Patent Application
20190153500
Sampling programs are used to monitor critical raw materials, in-process materials, finished goods, and processing environments in the food and beverage industry. Routine sampling and testing can allow quality assurance personnel to detect undesirable materials, such as microorganisms, at a very early stage and take steps to prevent subsequent contamination of equipment and/or products. A variety of tests can be performed to detect the unwanted materials. Examples of such tests include chemical residue tests (e.g., Adenosine triphosphate (ATP) bioluminescence tests and protein colorimetric tests), culture methods, genetic tests (e.g., PCR), immunodiagnostic tests, and bioluminescent tests.
Sample-collection devices typically are used to collect surface samples for environmental tests. Commercially-available sample-collection devices include absorbent devices such as sponges, swabs, and the like. In addition, certain sample-collection devices are capable of collecting a predetermined volume of a liquid sample.
ATP is used routinely to detect a presence or absence of microorganisms in a sample. The chemical energy produced from the breakdown of ATP is converted into light energy. Each molecule of ATP consumed in the reaction produces one photon of light. This light output can be quantified in a luminometer. The presence of ATP in a sample may be a direct indicator of the presence of a microorganism (i.e., the ATP is derived from a microorganism) or the ATP may be an indirect indicator of the presence of a microorganism (i.e., the ATP is derived from vegetative or animal matter and indicates that nutrients that support the growth of microorganisms may be present in the sample). In addition, the presence or absence of ATP in a sample is used routinely to assess the efficacy of cleaning processes in food, beverage, other industrial processed, healthcare (e.g. endoscopes) and for such as cooling and process waters and/or to determine whether biocide treatment has been effective in reducing the level of microorganisms.
The present disclosure generally relates to the detection of ATP in a sample using luciferase enzyme. In particular, in one aspect, the disclosure relates to a multi-purpose sampling device that comprises a dry coating comprising a pH-adjusting reagent. The pH-adjusting reagent is capable of causing a pH change when contacted with a liquid reagent composition comprising luciferin. The pH change can facilitate the achievement of a substantially steady-state light-emitting reaction in a shorter period of time than at a lower pH. Surprisingly, the sampling device can be contacted with a liquid sample and, even though the dry coating may be soluble in the liquid sample, an effective amount of the pH-adjusting reagent is retained on and/or in the sampling device and the effective amount can be transferred to the liquid reagent composition where it effects the aforementioned pH change. In addition, the sampling device obtains a predetermined volume of a liquid sample, thereby permitting the operator to detect and, optionally, quantitate the amount of ATP in the liquid sample.
In another aspect, the disclosure relates to a multi-purpose sampling device that comprises a liquid coating comprising a pH-adjusting reagent. The pH-adjusting reagent is capable of causing a pH change when contacted with a liquid reagent composition comprising luciferin. The pH change can facilitate the achievement of a substantially steady-state light-emitting reaction in a shorter period of time than at a lower pH. The sampling device can be contacted with a surface that may comprise liquid and/or solid residue. Surprisingly, after contact with the surface, an effective amount of the pH-adjusting reagent is retained on and/or in the sampling device and the effective amount can be transferred (with the residue collected by the sampling device) to the liquid reagent composition where the effective amount of pH-adjusting reagent effects the aforementioned pH change. After transferring the pH-adjusting reagent and the residue to the liquid reagent composition, the operator can detect and, optionally, quantitate the amount of ATP in the residue.
In one aspect, the present disclosure provides a kit. The kit can comprise a container with an opening and a cuvette portion that is adapted to be operationally coupled to a luminometer, a liquid reagent composition comprising luciferin, and a sampling device having a sampling portion. The liquid reagent composition can be disposed in a closed compartment. The pH of the liquid reagent composition is about 6.8 or lower. The sampling portion of the sampling device is adapted to acquire and releasably retain a sample. The sampling portion comprises a fibrous or a foam material. The sampling device comprises a coating that includes an effective amount of a pH-adjusting reagent that, when contacted with the liquid reagent composition, changes the pH of the liquid reagent composition to 6.9 or higher.
In another aspect, the present disclosure provides an apparatus. The apparatus can comprise a container with an opening and a cuvette portion that is adapted to be operationally coupled to a luminometer, a liquid reagent composition comprising luciferin, and a sampling device having a sampling portion. The liquid reagent composition can be disposed in a closed compartment. The pH of the liquid reagent composition is about 6.8 or lower. The sampling portion of the sampling device is adapted to acquire and releasably retain a sample. The sampling portion comprises a fibrous or a foam material. The sampling device comprises a coating that includes an effective amount of a pH-adjusting reagent that, when contacted with the liquid reagent composition, changes the pH of the liquid reagent composition to 6.9 or higher.
In any of the above embodiments of the kits or apparatuses, the sampling portion can be adapted to obtain a predetermined volume of liquid. In any of the above embodiments of the kits or apparatuses, at least a portion of the coating can be disposed on the sampling portion of the sampling device. In any of the above embodiments of the kits or apparatuses, the pH-adjusting reagent can comprise a water-soluble reagent. In any of the above embodiments of the kits or apparatuses, the coating can comprise a dry coating or a liquid coating. In any of the above embodiments of the kits or the apparatuses, the closed compartment can comprise a frangible wall. In any of the above embodiments, the frangible wall can be disposed in the container between the opening and the cuvette portion. In any of the above embodiments of the kits or apparatuses, the pH-adjusting reagent can comprise a buffer component selected from the group consisting of N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid] (HEPES), N-[tris(hydroxymethyl)methyl]glycine (TRICINE), and combinations thereof. In any of the above embodiments of the kits or apparatuses, the coating further can comprise an effective amount of a cell extractant. In any of the above embodiments of the kits or methods, the luciferase enzyme can comprise or consist essentially of a recombinant luciferase enzyme having luciferase activity that is less sensitive to variations in temperature, ionic detergents, and reducing agents than a corresponding non-recombinant luciferase enzyme. In any of the above embodiments of the kits or apparatuses, the predetermined volume can be about 0.01 milliliters to about 0.25 milliliters.
In yet another aspect, the present disclosure provides a method. The method can comprise using a sampling device to obtain a predetermined volume of sample, contacting the predetermined volume of sample with the pH-adjusting reagent and the liquid reagent composition in a container to form a reaction mixture, and using a luminometer to detect light emitted from the reaction mixture. The sampling device can comprise a sampling portion that comprises a fibrous material or a foam material. The sampling device comprises a coating that includes an effective amount of a pH-adjusting reagent that, when contacted with a liquid reagent composition having a pH of about 6.8 or lower, changes the pH of the liquid reagent composition to 6.9 or higher. The liquid reagent composition comprises a luciferin. The container comprises a cuvette portion adapted to be operationally coupled to the luminometer.
In yet another aspect, the present disclosure provides a method. The method can comprise using a sampling device with a surface to obtain a sample of residue from a surface; after obtaining the sample of residue, contacting the sample and the pH-adjusting reagent with the liquid reagent composition in a container to form a reaction mixture; and using a luminometer to detect light emitted from the reaction mixture. The sampling portion can comprise a fibrous material or a foam material. The sampling device comprises a coating that includes an effective amount of a pH-adjusting reagent that, when contacted with a liquid reagent composition having a pH of about 6.8 or lower, changes the pH of the liquid reagent composition to 6.9 or higher. The container can comprise a cuvette portion adapted to be operationally coupled to the luminometer.
In any of the above embodiments of the methods, the coating can comprise a dry coating or a liquid coating. In any of the above embodiments of the methods, the liquid reagent composition further can comprise a luciferase enzyme. In any of the above embodiments of the methods, the luciferase enzyme can consist essentially of a recombinant luciferase enzyme having luciferase activity that is less sensitive to variations in temperature, ionic detergents, and reducing agents than a corresponding non-recombinant luciferase enzyme. In any of the above embodiments of the methods, contacting the sampling device comprising the sample with liquid reagent composition can comprise contacting the sampling device with the liquid reagent composition at a temperature within a range of 10° C. and 35° C., inclusive. In any of the above embodiments of the methods, after contacting the sampling device with liquid reagent composition, a substantially steady-state amount of light can be emitted from the composition in less than 20 seconds.
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 cell extractant can be interpreted to mean “one or more” cell extractants.
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.
Additional details of these and other embodiments are set forth in the accompanying drawings and the description below. Other features, objects and advantages will become apparent from the description and drawings, and from the claims.
Before any embodiments of the present disclosure are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms “connected” and “coupled” and variations thereof are used broadly and encompass both direct and indirect connections and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure. Furthermore, terms such as “front,” “rear,” “top,” “bottom,” and the like are only used to describe elements as they relate to one another, but are in no way meant to recite specific orientations of the apparatus, to indicate or imply necessary or required orientations of the apparatus, or to specify how the invention described herein will be used, mounted, displayed, or positioned in use.
The present disclosure generally relates to an apparatus and method for detecting ATP from organic residues and/or cells in a sample. According to the present disclosure, the ATP from cells is detected by contacting the sample with a cell extractant to release ATP and detecting the released ATP using an enzymatic reaction that involves the reaction of the released ATP with luciferase enzyme activity in the presence of luciferin and results in the production of measurable light.
In particular, the present disclosure provides an apparatus and/or a kit with a sampling device that is adapted such that it 1) can acquire and releasably retain a predetermined volume of liquid sample, 2) can use the liquid sample to rehydrate an effective amount of dry, rehydratable pH-adjusting reagent, and 3) can deliver the liquid sample and effective amount of pH-adjusting reagent into a container in order to facilitate a luciferase-catalyzed light-emitting reaction. Advantageously, the present disclosure provides kits and apparatuses that can be used to store aqueous mixtures comprising luciferin for extended periods of time and instantly to adjust the pH of the mixtures in order to reduce the lag time required for a glow-type luciferase enzyme activity to achieve stable light output (e.g., maximum light output) in the presence of ATP and luciferin, particularly at lower temperatures (e.g., temperatures less than or equal to 20° C.).
The sampling portion 30 includes fibrous materials or foam materials capable of obtaining (e.g., via contact, capillary pressure and/or absorption) and retaining a sample from a sample source. In any embodiment, the sampling portion 30 can retain a liquid sample. In any embodiment, the sampling portion may comprise an applicator shaft (e.g., a solid molded plastic applicator shaft) having a tip coated with short fibers (e.g., nylon fibers) that are arranged in a perpendicular fashion such as, for example, the flocked swabs described in U.S. Pat. No. 8,114,027, which is incorporated herein by reference in its entirety. In any embodiment, the sampling portion may comprise an applicator shaft (e.g., a solid molded plastic applicator shaft) having a tip coated with a fibrous material such as, for example, the sea-island bicomponent fibers (e.g., polyester/polyester conjugated fiber yarn or polyester/nylon conjugated fiber yarn) described in U.S. Patent Publication Nos. 2011/0262951 and 2011/0262952, which are incorporated herein by reference in their entirety.
In any embodiment, the liquid sample may comprise a liquid sample having a predetermined maximum sample volume. The sampling portion 30 can be adapted to obtain and retain a predetermined volume of sample by selecting fibrous or foam materials having the appropriate composition, dimensions, porosity, hydrophilicity and combinations thereof. The maximum sample volume may be selected, for example, based on the desired sensitivity of the test performed with the sample. In any embodiment, the sampling portion may obtain (e.g., via contact capillary pressure and/or absorption) and retain residue (e.g., liquid and/or solid residue) from a surface.
In general, sampling portion 30 is comprised of a material that achieves the desired sample acquisition characteristics, which may depend upon the type of sample acquired (e.g., some liquid samples may include dissolved or suspended solids that affect the surface tension of the liquid). Material properties that may affect the ability of sampling portion 30 to acquire and retain a sample include, for example, surface energy. For example, the material may be selected to have a particular surface energy in order to draw the sample by capillary force or absorption, for example, into one or more spaces between the plurality of fibers disposed in the sampling portion 30. Other characteristics of the material from which the sampling portion is made may include substantial inertness relative to the sample or a relatively low rate of elution of chemicals or other contaminants that may affect luciferase enzyme activity, e.g., when the sample is released from sampling portion 30.
Foam articles (e.g., sponges, sponges mounted on a handle) for obtaining samples from environmental surfaces are known in the art.
Optionally, any sampling device according to the present disclosure further may comprise a coating area (not shown) on which a coating comprising a pH-adjusting reagent according to the present disclosure can be disposed. The coating area may be located, for example, on a portion of the stem and/or the optional piercing tip of the sampling device.
A sampling device of the present disclosure comprises a dry coating. In any embodiment, the dry coating can be disposed on and/or in (e.g., in the interstitial spaces between fibers of a fibrous material or in the individual cells of a foam material) the sampling portion of the device so that, when the device is dipped into the liquid sample, the coating is contacted with the liquid sample. The dry coating includes an effective amount of a pH-adjusting reagent that, when contacted with a liquid reagent composition of the present disclosure, changes the pH of the liquid reagent composition having an initial pH of about 6.8 or lower to an adjusted pH of about 6.9 or higher. In any embodiment, the pH-adjusting reagent comprises a water-soluble reagent. In any embodiment, the pH-adjusting reagent comprises a buffer component (e.g., N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid] (HEPES), N-[tris(hydroxymethyl)methyl]glycine (TRICINE), and combinations thereof). In some embodiments, the pH-adjusting reagent does not comprise an effective amount of a buffer component comprising phosphate. A buffer component comprising an amount of phosphate effective to change the pH of the liquid reagent composition from an initial pH 6.8 or lower to an adjusted pH of 6.9 or higher may also be sufficient to at least partially inhibit luciferase enzyme activity. In any embodiment, the pH-adjusting reagent further comprises a metallic base (e.g., sodium hydroxide, potassium hydroxide).
In any embodiment, the coating further can comprise a cell lysis agent that does not substantially inhibit luciferase enzyme activity. Examples of suitable cell lysis agents include, but are not limited to, chlorhexidine digluconate, TRITON X-100, lysozyme, lysostaphin, bacteriophage lysin, a quaternary ammonium compound (e.g., benzalkonium chloride, benzethonium chloride), cetyl trimethylammonium chloride (CTAB), dodecyltrimethylammonium chloride (DTAB), TWEEN 20, TWEEN 80, and a combination of any two or more of the foregoing.
The amount of pH-adjusting reagent coated onto the sampling device should be enough to change the pH of the liquid reagent composition from an initial (i.e., before contact with the pH-adjusting reagent) pH of 6.8 or lower to an adjusted pH of about 6.9 or higher (i.e., after contact with the pH-adjusting reagent). Preferably, the amount of pH-adjusting reagent coated on the sampling device should be enough to change the pH of the liquid reagent composition from an initial pH of 6.8 or lower to a pH about 7.2 or higher.
A non-limiting example of a coating solution that can be used to prepare sampling devices of the present disclosure is shown in Table 1. The coating can be applied to the sampling device using any suitable coating method. In any embodiment, the coating initially can be applied to the sampling device as a liquid coating (i.e., the pH-adjusting reagent is dissolved or suspended in a solvent such as water, for example). The solvent is subsequently dried to leave the substantially dry pH-adjusting reagent coated on the sampling device. In any embodiment, the liquid coating is applied by dipping the sampling device (e.g., the sampling portion of the sampling device) into a liquid comprising the pH-adjusting reagent. While immersed in the coating liquid, the sampling device can be agitated to facilitate movement of the coating liquid into the one or more cavity of the sampling portion. After coating the device, the coated portion can be dried (e.g., in a stream of air, a convection oven, or the like) under conditions (e.g., ambient or above-ambient (warm) temperatures) sufficient to evaporate the solvent without substantially degrading the pH-adjusting reagent and/or cell lysis agent.
In an alternative embodiment of the sampling device of the present disclosure, the coating can be applied to the sampling device as a liquid coating and held in a substantially liquid state (e.g., by packaging the device in a sealed container made from substantially moisture-resistant material) until use. Optionally, the liquid coating can comprise a humectant (e.g., propylene glycol) at a concentration that substantially does not leave a perdurable residue on a surface, as described in International Publication No. WO 2009/140356, which is incorporated herein by reference in its entirety. In some embodiments, the humectant comprises about 1 weight percent to about 50 weight percent of the liquid coating. Preferably, the humectant comprises about 2 weight percent to about 10 weight percent of the liquid coating. Sampling devices having a liquid coating may be used in any kit of the present disclosure.
Kits and apparatuses of the present disclosure further comprise a container. The container functions as a vessel in which to contact a sample with the liquid reagent composition described herein. The container comprises an opening and is configured (e.g., shaped and dimensioned) to receive at least a portion (e.g., the sampling portion) of the sampling device.
The container comprises at least one wall 50 and an opening 52, the opening configured to receive the sampling device 100. As discussed below, the apparatus 1000 with the opening 52 shown in the illustrated embodiment of
The container 200 may be fabricated as a unitary article (not shown), for example, using extrusion and/or molding processes known in the art. Alternatively, portions of the container 200, such as the cylindrically-shaped sleeve 56 and the cuvette 55 of the illustrated embodiment of
Kits and apparatuses of the present disclosure further comprise a liquid reagent composition (liquid reagent composition 70 shown in
A non-limiting example of a liquid composition according to the present disclosure is listed in Table 2 of the Examples.
In any embodiment of a kit or apparatus according to the present disclosure, the liquid reagent composition optionally may comprise a buffering agent. Suitable buffering agents have a pKa that is effective to maintain the pH of the liquid reagent composition at a pH of 6.8 or lower, preferably at a pH of 6.4 or lower. N-(2-acetamido)-iminodiacetic acid (ADA) is a nonlimiting example of a suitable buffering agent. The buffering agent should be present in the liquid reagent composition in an amount (e.g., about 16 mM ADA in 425 microliters of liquid reagent composition at pH 6.4-6.8) that is high enough to effectively maintain the pH of the liquid reagent composition, yet low enough so that it does not substantially resist a change in the pH of the liquid reagent composition mediated by the pH-adjusting reagent coated on the sampling portion of the sampling device. In any embodiment, the liquid reagent composition further may comprise a luciferase enzyme. In any embodiment, the luciferase enzyme can consist essentially of a recombinant luciferase enzyme having luciferase activity that is less sensitive to variations in temperature, ionic detergents, and reducing agents than a corresponding non-recombinant luciferase enzyme. The luciferase enzyme can be present in the liquid reagent composition at a concentration of about 9 mg/L, for example. In any embodiment, the liquid reagent composition further can comprise a source of magnesium ions (e.g., magnesium diacetate tetrahydrate). The source of magnesium ions can be present at a concentration (e.g., about 3 mM, for example) that does not substantially interfere with luciferase enzyme activity when the liquid reagent composition is mixed with the liquid sample. In any embodiment, the liquid reagent composition further can comprise a protein (e.g., bovine serum albumin). Without being bound by theory, the protein may stabilize the luciferase enzyme during prolonged storage and/or make the luciferase enzyme less susceptible to protease enzyme activities. At its working concentration, the protein should not substantially interfere with luciferase enzyme activity when the liquid reagent composition is mixed with the liquid sample. The protein may be present in the liquid reagent composition at a concentration of about 0.046 weight percent, for example. In any embodiment, the liquid reagent composition further can comprise a preservative (e.g., 0.046 weight percent sodium azide) at a concentration that does not substantially interfere with luciferase enzyme activity when the liquid reagent composition is mixed with the liquid sample. In any embodiment, the liquid reagent composition further may comprise ethylenediamine tetraacetic acid (EDTA). Without being bound by theory, the EDTA can act as a preservative in the liquid reagent composition and may also function to chelate ions present in the liquid reagent composition and/or the liquid sample.
In any embodiment of a kit or apparatus according to the present disclosure, the liquid reagent composition can be disposed in a closed compartment. In some embodiments, the closed compartment comprises a frangible wall. In the illustrated embodiment of
In an alternative embodiment (not shown) of a kit or apparatus according to the present disclosure, the liquid reagent composition may be disposed in a frangible ampoule (e.g., a glass or polymeric ampoule) which is disposed in the container. The liquid reagent composition can be released from the frangible ampoule by squeezing the container with enough force to fracture the ampoule or by urging the sampling device (e.g., the piercing tip of the sampling device) against the ampoule with enough force to fracture the ampoule.
In yet another embodiment (not shown) of a kit or apparatus according to the present disclosure, the liquid reagent composition may be disposed in a compartment disposed in the sampling device. Nason (U.S. Pat. No. 5,266,266, which is incorporated herein by reference in its entirety) discloses a swab device comprising a hollow stem and a liquid-containing closed compartment with an actuatable valve (break-off nib) that releases the liquid into the hollow stem for delivery into tube in which at least a portion of the swab is disposed. Any sampling device of the present disclosure may be modified to include the hollow stem and reagent chamber of Nason. The reagent chamber can contain the liquid reagent composition of the present disclosure and the liquid reagent composition can be transferred into the container of the present disclosure using the technique described by Nason. The liquid reagent composition can be transferred to the container before or after the liquid sample is acquired using the sampling device according to the present disclosure. Other compartments for temporarily storing liquid reagent compositions to be used in a reaction (e.g., a chemical and/or enzymatic reaction) are known in the art and a person of ordinary skill in the art will recognize such compartments can be used to temporarily contain the liquid reagent composition of the present disclosure. Non-limiting examples of such compartments can be found in U.S. Pat. Nos. 7,399,984; 6,548,018; 5,965,453; and 6,524,530, which are all incorporated herein by reference in their entirety.
The present disclosure provides a kit. The kit may be used, for example, to detect a presence or absence of ATP in a liquid sample according the method described herein. In any embodiment, the kit may comprise instructions disclosing an embodiment of the method disclosed herein.
Kits of the present disclosure comprise a container according to any of the embodiments disclosed herein. The container comprises an opening and a cuvette portion that is adapted to be operationally coupled to a luminometer. Kits of the present disclosure further comprise a liquid reagent composition comprising luciferin according to any of the embodiments disclosed herein. The initial pH (i.e., before contact with a pH-adjusting reagent of the present disclosure) of the liquid reagent composition is about pH 6.8 or lower; preferably, about pH 6.4 or lower. Kits of the present disclosure further comprise a sampling device having a sampling portion and a handling portion according to any of the embodiments disclosed herein. The liquid reagent composition is disposed in a closed compartment disposed in the container or the sampling device, as disclosed herein. The sampling portion of the sampling device comprises fibrous materials or foam materials and is configured to obtain (e.g., via capillary pressure or absorption) and retain a predetermined volume of liquid sample from a sample source. The sampling portion of the sampling device comprises a dry coating that includes an effective amount of a pH-adjusting reagent that, when contacted (e.g., mixed) with the liquid reagent composition, changes the pH of the liquid reagent composition to 6.9 or higher, preferably to about pH 7.2-8.0, more preferably to about pH 7.2-7.8. In any embodiment, the pH-adjusting reagent comprises a water-soluble reagent.
Kits and apparatuses of the present disclosure can be used methods of detecting a presence or an absence of ATP in a liquid sample. The presence of ATP in a sample can indicate a possible presence of organic residues and/or microorganisms (e.g., pathogenic microorganisms) in the sample. Moreover, the quantity of ATP in the sample can be an indicator of the relative number of microorganisms in the sample.
In one method of the present disclosure, a sampling device is used to obtain a liquid sample which is subsequently tested to detect a presence or an absence of ATP in the liquid sample. Because the amount of ATP in a sample can be an indicator of the cleanliness of the sample and/or the presence of microorganisms in the sample, it may be desirable to calculate the absolute or relative quantity of ATP in the sample. Therefore, it is desirable to test a predetermined volume of sample, so that the amount of ATP per unit volume of sample can be determined. The amount of ATP per unit volume of sample can be compared to a predetermined value to determine whether the sample is acceptable for use in a particular application (e.g., food or beverage production), whether a cleaning process has been effective in removing organic residues, and/or whether a biocide treatment has been effective in reducing the number of microorganisms.
Thus, a first method of the present disclosure comprises using a sampling device to obtain a predetermined volume of liquid sample, contacting the predetermined volume of sample with a liquid reagent composition in a container to form a reaction mixture, and using a luminometer to detect light emitted from the reaction mixture. The predetermined volume of liquid sample can be about 10 microliters to about 250 microliters. In some embodiments, the predetermined volume of liquid sample can be about 100 microliters to about 200 microliters. In some embodiments, the predetermined volume of liquid sample can be about 125 microliters to about 175 microliters.
According to the first method of the present disclosure, the sample comprises a liquid. The liquid may comprise water. The sample further may comprise solids that are dissolved, dispersed, and/or suspended in the liquid. Nonlimiting examples of suitable samples include clean-in-place (CIP) rinse-water samples, process and cooling water samples, and endoscope rinse water samples.
The sampling device used in the first method of the present disclosure can be any sampling device disclosed herein. The sampling device comprises a sampling portion and a handling portion. The sampling portion is adapted to acquire and releasably retain a predetermined volume of liquid sample. The sampling portion includes fibrous materials or foam materials capable of obtaining (e.g., via capillary pressure or absorption) and retaining a liquid sample from a sample source. The sampling portion retains a predetermined maximum sample volume. In any embodiment of the first method, the sampling portion of the device comprises a dry coating that includes an effective amount of a pH-adjusting reagent that, when contacted (e.g., mixed) with a liquid reagent composition having an initial pH of 6.8 or lower (i.e., before contacting the pH-adjusting reagent), adjusts the pH of the liquid reagent composition to 6.9 or higher. In some embodiments of the first method, the sampling portion of the device comprises a moist (i.e., liquid) coating that includes an effective amount of a pH-adjusting reagent that, when contacted with a liquid reagent composition having an initial pH of 6.8 or lower, adjusts the pH of the liquid reagent composition to 6.9 or higher.
The present disclosure further provides a second method of detecting ATP in a sample. The second method is used to detect ATP that is present in residue on a surface (e.g., liquid and/or solid residue found on a solid surface. The second method is particularly useful, for example, to detect the efficacy of a process that is used to clean the surface. In many environments (e.g., food-processing facilities, hospitals), a cleaning process should substantially reduce or remove detectable ATP from the surface that is to be tested. Suitable samples for the second method of the present disclosure include, but are not limited to, processing equipment (e.g., food-handling surfaces, tubes, drains, conveyors, storage containers), environmental surfaces (e.g., sinks, countertops, drawers, floors, walls, ceilings, doors, bedrails, linens, computer touch screens, keyboards, monitors), and medical equipment or devices (e.g., endoscopes, retractors, trocars, scalpels, trays). According to the second method of the present disclosure, the sample may comprise liquid and/or solid residue present on a surface.
The sampling device used in the second method of the present disclosure can be any sampling device disclosed herein. The sampling device comprises a sampling portion and a handling portion. The sampling portion is adapted to acquire a sample of surface residue. The sampling portion includes fibrous materials or foam materials capable of obtaining and retaining the surface residue. In any embodiment of the second method, the sampling portion of the device comprises a dry coating that includes an effective amount of a pH-adjusting reagent that, when contacted with a liquid reagent composition having an initial pH of 6.8 or lower, adjusts the pH of the liquid reagent composition to 6.9 or higher. In some embodiments of the second method, the sampling portion of the device comprises a moist (i.e., liquid) coating that includes an effective amount of a pH-adjusting reagent that, when contacted with a liquid reagent composition having an initial pH of 6.8 or lower, adjusts the pH of the liquid reagent composition to 6.9 or higher.
The liquid reagent composition used in any embodiment of the first or second method of the present disclosure comprises a luciferin (e.g., firefly luciferin). Luciferin is a compound that undergoes oxidation by luciferase enzyme activity to produce oxyluciferin and light. Luciferin compounds can be unstable when stored for extended periods of time in liquid solutions at a pH above 6.8. Thus, the liquid reagent composition used in the method of the present disclosure has an initial pH of 6.8 or lower. Preferably, liquid reagent composition has an initial pH of 6.4 or lower.
In any embodiment, the liquid reagent composition may further comprise a luciferase enzyme. In a preferred embodiment, the luciferase enzyme comprises a recombinant luciferase enzyme having luciferase activity that is less sensitive than a corresponding non-recombinant luciferase enzyme to variations in temperature, ionic detergents, and reducing agents. In some embodiments, the luciferase enzyme consists essentially of a recombinant luciferase enzyme having luciferase activity that is less sensitive to variations in temperature, ionic detergents, and reducing agents than a corresponding non-recombinant luciferase enzyme.
In any embodiment, the liquid reagent composition optionally may comprise a buffering agent. The buffering agent can be used to maintain the liquid reagent composition at an initial pH (e.g., ≤6.8) that is suitable to store a liquid solution containing luciferin. N-(2-acetamido)-iminodiacetic acid (ADA) is a nonlimiting example of a suitable buffering agent. The buffering agent should be present in the liquid reagent composition in an amount (e.g., about 16 mM ADA in 425 microliters of liquid reagent composition at pH 6.4-6.8) that is high enough to effectively maintain the pH of the liquid reagent composition, yet low enough so that it does not substantially resist a change in the pH of the liquid reagent composition mediated by the pH-adjusting reagent coated on the sampling portion of the sampling device.
The container used in any embodiment of the first or second method of the present disclosure comprises a cuvette portion. The cuvette portion permits the transmission there through of light emitted as a product of the reaction catalyzed by luciferase enzyme. The cuvette portion is adapted (e.g., shaped and dimensioned) to be operationally coupled to the luminometer. In some embodiments, using a luminometer to detect light emitted from the reaction mixture comprises detecting a presence or an absence of ATP in the liquid sample. In some embodiments, using a luminometer to detect light emitted from the reaction mixture comprises measuring a quantity (e.g., a relative quantity or an absolute quantity) of ATP present in the liquid sample.
In any embodiment of the first or second method of the present disclosure, contacting the predetermined volume of sample with a liquid reagent composition in a container to form a reaction mixture comprises bringing the sampling portion of the sampling device into contact with the liquid reagent composition in a container. The contact may be achieved, for example, by immersing at least a part; preferably, the entire sampling portion; of the sampling device in the liquid reagent composition. The contact forms a reaction mixture (e.g., by diffusion of at least part of the liquid sample into the liquid reagent composition). If it is not already present in the liquid reagent composition, luciferase can be added to the reaction mixture. Thus, if ATP is present in the liquid sample, the ATP can facilitate a light-emitting reaction catalyzed by luciferase enzyme. The light-emitting reaction can be detected in the aforementioned luminometer.
In any embodiment of the first or second method according to the present disclosure, contacting the predetermined volume of sample with a liquid reagent composition in a container to form a reaction mixture further can comprise the step of agitating the sample and the liquid reagent composition in the container. This may be done manually or mechanically, for example, by rapidly shaking the container in a swirling motion, a pendulum-like motion, or by contacting the container with a machine that vibrates the container.
In any embodiment of the first or second method of the present disclosure, the cuvette portion of the container is operationally coupled to the luminometer in order to detect the light emission resulting from luciferase enzyme activity. Commercially-available luminometers are suitable for use in any embodiment of the method of the present disclosure. A non-limiting example of a suitable luminometer is the 3M™ Clean-Trace™ NG Luminometer commercially available from 3M Company, St. Paul, Minn. Typically, the cuvette is operationally coupled by placing a portion (e.g., the cuvette portion) of the container or the entire container into a corresponding receiving compartment of the luminometer. The cuvette portion may be operationally coupled to the luminometer before or after forming the reaction mixture. Typically, the cuvette portion is operationally coupled to the luminometer after the reaction mix is formed. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 5 seconds after forming the reaction mixture. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 10 seconds after forming the reaction mixture. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 15 seconds after forming the reaction mixture. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 20 seconds after forming the reaction mixture. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 30 seconds after forming the reaction mixture. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 45 seconds after forming the reaction mixture. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 60 seconds after forming the reaction mixture. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 90 seconds after forming the reaction mixture. In some embodiments the cuvette portion is operationally coupled to the luminometer up to 2 minutes after forming the reaction mixture. Preferably, the cuvette is operationally coupled to the luminometer within about 5 to 10 seconds after forming the reaction mixture.
In any embodiment of the first or second method of the present disclosure, after operationally coupling the cuvette portion to the luminometer, the light-emission is monitored by the luminometer. After the reaction mixture is formed, it may take a period of time for the reaction to reach a relatively stable quantity of light emission.
In any embodiment of the first or second method of the present disclosure, the liquid reagent composition comprises luciferin at a concentration (e.g., about 0.3-0.4 mg/L luciferin) sufficient to facilitate a light-emitting reaction in the presence of luciferase and a source of ATP. In the method of the present disclosure, the source of ATP can be a liquid sample as disclosed herein or a solution containing a predefined amount or concentration of ATP (i.e., a positive control or an ATP standard). In any embodiment, the liquid reagent composition can comprise an aqueous solution. The liquid reagent composition used in the apparatus or kit of the present disclosure has an initial pH of 6.8 or lower. Preferably, liquid reagent composition has an initial pH of 6.4 or lower.
In any embodiment of the first or second method of the present disclosure, the liquid reagent composition optionally may comprise a buffer component. Suitable buffer components have a pKa that is effective to maintain the initial pH of the liquid reagent composition at a pH of 6.8 or lower, preferably at a pH of 6.4 or lower. N-(2-acetamido)-iminodiacetic acid (ADA) is a nonlimiting example of a suitable buffering agent. The buffer component should be present in the liquid reagent composition in an amount (e.g., about 16 mM ADA in 425 microliters of liquid reagent composition at pH 6.4-6.8) that is high enough to effectively maintain the pH of the liquid reagent composition, yet low enough so that it does not substantially resist a change in the pH of the liquid reagent composition mediated by the pH-adjusting reagent coated on the sampling portion of the sampling device. In any embodiment of the first or second method, the liquid reagent composition further may comprise a luciferase enzyme. In any embodiment of the first or second method, the luciferase enzyme can consist essentially of a recombinant luciferase enzyme having luciferase activity that is less sensitive to variations in temperature, ionic detergents, and reducing agents than a corresponding non-recombinant luciferase enzyme. The luciferase enzyme can be present in the liquid reagent composition at a concentration of about 9 mg/L, for example. In any embodiment of the first or second method, the liquid reagent composition further can comprise a source of magnesium ions (e.g., magnesium diacetate tetrahydrate). The source of magnesium ions can be present at a concentration (e.g., about 3 mM, for example) that does not substantially interfere with luciferase enzyme activity when the liquid reagent composition is mixed with the liquid sample. In any embodiment of the first or second method, the liquid reagent composition further can comprise a protein (e.g., bovine serum albumin). The protein may be present in the liquid reagent composition at a concentration of about 0.046 weight percent, for example. In any embodiment of the first or second method, the liquid reagent composition further can comprise a preservative (e.g., 0.046 weight percent sodium azide) at a concentration that does not substantially interfere with luciferase enzyme activity when the liquid reagent composition is mixed with the liquid sample.
In any embodiment of the first or second method of the present disclosure, contacting the sampling device comprising the liquid sample with liquid reagent composition comprises contacting the sampling device with the liquid reagent composition at a particular temperature (e.g. ambient temperature). In general, when the contact occurs at lower temperatures, a longer period of time is required for the light-emitting reaction to reach a relatively stable amount of light emission. Conversely, when the contact occurs at higher temperatures, a shorter period of time is required for the light-emitting reaction to reach a relatively stable amount of light emission. In any embodiment of the method of the present disclosure, contacting the sampling device comprising the liquid sample with liquid reagent composition comprises contacting the liquid sample with the liquid reagent composition at temperature within a range of 10° C. and 35° C., inclusive. Preferably, contacting the sampling device comprising the liquid sample with liquid reagent composition comprises contacting the liquid sample with the liquid reagent composition at temperature within a range of 15° C. and 30° C., inclusive.
In any embodiment of the first or second method, after contacting the sampling device with the liquid reagent composition, a substantially steady-state amount of light is emitted from the composition after a period of time. As discussed above, the period of time may be affected by the temperature at which the contact occurs. In any embodiment of the first or second method of the present disclosure, a substantially steady-state amount of light is emitted from the composition in less than 25 seconds (e.g., when contact occurs at a temperature of about 10° C. to about 20° C.). In any embodiment of the first or second method of the present disclosure, a substantially steady-state amount of light is emitted from the composition in about 22 seconds or less (e.g., when contact occurs at a temperature of about 10° C. to about 20° C.). In some embodiments, a substantially steady-state amount of light is emitted from the composition in less than 20 seconds (e.g., when contact occurs at a temperature of about 14° C. to about 20° C.). In some embodiments, a substantially steady-state amount of light is emitted from the composition in about 10 seconds or less (e.g., when contact occurs at a temperature of about 20° C.).
The methods of the present invention substantially reduces the amount of time necessary for a luciferase enzyme, in contact with a sample comprising ATP and a liquid reagent composition according to the present disclosure, to catalyze a light-emitting reaction that is substantially steady state.
Embodiment A is a kit, comprising:
a container with an opening and a cuvette portion that is adapted to be operationally coupled to a luminometer;
a liquid reagent composition comprising luciferin, wherein the liquid reagent composition is disposed in a closed compartment, wherein the pH of the liquid reagent composition is about 6.8 or lower; and
a sampling device having a sampling portion;
Embodiment B is an apparatus, comprising:
a container with an opening and a cuvette portion that is adapted to be operationally coupled to a luminometer;
a liquid reagent composition comprising luciferin, wherein the liquid reagent composition is disposed in a closed compartment, wherein the pH of the liquid reagent composition is about 6.8 or lower; and
a sampling device having a sampling portion;
Embodiment C is the kit of Embodiment A or the apparatus of Embodiment B, wherein the sampling portion is adapted to obtain a predetermined volume of a liquid.
Embodiment D is the kit or the apparatus of any one of the preceding Embodiments, wherein at least a portion of the coating is disposed on the sampling portion of the sampling device.
Embodiment E is the kit or the apparatus of any one of the preceding Embodiments, wherein the pH-adjusting reagent comprises a water-soluble reagent.
Embodiment F is the kit or the apparatus of any one of the preceding Embodiments, wherein the coating comprises a dry coating.
Embodiment G is the kit or the apparatus of any one of Embodiments A through E, wherein the coating comprises a liquid coating.
Embodiment H is the kit or the apparatus of Embodiment G, wherein the liquid coating comprises a humectant.
Embodiment I is the kit or the apparatus of any one of the preceding Embodiments, wherein the closed compartment comprises a frangible wall.
Embodiment J is the kit or the apparatus of Embodiment I, wherein the frangible wall is disposed in the container between the opening and the cuvette portion.
Embodiment K is the kit or the apparatus of any one of Embodiments A through H, wherein the sampling device comprises the closed compartment, wherein the sampling device further comprises a structure that selectively releases the fluid composition from the container.
Embodiment L is the kit or the apparatus of any one of the preceding Embodiments, wherein the pH-adjusting reagent comprises a buffer component selected from the group consisting of N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid] (HEPES), N-[tris(hydroxymethyl)methyl]glycine (TRICINE), and combinations thereof.
Embodiment M is the kit or the apparatus of any one of the preceding Embodiments, wherein the coating further comprises an effective amount of a cell extractant.
Embodiment N is the kit or the apparatus of any one of the preceding Embodiments, wherein the luciferase enzyme comprises a recombinant luciferase enzyme having luciferase activity that is less sensitive to variations in temperature, ionic detergents, and reducing agents than a corresponding non-recombinant luciferase enzyme.
Embodiment O is the kit or the apparatus of any one of Embodiments A through M, wherein the luciferase enzyme consists essentially of a recombinant luciferase enzyme having luciferase activity that is less sensitive to variations in temperature, ionic detergents, and reducing agents than a corresponding non-recombinant luciferase enzyme.
Embodiment P is the kit or the apparatus of any one of the preceding Embodiments, wherein the coating does not comprise an effective amount of a buffer component comprising phosphate.
Embodiment Q is the kit or the apparatus of any one of the preceding Embodiments, wherein the predetermined volume is about 0.01 milliliters to about 0.25 milliliters.
Embodiment R is a method, comprising:
using a sampling device to obtain a predetermined volume of liquid sample;
contacting the predetermined volume of sample with the pH-adjusting reagent and the liquid reagent composition in a container to form a reaction mixture; and
using a luminometer to detect light emitted from the reaction mixture;
wherein the container comprises a cuvette portion adapted to be operationally coupled to the luminometer.
Embodiment S is a method, comprising:
using a sampling device to obtain a sample of residue from a surface;
after obtaining the sample of residue, contacting the sample and the pH-adjusting reagent with the liquid reagent composition in a container to form a reaction mixture; and
using a luminometer to detect light emitted from the reaction mixture;
wherein the container comprises a cuvette portion adapted to be operationally coupled to the luminometer.
Embodiment T is the method of Embodiment R or Embodiment S, wherein the coating comprises a dry coating.
Embodiment U is the method of Embodiment R or Embodiment S, wherein the coating comprises a liquid coating.
Embodiment V is the method of any one of Embodiments Q through T, further comprising the step of agitating the sampling portion and the liquid reagent composition in the container.
Embodiment W is the method of any one of Embodiments R through V, wherein the liquid reagent composition further comprises a luciferase enzyme.
Embodiment X is the method of Embodiment W, wherein the luciferase enzyme consists essentially of a recombinant luciferase enzyme having luciferase activity that is less sensitive to variations in temperature, ionic detergents, and reducing agents than a corresponding non-recombinant luciferase enzyme.
Embodiment Y is the method of any one of Embodiments R through X, wherein contacting the sampling device comprising the sample with liquid reagent composition comprises contacting the sampling device with the liquid reagent composition at a temperature within a range of 10° C. and 35° C., inclusive.
Embodiment Z is the method of any one of Embodiments R though Y wherein, after contacting the sampling device with liquid reagent composition, a substantially steady-state amount of light is emitted from the composition in less than 20 seconds.
Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention.
Coating Formulation 1:
Coating Formulation 1 was prepared as a solution from the components listed in Table 1 and was used to coat the fibrous sampling portions of the sampling devices in Examples 1-5 and 8-12. The pH of Coating Formulation 1 was about 8.5.
Liquid Reagent Composition:
The liquid reagent composition described herein comprises luciferin and a luciferase enzyme. The liquid reagent composition was prepared as a solution using the components listed in Table 2. The pH of the formulation was about 6.4. The total volume of liquid reagent composition contained in the sealed cuvette portion of each container of the present disclosure was about 425 microliters.
Coating Formulation 2:
Coating Formulation 2 was prepared as a solution from the components listed in Table 3 and was used to coat the fibrous sampling portions of the sampling devices in Comparative Examples 1-5 and 8-12. The pH of Coating Formulation 2 was about 7.2.
Preparation of ATP Test Apparatus 1:
An apparatus similar to the design described in
The container component of the apparatus was obtained from a 3M Clean-Trace™ Water Plus-Total ATP Test Device (product number AQT200, 3M Company, St. Paul, Minn.). The sampling device component was removed from the device and discarded, while the container component was saved. The cuvette portion of the container contained the liquid reagent composition described in Table 2.
The sampling device with a fibrous sampling portion was obtained from a 3M Clean-Trace™ Surface ATP Test Device (product number UXL-100, 3M Company, St. Paul, Minn.). The fibrous sampling portion was prepared from polyethylene terephthalate fibers. The sampling device component was removed from the test device and the rest of the test device (i.e. the container component) was discarded. The sampling device was then coated using Coating Formulation 1 (Table 1). The sampling device was coated by holding the handling portion of the sampling device and dipping the opposite end of the device into a vessel containing Coating Formulation 1 to a point where the entire fibrous sampling portion was immersed in the coating formulation for a period of 15 seconds. The sampling device was withdrawn from the coating formulation. Excess coating formulation was removed from the fibrous sampling portion by gently tapping the stem portion of the sampling device against the side of the container. The sampling device retained about 170 microliters of the formulation. The coated sampling device was immediately combined with the container portion of the 3M Clean-Trace™ Water Plus-Total ATP Test Device so that the sampling device and container were assembled in the first operational position with respect to each other (
Preparation of ATP Test Apparatus 2:
The ATP Test Apparatus 2 was prepared in the same manner as ATP Test Apparatus 1 except for the procedure used to coat the sampling device. For ATP Test Apparatus 2, the sampling device was coated by holding the handling portion of the sampling device and dipping the opposite end of the device into a vessel containing Coating Formulation 1 to a point where the entire fibrous sampling portion was immersed in the coating formulation for a period of 15 seconds. The sampling device was withdrawn from the coating formulation. Excess coating formulation was removed from the fibrous sampling portion by gently tapping the stem portion of the sampling device against the side of the container. The sampling device was then placed in a laminar flow cabinet and air dried at 20° C. for a period of at least 3 hours. After the drying step, the coated sampling device was combined with the container portion of the 3M Clean-Trace™ Water Plus-Total ATP Test Device so that the sampling device and container were assembled in the first operational position with respect to each other (
The ATP Test Apparatus 1 described above was used. The sampling device was removed from the ATP test apparatus and 10 microliters of a 1×10−7 M aqueous solution of adenosine triphosphate (ATP) was added to the midsection of the fibrous sampling portion using a micropipette. The sampling device was reinserted into the container. The handle of the sampling device was pressed in order to move the sampling device from the first operational position to the second operational position. The movement of the sampling device in the container caused the frangible seal to be broken and the fibrous sampling portion to be positioned in direct contact with the liquid reagent composition. The container was held in the vertical position and shaken rapidly from side to side (pendulum motion) for about 5 seconds. After shaking the container, the cuvette portion of the apparatus was immediately inserted into a 3M Clean-Trace™ NG Luminometer (commercially available from 3M Company, St. Paul, Minn.) and measurements were recorded in relative light units (RLU). The RLU measurements were taken approximately every 10 seconds over a 70 second time period. This was accomplished by initiating a new reading for the luminometer immediately after the previous reading. Because the instrument takes approximately 10 seconds to obtain and present each new reading, this resulted in RLU measurements taken about every 10 seconds. All testing was conducted in an environmental chamber at 10° C. All materials and equipment were equilibrated in the chamber prior to testing. A total of five replicate samples were tested and the results are presented in Table 4.
Movement of the sampling device from the first operational position to the second operational position resulted in the pH of the liquid reagent composition in the cuvette increasing from a pH of approximately 6.4 to a pH of approximately 7.2 to 7.6.
The same procedure was used as in Example 1 with the exception that the sampling device was coated with Coating Formulation 2 instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 5.
The same procedure as described in Example 1 was followed with the exception that the tests were conducted at 15° C. A total of five replicate samples were tested and the results are presented in Table 6.
The same procedure was used as in Example 2 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 7.
The same procedure as described in Example 1 was followed with the exception that the tests were conducted at 20° C. A total of five replicate samples were tested and the results are presented in Table 8.
The same procedure was used as in Example 3 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 9.
The same procedure as described in Example 1 was followed with the exception that the tests were conducted at 25° C. A total of five replicate samples were tested and the results are presented in Table 10.
The same procedure was used as in Example 4 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 11.
The same procedure as described in Example 1 was followed with the exception that the tests were conducted at 30° C. A total of five replicate samples were tested and the results are presented in Table 12.
The same procedure was used as in Example 5 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 13.
For the test samples of Examples 1-5 and the corresponding Comparative Examples 1-5, the percent increase in the RLU measurement between the first time point (10 seconds) and the second time point (20 seconds) was calculated using the following equation:
% Increase in RLU={[(RLU at 20 sec)−(RLU at 10 sec)]/(RLU at 10 sec)}×100
The mean percent increase (n=5) in the RLU measurement is reported in Table 14.
For the test samples of Examples 1-5 and the corresponding Comparative Examples 1-5, the mean time (n=5) to reach the maximum RLU measurement was determined. The results are reported in Table 15.
The same procedure as described in Example 1 was followed with the exception that ATP Test Apparatus 2 was used instead of ATP Test apparatus 1. A total of five replicate samples were tested and the results are presented in Table 16.
The same procedure was used as in Example 8 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 17.
The same procedure as described in Example 8 was followed with the exception that the tests were conducted at 15° C. A total of five replicate samples were tested and the results are presented in Table 18.
The same procedure was used as in Example 9 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 19.
The same procedure as described in Example 8 was followed with the exception that the tests were conducted at 20° C. A total of five replicate samples were tested and the results are presented in Table 20.
The same procedure was used as in Example10 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 21.
The same procedure as described in Example 8 was followed with the exception that the tests were conducted at 25° C. A total of five replicate samples were tested and the results are presented in Table 22.
The same procedure was used as in Example 11 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 23.
The same procedure as described in Example 8 was followed with the exception that the tests were conducted at 30° C. A total of five replicate samples were tested and the results are presented in Table 24.
The same procedure was used as in Example 12 with the exception that the sampling device was coated with Coating Formulation 2, instead of Coating Formulation 1. The RLU measurements were taken every 10 seconds over a 70 second time period. A total of five replicate samples were tested and the results are presented in Table 25.
For the test samples of Examples 8-12 and the corresponding Comparative Examples 8-12, the percent increase in the RLU measurement between the first time point (10 seconds) and the second time point (20 seconds) was calculated using the following equation:
% Increase in RLU={[(RLU at 20 sec)−(RLU at 10 sec)]/(RLU at 10 sec)}×100
The mean percent increase (n=5) in the RLU measurement is reported in Table 26.
For the test samples of Examples 8-12 and the corresponding Comparative Examples 8-12, the mean time (n=5) to reach the maximum RLU measurement was determined. The results are reported in Table 27.
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.
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.
Various modifications may be made without departing from the spirit and scope of the invention. These and other embodiments are within the scope of the following claims.
This application is a division of application of application Ser. No. 14/412,143 filed Dec. 30, 2014, which is a 371 of International PCT/US2013/027631, filed Feb. 25, 2013, and claims the benefit of U.S. Provisional Patent Application No. 61/668,526, filed Jul. 6, 2012, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61668526 | Jul 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14412143 | Dec 2014 | US |
| Child | 16259234 | US |
