Patent Application
20080176255
This invention is directed to compositions and methods for the detection of mold. Specifically, provided herein are compositions and methods for the detection of mold by detecting differences in light intensity of fluoresence resulting from interaction between the mold and the compositions described herein.
Various fluorescent stains have been used to identify fungal particles in clinical and laboratory settings. However, the techniques and stains employed require extensive preparation, are slow acting, and are often ineffective at staining the various asexual and sexual spore types produced by ascomycetes, zygomycetes and basidiomycetes. These may comprise the vast majority of fungi having clinical, agricultural, and/or industrial application. Therefore, a need exists for a dye which is rapid acting for detecting such mold spores.
In one embodiment, the invention provides a fluorescent staining composition for mold detection, comprising: 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD).
In another embodiment, provided herein is a method for producing a high-viscosity fluorescent staining composition for mold detection, comprising: mixing 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD), a gelling agent, a solvent, and a diluent together solubilizing the mixture in the diluent; and cooling the staining solution below the gelling temperature of the gelling agent.
In one embodiment, provided herein is a method for producing a low-viscosity fluorescent staining composition for a mold detection, comprising: mixing, 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD), a solvent, and a diluent togther; and solubilizing the mixture in the diluent.
In another embodiment, provided herein is a method of detecting a mold in solution comprising: contacting the solution with a dye comprising 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD), a solvent, and a diluent; exposing the contacted solution to electromagnetic radiation; and analyzing the difference in fluorescence between the contacted and uncontacted solution, whereby an increase in light intensity of fluorescence of above about 45% indicates the presence of mold.
In one embodiment, provided herein is a method of detecting a mold on a surface comprising the steps of: collecting a sample; contacting the sample with a dye comprising 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD), a gelling agent, a solvent, and a diluent; exposing the contacted sample to electromagnetic radiation; and analyzing the difference in fluorescence between the collected sample and the contacted sample, whereby an increase in the light intensity of fluorescence of above about 45% indicates the presence of mold.
In another embodiment, provided herein is a method of quantifying mold concentration in a sample, comprising: contacting the sample containing the mold with 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD); and comparing the observed light intensity of fluorescence to a standard.
The method and apparatus of the present invention will be better understood by reference to the following detailed discussion of specific embodiments and the attached figures which illustrate and exemplify such embodiments.
A specific embodiment of the present invention will be described with reference to the following drawings, wherein:
This invention relates in one embodiment to compositions and methods for the detection of mold. Specifically, provided herein are compositions and methods for the detection of mold by detecting the light intensity of fluoresence resulting from interaction between the mold and the compositions are described herein.
In another embodiment, the novel fluorescent fungal staining gel described herein, may be used to capture and stain fungal spores and particles from the air by using a wide variety of spore collecting devices and pumps. Such spore collecting devices are well known to those skilled in the art. Moreover, the capture gel may allow for on-site or field quantification of fungal spores and particles. This may be accomplished by using standard detection devices outfitted with a light source, excitation/emission filters, and/or a fluorescent light detector.
According to one aspect of the invention and in one embodiment, a fluorescent staining composition for mold detection, may comprise: 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD). In another embodiment, 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD) is represented by the following structure:
2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD) is anionic and has a solubility in distilled water of 25 g/l at 25° C., and 300 g/l at 95° C. In one embodiment, the compositions described herein may further comprise a solvent; a gelling agent; and a diluent.
In one embodiment, the solvent used may be Dimethyl Sulfoxide (DMSO), absolute methanol, or other solvents capable of improving the availability of the dye in the compositions used herein. In one embodiment, the gelling agent used in the compositions described herein is glycerol, gellatine, agar, a synthetic hydrocolloid, or a natural hydrocolloid. It has been found that any appropriate material capable of increasing the viscosity of the composition may be used as a gelling agent. The increase in viscosity may be accomplished by the introduction of inert solids, or with any compound acquiring waters of hydration, or through chain entanglement. The gelling agent encompassed in the compositions described herein are those compounds that will delay the flow of the compositions off the samples or surfaces to which they are applied. In another embodiment, the diluent used is pyrogen-free water. Any compound that will increase the viscosity of the solution having the dye is encompassed by the term, “gelling agent”.
In one embodiment, the gelling agent used in conjunction with the methods and compositions provided herein is Alginic acid, or Sodium alginate, Potassium alginate, Calcium alginate, Agar, Carrageenan, Carob gum, Gelatine, Propylene glycol alginate, gum Arabic, Tragacanth, Guar gum, Xanthan gum, Karaya gum, Tara gum, Gellan gum, Polydextrose, Dextrin, Modified starch, Alkaline modified starch, Bleached starch, Oxidized starch, Monostarch phosphate, Distarch phosphate, Distarch phosphate, Phosphated distarch phosphate, Acetylated distarch phosphate, Acetylated starch mono starch acetate, Acetylated starch, Acetylated distarch adipate, Distarch glycerine, Hydroxy propyl starch, Hydroxy propyl distarch glycerine, Hydroxy propyl distarch phosphate, Starch sodium octenyl succinate, Acetylated oxidised starch, Polyvinyl Alcohol, Phytagel, Transfergel, and their combination in other embodiments.
In one embodiment, the mold sought to be detected or quantified using the compositions and methods described herein, is Stachybotrys chartarum, or Penicillium chrysogenum, Penicillium islandicum, Penicillium sp., Aspergillus niger, Rhizopus nigricans or any combination thereof. In other embodiments, other fungi species may be detected or quantified using the compositions and methods described herein. In another embodiment, the mold sought to be detected or quantified using the compositions and methods described herein, are listed in Table 3.0 provided hereinbelow.
Absidia corymbifera
Aspergillus unguis
Eurotium repens
Acremonium strictum
Aspergillus ustus
Epicoccum nigrum
Alternaria alternata
Aspergillus versicolor
Fusarium solani
Aspergillus auricomus
Aspergillus wentii
Geotrichum candidum
Aspergillus caespitosus
Aureobasidium pullulans
Geotrichum klebahnii
Aspergillus candidus
Candida albicans
Memnoniella echinata
Aspergillus carbonarius
Candida dubliniensis
Mucor amphibiorum
Aspergillus cervinus
Candida glabrata
Mucor circinelloides
Aspergillus clavatus
Candida haemulonii
Mucor hiemalis
Aspergillus flavus
Candida krusei
Mucor indicus
Aspergillus giganteus
Candida lipolytica
Mucor mucedo
Aspergillus oryzae
Candida lusitaniae
Mucor racemosus
Aspergillus fumigatus
Candida maltosa
Mucor ramosissimus
Neosartorya fischeri
Candida parapsilosis
Rhizopus azygosporus
Aspergillus flavipes
Candida sojae
Rhizopus homothalicus
Aspergillus niger
Candida tropicalis
Rhizopus microsporus
Aspergillus awamori
Candida viswanathii
Rhizopus oligosporus
Aspergillus foetidus
Candida zeylanoides
Rhizopus oryzae
Aspergillus phoenicis
Chaetomium globosum
Myrothecium verrucaria
Aspergillus niveus
Cladosporiunm
Paecilomyces lilacinus
Aspergillus paradoxus
cladosporioides
Paecilomyces variotii
Aspergillus parasiticus
Cladosporium herbarum
Penicillium aethiopicum
Aspergillus sojae
Cladosporium
Penicillium
Aspergillus penicillioides
sphaerospermum
atramentosum
Aspergillus puniceus
Emericella nidulans
Penicillium
Aspergillus restrictus
Emericella rugulosa
aurantiogriseum
Aspergillus caesillus
Emericella quadrilineata
Penicillium freii
Aspergillus conicus
Emericella variecolor
Penicillium polonicum
Aspergillus sydowii
Eurotium amstelodami
Penicillium tricolor
Aspergillus sclerotiorum
Eurotium chevalieri
Penicillium viridicatum
Aspergillus tamarii
Eurotium herbariorum
Penicillium verrucosum
Aspergillus terreus
Eurotium rubrum
Scopulariopsis
Penicillium
Penicillium miczynskii
brevicaulis
brevicompactum
Penicillium olsonii
Scopulariopsis fusca
Penicillium stoloniferum
Penicillium oxalicum
Scopulariopsis brumptii
Penicillium canescens
Penicillium
Scopulariopsis chartarum
Penicillium chrysogenum
purpurogenum
Scopulariopsis
Penicillium griseofulvum
Penicillium restrictum
sphaerospora
Penicillium glandicola
Penicillium raisitrickii
Stachybotrys chartarum
Penicillium coprophilum
Penicillium roquefortii
Trichoderma asperellum
Penicillium expansum
Penicillium sclerotiorum
Trichoderma hamatum
Eupenicillium crustaceum
Penicillium
Trichoderma harzianum
Eupenicillium egyptiacum
simplicissimum
Trichoderma longibrachiatum
Penicillium verrucosum
Penicillium ochrochloron
Trichoderma citrinoviride
Penicillium variabilis
Penicillium lividum
Trichoderma viride
Penicillium thomii
Penicillium pupurescens
Trichoderma atroviride
Penicillium spinulosum
Penicillium spinulosum
Trichoderma koningii
Penicillium pupurescens
Rhizomucor meihei
Ulocladium botrytis
Penicillium lividum
Rhizomucor pusillus
Ulocladium chartarum
Penicillium islandicum
Rhizomucor variabilis
Wallemia sebi
Penicillium italicum
Rhizopus stolonifer
Penicillium melinii
Scopulariopsis asperula
In one embodiment, the compositions for producing a high-viscosity fluorescent staining composition for mold detection or quantification, may comprise: mixing 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD), a gelling agent, a solvent, and a diluent together for solubilizing the mixture in the diluent; and cooling the staining solution below the gelling temperature of the gelling agent. In certain embodiments, whereby the gelling agent may be a solid, the step of cooling may not be necessary. Alternatively, cooling may be used and still be encompassed by the methods provided herein. As used herein, the term “high viscosity” refers to any composition where the measured viscosity is more than about 9 cP·s. Conversely, non-viscous compositions described herein, will have a viscosity of less than about 9 cP·s.
According to one embodiment, a method of producing a low-viscosity fluorescent staining composition for mold detection or quantification, may comprise mixing 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD), a solvent, and a diluent together; and solubilizing the mixture in the diluent.
In another embodiment, compositions made using the methods described above may be for detecting and/or quantifying mold or fungi. According to this aspect of the invention, a method of detecting a mold in solution may comprise contacting the solution with a dye comprising 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD), a solvent, and a diluent; exposing the contacted solution to electromagnetic radiation; and analyzing the difference in fluorescence intensity between the contacted and uncontacted solution, whereby an increase in light intensity of fluorescence of above about 45% indicates the presence of mold in the solution.
The fluorescence characteristics of the dyes described herein, are readily determined according to standard methods known in the art. In one embodiment, the excitation spectrum of the dye is determined by monitoring its emission at a constant wavelength while the excitation wavelength is varied, thereby generating a curve resembling an absorption spectrum. The emission spectrum of the dye may be determined by exciting the dye at a constant wavelength and analyzing the emitted spectrum. This may be done either directly or by analyzing the degree of transmission by a series of filters. The true spectra may be determined by normalizing for the wavelength-dependent intensity of the light source and the wavelength-dependent variation of the detector response. Such normalization is typically performed by comparing the detected spectra with corrected spectra of a known standard (e.g., quinine in sulfuric acid). In one embodiment, the excitation spectrum used in the methods provided herein is between about 330 nm and about 460 nm. In another embodiment, the excitation spectra may be between 350 nm and 455 nm. In still another embodiment, the excitation spectra may be between 360 nm and 400 nm. In a further embodiment, the excitation spectra may be between 360 nm and 480 nm. In a still further embodiment, the excitation spectra may be between 360 nm and 370 nm.
The term “fluorescent” refers to the property of a molecule which becomes excited and emits light of a longer wavelength or wavelengths upon irradiation with light of a given wavelength or wavelengths. The term “fluorophore” as used herein refers to a fluorescent molecule. There are a number of parameters which together describe the fluorescence characteristics of a fluorophore. These include, for example, the maximum wavelengths of excitation and emission, the breadth of the peaks for excitation and emission, the difference between the excitation and emission maxima (the “Stokes shift”), fluorescence intensity, quantum yield, and the extinction coefficient. For biological or biochemical applications, longer Stokes shifts may be generally preferred to shorter ones.
Fluorescence intensity may be determined as the product of the extinction coefficient and the fluorescence quantum yield. The fluorescence quantum yield is a measure of the relative efficiency or extent to which absorbed light energy is re-emitted as fluorescence. It is defined as the ratio of the number of fluorescence photons emitted, “F” to the number of photons absorbed, “A”. Molecules with larger quantum yields exhibit greater fluorescence intensity. The molar extinction coefficient is a measure of a fluorophore's ability to absorb light. Commonly used fluorophores tend to have molar extinction coefficients (at their absorption maximum) between 5,000 and 200,000 cm−1 M−1 (Haugland, R. P. (1996) Molecular Probes Handbook for Fluorescent Probes and Research Chemicals, 6th Edition). Higher extinction coefficients also correlate with greater fluorescence intensity because fluorescence intensity is the product of quantum yield and the extinction coefficient.
In one embodiment, the methods and compositions described hereinabove may be a whole or a part of the methods described herein. A method of detecting a mold on a surface may comprise the steps of collecting a sample; contacting the sample with a dye comprising 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD), a gelling agent, a solvent, and a diluent; exposing the contacted sample to electromagnetic radiation; and analyzing the difference in fluorescence intensity between the collected sample and the contacted sample. An increase in the light intensity of fluorescence of above about 45% may generally indicate the presence of mold. The fluorescent dye used uin the compositions and methods provided herein, reacts in one embodiment with spore wall glucans, carbohydrates and glycoproteins. The dye is rapidly bound to the aforementioned molecules. The binding is irreversible and permanent. Unlike most fluorescent dyes that “wash-out” or fade with exposure to fluorescent light at the excitation peak, the dyes described herein is stable for at least 6 months when stored at room temperature and years in other embodiments if refrigerated and kept in the dark.
In one embodiment, detection and quantification of the mold or fungi using the methods and compositions provided herein is done using Fluorescent microscopes (e.g. Leica 5500), or plate readers (e.g. Tecan Infinite M200), imaging systems (e.g. Kodak Imaging Station 4000MM), Quantitative PCR machines (e.g. Corbett RotoGene 6000), or their combination in other embodiments.
Collecting a sample may comprise any collection method now known in the art or developed in the future, so long as it lends itself to be contacted with the compositions described herein. These methods include but are not limited to swabbing, vaccuum-assisted, wash-away, wiping, filtering, and the like as is well known to those skilled in the art.
In one embodiment, several fluorescence readouts may be used for quantifying a mold's intensity, anisotropy, or spectral characteristics. In other embodiments, a variety of measurement techniques may be used including, for example, confocal microscopy, flow cytometry, and the like.
In another embodiment, the methods may be used for quantifying mold in a sample. According to this aspect, the method may comprise contacting the sample containing the mold with 2,2′-([1,1′-Biphenyl]-4,4′-diyldi-2,1-ethenediyl)bis-benzenesulfonic acid disodium salt (BBD); and comparing the observed light intensity of fluorescence to a standard. In one embodiment, the standard may be obtained from a sample containing a known concentration. In another embodiment, the standard may be a range of concentrations in solution together with their accompanying light intensity.
In one embodiment, a solution comprising the compositions provided herein is directly applied to a surface, in a field setting, and thereafter, exposed to electromagnetic radiation in order determine the presence and quantity of mold. Detection is done either by a direct visual examination with the naked eye in one embodiment, or viewed by the naked eye through a emission filter (e.g. glasses or goggles), or detected with a electromagnetic radiation detector in other embodiments.
In still another embodiment, a kit may be used for detecting or quantifying mold in a sample comprising the compositions described herein. Instructions, packaging materials, and standards may also be included in such a kit. Optimal excitation wavelengths and the expected shift when a given mold is reacted or contacted with the compositions described herein may be included as part of the instructions. Such kits may be adapted for a specific mold, which may be Stachybotrys chartarum in one embodiment, or Penicillium chrysogenum, Penicillium islandicum, Penicillium sp., Aspergillus niger, or Rhizopus nigricans, for example. Such kits may also comprise concentration-intensity curves that were obtained using the methods described herein.
The term “about” as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.
The term “subject” refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term “subject” does not exclude an individual that is normal in all respects.
The following examples are presented to illustrate preferred embodiments of the invention. It is to be understood that the following example of the present invention is not intended to restrict the present invention since many more modifications may be made within the scope of the claims without departing from the spirit thereof.
Materials and Methods
Detection Systems For Testing Captured Spores
1. UV Source: Light Emitting Diode (LED)
2. Peak Transmission: 360 nm
Note: The desired wavelength reaching the detector from the fluorescing sample should be larger than about 430.
1. UV Source: Halogen 100 watt fiber optic system
2. Excitation Filter: ex385 (Kodak Cat#1829308)
3. Emission Filter: em440WA (Kodak Cat#8739518)
Fluorescent Microscope (Riechert MicroStar IV with DAPI Cluster K2834)
1. UV Source: Metal Halide Lamp 50 watt
2. Excitation: 365 nm
3. Emission: 390 nm
4. Dichloric Mirror: 395 nm
1. Stachybotrys chartarum
2. Penicillium chrysogenum
3. Penicillium islandicum
4. Penicillium sp.
5. Aspergillus niger
Methods
A variety of dye formulations were tested, but the following formulation has given the best results to date
1. 0.01% BBD w/v
2. 25% Glycerol v/v
3. 0.1% DMSO
4. Pyrogen Free MilliQ water as solvent base
1. 0.01% BBD w/v
2. 0.1% DMSO
3. Pyrogen Free MilliQ water as solvent base
All glassware was baked at 200° C. for 2 hours to ensure that no fungal spores would contaminate the experiments and all plastic disposables were certified sterile.
A. Direct spore release and capture using the SRCT system.
B. Direct Application/Quick Wash (Table 1.0 and
Aspergillus niger Stained Spores detected with Photomultiplier Tube System
Direct Fluorescent Microscopic examination of Gel Formulation (Table 2.0)
In a separate experiment spores were examined under a fluorescent microscope to confirm that they were indeed adsorbing and emitting a fluorescent signal.
Stachybotrys chartarum
Penicillium chrysogenum
Penicillium islandicum
Aspergillus niger
Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.
