Culture device containing oxygen-sensitive luminescent phosphatids and method of use

Abstract

This invention provides a culture device containing an oxygen-sensitive luminescent phosphine and a method for using it. [Solution] The present invention provides a culture device comprising an oxygen-sensitive phosphorescent phosphodiol, typically such as a porphyrin, and a method for using it to culture and count microorganisms.

Description

Background Art

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[0001] The paper "Non-invasive transdermal two-dimensional mapping of cutaneous oxygenation with a rapid drying liquid bandage" (Li et al.) discloses quantifying and mapping both the pO2 and oxygen consumption of the underlying tissue and oxygen consumption using the oxygen-dependent phosphorescence of a bandage.

[0002] The paper "The triplet state in Pt-acetylide oligomers, polymers and copolymers" (Silverman et al.) discloses that platinum acetylide oligomers and polymers are π-conjugated materials that exhibit luminescence from triplet excitons. [[ID=​​​​​​​​​​​​​U.S. Patent Publication No. 20180312895 discloses a device for counting microbial colonies. The growth compartment of the device contains a cold water-soluble gelling agent, a dry oxygen scavenger, a dry buffer system, and an effective amount of dry carbon dioxide generator. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 3338794 [Patent Document 2] U.S. Patent Application Publication No. 20180312895 [Overview of the project]

[0008] Throughout this disclosure, singular forms such as “a,” “an,” and “the” are often used for convenience, but unless explicitly specified to be singular only or clearly indicated by the context, singular forms are intended to include plurals. When referring to singular only, the term “one and only one” is typically used.

[0009] Some terms used in this disclosure are defined below. Other terms are well known to those skilled in the art, and those terms are to be given the meanings that those skilled in the art have assigned to them.

[0010] Terms indicating high frequency, such as “common / general,” “typical,” and “usual,” as well as “commonly / generally,” “typically,” and “usual,” are used in the present invention to refer to features that are frequently used, and these are not intended to mean that the features are present in the prior art unless used specifically in reference to the prior art, nor do they mean that these features are common, common, or typical in the prior art.

[0011] The term "oxygen-sensitive dye" refers to a chemical substance that changes the wavelength or intensity of light it absorbs or emits in the presence of oxygen. Compounds that do not absorb or emit light in the absence of oxygen, but absorb or emit light in the presence of oxygen, are a type of "oxygen-sensitive dye." Oxygen-sensitive chromophores (as defined herein), as well as oxygen-sensitive phosphors (as defined herein) and colorimetric oxygen dyes (as defined herein), are examples of oxygen-sensitive dyes.

[0012] The term "colorimetric oxygen dye" refers to a chemical substance that, in the presence of oxygen (as opposed to the absence of oxygen), changes the wavelength of light it absorbs, particularly ultraviolet or visible light (such as the maximum absorption wavelength or λmax). This change does not necessarily have to be reversible. Certain colorimetric oxygen dyes do not absorb enough light to be visible to the human eye in the absence of oxygen, but absorb enough light to be visible to the human eye when exposed to oxygen, while other specific colorimetric oxygen dyes have a first λmax in the absence of oxygen and a second, different λmax after exposure to oxygen. In any case, the change may be reversible in that the colorimetric oxygen dye can return to its pre-oxygen-exposed state if the oxygen is removed or if the change is irreversible.

[0013] The term "luminescent group" refers to a chemical substance that exhibits light emission.

[0014] The term "oxygen-sensitive luminescent phosphatid" refers to a luminescent phosphatid that exhibits quenching in the presence of oxygen.

[0015] The term "phosphor" refers to a luminescent group that exhibits phosphorescence. Phosphorers can also exhibit fluorescence, but this is not essential.

[0016] The term "oxygen-sensitive phosphorescent material" refers to a phosphorescent material whose phosphorescence is quenched in the presence of oxygen. If a phosphorescent material exhibits fluorescence, the fluorescence may also be quenched in the presence of oxygen, but this is not essential.

[0017] "Oxygen-scavenging system" refers to a chemical, biological, or mechanical system, which may be an enzymatic or other chemical system designed to consume substantially all of the oxygen in the growth compartment of a culture device. However, the oxygen-scavenging system does not include microorganisms cultured on the culture device, such as in the growth compartment of a culture device.

[0018] The verb "to extinguish" and its conjugations mean to cause a decrease in luminescence intensity, and when used in relation to phosphorescent materials or phosphorescence, more specifically, it means to cause a decrease in phosphorescence intensity. Therefore, when a phosphorescent material is extinguished by oxygen, the phosphorescence intensity of the phosphorescent material decreases with increasing partial pressure of oxygen.

[0019] This disclosure acknowledges that in techniques for culturing and detecting microorganisms, there is often a problem in that it is necessary to stain the cultured microorganisms or otherwise impart color to them. Even when staining is not necessary, it may be necessary to rely on detecting the intrinsic color of the microorganisms. In either case, detection relies on a light source outside the culture device, such as a lamp in the detector or another illumination source. This not only needs to be constructed to have a special lamp to illuminate the culture device, but also needs to be configureable to repeatedly provide the same illumination conditions in order to provide consistent results. When microorganisms are to be counted, the problem is even more difficult because the illumination conditions must be repeatable in luminosity to ensure that the count is correct.

[0020] A related issue is the use of oxygen-sensitive dyes for detecting cultured microorganisms, and more specifically, for counting cultured microorganisms.

[0021] A related issue is the use of synchrotron radiation for detecting cultured microorganisms, and more specifically, for counting cultured microorganisms.

[0022] The present disclosure also recognizes problems in the field of air-sensitive phosphors, more specifically oxygen-sensitive phosphors. Thus, another problem is the use of an oxygen-sensitive luminophore, more specifically an oxygen-sensitive phosphor, to detect the presence of cultured microorganisms. A related problem is the use of a porphyrin-containing material to detect, more specifically to count, cultured microorganisms.

[0023] The present disclosure also recognizes problems in the field of colorimetric oxygen dyes. Thus, another problem is the use of a colorimetric oxygen dye to detect the presence of cultured microorganisms.

[0024] These and related problems are addressed by the use of the culture device described herein. The culture device has a growth compartment surrounded by one or more oxygen-impermeable barriers. At least one of the oxygen-impermeable barriers is configurable between an open configuration and a closed configuration. In the open configuration, the growth compartment is exposed to the environment outside the growth compartment. In the closed configuration, the growth compartment is sealed from exchanging oxygen with the environment outside the growth compartment.

[0025] The culture device also includes a medium that can support the replication of at least one microorganism disposed within the growth compartment. Also, an oxygen-sensitive dye, particularly a colorimetric oxygen dye or an oxygen-sensitive luminophore, more specifically an oxygen-sensitive luminophore, is disposed within the growth compartment.

[0026] The various embodiments of the culture device and method described herein can be used to address the aforementioned problems and other problems.

[0027] In any of the culture devices described herein, one or more oxygen-impermeable barriers can be those used in 3M (trademark) Petrifilm (trademark) plates for measuring the number of lactic acid bacteria (available from 3M Company, St. Paul MN, USA). The oxygen-impermeable barrier can include such materials as polyethylene, for example, low-density polyethylene, linear low-density polyethylene, etc., foils such as aluminum foil, and other oxygen-impermeable materials known in the art, and one material or a combination of materials can be used to create the oxygen-impermeable barrier.

[0028] In relation to any of the aforementioned culture devices, at least one of the oxygen-impermeable barriers preferably includes a coverslip. In any culture device where a coverslip is present, the open configuration can be a configuration where the coverslip is on the growth section, and the closed configuration can be a configuration where the coverslip is at least partially removed from the growth section.

[0029] In any of the aforementioned culture devices, a port can be present in at least one of the one or more oxygen-impermeable barriers, whereby the port can be converted between an open configuration and a closed configuration. For example, when the port is in the open configuration, the growth section can be inoculated, and then the port can be closed.

[0030] In relation to any of the aforementioned culture devices, the medium can be any type of medium and can vary depending on the type of microorganism to be cultured, the detection method used, or other practical considerations. For example, in any of the aforementioned embodiments of the culture device, the medium can be a thin-film medium, more specifically, a cold-water gelling thin film medium. This type of medium is commercially available, such as those sold under the PETRIFILM (trademark) brand by 3M Company St. Paul MN USA. Alternatively, agar can be used as the medium in any of the aforementioned culture devices.

[0031] Any suitable oxygen-sensitive dye can be used with respect to any of the culture devices described herein. Examples of oxygen-sensitive dyes include colorimetric oxygen dyes and oxygen-sensitive luminescent phospholipids.

[0032] An oxygen-sensitive luminescent phore is a specific oxygen-sensitive dye that can be used. With respect to any of the culture devices described herein, the oxygen-sensitive luminescent phore may be any luminescent phore that is quenched by oxygen. Preferably, in any culture device, the oxygen-sensitive luminescent phore is an oxygen-sensitive phosphor. With respect to any of the aforementioned culture devices, the oxygen-sensitive phosphor may advantageously include at least one of porphyrin, or a π-conjugated molecule, or a π-conjugated polymer. With respect to any of the culture devices described herein, the oxygen-sensitive phosphor may include a dendrimer. With respect to any of the culture devices described herein, the oxygen-sensitive phosphor may include a porphyrin. With respect to any of the culture devices described herein, the oxygen-sensitive phosphor may include a π-conjugated molecule. In any of the disclosed culture devices in which a π-conjugated molecule is used, the π-conjugated molecule may advantageously include a π-conjugated ligand to a transition metal or lanthanide. Examples of these include cyclometalated complexes of iridium(III) or platinum(II), particularly disubstituted pyridines, especially pyridines such as aryl or cycloarylpyridines, and more specifically, phenylpyridine complexes of iridium(III) or platinum(II). Other examples include pyridine systems, more specifically, polypyridyl complexes of ruthenium(II), osmium(II), or rhenium(II). The π-conjugated ligand may be a bipyridine in any of the culture devices in which it is used. "Bipyridine" means that a bipyridine moiety is present in the molecule, but other moieties may be present or not present, and if other moieties are present, they are directly or indirectly bound to the bipyridine moiety. The π-conjugated ligand may be an acetylide in any of the culture devices in which it is used. In any of the culture devices in which an acetylide is used, the acetylide may be phenyleneethynylene or polyphenyleneethynylene."Phenyleneethynylene or polyphenyleneethynylene" means that a phenyleneethynylene or polyphenyleneethynylene moiety is present in the molecule, but other moieties may be present or not present at all, and if other moieties are present, they are directly or indirectly bound to the phenyleneethynylene or polyphenyleneethynylene moiety. The π-conjugated ligand may be a porphyrin in any of the culture devices in which it is used. The π-conjugated ligand may be a dendrimer in any of the culture devices in which it is used. Advantageously, the π-conjugated ligand may be a porphyrin-containing dendrimer in any of the culture devices in which it is used.

[0033] With respect to any of the culture devices referred to herein, a metal may be conjugated to an oxygen-sensitive luminescent phore, which may be any of the oxygen-sensitive luminescent phores referred to herein, more specifically, to a π-conjugated molecule. In any case in which a metal is conjugated to a π-conjugated molecule, the metal is advantageously a transition metal or a lanthanide, but other metals such as actinides may also be used. Transition metals are most commonly used when a metal is conjugated to a π-conjugated molecule. In any culture device in which a metal is conjugated to any luminescent phore, the conjugation may be by any type of chemical interaction, such as ligation, covalent bonding, ionic bonding, or van der Waals interactions.

[0034] With respect to any of the aforementioned culture devices, the transition metal conjugated to the π-conjugated molecule, if used, is preferably selected from palladium, platinum, rhenium, or ruthenium. However, it should be understood that other transition metals may also be used. In any culture device in which a lanthanide is used, the lanthanide is most commonly iridium. In all cases, including when the oxygen-sensitive phosphorus contains a metal and the metal is a transition metal, a lanthanide, or other such metal as palladium, platinum, rhenium, ruthenium, or iridium, it should be understood that the metal can be in any oxidation state that provides the oxygen-sensitive phosphorus, and is not necessarily zero-oxidation.

[0035] In any culture device described herein, when acetylide is used as a π-conjugated ligand, the acetylide is preferably conjugated to a platinum metal.

[0036] In particular, porphyrins containing oxygen-sensitive phosphors can be used in the culture devices described herein. In any culture device using a porphyrin-containing oxygen-sensitive phosphor, the porphyrin may be conjugated to a metal, such as one of the metals described above. A porphyrin containing an oxygen-sensitive phosphor in any culture device disclosed herein may be a porphyrin dendrimer. Most particularly, a porphyrin dendrimer in any culture device described herein may coordinate to a metal, the metal being a transition metal or a lanthanide, and most particularly platinum or palladium. Porphyrin-containing dendrimers are disclosed. A specific porphyrin-containing dendrimer that can be used in any of the aforementioned culture devices is the Pd-meso-tetra-(4-carboxyphenyl)porphyrin dendrimer, which is known in the art and can be prepared by methods known and approved in the art. Other porphyrins and porphyrin-containing dendrimers, as well as other types of oxygen-sensitive phosphors described herein for use with culture devices, can also be prepared according to methods recognized in the art.

[0037] Other examples of oxygen-sensitive phosphors that can be used include, but are not limited to, phosphorescent Al(III)-feron complexes, phosphorescent boron complexes, complexes of rare earth elements or their salts, Cu(I), Au(I), etc.

[0038] Examples of oxygen-sensitive dyes that are not luminescent include, but are not limited to, leuco-type indigo dyes, leuco-type thioindigo dyes, or one or more complexes of bis(histazino)cobalt-meso-tetra(α-α-α-α-o-pivalminophenyl)porphyrinatocobalt, and fullerenes such as Buckminsterfullerene. Further examples include polycyclic aromatic compounds such as 1-pyrendecanoic acid and decacycline.

[0039] In any of the culture devices described herein, any of the aforementioned oxygen-sensitive dyes, particularly any of the aforementioned oxygen-sensitive luminescent phospholipids, can be placed in the culture medium.

[0040] In any of the culture devices described above, the adhesive may be present in the growth compartment, and if the adhesive is present, one of the oxygen-sensitive dyes or luminescent phores described herein may be placed in or on the adhesive.

[0041] None of the aforementioned oxygen-sensitive dyes, or any of the aforementioned culture devices that may contain an oxygen-sensitive luminescent phosphate, advantageously contain an oxygen-scavenging system within the growth compartment. As stated above, cultured microorganisms, such as microorganisms that can be used to inoculate any of the culture devices described herein, are not considered oxygen-scavenging systems in this disclosure. The volume of oxygen is advantageously present in the atmosphere of the growth compartment in any of the culture devices described herein. In particular, if the growth compartment is arranged in a closed configuration, the atmosphere within the growth compartment cannot communicate with the atmosphere outside the growth compartment. As a result, any oxygen in a depleted growth compartment cannot be restored by the diffusion of oxygen from outside the growth compartment to inside the growth compartment.

[0042] When in use, any of the aforementioned culture devices, which may contain any of the oxygen-sensitive luminescent phores described herein, may be provided in an open configuration, with the growth compartment inoculated with a sample containing one or more microorganisms. In any method of use, the sample may be a liquid sample, particularly an aqueous liquid sample, which can be added to the growth compartment. Alternatively, in any method of use, the sample may be a swab, such as one located on an absorbent swab, which can be inoculated into the growth compartment by bringing the swab into contact with the culture medium in the growth compartment.

[0043] With respect to any of the methods described herein, the microorganisms that can be used in any of the culture devices described herein may be any oxygen-consuming microorganisms. Typically, this means that the microorganisms are aerobic or facultative anaerobic. However, it may also be possible to culture microaerophilic bacteria using the methods described herein.

[0044] After inoculation, the culture device can be converted to a closed configuration. In the closed configuration, the growth compartment initially has an oxygen content, which can be referred to or measured as, for example, a different oxygen partial pressure from that of the environment outside the growth compartment. This is because the culture device was configured in an open configuration during the inoculation process.

[0045] Next, the culture device is incubated for a sufficient time and at a sufficient temperature for the oxygen-sensitive dye, which may be any of the aforementioned oxygen-sensitive dyes, and in particular any of the aforementioned oxygen-sensitive luminescent phosphatids, to undergo a change in absorption or emission (in the case of an oxygen-sensitive luminescent phosphatid, typically the emission of the oxygen-sensitive luminescent phosphatid). The time and temperature vary depending on the specific microorganism being cultured. Typical times range from 1 hour to 7 days, and typical temperatures range from 20°C to 60°C. If the oxygen-sensitive dye is an oxygen-sensitive phosphorus, in particular one of the oxygen-sensitive phosphorus described above, the oxygen-sensitive phosphorus will emit phosphorescence.

[0046] While we do not wish to be bound by theory, when one or more microorganisms inoculated into a growth compartment respire and reproduce, they can consume the oxygen within the growth compartment. Because the culture device is in a closed configuration, the consumed oxygen cannot be replaced by oxygen from outside the growth compartment, and therefore the partial pressure of oxygen within the growth compartment decreases. When the partial pressure decreases sufficiently, oxygen-sensitive dyes undergo a color change and, if oxygen-sensitive luminescent phosphores, particularly oxygen-sensitive phosphorescent media, exhibit detectable luminescence such as phosphorescence.

[0047] Color changes, particularly luminescence such as phosphorescence, can occur at any detectable wavelength and do not need to be in the visible spectrum. A detectable wavelength is any wavelength that can be detected by a detector. Various photodetectors are known to those skilled in the art, and suitable detectors, such as charge-coupled devices (CCDs), photodiodes, or even the human eye, can be selected depending on the wavelength of the emission. If the color change is due to a change in absorption, it can be measured by absorption spectroscopy, such as UV / VIS absorption or IR absorption.

[0048] It is also possible to count microorganisms. This can be achieved with any of the culture devices or methods described above, and is simplest when the oxygen-sensitive dye is an oxygen-sensitive luminescent phosphonate homogeneously distributed in the culture medium, adhesive, or on the adhesive. Counting can be performed, for example, by recording a photograph of the entire growth compartment of the culture device, measuring the intensity, location, or both of the intensity and location of the luminescence using a detector such as a CCD camera. The number of colony-forming units can then be counted from the photograph, for example, by assigning areas with an intensity higher than the threshold intensity to represent colonies. The threshold intensity is the intensity that distinguishes the presence of microorganisms from noise, although this depends on the specific culture device and microorganisms. The oxygen concentration in any area of ​​the growth compartment can also be determined indirectly, for example, by measuring the oxygen concentration at a specific location within the growth compartment. The oxygen concentration at any location within the growth compartment, which may be related to the amount of microorganisms at that location, can be calculated using the Stern-Volmer relationship.

[0049] In particular, these methods are preferably carried out without placing the culture device, or more specifically, the growth compartment of the culture device, in a low-oxygen atmosphere such as a glove box. Furthermore, these methods are preferably carried out without activating the oxygen-scavenging system within the culture device, or more specifically, within the growth compartment of the culture device.

Claims

1. A culture device, A growth compartment surrounded by one or more oxygen-impermeable barriers, wherein at least one of the oxygen-impermeable barriers can be configured between an open configuration in which the growth compartment is exposed to the environment outside the growth compartment and a closed configuration in which the growth compartment is prevented from exchanging oxygen with the environment outside the growth compartment, A culture medium capable of supporting the replication of at least one microorganism placed within the growth compartment, A culture device comprising an oxygen-sensitive dye placed within the growth compartment.

2. The culture device according to claim 1, wherein the oxygen-sensitive dye includes an oxygen-sensitive luminescent phosphine.

3. The culture device according to claim 1 or 2, wherein at least one of the oxygen-impermeable barriers is a coverslip, and the coverslip is configurable between a first position in which the coverslip is located on the growth compartment and a second configuration in which the coverslip is at least partially removed from the growth compartment.

3. The culture device according to claim 1 or 2, wherein the culture medium comprises agar or a water-gelled thin film.

4. The culture device according to any one of claims 1 to 3, wherein the oxygen-sensitive luminescent phosphatid is an oxygen-sensitive phosphorescent body.

5. The culture device according to any one of claims 1 to 4, wherein the oxygen-sensitive phosphorescent material comprises at least one of porphyrin, a π-conjugated molecule, or a π-conjugated polymer.

6. The culture device according to claim 5, wherein the oxygen-sensitive phosphorescent material comprises porphyrin.

7. The culture device according to any one of claims 1 to 6, wherein the oxygen-sensitive phosphorescent material comprises a π-conjugated ligand, and optionally comprises a π-conjugated ligand for a transition metal or a lanthanide.

8. A culture device according to any one of claims 1 to 7, further comprising a metal conjugated to the oxygen-sensitive luminescent phosphatid, wherein the metal is optionally a transition metal or a lanthanide, and the transition metal is optionally ruthenium, rhenium, palladium, or platinum.

9. The culture device according to any one of claims 1 to 8, wherein the π-conjugated molecule comprises porphyrin.

10. The culture device according to any one of claims 1 to 9, wherein the oxygen-sensitive luminescent phore is a Pd-meso-tetra-(4-carboxyphenyl)porphyrin dendrimer.

11. The culture device according to any one of claims 1 to 10, wherein the oxygen-sensitive dye comprises one or more complexes of a leuco-type indigo dye, a leuco-type thioindigo dye, or bis(histazino)cobalt / meso-tetra(α-α-α-α-o-pivalminophenyl)porphyrinatocobalt.

12. The culture device according to any one of claims 1 to 11, wherein the oxygen-scavenging system is not present in the growth compartment.

13. The culture device according to any one of claims 1 to 12, wherein the oxygen-sensitive luminescent phospholipid is present in the culture medium and optionally homogeneously dispersed in the culture medium.

14. The culture device according to any one of claims 1 to 13, further comprising one or more adhesive matrices within the growth compartment, wherein the oxygen-sensitive luminescent phosphodiol is dispersed in at least one of the one or more adhesive matrices.

15. A method for detecting microorganisms, The culture device according to any one of claims 1 to 14 is inoculated with a sample containing microorganisms while the culture device is in the open configuration, Converting the culture device to the closed configuration, The culture device is incubated for a sufficient time and at a sufficient temperature so that the oxygen-sensitive phosphorescent material emits phosphorescence. A method comprising detecting a change in the absorption or emission of the oxygen-sensitive dye.

16. The method according to claim 15, wherein the change in luminescence absorption is a change in luminescence, in particular a change in the luminescence intensity of an oxygen-sensitive luminescent phosphatid, more specifically an oxygen-sensitive phosphorescent body.

17. The method according to claim 15 or 16, wherein the step of inoculating the culture device includes adding a liquid sample containing the microorganism, optionally an aqueous sample, to the culture medium.

18. The method according to any one of claims 15 to 17, wherein the microorganism is an aerobic microorganism, a facultative anaerobic microorganism, or a microaerophilic microorganism, and optionally the microorganism is an aerobic microorganism.

19. The method according to any one of claims 15 to 18, further comprising counting the microorganisms.

20. The method according to claim 19, wherein counting the microorganisms includes measuring the magnitude of luminescence.

21. The method according to any one of claims 15 to 20, wherein the step of placing the culture device in a low-oxygen atmosphere is not included.

22. The method according to any one of claims 15 to 21, wherein the step of activating an oxygen-scavenging reagent in the growth compartment is not included.