection of a biological analyte associated with a live microorganism.
The frangible seal 460 forms a barrier between the upper compartment 426 (which includes the opening 422 of the housing 420) and the reaction well 428. In some embodiments, the frangible seal 460 forms a water-resistant barrier. The frangible seal 460 can be constructed from a variety of frangible materials including, for example polymer films, metal-coated polymer films, metal foils, dissolvable films (e.g., films made of low molecular weight polyvinyl alcohol or hydroxypropyl cellulose (HPC) and combinations thereof.
Frangible seal 460 may be connected to the wall 424 of the housing 420 using a variety of techniques. Suitable techniques for attaching a frangible seal 460 to a wall 424 include, but are not limited to, ultrasonic welding, any thermal bonding technique (e.g., heat and/or pressure applied to melt a portion of the wall 424, the frangible seal 460, or both), adhesive bonding, stapling, and stitching. In one desired embodiment of the present invention, the frangible seal 460 is attached to the wall 424 using an ultrasonic welding process.
The housing 420 can be used in methods to detect cells in a sample. Methods of the present disclosure include the formation of a liquid mixture comprising the sample material and the release element 440 comprising a cell extractant and include the detection of a biological analyte, as described herein.
If the sample is a liquid sample (e.g., water, juice, milk, meat juice, vegetable wash, food extracts, body fluids and secretions, saliva, wound exudate, and blood), the liquid sample can be transferred (e.g., poured pipetted, or released from a sample acquisition device) directly into the upper compartment 426. A detection reagent can be added to the sample before the sample is transferred to the housing 420. A detection reagent can be added to the sample after the sample is transferred to the housing 420. A detection reagent can be added to the sample while the sample is transferred to the housing 420. The frangible seal 460 can be ruptured (e.g., by piercing it with a pipette tip or a sample acquisition device) before the liquid sample is transferred to the housing 420. The frangible seal 460 can be ruptured after the liquid sample is transferred to the housing 420. The frangible seal 460 can be ruptured while the liquid sample is transferred to the housing 420. When the liquid sample is in the housing 420 and the frangible seal is ruptured, a liquid mixture comprising the sample and the release element 440 comprising a cell extractant is formed. The liquid mixture comprising the sample and the release element 440 can be used for the detection of a biological analyte associated with a live microorganism.
If the sample is a solid sample (e.g., powder, particulates, semi-solids, residue collected on a sample acquisition device, air filter), the housing 420 can advantageously be used as a vessel in which the sample can be mixed with a liquid suspending medium such as, for example, water or a buffer. Preferably, the liquid suspending medium is substantially free of microorganisms. More preferably, the liquid suspending medium is sterile. Before, after or during the process of mixing the solid sample with the liquid suspending medium, a detection reagent can be added to the liquid suspending medium. Either before, after, or during the process of mixing the solid sample with the liquid suspending medium, the frangible seal 460 can be ruptured (e.g., by piercing with a pipette tip or a swab), thus forming a liquid mixture comprising the sample and the release element 440 comprising a cell extractant. The liquid mixture comprising the sample and the release element 440 can be used in a method for the detection of a biological analyte associated with a live cell.
In
The reagent well 528 of housing 520 comprises a detection reagent 570. Optionally, the detection reagent 570 can comprise a detection reagent (i.e., a detection reagent may be dissolved and/or suspended in the detection reagent 570). In other embodiments (not shown), the reagent well 528 can comprise a dry detection reagent (e.g., a powder, particles, microparticles, a tablet, a pellet, and the like) instead of the detection reagent 570.
The housing 520 can be used in methods to detect cells in a sample. Methods of the present disclosure include the formation of a liquid mixture comprising the sample material and the release element 440 comprising a cell extractant and include the detection of a biological analyte, as described herein.
If the sample is a liquid sample (e.g., water, juice, milk, meat juice, vegetable wash, food extracts, body fluids and secretions, saliva, wound exudate, and blood), the liquid sample can be transferred (e.g., poured, pipetted, or released from a sample acquisition device) directly into the upper compartment 526, thus forming a liquid mixture comprising the sample and the release element 540 comprising a cell extractant. Before, after or during the transfer of the sample into the housing 520, a detection reagent can be added to the liquid sample. Before, after, or during the transfer of the liquid sample to the housing 520, the frangible seal 560 can be ruptured (e.g., by piercing with a pipette tip or a swab). The liquid mixture comprising the sample and the release element 540 can be used for the detection of a biological analyte associated with a live microorganism before and/or after the frangible seal 560 is ruptured.
If the sample is a solid sample (e.g., powder, particulates, semi-solids, residue collected on a sample acquisition device), the housing 520 can advantageously be used as a vessel in which the sample can be mixed with a liquid suspending medium such as, for example, water or a buffer. Preferably, the liquid suspending medium is substantially free of microorganisms. More preferably, the liquid suspending medium is sterile.
Mixing the solid sample with a liquid suspending medium forms a liquid mixture comprising the sample and the release element 540 comprising a cell extractant. Before, after or during the process of mixing the solid sample with the liquid suspending medium, a detection reagent can be added to the liquid suspending medium. Before, after, or during the process of mixing the solid sample with the liquid suspending medium, the frangible seal 560 can be ruptured (e.g., by piercing with a pipette tip or a swab). The liquid mixture comprising the sample and the release element 540 can be used for the detection of a biological analyte associated with a live microorganism, as described herein.
The sample acquisition device 630 comprises a handle 631 which can be grasped by the operator while collecting a sample. The sample acquisition device 630 is shown in
In use, the tip 739 of a sample acquisition device 730 is contacted with a sample material (e.g., a solid, a semisolid, a liquid suspension, a slurry, a liquid, a surface, and the like), as described above. After collecting the sample, the sample acquisition device 730 is reinserted into the housing 720 and the handle is urged into the housing 720, as described above, thereby causing the tip 739 to pass through frangible seals 760a and 760b and into the detection reagent in the reaction well 728. As the tip 739 passes through frangible seals 760a and 760b, the release element 740 is also moved into the detection reagent 770 in the reaction well 728. This process forms a liquid mixture that includes a sample and a release element 740 comprising a cell extractant. The liquid mixture comprising the sample and the cell extractant can be used for the detection of a biological analyte associated with a live microorganism, as described herein.
In use, the sample acquisition device 830 is removed from the detection device 810 and a sample is collected as described herein on the tip 839. The sample acquisition device 830 is reinserted into the housing 820 and the handle 831 is urged into the housing 820, as described for the detection device in
Devices of the present disclosure may include a detection system. In some embodiments, the detection system comprises a detection reagent, such as an enzyme or an enzyme substrate. In certain embodiments, the detection reagent can be used for detecting ATP. The detection reagent may be loaded into a delivery element. Such delivery elements can be used conveniently to store and/or deliver the detection reagent to a liquid mixture, comprising a sample and a cell extractant, for the detection of live cells in the sample.
Delivery elements, as used herein, include encapsulating agents, matrices, shell structures with a core, and coated substrates, as described herein. A detection reagent comprising a protein, such as an enzyme or an antibody, can be incorporated into the delivery element using the similar processes as those described for the incorporation of cell extractants into a release element. For example, luciferase can be incorporated into a delivery element during the synthesis of a polymer matrix, as described in Preparative Examples 4 and 5 below.
An enzyme substrate can be incorporated into a delivery element during the synthesis of the delivery element. For example, luciferin can be incorporated into a delivery element during the synthesis of a polymer matrix delivery element, as described in Preparative Examples 2 and 3 below.
Although proteins may be incorporated into a delivery element (e.g., a hydrogel) during the synthesis of the delivery element, chemicals and or processes (e.g., u.v. curing processes) used in the synthesis process (e.g., polymerization) can potentially cause the loss of some biological activity by certain proteins (e.g. certain enzymes or binding proteins such as antibodies). In contrast, incorporation (e.g., by diffusion) of a detection reagent protein into the delivery element after synthesis of the delivery element can lead to improved retention of the protein's biological activity.
In some applications, it may be desirable that the delivery element containing a detection reagent is in a dry or partially-dried state. Certain delivery elements (e.g., swollen hydrogels) can be dried, for example, by methods known to those skilled in the art, including evaporative processes, drying in convection ovens, microwave ovens, and vacuum ovens as well as freeze-drying. When the dried delivery element is exposed to a liquid or aqueous solution, the detection reagent can diffuse out of the delivery element. The detection reagent can remain essentially dormant in the delivery element until exposed to a liquid or aqueous solution. That is, the detection reagent can be stored within the dry or partially-dried delivery element until the element is exposed to a liquid. This can prevent the waste or loss of the detection reagent when not needed and may improve the stability of moisture sensitive detection reagents that may degrade by hydrolysis, oxidation, or other mechanisms.
Methods of the present disclosure include methods for the detection of biological analytes that are released from live cells including, for example, live microorganisms, after exposure to an effective amount of cell extractant.
Methods of the present disclosure allow an operator instantaneously to form a liquid mixture containing a sample and a release element comprising a cell extractant. In some embodiments, the methods provide for the operator to, within a predetermined period of time after the liquid mixture is formed, measure the amount of a biological analyte in the mixture to determine the amount of acellular biological analyte in the sample. Advantageously, in some embodiments, the release of the cell extractant from the release element is triggered by a release factor and/or a process step causing the release of the cell extractant. Non-limiting examples of a release factor causing the release of the cell extractant include a base, an acid, and an enzyme or a chemical (e.g., a metal or salt ion) to solublize the release element. Factors can also include mechanically disrupting (e.g., compressing or crushing) the release element, and thermally disrupting (e.g., freezing, freeze-thawing, or melting) the release element.
In some embodiments, the methods provide for the operator to, after a predetermined period of time during which an effective amount of cell extractant is released from the release element into the liquid mixture, measure the amount of a biological analyte to determine the amount of biological analyte from acellular material and live cells in the sample. In some embodiments, the methods provide for the operator, within a first predetermined period of time, to perform a first measurement of the amount of a biological analyte and, within a second predetermined period of time during which an effective amount of cell extractant is released from the release element, perform a second measurement of the amount of biological analyte to detect the presence of live cells in the sample. In some embodiments, the methods can allow the operator to distinguish whether biological analyte in the sample was released from live plant or animal cells or whether it was released from live microbial cells (e.g., bacteria). The present invention is capable of use by operators under the relatively harsh field environment of institutional food preparation services, health care environments and the like.
The detection of the biological analytes involves the use of a detection system. Detection systems for certain biological analytes such as a nucleotide (e.g., ATP), a polynucleotide (e.g., DNA or RNA) or an enzyme (e.g., NADH dehydrogenase or adenylate kinase) are known in the art and can be used according to the present disclosure. Methods of the present disclosure include known detections systems for detecting a biological analyte. Preferably, the accuracy and sensitivity of the detection system is not significantly reduced by the cell extractant. More preferably, the detection system comprises a homogeneous assay.
In some embodiments, the detection system comprises a detection reagent. Detection reagents include, for example, dyes, enzymes, enzyme substrates, binding partners (e.g., an antibody, a monoclonal antibody, a lectin, a receptor), and/or cofactors. In some embodiments, the detection system comprises an instrument. Nonlimiting examples of detection instruments include a spectrophotometer, a luminometer, a plate reader, a thermocycler, an incubator.
Detection systems are known in the art and can be used to detect biological analytes colorimetrically (i.e., by the absorbance and/or scattering of light), fluorescently, or lumimetrically. Examples of the detection of biomolecules by luminescence are described by F. Gorus and E. Schram (Applications of bio- and chemiluminescence in the clinical laboratory, 1979, Clin. Chem. 25:512-519).
An example of a biological analyte detection system is an ATP detection system. The ATP detection system can comprise an enzyme (e.g., luciferase) and an enzyme substrate (e.g., luciferin). The ATP detection system can further comprise a luminometer. In some embodiments, the luminometer can comprise a bench top luminometer, such as the FB-12 single tube luminometer (Berthold Detection Systems USA, Oak Ridge, Tenn.). In some embodiments, the luminometer can comprise a handheld luminometer, such as the NG Luminometer, UNG2 (3M Company, Bridgend, U.K.).
Methods of the present disclosure include the formation of a liquid mixture comprising a sample suspected of containing live cells and a release element comprising a cell extractant. Methods of the present disclosure further include detecting a biological analyte. Detecting a biological analyte can further comprise quantitating the amount of biological analyte in the sample.
In some embodiments, detecting the biological analyte can comprise detecting the analyte directly in a vessel (e.g., a tube, a multi-well plate, and the like) in which the liquid mixture comprising the sample and the release element comprising a cell extractant is formed. In some embodiments, detecting the biological analyte can comprise transferring at least a portion of the liquid mixture to a container other than the vessel in which the liquid mixture comprising the sample and the release element comprising a cell extractant is formed. In some embodiments, detecting the biological analyte may comprise one or more sample preparation processes, such as pH adjustment, dilution, filtration, centrifugation, extraction, and the like.
In some embodiments, the biological analyte is detected at a single time point. In some embodiments, the biological analyte is detected at two or more time points. When the biological analyte is detected at two or more time points, the amount of biological analyte detected at a first time (e.g., before an effective amount of cell extractant is released from a release element to effect the release of biological analytes from live cells in at least a portion of the sample) point can be compared to the amount of biological analyte detected at a second time point (e.g., after an effective amount of cell extractant is released from a release element to effect the release of biological analytes from live cells in at least a portion of the sample). In some embodiments, the measurement of the biological analyte at one or more time points is performed by an instrument with a processor. In certain preferred embodiments, comparing the amount of biological analyte at a first time point with the amount of biological analyte at a second time point is performed by the processor.
For example, the operator measures the amount of biological analyte in the sample after the liquid mixture including the sample and the release element comprising a cell extractant is formed. The amount of biological analyte in this first measurement (T0) can indicate the presence of “free” (i.e. acellular) biological analyte and/or biological analyte from nonviable cells in the sample. In some embodiments, the first measurement can be made immediately (e.g., about 1 second) after the liquid mixture including the sample and the release element comprising a cell extractant is formed. In some embodiments, the first measurement can be at least about 5 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 60 seconds, at least about 80 seconds, at least about 100 seconds, at least about 120 seconds, at least about 150 seconds, at least about 180 seconds, at least about 240 seconds, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes after the liquid mixture including the sample and the release element comprising a cell extractant is formed. These times are exemplary and include only the time up to that the detection of a biological analyte is initiated. Initiating the detection of a biological analyte may include diluting the sample and/or adding a reagent to inhibit the activity of the cell extractant. It will be recognized that certain detection systems (e.g., nucleic acid amplification or ELISA) can generally take several minutes to several hours to complete.
The operator allows the sample to contact the release element comprising the cell extractant for a period of time after the first measurement of biological analyte has been made. After the sample has contacted the release element for a period of time, a second measurement of the biological analyte is made. In some embodiments, the second measurement can be made up to about 0.5 seconds, up to about 1 second, up to about 5 seconds, up to about 10 seconds, up to about 20 seconds, up to about 30 seconds, up to about 40 seconds, up to about 60 seconds, up to about 90 seconds, up to about 120 seconds, up to about 180 seconds, about 300 seconds, at least about 10 minutes, at least about 20 minutes, at least about 60 minutes or longer after the first measurement of the biological analyte. These times are exemplary and include only the interval of time from which the first measurement for detecting the biological analyte is initiated and the time at which the second measurement for detecting the biological analyte is initiated. Initiating the detection of a biological analyte may include diluting the sample and/or adding a reagent to inhibit the activity of the cell extractant.
Preferably, the first measurement of a biological analyte is made about 1 seconds to about 240 seconds after the liquid mixture including the sample and the release element comprising a cell extractant is formed and the second measurement, which is made after the first measurement, is made about 1.5 seconds to about 540 seconds after the liquid mixture is formed. More preferably, the first measurement of a biological analyte is made about 1 second to about 180 seconds after the liquid mixture is formed and the second measurement, which is made after the first measurement, is made about 1.5 seconds to about 120 seconds after the liquid mixture is formed. Most preferably, the first measurement of a biological analyte is made about 1 second to about 5 seconds after the liquid mixture is formed and the second measurement, which is made after the first measurement, is made about 1.5 seconds to about 10 seconds after the liquid mixture is formed.
The operator compares the amount of a biological analyte detected in the first measurement to the amount of biological analyte detected in the second measurement. An increase in the amount of biological analyte detected in the second measurement is indicative of the presence of one or more live cells in the sample.
In certain methods, it may be desirable to detect the presence of live somatic cells (e.g., nonmicrobial cells). In these embodiments, the release element comprises a cell extractant that selectively releases biological analytes from somatic cells. Nonlimiting examples of somatic cell extractants include nonionic detergents, such as non-ionic ethoxylated alkylphenols, including but not limited to the ethoxylated octylphenol Triton X-100 (TX-100) and other ethoxylated alkylphenols; betaine detergents, such as carboxypropylbetaine (CB-18), NP-40, TWEEN, Tergitol, Igepal, commercially available M-NRS (Celsis, Chicago, Ill.), M-PER (Pierce, Rockford, Ill.), CelLytic M (Sigma Aldrich). Cell extractants are preferably chosen not to inactivate the analyte and its detection reagents.
In certain methods, it may be desirable to detect the presence of live microbial cells. In these embodiments, the release element can comprise a cell extractant that selectively releases biological analytes from microbial cells. Nonlimiting examples of microbial cell extractants include quaternary ammonium compounds, including benzalkonium chloride, benzethonium chloride, ‘cetrimide’ (a mixture of dodecyl-, tetradecyl- and hexadecyl-trimethylammoium bromide), cetylpyridium chloride; amines, such as triethylamine (TEA) and triethanolamine (TeolA); bis-Biguanides, including chlorhexidine, alexidine and polyhexamethylene biguanide Dialkyl ammonium salts, including N-(n-dodecyl)-diethanolamine, antibiotics, such as polymyxin B (e.g., polymyxin B1 and polymyxin B2), polymyxin-beta-nonapeptide (PMBN); alkylglucoside or alkylthioglucoside, such as Octyl-β-D-1-thioglucopyranoside (see U.S. Pat. No. 6,174,704 herein incorporated by reference in its entirety); nonionic detergents, such as non-ionic ethoxylated alkylphenols, including but not limited to the ethoxylated octylphenol Triton X-100 (TX-100) and other ethoxylated alkylphenols; betaine detergents, such as carboxypropylbetaine (CB-18); and cationic, antibacterial, pore forming, membrane-active, and/or cell wall-active polymers, such as polylysine, nisin, magainin, melittin, phopholipase A2, phospholipase A2 activating peptide (PLAP); bacteriophage; and the like. See e.g., Morbe et al., Microbiol. Res. (1997) vol. 152, pp. 385-394, and U.S. Pat. No. 4,303,752 disclosing ionic surface active compounds which are incorporated herein by reference in their entirety. Cell extractants are preferably chosen not to inactivate the biological analyte and/or a detection reagent used to detect the biological analyte.
In certain alternative methods to detect the presence of live microbial cells in a sample, the sample can be pretreated with a somatic cell extractant for a period of time (e.g., the sample is contacted with a somatic cell extractant for a sufficient period of time to extract somatic cells before a liquid mixture including the sample and a release element comprising a microbial cell extractant is formed). In the alternative embodiment, the amount of biological analyte detected at the first measurement will include any biological analyte that was released by the somatic cells and the amount of additional biological analyte, if any, detected in the second measurement will include biological analyte from live microbial cells in the sample.
The present invention has now been described with reference to several specific embodiments foreseen by the inventor for which enabling descriptions are available. Insubstantial modifications of the invention, including modifications not presently foreseen, may nonetheless constitute equivalents thereto. Thus, the scope of the present invention should not be limited by the details and structures described herein, but rather solely by the following claims, and equivalents thereto.
Hydrogel beads containing luciferin were made similarly by mixing 20 parts of EO20-TMPTA with 30 parts of luciferin (2 mg in 30 ml of 14 mM of phosphate buffer, pH 6.4) and 0.4 parts photoinitiator (IRGACURE 2959) and exposed to UV light to prepare beads as described in example 1 in International Patent Publication No. WO 2007/146722 A1. The beads were then stored in a jar at 4° C. and designated as Luciferin-1s.
Hydrogel beads (1× gram) were dried at 60° C. for 2 h and dipped in 2× grams of luciferin solution (2 mg in 30 ml of 14 mM of phosphate buffer, pH6.4) for at least 16 h at 4° C. After soaking, the beads were poured into a Buchner funnel to drain the beads and then rinsed with distilled water. The excess water was removed from the surface of the beads by blotting them with a paper towel. The beads were then stored in a jar at 4° C. and designated as Lucifein-1p.
Hydrogel beads containing luciferase were made by mixing 20 parts of polymer with 30 parts of luciferase (150 μl of 6.8 mg/ml in 30 ml of 14 mM of phosphate buffer, pH 6.4) and 0.4 parts photoinitiator (IRGACURE 2959) and exposed to UV light to prepare beads as described in example 1 in International Patent Publication No. WO 2007/146722 A1. The beads were then stored in a jar at 4° C. and designated as Luciferase-1s.
A luciferase working solution was prepared by adding 150 μl of 6.8 mg/ml luciferase stock solution to 30 ml of 14 mM phosphate buffer, pH6.4. A lysozyme working solution was prepared by adding the enzyme to 50 mM TRIS buffer (pH 8.0) to a final concentration of 0.5 mg/mL. A lysostaphin working solution was prepared by adding the enzyme to 50 mM TRIS buffer (pH 8.0) to a final concentration of 0.05 mg/mL. Hydrogel beads were dried at 60° C. for 2 h. One-gram aliquots of the dried beads were dipped into 2 milliliters of one of the working solutions (luciferase, lysozyme, or lysostaphin) described above. The beads were allowed to soak in the solution for at least 16 h at 4° C. After the beads were saturated with the solution, the beads were poured into a Buchner funnel to drain the beads and then rinsed with distilled water. The excess water was removed from the surface of the beads by blotting them with a paper towel. The beads were then stored in a jar at 4° C. and designated as Luciferase-1p, Lysozyme-1p and Lysostaphin-1p.
Microtablets were formed from a mixture containing luciferase and luciferin, sorbitol (Sigma-Aldrich), leucine and Cab-O-Sil (Table 2) using a hand operated Arbor Press. Twenty ml of UltraGlo luciferase (9 mg/lit, Promega, Madison, Wis.) and luciferin (0.05 mg/lit, Promega)) in 16 mM ADA (N-(2-Acetamido) Iminodiacetic Acid; N—(Carbamoylmethyl) Iminodiacetic Acid) buffer and 20 ml of luciferase (7.8 mg/lit, 3M Bridgend, UK) and luciferin (5.5 mg/lit, Promega) in 14 mM in Phosphate buffer were lyophilized.
The lyophilized enzyme mixture was placed in a mortar and ground with a pestle and added to a scintillation vial. Pre-ball milled sorbitol (sieved to <300 μm) was added to the glass scintillation vial and the formulation was vortexed for 2 minutes. Later Cab-O-Sil was added and vortexed for 2 minutes. L-Leucine (jet-milled to <10 μm) was weighed out and added to the vial and vortexed for 2 minutes to provide a well mixed powder exhibiting substantially uniform distribution of the reagents. The resulting mixture was formed into microtablets using a single leverage lab Arbor Press fitted with a custom made 3 mm diameter stainless steel punch and die set equipped with spacers for adjusting fill volume. The Arbor Press was operated using an electronic torque wrench. The fill volume was adjusted to obtain a compressed microtablet weight of 20 or 30 milligrams. The microtablets were compressed at a pressure of 155 MPa.
Polyvinyl alcohols (PVOH 26-88 and PVOH 403) were obtained from Kuraray America; Houston, Tex. A 3% polyvinyl alcohol solution (1.5% of PVOH-26-88 and 1.5% of PVOH 403) was prepared in deionized water and the solution was agitated on a shaker in a warm bath for 24 hours to allow the PVOH to fully dissolve. An antimicrobial film forming solution containing 0.5% carboquat (Lonza; Basel, Switzerland), 0.5% VANTOCIL (Arch Chemicals; Norwalk, Conn.) and 0.5% GLUCOPON 425N (Cognis; Monheim, Del.) was made in the 3% PVOH solution. Polyethylene terephthalate (PET) films were coated with the antimicrobial solution using a Meyer rod #6. The coating was allowed to dry on the substrate at room temperature and the dried films were stored at room temperature. The coated film, dried film was die-cut into circular disks having approximately 7 mm diameter. Negative controls disks were prepared by coating PET film with a 3% PVOH solution containing no cell extractant, drying the coated film, and die-cutting the coated film into 7 mm disks.
Various matrices were dipped in the extractant solution containing polyvinyl alcohol, VANTOCIL and CARBOSHIELD (as made in Preparative Example 6) or 5% benzalkonium chloride solution (Alfa Aesar). The matrices were removed and dried at 70° C. for about an hour and stored at room temperature. The matrices used were Grade 54, 22 μm Quantitative Filter Paper, Grade 4, 20-25 μm Qualitative Filter Paper, Grade 30, Glass-Fiber Filter Paper, Grade GB005, a thick (1.5 mm) highly absorbent blotting paper (all obtained from Whatman, Inc, Florham Park, N.J.), Zeta Plus Virosorb 1MDS discs (CUNO, Inc, Meriden, Conn.) and 0.45 μm MF-Millipore membrane (Millipore, Billerica, Mass.)
S. aureus ATCC 6538 and E. coli ATCC 51183 were obtained from the American Type Culture Collection (Manassas, Va.). 3M™ Clean-Trace™ Surface ATP system swabs were obtained from 3M Company (St. Paul, Minn.). A bench top luminometer (20/20n single tube luminometer) with 20/20n SIS software was obtained from Turner Biosystems (Sunnyvale, Calif.). Cell extractant-loaded disks and negative control disks were prepared as described in Preparative Example 1.
Reagent (300 μl) from Clean Trace ATP system was removed and added to 1.5 ml microfuge tube. Pure cultures of the bacterial strains were inoculated into tryptic soy broth and were grown overnight at 37° C. The bacteria were diluted in Butterfield's diluent to obtain suspensions containing approximately 107 and 108 colony-forming units (CFU) per milliliter, respectively. Ten microliter aliquots of the diluted suspensions were added to separate microfuge tubes containing the ATP detection reagent.
Immediately after adding the bacterial suspension, the microfuge tube was placed into the luminometer and an initial (T0) measurement of RLUs was recorded. The initial measurement and all subsequent luminescence measurements were obtained from the luminometer using the 20/20n SIS software. The light signal was integrated for 1 second and all results are expressed in RLU/sec.
After taking T0 measurement, a disk containing cell extractant was added to the tube and RLU measurements were recorded at 10 sec intervals until the number of RLUs reached a plateau (Table 2). The film containing cell extractants cause the release of ATP from the S. aureus and E. coli and the ATP reacted with the ATP-detection reagents to elicit bioluminescence. Tubes receiving the extractant-loaded films showed a relatively large increase in RLU during the observation period. The magnitude of the increase was related to the number of bacteria inoculated into the tube. In contrast, tubes receiving the negative control film showed a relatively small increase in RLU during the observation period.
S. aureus
E. coli
Cell extractant-loaded matrices were prepared as described in Preparative Example 8. S. aureus and E. coli overnight cultures were prepared as described in Example 1. Reagent (600 μl) from Clean Trace ATP system was removed and added to 1.5 ml microfuge tube. About 106 cfu of bacteria in Butterfield's buffer (10 μl) were added directly to the microfuge tube. Immediately after adding the bacterial suspension, a disk (ca. 7 mm) of various matrices containing cell extractant was added to the tube. The tube was immediately placed into a bench-top luminometer (20/20n single tube luminometer) and RLU measurements were recorded at 10 sec intervals until the number of RLUs reached a plateau (Tables 9-12). All luminescence measurements were obtained from the luminometer using 20/20n SIS software. The light signal was integrated for 1 second and the results are expressed in RLU/sec.
All the matrices, except grade 54 filter paper and grade 30 glass-fiber filter paper, coated with VANTOCIL_CARBOSHIELD solution with a binder showed a gradual release of ATP as the RLU increased over time. In case of matrices coated with 5% benzalkonium chloride, only few matrices (grade 4, grade 54 and GB005 blotting paper) showed some gradual release of ATP.
Microfuge tubes were set up containing 100 μl of PBS, 10 μl of 1 μM ATP and 1 μl of 6.8 mg/ml luciferase. Background reading was taken in a bench top luminometer (20/20n single tube luminometer with software), as described in Example 1, and hydrogel beads containing luciferin were added to the tube and reading was followed at 10 sec interval. The post-absorbed (1s) beads were more active than the preparative (1p) beads (Table 7).
Hydrogel Beads Containing Luciferase were Made Either Using Direct Method (Preparative Example 4) or by Post-Absorption (Preparative Example 5)
Microfuge tubes were set up containing 100 microliter of luciferase assay substrate buffer (Promega Corporation, Madison, Wis.) Background reading was taken in a bench top luminometer (20/20n single tube luminometer with software, as described in Example 1) and hydrogel beads containing luciferase were added to the tube and reading was followed at 10 sec interval. Both types of beads showed good activity (Table 8).
In a similar experiment, effect of increasing number of post-absorbed luciferase beads was tested. Microfuge tubes containing 100 microliter of luciferase assay substrate buffer (Promega) were set up and luciferase hydrogel beads (1-4 beads per tube) were added. The luminescence was monitored immediately in a bench top luminometer (FB-12 single tube luminometer, Berthold Detection Systems USA, Oak Ridge, Tenn.) and an initial (T0) measurement of RLUs was recorded. The initial (and all subsequent luminescence measurements) were obtained from the luminometer using FB12 Sirius PC software that was provided with the luminometer. The light signal was integrated for 1 second and the results are expressed in RLU/sec. The experiment was done in triplicate. The results, shown in Table 9, indicate a generally linear relationship between the number of beads per tube and the amount of luciferase activity.
Microtablets containing luciferase and luciferin were prepared as described in Preparative Example 10. Microfuge tubes were set up containing 190 μl of Butterfield's buffer. Ten microliters of 1 μM ATP (Sigma-Aldrich) solution in sterile water was added to the tube. The microtablets containing luciferase and luciferin were added to the tube and the tube was placed into a bench-top luminometer (20/20n single tube luminometer). Measurement of RLUs was recorded at 10 sec interval using 20/20n SIS software. The light signal was integrated for 1 second and the results are expressed in RLU/sec. The bioluminescence (RLU) increased with addition of microtablets while without microtablets the back ground did not increase. ATP bioluminescence was also measured using the formulation used for lyophilization. ATP bioluminescence gradually increased in tubes with enzyme microtablets, while the relative light units peaked with in 10 to 20 sec with liquid formulation (Table 10 and Table 11).
Microfuge tubes were set up containing 190 μl of Butterfield's buffer. Ten microliters of 1 μM ATP (Sigma-Aldrich) solution in sterile water was added to the tube. A solution containing luciferase (7.8 mg/lit, 3M Bridgend, UK) and luciferin (5.5 mg/lit, Promega) in 14 mM in Phosphate buffer was prepared. A known amount of the luciferin-luciferase solution was added to the tube and the tube was placed into a bench-top luminometer (20/20n single tube luminometer). Measurement of RLUs was recorded at 10 sec interval using 20/20n SIS software. The light signal was integrated for 1 second and the results are expressed in RLU/sec. The relative light units peaked with in 10 to 20 sec with liquid formulation (Table 12).
The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application claims the benefit of U.S. Provisional Patent Application No. 61/175,987, filed May 6, 2009, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2010/033804 | 5/6/2010 | WO | 00 | 12/21/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61175987 | May 2009 | US |
