ATP detection

Abstract

The present disclosure relates to devices, methods, and systems or kits for detecting ATP in a sample. In particular, the present disclosure relates to methods for removing ATP contamination from a sample and for detecting intracellular ATP in a sample (e.g., as a surrogate for detecting live cells in a sample).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 328,070, filed April 6, 2022, the contents of which are incorporated by reference herein in their entirety.

[0002] The present disclosure relates to the detection of adenosine triphosphate (ATP) in a sample. In particular, the present disclosure relates to devices, methods, and systems or kits for detecting intracellular ATP in a sample, for example as a determination of viable cells in the sample. [Background technology]

[0003] Millimolecular intracellular ATP concentrations are a relatively constant feature of all living cells, and this ATP is rapidly degraded upon cell death. Therefore, measurements of intracellular ATP are performed as a means to detect and quantify the presence of live cells, such as pathogenic and non-pathogenic bacteria. Traditional approaches for ATP detection require the growth of contaminating cells in or on nutrient media, which can take up to several days, and are unable to detect viable but non-culturable cells. False-positive signals due to the presence of free ATP are also frequently encountered. Current methods for minimizing or eliminating free ATP-dependent false positives by ATPase treatment are not directly compatible with subsequent live cell ATP measurements. Summary of the Invention

[0004] Disclosed herein are methods that include fully or partially removing ATP contamination from a sample. In some embodiments, the methods include incubating the sample with a conditionally active ATPase selected from the group consisting of Cimex lectularius apyrase, Phlebotomus papatasi apyrase, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), and combinations thereof.

[0005] In some embodiments, the sample is incubated for less than about 5 minutes (e.g., less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute). In some embodiments, the sample is incubated for about 5 to about 60 seconds.

[0006] In some embodiments, the method further comprises performing a biological assay using the sample obtained after incubating. In some embodiments, the biological assay comprises detecting remaining ATP in the sample obtained after incubating. In some embodiments, detecting remaining ATP in the sample comprises a bioluminescence assay. In some embodiments, the presence of ATP indicates the presence of live cells in the sample.

[0007] In some embodiments, the method includes inhibiting the conditionally active ATPase in the sample obtained after incubation, hi some embodiments, inhibiting includes contacting the sample with EGTA, a zinc chelator, a small molecule inhibitor, or a combination thereof.

[0008] In some embodiments, the method further comprises lysing cells in the obtained sample after incubating and / or before performing the biological assay.

[0009] Further disclosed herein is a method for detecting intracellular ATP. The method may include at least one or all of the following: collecting a sample containing or suspected to contain cells; incubating the sample with a conditionally active ATPase to substantially degrade any extracellular ATP in the sample; inhibiting the conditionally active ATPase; lysing the cells; and determining the presence or amount of ATP. In some embodiments, the conditionally active ATPase is selected from the group consisting of Cimex lectularius apyrase, Phlebotomus papatasi apyrase, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), and combinations thereof.

[0010] In some embodiments, the sample contains or is suspected of containing a microorganism. In some embodiments, the sample is collected on an absorbent pad. In some embodiments, the absorbent pad is pre-wetted with a solution containing a conditionally active ATPase. In some embodiments, the sample is a liquid sample. In some embodiments, the sample is an environmental sample.

[0011] In some embodiments, the inhibiting comprises contacting the sample with EGTA, a zinc chelator, a small molecule inhibitor, or a combination thereof.

[0012] In some embodiments, determining the presence or amount of ATP comprises a bioluminescence assay, hi some embodiments, the ATP detection reagent comprises a luciferase enzyme and a luciferin substrate.

[0013] In some embodiments, the sample is incubated for less than about 5 minutes (e.g., less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute). In some embodiments, the sample is incubated for about 5 to about 60 seconds.

[0014] In some embodiments, the method is performed in less than about 10 minutes (e.g., less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute).

[0015] Also disclosed herein is an assay device. The assay device may include at least one or all of a swab assembly including a collection swab configured to receive and absorb a sample, a cap with a reservoir containing a reaction buffer, and a reaction chamber configured to receive the swab assembly. In some embodiments, the collection swab is pre-wetted with a solution including a conditionally active ATPase. In some embodiments, the conditionally active ATPase is selected from the group consisting of: Cimex lectularius apyrase, Phlebotomus papatasi apyrase, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), and combinations thereof.

[0016] In some embodiments, the reservoir comprises an articulatable element for releasing a reaction buffer into the reaction chamber, hi some embodiments, the reaction buffer comprises a cell lysis reagent, an inhibitor of the conditionally active ATPase, an ATP detection reagent, or a combination thereof.

[0017] In some embodiments, the conditionally active ATPase inhibitor comprises EGTA, a zinc chelator, a small molecule inhibitor, or a combination thereof.

[0018] In some embodiments, the solution containing the conditionally active ATPase further comprises a divalent cation.

[0019] Also disclosed is a method of detecting intracellular ATP using the disclosed assay device. In some embodiments, the method includes at least one or all of: collecting a sample with a collection swab of the disclosed assay device; placing the swab assembly in a reaction chamber, deploying a reaction buffer from a cap, and determining the presence or amount of intracellular ATP in the sample.

[0020] In some embodiments, the method is performed in less than about 10 minutes (e.g., less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute).

[0021] In some embodiments, the sample is a liquid sample, hi some embodiments, the sample is an environmental sample.

[0022] In some embodiments, determining the presence or amount of ATP comprises a bioluminescence assay, hi some embodiments, the ATP detection reagent comprises a luciferase enzyme and a luciferin substrate.

[0023] Disclosed herein are kits that include at least one or all of a swab assembly comprising a conditionally active ATPase, a collection swab configured to receive and absorb a sample, and a cap, a reaction chamber configured to receive the swab assembly, and a reaction buffer containing a cell lysis reagent, an inhibitor of the conditionally active ATPase, an ATP detection reagent, or a combination thereof.

[0024] In some embodiments, the conditionally active ATPase is on a collection swab.

[0025] In some embodiments, the reaction buffer is in a reservoir in the cap.

[0026] In some embodiments, the conditionally active ATPase is selected from the group consisting of: Cimex lectularius apyrase, Phlebotomus papatasi apyrase, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), and combinations thereof.

[0027] Other aspects and embodiments of the present disclosure will become apparent in light of the following detailed description and accompanying figures. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram of a system and workflow for ATP detection using the cartridge of an exemplary sample collection and assay device. [Diagram 2] 1 is a graph of the luminescence output from a Ca2+-dependent apyrase ATP scavenger. [Diagram 3] 1 is a graph of the inhibition by EGTA of Ca-apyrase from Cimex lectularus (Cl) and Ca2+ / Mg2+-apyrase from potato Solanum tuberosum (St-apyrase). [Figure 4] Graph of inhibition of ENPP1 (a zinc-dependent, metal chelator-sensitive ectonucleotide pyrophosphatase / phosphodiesterase) by various amounts of zinc chelators (EGTA, TPEN) or a direct small molecule ENPP1 inhibitor (inhibitor C), as indicated. [Figure 5A] 1 is a graph of the Ca2+ dependence of sand fly Phlebotomus papatasi (Pp-apyrase) at 0.2 mg / 100 mL each to degrade ATP. [Figure 5B] 1 is a graph of the Ca2+ dependence of sand fly Phlebotomus papatasi (Pp-apyrase) at 2.0 μg / 100 μL, respectively, to degrade ATP. [Figure 6] Graph showing exposure of 1 ng of ATP to various amounts of Pp-apyrase for 20 seconds followed by addition of cell lysis / ATP detection reagent. [Figure 7] 1 is a graph of time-dependent ATP elimination by Pp-apyrase. [Figure 8] 7 is a graph similar to that of FIG 6, in which 10 mM EGTA present in the Cell Lysis / ATP Detection Reagent inactivates Pp-apyrase. [Figure 9] 1 is a bar graph of results for cartridge configurations in which ATP was applied to absorbent swabs with or without Pp-apyrase. [Figure 10] 10 is a bar graph of results from a setup similar to that used in FIG. 9 but without the absorbent swab. [Figure 11] 1 is a bar graph showing the dependence of ATP elimination on Pp-apyrase concentration. [Figure 12] 1 is a bar graph showing the effectiveness of EGTA in inhibiting Pp-apyrase after the initial ATP elimination step.

[0029] It should be understood that the figures are not necessarily drawn to scale, nor are objects in the figures necessarily drawn to scale in relation to each other. The figures are representations intended to provide clarity and understanding of various embodiments of the devices, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, the drawings are not intended to limit the scope of the present teachings in any way. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present disclosure provides techniques relating to the detection of ATP in a sample. In particular, the present disclosure provides devices, systems, kits, and methods for sample collection and live cell ATP detection.

[0031] Conventional devices and configurations for detecting surface contamination detect total ATP, which is free ATP plus ATP contained in live cells. Free ATP limits the sensitivity of cell detection and represents background that may be present as a false positive signal. Two-cartridge approaches have been applied to liquid samples to selectively detect only live cell ATP versus total cell and free ATP. In these cases, substantially identical aliquots of the liquid sample are captured by each of the two cartridges, and the free ATP value is subtracted from the total to obtain a live cell only value. This approach is unsustainable for surface testing, as it is highly likely that two substantially identical samples will be collected. The methods, systems, and devices disclosed herein enable the integration of live cell ATP detection and free ATP exclusion in a single sample (e.g., a surface swab), solving the major challenge of providing a matched free ATP control via an orthogonal sample.

[0032] The disclosed devices and methods also enable live cell ATP detection with improved sensitivity and selectivity, reduced cost, and simplified workflow. The improved methods and devices use a conditionally active ATPase that is cost-effective in the quantities required to deplete ATP contamination in a short time, does not inhibit either activity, is sensitive to inhibitors suitable for incorporation into dual-purpose reagents for cell lysis and luciferase-dependent ATP detection, and is stable to storage and formulation in preloaded swabs. As shown, extracellular ATP was substantially depleted in about 5 to about 60 seconds, and the ATPase was rapidly inhibited to such an extent that detection of the ATP of interest (e.g., live cell ATP) was selective and sensitive. Exemplary assay devices described herein can provide results in less than one minute from sample collection. Previous devices, compositions, and / or reagents have primarily used Solanum tuberosum (potato) apyrase as an extracellular ATP scavenger, the inactivation of which requires means such as high temperature or ultra-pH treatment that are not suitable for use with the sensitivity and selectivity, cost reduction, workflow simplification, and speed of the disclosed methods and devices.

[0033] 1. Definition As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" also include plural referents unless the context clearly indicates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0034] With respect to references to numerical ranges herein, each intervening numerical value therebetween is expressly contemplated to the same degree of precision, for example, with respect to the range from 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and with respect to the range from 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0035] As used herein, the term "bioluminescence" refers to the production and emission of light by a chemical reaction catalyzed or enabled by an enzyme, protein, protein complex, or other biological molecule (e.g., a bioluminescent complex). In a typical embodiment, a substrate for a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted by the bioluminescent entity to an unstable form; the substrate subsequently emits light.

[0036] As used herein, the term "reagent" refers to compositions including, for example, chemicals (e.g., organic and inorganic compounds), enzymes, and combinations thereof. Reagents can be provided in gaseous, solid, or liquid form, or any combination thereof, and can be components of a solution or suspension. In some embodiments, reagents include fluids useful in methods of detecting ATP in a sample, such as cell lysis buffers, bioluminescence reagents, ATP-removing enzyme inactivators, buffers, etc.

[0037] As used herein, the term "detection" refers to the qualitative determination of the presence or absence of an analyte in a sample. The term "detection" further includes the quantification of an analyte in a sample, e.g., the amount and / or concentration of the analyte in a sample. The term "detection" also includes the "identification" of an analyte. As used herein, the terms "present" or "absent" (or alternatively "present" or "absent") are used in a relative sense to describe the amount or level of a particular entity (e.g., an analyte, e.g., ATP). For example, when an analyte is said to be "present" in a test sample, it means that the level or amount of the analyte is above a predetermined threshold, and conversely, when an analyte is said to be "absent" in a test sample, it means that the level or amount of the analyte is below a predetermined threshold. The predetermined threshold may be a detectability threshold associated with the particular test used to detect the analyte, or any other threshold. When an analyte is "detected" in a sample, the analyte is "present" in the sample. When an analyte is "not detected", the analyte is "absent" from the sample. Furthermore, a sample in which the analyte is "detected" or in which the analyte is "present" is a sample that is "positive" for the analyte. A sample in which the analyte is "not detected" or in which the analyte is "absent" is a sample that is "negative" for the analyte.

[0038] As used herein, the term "contacting" refers to bringing into contact or causing contact, being in contact, or coming into contact. As used herein, the term "contact" refers to the state or condition of being in contact or in direct or local proximity.

[0039] "Variant" is used herein to describe a peptide or polypeptide that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. Variant is also used to describe a protein with an amino acid sequence substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with an amino acid of different similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. "Variant" can also be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, but retains its biological activity.

[0040] As used herein, the term "sample" is used in the broadest sense. In one sense, it is meant to include specimens obtained from any source, including biological samples and environmental samples. In another sense, it refers to any sample that contains or is suspected of containing ATP. Biological samples can be obtained from animals (including humans) and can include fluids, solids, tissues, and gases. However, such examples should not be construed as limiting the type of sample. In some embodiments, the sample is a fluid sample, such as a liquid sample. Examples of liquid samples include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, and spinal fluid), water samples (e.g., water samples from the ocean, sea, lakes, rivers, etc.), samples from domestic, municipal, or industrial water sources, runoff, or sewage samples; and food samples (e.g., milk, beer, juice, or wine). Viscous liquid, semi-solid, or solid specimens can be used to create liquid solutions, eluates, suspensions, or extracts that can be samples. Liquid samples can be made from solid, semi-solid, or highly viscous materials, such as soil, fecal matter, tissues, organs, biological fluids, or other samples that are not liquid in nature. For example, a solid or semi-solid sample can be mixed with an appropriate solution, such as a buffer, diluent, and / or extraction buffer. The sample can be macerated, frozen and thawed, or otherwise extracted to form a liquid sample. In some embodiments, the sample is a wipe from an environmental surface. In environmental or other non-clinical applications, the sample can be derived from soil, dust, plants, or food, or an environmental swab (e.g., food and beverage manufacturing / preparation environments, clinical hygiene laboratory environments, etc.).

[0041] Samples may include biological material, such as cells, microorganisms, organelles, and biochemical complexes. In some embodiments, the sample is suspected of containing microorganisms. Non-limiting examples of suitable samples suspected of containing microorganisms include environmental samples (e.g., surface swabs / sponges, soil, sediment, fomites), food (e.g., raw materials, in-process samples, and finished product samples), beverages, clinical / veterinary samples (e.g., blood, serum, plasma, urine, sputum, tissue, mucus, feces, wound exudate, pus, cerebrospinal fluid), and water (e.g., surface water, drinking water, process water). In some embodiments, samples include food handling surface samples (e.g., conveyor belts, blades, cutting surfaces, mixing equipment surfaces, filters, storage containers), room samples (e.g., walls, floors, drains, ventilation systems), and cleaning equipment (e.g., hoses, cleaning tools). Microorganisms may indicate specific contamination or may be an indicator of general hygiene.

[0042] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms shall be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0043] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not limiting.

[0044] 2. Method The present disclosure provides a method comprising fully or partially removing ATP contamination from a sample by incubating the sample with a conditionally active ATPase. In some embodiments, the ATPase may be selected from the group consisting of apyrase from Cimex lectularus (e.g., NCBI Reference Sequences: NP_001303629 and XP_024081705 and GenBank: AAD09177.1), apyrase from Phlebotomus papatasi (e.g., GenBank: AAG17637.1), ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) (e.g., NCBI Reference Sequence: NP_006199.2), and combinations thereof. In selected embodiments, the ATPase is apyrase from Phlebotomus papatasi.

[0045] The sample may be incubated with the conditionally active ATPase for a period of time necessary to substantially deplete the sample of ATP contamination. For example, the incubation may remove more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more of the ATP contamination. The level of depletion of the sample of ATP contamination may be determined using the methods described in the Examples herein, particularly Examples 1 and 2.

[0046] In some embodiments, the sample is incubated for less than about 5 minutes (e.g., about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute, or less), In some embodiments, the sample is incubated for about 5 to about 60 seconds (e.g., about 5 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, or about 60 seconds).

[0047] Incubations are typically carried out at room temperature, however, any temperature necessary to substantially deplete the sample of ATP contamination is suitable for use in the methods of the present disclosure.

[0048] In some embodiments, incubating the sample with the conditionally active ATPase comprises incubating the sample with a solution comprising the conditionally active ATPase and other components (e.g., including buffers, additives, salts, stabilizers, etc.). In some embodiments, the solution further comprises a divalent cation, e.g., calcium ions, magnesium ions, and / or zinc ions, as a cofactor for the conditionally active ATPase. Preferably, the divalent cation is provided as a salt, e.g., CaCl2, ZnCl2, or MgCl2. A suitable concentration of the divalent cation is in the range of 1-6 mM.

[0049] The particular concentration and / or amount of the conditionally active ATPase will vary depending on the enzyme used, as well as the amount and type of sample, in some embodiments, about 0.1 to about 20 μg of the conditionally active ATPase is incubated with the sample. In some embodiments, about 0.1-15 μg, about 0.1-10 μg, about 0.1-5 μg, about 0.1-2 μg, about 0.1-1 μg, about 0.5-20 μg, about 0.5-15 μg, about 0.5-10 μg, about 0.5-5 μg, about 0.5-2 μg, about 0.5-1 μg, about 1-20 μg, about 1-15 μg, about 1-10 μg, about 1-5 μg, about 1-2 μg, about 5-10 μg, about 5-15 μg, about 5-20 μg, about 10-20 μg, or about 15-20 μg of the conditionally active ATPase is incubated with the sample.

[0050] The method may further comprise performing a biological assay using the sample obtained after incubation. In some embodiments, the biological assay detects the presence, absence, or amount of at least one analyte or biomarker. The analyte may be a protein, a nucleic acid, an antigen, a metabolite, or a fragment thereof.

[0051] In some embodiments, the biological assay comprises determining the presence or amount of ATP in the sample obtained after incubation with the conditionally active ATPase. Detecting or determining the amount of ATP in the sample can include any method that qualitatively or quantitatively determines the presence of ATP in a sample.

[0052] In some embodiments, detecting ATP comprises a bioluminescence assay or an assay with a detectable light product. In particular, any bioluminescence generating enzyme that is ATP-dependent may be suitable for use in the methods of the present invention. In some embodiments, detecting ATP comprises detecting the luminescence generated by the luciferase-luciferin reaction with a luminometer, although other detection means may be used. The presence of light above background levels indicates the presence of ATP in the sample. Suitable control reactions are easily designed by those skilled in the art.

[0053] In some embodiments, the method further comprises inhibiting the conditionally active ATPase before performing the bioassay and / or after incubation with the conditionally active ATPase. The method of inhibiting the conditionally active ATPase depends on the choice of the conditionally active ATPase and the downstream application of the sample (e.g., biological assay). In some embodiments, inhibiting comprises contacting the sample with an inhibitor of the conditionally active ATPase, including, for example, any small molecule, ion, protein, ligand, etc., that effectively stops the enzymatic reaction with ATP. In some embodiments, inhibiting comprises contacting the sample with EGTA, a zinc chelator, a small molecule inhibitor, or a combination thereof. In some embodiments, the inhibitor of the conditionally active ATPase comprises EGTA, which is suitable for inhibiting any of the calcium-dependent and / or zinc-dependent ATPases, such as Cimex lectularus apyrase, Phlebotomus papatasi apyrase, and ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1). In some embodiments, the inhibitor of the conditionally active ATPase comprises a zinc chelator suitable for inhibiting zinc-dependent ATPases such as ENPP1. In selected embodiments, the zinc chelator comprises N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). In some embodiments, the inhibitor of the conditionally active ATPase encompasses the small molecule inhibitor 6-[(3-aminophenyl)methyl]-N,N,5-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (also known as Inhibitor C), suitable for use in inhibiting ENPP1.

[0054] The particular concentration and / or amount of an inhibitor of a conditionally active ATPase will depend on the particular conditionally active ATPase used. Preferably, the IC 50 At least about 5-fold higher concentrations of the conditionally active specific ATPase are used.

[0055] The method may further include lysing the cells in the sample obtained after incubation prior to performing the biological assay. In some embodiments, the cells are lysed by chemical or enzymatic methods, such as treatment with detergents, pH adjusters, or membrane-disrupting small molecules or enzymes.

[0056] Thus, the present disclosure further provides a method for detecting intracellular ATP in a sample, for example, a sample suspected of containing cells. In some embodiments, a suitable method for detecting intracellular ATP in a sample may include collecting a sample suspected of containing cells, incubating the sample with a conditionally active ATPase, inhibiting the conditionally active ATPase, lysing the cells, and determining the presence or amount of ATP.

[0057] In some embodiments, the sample is collected on an absorbent pad. In some embodiments, the absorbent pad is pre-wetted with a solution containing a conditionally active ATPase. Thus, in some embodiments, incubation with the conditionally active ATPase is performed on the absorbent pad.

[0058] Since the concentration of ATP is relatively constant within a cell, measuring the ATP content in a sample can be used as a proxy for detecting or determining the number of live cells.Therefore, detecting intracellular ATP in a sample can provide an indication of live cells (e.g., microbial contamination) in the sample.Therefore, the present disclosure also provides a method for detecting or determining live cells (e.g., microbial contamination) in a sample of a sample.

[0059] In some embodiments, the method may use an assay device described herein. In some embodiments, the method includes collecting a sample on a collection swab of the assay device. First, the swab is removed from the empty reaction chamber and the tip of the swab is directly loaded by streaking across the surface or, in the case of a liquid sample, by dipping, wicking, or placing the liquid sample on the swab using a dropper or pipette. After the swab is inserted or placed in the reaction chamber, a reaction buffer is deployed from a reservoir into the container to contact the swab. Contacting the reagent with the swab results in a luminescent reaction with the luciferase enzyme and its luciferin substrate to determine the presence or amount of ATP in the sample.

[0060] The disclosed methods allow for complete or partial removal of ATP contamination and / or improved sensitivity and selectivity in ATP detection in a short period of time. In some embodiments, the methods are performed in less than about 10 minutes, e.g., 10 seconds to 10 minutes, 10 seconds to 5 minutes, 10 seconds to 2 minutes, 10 seconds to 1 minute, 30 seconds to 10 minutes, 30 seconds to 5 minutes, 30 seconds to 2 minutes, 30 seconds to 1 minute, 1 to 5 minutes, or 1 to 2 minutes. The methods can be performed in about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes.

[0061] 3. Assay Equipment The present technology relates to an assay device 100 (FIG. 1). In some embodiments, the present technology relates to analytical devices suitable for use in a variety of environments that enable rapid delivery of analytical results with minimal skill and user involvement, including, but not limited to, food and beverage production / preparation environments, clinical hygiene testing environments (e.g., surgical equipment and patient-exposed materials and surfaces), clinical and consumer product safety testing, environmental testing applications (e.g., water contamination), sterility testing, etc. As used herein, the terms "assay device," "assay device," and "detection device" are used interchangeably and refer to devices for detecting the presence, concentration, and / or amount of ATP in a sample or specimen.

[0062] 1, an exemplary assay device 100 is shown. The device 100 includes a reaction chamber 110 and a swab assembly 120. In the illustrated embodiment, the swab assembly 120 includes a cap 130 with a deployable reagent-filled reservoir, a swab shaft 140, and a collection swab 150. The assay device can be of any size or shape. In some embodiments, the size, shape, and orientation are configured for use in or with an optical detection device.

[0063] With continued reference to FIG. 1 , the collection swab 150 comprises an absorbent pad (e.g., a cotton pad) configured to receive and absorb a sample. In some embodiments, the collection swab 150 is pre-wetted with a solution containing a conditionally active ATPase. The conditionally active ATPase is selected from the group consisting of: Cimex lectularius apyrase, Phlebotomus papatasi apyrase, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), and combinations thereof. In some embodiments, the conditionally active ATPase is Phlebotomus papatasi apyrase.

[0064] In some embodiments, about 0.1-20 μg, about 0.1-15 μg, about 0.1-10 μg, about 0.1-5 μg, about 0.1-2 μg, about 0.1-1 μg, about 0.5-20 μg, about 0.5-15 μg, about 0.5-10 μg, about 0.5-5 μg, about 0.5-2 μg, about 0.5-1 μg, about 1-20 μg, about 1-10 μg, about 1-5 μg, about 1-2 μg, about 5-10 μg, about 5-15 μg, about 5-20 μg, about 10-20 μg, or about 15-20 μg of the conditionally active ATPase is pre-wetted onto the collection swab.

[0065] The solution containing the conditionally active ATPase may further comprise buffers, additives, salts, stabilizers, etc. that do not adversely affect downstream detection of ATP in the sample and / or lysis of cells. In some embodiments, the solution further comprises a divalent cation, e.g., calcium ions, magnesium ions, and / or zinc ions, as a cofactor for the conditionally active ATPase. In some embodiments, the divalent cation is provided as a salt, e.g., CaCl2, ZnCl2, or MgCl2. In some embodiments, the solution comprises 1-6 mM (e.g., about 1 mM, about 3 mM, about 5 mM) of divalent cation.

[0066] The device further includes a cap 130. The cap 130 may comprise a reservoir containing a reaction buffer. In some embodiments, the reservoir includes an articulatable element for releasing the reaction buffer into the reaction chamber. In some embodiments, the articulatable element includes a seal that can be broken or a valve that can be opened to dispense the reaction buffer into the reaction chamber. Any means for sealing the reaction buffer in the reservoir that allows a user to release the reaction buffer into the reaction chamber is suitable for use with the device.

[0067] The reaction buffer comprises a cell lysis reagent, an inhibitor of the conditionally active ATPase, an ATP detection reagent, or a combination thereof.

[0068] Lysis reagents (e.g., lysis reagents in lysis buffers) are known to those of skill in the art. In some embodiments, the cell lysis reagent comprises a detergent, such as a mild non-denaturing detergent (e.g., Triton® X-100 or CHAPS) or other detergent with lytic activity against microorganisms. In some embodiments, the cell lysis reagent comprises an enzyme(s) or other agent(s) that promotes lysis, including, for example, lysozyme, labiase, lysostaphin, achromopeptidase, alpha-hemolysin, chitinase, streptolysin O, tetanolysin, and mutatricin.

[0069] The conditionally active ATPase inhibitor may include one or more small molecules, ions, proteins, ligands, etc. that effectively stop the enzymatic reaction with ATP. In some embodiments, the conditionally active ATPase inhibitor includes EGTA. In some embodiments, the conditionally active ATPase inhibitor includes a zinc chelator. In selected embodiments, the zinc chelator includes N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). In some embodiments, the conditionally active ATPase inhibitor includes a small molecule inhibitor. In selected embodiments, the small molecule inhibitor includes 6-[(3-aminophenyl)methyl]-N,N,5-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine, also known as inhibitor C. In some embodiments, the conditionally active ATPase inhibitor includes any two or more of EGTA, a zinc chelator, and a small molecule inhibitor.

[0070] ATP detection reagents may include any reagent useful for measuring the presence of ATP in a qualitative or quantitative manner, including, for example, a product that provides a detectable light product. In particular, any bioluminescence generating enzyme that is ATP-dependent may be suitable for use in the methods and devices of the present invention.

[0071] In some embodiments, the ATP detection reagent comprises a luciferase enzyme and a luciferin substrate. The luminescence produced by the luciferase-luciferin reaction is typically detected with a luminometer, although other detection means can be used. The presence of a light level higher than background level indicates the presence of ATP in the sample, and in some embodiments, the presence of live cells. Suitable control reactions are readily designed by one of skill in the art.

[0072] The materials required, as well as the specific concentrations and / or amounts of materials required to generate a luminescent signal, will vary depending on the luciferase enzyme used. In some embodiments, the ATP detection reagent further comprises cofactors and other molecules useful for the reaction. Generally, for beetle luciferases, the additional materials include ATP and magnesium (Mg), such as magnesium sulfate. 2+ In some embodiments, other substances can be added to the solution, including buffers to maintain the reaction at the appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) to help maintain luciferase activity, reducing agents, detergents, esterases, salts, amino acids (e.g., D-cysteine), and the like.

[0073] As used herein, the term "luciferin substrate" refers to a molecule that can generate light through a chemical or biochemical reaction (e.g., luciferin, a luciferin derivative, or a functional analog thereof). A luciferin substrate can be a molecule that can generate the light generated by luciferase. Suitable luciferin substrates for luciferase enzymes include luciferin, luciferin derivatives, and functional analogs of luciferin. A naturally occurring substrate for beetle luciferase is firefly luciferin, D-(-)-2-(6'-hydroxy-2'-benzothiazolyl)-Δ2-thiazoline-4-carboxylic acid (D-luciferin), a polyheterocyclic organic acid. Luciferin can be isolated from nature (e.g., fireflies) or can be synthesized. Synthetic luciferin can have the same structure as naturally occurring luciferin or can be derivatized as long as it functions similarly. Examples of derivatives of luciferin include D-luciferin methyl ester and other esters of luciferin, which are hydrolyzed or acted upon by esterases in the sample to produce luciferin, as well as naphthyl-luciferin and quinolyl-luciferin (Branchini et al., 1989). There are several commercial sources of luciferin (e.g., Promega Corp. Madison, Wis.).

[0074] Any luciferase enzyme or its variant or derivative that meets the limitations described herein, such as the luciferase that uses ATP as substrate, can be used in the method, device, or kit disclosed herein.To date, several classes of luciferase have been identified.Among these, beetle luciferases, such as the luciferase of the common firefly (family Lampyridae), form a separate class with unique evolutionary origin. Suitable luciferase enzymes include, but are not limited to, those selected from the group consisting of Photinus pyrali, i.e. North American firefly luciferase; Luciola cruciata, i.e. Japanese firefly or Genji firefly luciferase; Luciola italic, i.e. Italian firefly luciferase; Luciola lateralis, i.e. Japanese firefly or Heike luciferase; Luciola mingrelica, i.e. Eastern European firefly luciferase; Photris Pennsylvaniaca, i.e. Pennsylvania firefly luciferase; Pyrophorus plagiophthalamus, i.e. click beetle luciferase; and Phrixhothrix hirtus, i.e. railroad worm luciferase. Optionally, the luciferase used in the compositions and methods of the present invention has enhanced thermostability and / or chemical stability properties.

[0075] In some embodiments, Photuris Pennsylvanianica firefly luciferase (LucPpe2; 545 amino acid residues; GenBank2190534), or a derivative or variant thereof, is used. Thermostable and / or chemically stable mutant luciferases derived from LucPpe2 (e.g., LucPpe2m78 (also known as 78-0B10); LucPpe2m90 (also known as 90-1B5); LucPpe2m133 (also known as 133-1B2); LucPpe2m146 (also known as 146-1H2) may also be used (see U.S. Patent No. 8,603,767, which is incorporated herein by reference in its entirety).

[0076] In some embodiments, the assay device 100 includes one or more labels or other scribable or scribed surface(s) on which information is printed, written, or displayed. The labels may be attached to the exterior surface by gluing, imprinting, texturing, scribing, etching, surface treating, impregnating, painting, screen printing, staining, coloring, embossing, or other suitable methods. In some embodiments, the labels include instructions for using the device.

[0077] In some embodiments, the assay device 100 and its components described herein are constructed using construction methods known in the mechanical arts or medical device construction arts. The materials from which the assay device 100 is manufactured can vary. In some embodiments, the assay device 100 includes materials that are metals, silicon, glass, ceramics, plastics, and synthetic and natural polymers, as well as combinations and mixtures thereof. In some embodiments, the assay device 100 includes polypropylene and / or high impact polystyrene compositions using suitable manufacturing methods (e.g., pressure injection molding, machining, three-dimensional printing, etc.). In some embodiments, the assay device 100 or its components are constructed using other suitable manufacturing methods such as milling, casting, spraying, spinning, and other methods known in the mechanical arts and medical device construction.

[0078] 4. Kits and Systems The present disclosure provides kits that include the devices disclosed herein or their separate components. In some embodiments, the kits include a swab assembly that includes a conditionally active ATPase, a collection swab configured to receive and absorb a sample, and a cap, a reaction chamber configured to receive the swab assembly, and a reaction buffer that includes a cell lysis reagent, an inhibitor of the conditionally active ATPase, an ATP detection reagent, or a combination thereof. The descriptions of the conditionally active ATPase, the swab assembly, the collection swab, the cap, the reaction chamber, the reaction buffer, the inhibitor of the conditionally active ATPase, and the ATP detection reagent provided elsewhere herein are suitable for use in the disclosed kits. In some embodiments, the conditionally active ATPase is provided on a collection swab that is pre-wetted. In some embodiments, the reaction buffer is provided pre-filled in a reservoir in the cap.

[0079] The reagents included in the kit may be provided in any type of container such that the activity of the different components is preserved and the components are not adsorbed or altered by the material of the container. For example, a sealed glass ampoule may contain lyophilized luciferase or buffers packaged under a neutral non-reactive gas such as nitrogen. The ampoules may be composed of any suitable material such as glass, organic polymers such as polycarbonate, polystyrene, ceramics, metals, or any other material typically used to hold reagents. Other examples of suitable containers include simple bottles that may be manufactured from similar materials as ampoules, and envelopes that may consist of a foil-lined interior such as aluminum or alloys. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container may have a sterile access port, such as a bottle with a stopper that can be pierced by a hypodermic needle. Other containers may have two compartments separated by an easily removable membrane, which, upon removal of the membrane, allows the components to be mixed. The removable membrane may be glass, plastic, rubber, and the like. The individual member components of the system or kit may be physically packaged together or separately.

[0080] The kit may also include instructions for using the components of the kit. Instructions are materials or methodologies related to the kit. The materials may include background information, a list of components, simple or detailed methods for using the system, troubleshooting, technical support, and any other related documentation thereof. The instructions may be provided with the kit or as a separate member component, in paper or electronic form, provided on a computer readable memory device, downloaded from an internet website, or provided as a recorded presentation.

[0081] The present disclosure also provides a system including the assay device or kit described herein. For example, in some embodiments, the present technology provides a system including the assay device or kit described herein and an optical reader (e.g., a luminometer) configured to record, calculate, display, or communicate results. In some embodiments, the system and / or the optical reader includes a computer-based analysis program that converts the results (e.g., the presence, absence, concentration, and / or amount of ATP) into an index for a user. EXAMPLES

[0082] Example 1 Sample Collection and Assay Cartridge Device To improve the sensitivity and selectivity of the live cell ATP detection cartridge design, an ATP degrading enzyme (ATPase) was incorporated into the non-lytic absorbent swab wetting solution, combining the cell lysis function in the ATP detection reagent (Figure 1). ATPase degrades extracellular ATP while leaving intracellular ATP intact by sequestration of ATP within intact cell walls and membranes that exclude apyrase.

[0083] The device uses a swab with an absorbent tip to collect a sample, for example, from a potentially dirty surface by streaking the swab across its surface, or from a potentially contaminated liquid applied directly to the swab. Prior to sample collection, the swab is pre-wetted with a solution containing a conditionally active ATP-degrading enzyme (e.g., ATPase) that substantially removes free ATP while leaving intracellular ATP intact. The ATPase is added to the tip of the swab in a stable solution suitable for long-term storage at 4°C or short-term storage at room temperature. After a short holding time (e.g., 10-20 seconds) to allow for sample collection and removal of free ATP, the swab is immersed in a cell lysis solution. This cell lysis solution also contains an ATPase inactivator and a luciferase-based ATP detection formulation. The lysis ability of the formulation releases ATP from viable cells, the ATPase inhibitor protects the released ATP from the applied ATPase, and the luciferase reaction produces an ATP-dependent luminescent signal proportional to the number of viable cells. The elimination of free ATP by ATPase has the effect of reducing non-cellular background ATP, thereby increasing the sensitivity for detecting cells and reducing the probability of false positive signals caused by free ATP.

[0084] Example 2 ATP exclusion Ca2 for use as a conditionally active ATP scavenger + To test for dependent apyrases, apyrase from the bedbug Cimex lectularius (Cl-apyrase) was expressed as a His-tagged fusion protein in E. coli and purified by nickel affinity. Cl-apyrase was serially diluted in 25 μl of buffer containing 50 mM Tris-HCl (pH 6.5), 50 mM NaCl, and 5 mM CaCl2 over the range of 20 to 0.02 μg / ml. The diluted enzyme was dispensed into wells of a 96-well plate. 25 μl of 10 nM ATP was dispensed into all wells and incubated for 1, 2, or 5 min. Luciferase / luciferin detection reagent was added and luminescence output was recorded. Approximately 3 μg / ml of Cl-apyrase removed all ATP within 1 min (Figure 2).

[0085] In addition to Cl-apyrase, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) was identified and tested as another enzyme that meets the criteria for use as an ATPase. ENPP1 converts ATP to AMP and pyrophosphate, is zinc-dependent, and is inactivated by EGTA, by the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and by certain small molecule inhibitors.

[0086] ENPP1 (100 ng / reaction) was incubated with 10 μM ATP and various amounts of chelators (EGTA, TPEN) or an ENPPI-selective inhibitor, termed inhibitor C, in a reaction buffer containing 50 mM Tris HCl (pH 7.5), 250 mM NaCl, 5 uM ZnCl2, 0.1 mg / ml BSA. The reaction was allowed to proceed for 60 min. The amount of remaining ATP was measured by the addition of a commercial ATP detection reagent (Kinase-Glo®, Promega Corp.) containing luciferase and luciferin, as shown in FIG. 4. ENPP1 qualitatively exhibited conditionally active ATP scavenging properties.

[0087] Ca from potato Solanum tuberosum 2+ / Mg 2+ -apyrase (St-apyrase) is the most commonly used apyrase for ATP scavenging. However, its inactivation requires measures such as high temperature or extreme pH treatment, which are not compatible with the simple cartridge design disclosed herein. The need for extended heating times for apyrase inactivation is also not compatible with the goal of rapid sample collection and analysis. For comparison with Cl-apyrase (Ca-apyrase), a commercially available St-apyrase preparation (Sigma-Aldrich) was used. Cl-apyrase and St-apyrase were compared for their ability to scavenge extracellular ATP and for Ca 2+ The ability to be inhibited by the selective chelator EGTA was compared.2+ is essential for luciferase activity, so Mg 2+ The selective chelator EDTA was not tested. In the absence of EGTA, both enzymes excluded ATP, as shown by a time-dependent decrease in luminescence (Figure 3). In the presence of EGTA, St-apyrase was only partially inhibited and therefore remained substantially active (luminescence decreased over time). In contrast, with the same amount of EGTA present, no Cl-apyrase activity was detected (there was no decrease in luminescence over time), thus demonstrating the superior sensitivity of Cl-apyrase to inactivation by EGTA.

[0088] Example 3 Phlebotomus papatasi apyrase Apyrase from the sand fly Phlebotomus papatasi (Pp-apyrase) also reacts with Ca2 + Pp-apyrase was characterized for its ATP-dependent activity and sensitivity to inhibition by EGTA. Pp-apyrase was expressed as a glutathione S-transferase (GST) fusion in an E. coli expression system and purified by glutathione affinity. Pp-apyrase was then formulated with 100 mM HEPES (pH 7.5), 0.2 mg / ml BSA, 0.1% NaN3, and 10% glycerol. CaCl2 was included at 5 mM or in the range of 1 to 6 mM. Figures 5A and 5B show the CaCl2-dependent activity of 0.2 or 2.0 mg / 100 mL of Pp-apyrase to catalyze ATP decomposition. 2+ The results show that Pp-apyrase is a 1- or 2-minute incubation of 5 ng of ATP followed by the addition of a commercial ATP detection reagent (CellTiter-Glo®, Promega Corp.). Reactions were performed in microcentrifuge tubes and luminescence was analyzed in a single-tube reading luminometer (GloMax® 20 / 20, Promega Corp.). Reactions using 0.2 mg of Pp-apyrase demonstrated that the enzyme is sensitive to >4 mM Ca 2+ It is clear that the most active is at 0.05 mg, with the 2.0 mg reaction showing that nearly all of the ATP can be eliminated in less than 1 min.

[0089] Figure 6 shows the results of exposing 1 ng ATP to various amounts of Pp-apyrase for 20 seconds, followed by addition of a lysis / ATP detection reagent consisting of 100 mM HEPES (pH 7.5), 10 mM MgCl2, 5 mM Na-citrate, 10% glycerol, 0.2 mg / ml BSA, 10 mM EGTA, 0.02% CTAB, 0.2% Thesit, 0.002% CHEX, 200 ug / ml ultraGlo luciferase, 3 mM D-luciferin, and 0.1% NaN3. Luminescence signal was measured over time starting immediately after addition of the lysis / ATP detection reagent. Reactions were performed in microcentrifuge tubes and luminescence was analyzed with a single tube reading luminometer (GloMax® 20 / 20, Promega Corp.).

[0090] Figure 7 shows the time-dependent ATP clearance by Pp-apyrase, where the formulation described in Figure 6 was supplemented with 5 mM Ca. 2+ Two micrograms of enzyme containing Pp eliminated all or most of the 1 ng ATP sample within 10 seconds. The legend indicates the incubation time with Pp-apyrase before measuring luminescence. The graph shows the luminescence after Pp incubation when the tubes were placed in the luminometer. Reactions were performed in microcentrifuge tubes and luminescence was analyzed in a single tube reading luminometer (GloMax® 20 / 20, Promega Corp.).

[0091] Figure 8 shows that 10 mM EGTA present in the cell lysis / ATP detection reagent described in Figure 6 effectively inactivated Pp-apyrase. This was demonstrated by showing that apyrase degrades ATP before EGTA is added, but does not degrade ATP added after EGTA is introduced into the mixture. Reactions were performed in microcentrifuge tubes and luminescence was analyzed with a single-tube reading luminometer (GloMax® 20 / 20, Promega Corp.).

[0092] Thus, both the calcium-dependent apyrase (Pp-apyrase) from the sand fly Phlebotomus papatasi and the calcium-dependent apyrase (Cl-apyrase) from the bedbug Cimex lectularus tested herein demonstrated rapid and substantial ATP elimination capabilities. Both were substantially inactivated by the Ca2+-selective divalent metal chelator EGTA incorporated into the lytic detection reagent without substantially reducing its lysis or ATP detection capabilities. Each was also stable in a HEPES-buffered formulation containing 10% glycerol, sodium azide, and bovine serum albumin, which did not substantially affect the lysis or ATP detection capabilities of the luciferase-based lytic detection reagent. The stability of the lytic detection reagent at room temperature and 4°C allows the use of a thermostable firefly luciferase enzyme (UltraGlo™ luciferase, Promega Corp.). Example 4 Cartridge detection

[0093] Apyrase from the sand fly Phlebotomus papatasi (Pp-apyrase) was also characterized for use in the exemplary cartridge configuration. When ATP was added to an absorbent swab with or without Pp-apyrase, the method tested shows subsequent ATP detection in the exemplary cartridge (Figure 9). Empty plastic cartridges were obtained from Empire Bio Diagnostics. An aliquot of ATP solution was pipetted onto the tip of a pre-wetted swab with or without Pp-apyrase in the ATP displacement reagent formulation described above. After a 20 second exposure of ATP within the tip of the swab in the cartridge receptacle, the tip of the swab was subsequently immersed in the ATP detection / cell lysis formulation with 10 mM EGTA described in Figure 6. The cartridge receptacle is then inserted into a handheld luminometer (luminometer prototype by Empire Bio Diagnostics). Luminescence readings from the cartridge reflected the detection of a range of ATP concentrations that were effectively eliminated by 20 seconds of exposure to Pp-apyrase. Some of the values ​​shown above the vertical axis represent luminescence values ​​so low that they cannot be seen as bars on the graph. These results are similar to those obtained in solution in microcentrifuge-type tubes (see, for example, Figures 6-8), indicating that the absorbent matrix at the tip of the swab does not substantially reduce apyrase activity or the detection of bioluminescent ATP. A similar experiment was performed without an absorbent swab (Figure 10), in which ATP and Pp-apyrase formulations were added directly to the cartridge receptacle. In comparison to Figure 9, these results confirmed that the ATP elimination and detection reactions were not significantly affected by the swab matrix.

[0094] The dependence of ATP elimination on Pp-apyrase concentration was investigated, as shown in Figure 11. In a 20 s reaction, 1-2 μg of enzyme was required to eliminate most or all of the ATP.

[0095] In the cartridge configuration, EGTA was effective in inhibiting Pp-apyrase after the initial ATP elimination step (Figure 12). As shown in the test tube format in Figure 8, Pp-apyrase present in the absorbent swab does not degrade ATP that is added after the lysis / ATP detection reagent containing EGTA is introduced into the mixture.

[0096] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be construed as limiting the scope of the disclosure, which is defined solely by the appended claims and equivalents thereof.

[0097] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and can be made without departing from the spirit and scope thereof.

Claims

1. A method for completely or partially removing ATP contamination from a sample by incubating the sample with a conditionally active ATP-degrading enzyme selected from the group consisting of Cimex lectularius apirase, Phlebotomus papatasi apirase, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), and combinations thereof, The method further includes performing a bioluminescence assay using the sample obtained after incubation, A method comprising the bioluminescence assay detecting residual ATP in the sample obtained after incubation.

2. The method according to claim 1, wherein the sample is incubated for less than approximately 5 minutes.

3. The method according to claim 1 or claim 2, wherein the sample is incubated for about 5 to about 60 seconds.

4. The method according to claim 1, further comprising inhibiting the conditionally active ATP-degrading enzyme in the sample obtained after incubation.

5. The method according to claim 4, wherein the inhibition comprises contacting the sample with EGTA, a zinc chelating agent, a small molecule inhibitor, or a combination thereof.

6. The method according to claim 1, further comprising lysing cells in a sample obtained after incubation and / or before performing the bioluminescence assay.

7. The method according to claim 1, wherein the presence of ATP indicates the presence of living cells in the sample.

8. A method for detecting intracellular ATP, Collecting samples that contain or are suspected of containing cells; The aforementioned sample is incubated with a conditionally active ATP-degrading enzyme to substantially degrade all extracellular ATP in the aforementioned sample; Inhibiting the active ATP-degrading enzyme under the aforementioned conditions; To dissolve the aforementioned cells; This includes determining the presence or amount of ATP, The method wherein the conditionally active ATP-degrading enzyme is selected from the group consisting of: apirase from Cimex lectularius, apirase from Phlebotomus papatasi, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), and combinations thereof.

9. The method according to claim 8, wherein the sample contains or is suspected to contain microorganisms.

10. The method according to claim 8 or 9, wherein the sample is collected on an absorbent pad or a collection swab.

11. The method according to claim 10, wherein the absorbent pad is pre-moistened with a solution containing the conditionally active ATP-degrading enzyme.

12. The method according to claim 8, wherein the sample is a liquid sample or an environmental sample.

13. The method according to claim 8, wherein the inhibition comprises contacting the sample with EGTA, a zinc chelating agent, a small molecule inhibitor, or a combination thereof.

14. The method according to claim 8, wherein determining the presence or amount of the ATP includes a bioluminescence assay.

15. The method according to claim 8, wherein the sample is incubated for less than approximately 5 minutes.

16. The method according to claim 8, wherein the sample is incubated for about 5 to about 60 seconds.

17. The method according to claim 8, wherein the method is performed in less than approximately 10 minutes.

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