WO2016164488A1 - Étude basée sur la cytométrie de flux pour zinc biodisponible - Google Patents

Étude basée sur la cytométrie de flux pour zinc biodisponible Download PDF

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WO2016164488A1
WO2016164488A1 PCT/US2016/026255 US2016026255W WO2016164488A1 WO 2016164488 A1 WO2016164488 A1 WO 2016164488A1 US 2016026255 W US2016026255 W US 2016026255W WO 2016164488 A1 WO2016164488 A1 WO 2016164488A1
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zinc
binding
binding complex
bis
fluorophore
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PCT/US2016/026255
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English (en)
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Chad ROBINSON
George BABCOCK
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Shriners Hospital For Children
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • the presently disclosed subject matter relates to complexes and methods for detecting bioavailable zinc.
  • Zinc is an abundant transition metal ion that plays an important role in intra- and extra- cellular functions, including protein and enzyme structure, gene regulation, protein synthesis, intracellular protein trafficking, hormone function, neurotransmission, and immune function.
  • An abnormal level and/or distribution of zinc ions is therefore associated with diseases and conditions such as increased infection, weakened immune system, reduced growth in children, delayed wound healing, Alzheimer's disease, amyotropic lateral sclerosis (ALS), Parkinson's disease, epilepsy, ischemia, prostate cancer, and anemia.
  • ALS amyotropic lateral sclerosis
  • Parkinson's disease epilepsy
  • ischemia ischemia
  • prostate cancer and anemia.
  • the broad scope of zinc's effects demands sensitive and accurate techniques for binding, detecting, and quantifying zinc ions in a sample.
  • ICP-MS inductively coupled plasma mass spectrometry
  • a sample can be generated into an aerosol, ionized using an inductively coupled argon plasma, and separated and measured for zinc ions with a mass spectrometer.
  • This method is slow, susceptible to various interferences, and dependent on an uneconomical mass spectrometer not commonly found in clinical laboratories. Without further processing, this method is only able to measure a total concentration of zinc, which includes both bioavailable zinc and bound zinc.
  • Methods for using ICP-MS to detect metal ions are known in the art. For example, U.S. Patent No. 5,767,512 discloses an improved method and system of utilizing ICP-MS.
  • WO 2014/095375 discloses a use of ICP- MS to measure concentrations of soluble zinc.
  • Another method for detecting zinc is through colorimetric plate assays. During this method, a zinc-specific chromogen is added to a deproteinized sample and then measured for zinc ions with a spectrophotometer. This method, however, is slow, does not work with protein-containing samples, can only be used for measuring zinc, has low sensitivity, and requires setting up a standard curve each time for measuring the amount of zinc present in the sample.
  • Colorimetric plate assays to detect zinc ions are known in the art.
  • Sigma-Aldrich's Zinc Assay Kit, Abnova's Zinc Assay Kit, ImmunoWay's Zinc Colorimetric Assay Kit, and QuantiChrom' s Zinc Assay Kit are commercial colorimetric plate assays for detecting zinc.
  • these plate methods are not used to study single samples due to the impracticality, cost-efficiency, or simply their inability to do so. Plate methods are known to be carried out on batches of samples to reduce costs.
  • U.S. Patent No. 8,344,150 discloses a pyrrole end-capped derivative that can be used in spectroscopic-based assays for detecting zinc ions.
  • U.S. Patent No. 8,445,702 discloses a compound with a modified metal chelator that can selectively bind zinc ions and be used in spectroscopic-based assays for detecting zinc ions.
  • U.S. Patent Pub. No. 2014/0134665 discloses a zinc-responsive probe for detecting changes in zinc concentration that can be used in spectroscopy and microscopy assays for detecting zinc ions.
  • the presently disclosed subject matter provides zinc-binding complexes and methods for detecting bioavailable zinc.
  • the presently disclosed subject matter provides zinc -binding complexes and methods for more accurately and quickly detecting zinc ions that can be used in a wide range of analytical assays.
  • the zinc -binding complex includes a biotinylated zinc-binding protein and a polymeric microsphere.
  • the zinc-binding protein can be attached to the polymeric microsphere.
  • the biotinylated zinc -binding protein can include Metallothionein-1, Metallothionein-2, Metallothionein-3, Metallothionein-4, alpha-2-Macroglobulin, S 100 proteins, albumin, or a combination thereof.
  • the biotinylated zinc-binding protein includes Metallothionein-2.
  • the polymeric microsphere includes a particulate material, which can include silica gel, glass, nylon, latex, polystyrene, Sephadex, Sepharose, cellulose, metal, polyethylene, polypropylene, or a combination thereof.
  • the polymeric microsphere includes polystyrene.
  • the polymeric microsphere can include a biotin-binding protein, which can include streptavidin, avidin, ferritin avidin, nitroavidin, nitrostreptavidin, neutravidin, captavidin, or an analog or derivative thereof.
  • the polymeric microsphere includes streptavidin.
  • the polymeric microsphere includes streptavidin coated polystyrene beads.
  • the polymeric microsphere can have a diameter of from about 1 ⁇ to about 20 ⁇ .
  • the zinc -binding complex further includes a fluorophore, which can include a fluoresceine derivative, rhodamine derivative, cyanine, alexa, phenanthridine, ethidium, acridine, carbazole, phenoxazine, porphyrin, polymethine, boron-dip yrromethene, quinoline, pyrene derivatives, pyridyloxazole derivative, indicator based on the N-(2-methoxyphenyl) iminodiacetate chelator, BAPTA-based indicator, fluorescamine, or a combination thereof.
  • the fluorophore can be modified with a reactive group, which can be dichlorotriainyl, isothiocyanate, succinimidyl ester, or sulfonyl chloride.
  • the presently disclosed subject matter further provides a method for detecting zinc ions in a sample.
  • the method includes providing a zinc-binding complex including a biotinylated zinc -binding protein and a polymeric microsphere.
  • the method can further include contacting the sample with the zinc-binding complex and adding a zinc -binding fluorophore to the sample.
  • the method can further include detecting a fluorescence signal emitted by the fluorophore.
  • the method can further include measuring the fluorescence signal, wherein intensity of the fluorescence signal indicates zinc ion levels.
  • the biotinylated zinc-binding protein can be attached to the polymeric microsphere.
  • the biotinylated zinc -binding protein can include Metallothionein-2.
  • the polymeric microsphere includes streptavidin coated polystyrene beads.
  • the zinc-binding complex can be present in a concentration of from about 100 complexes/mL to about 100,000 complexes/mL.
  • the fluorophore can include 4 ' ,5 ' -Bis[bis(2- pyridylmethyl)aminomethyl]-2' ,7 ' -dichlorofluorescein (Zinpyr-1 or ZP-1), zinpyr dyes (e-g-, 9-(0-carboxyphenyl)-2,7-dichloro-4,5-bis[bis(2-pyridylmethyl)- aminomethyl]-6-hydroxy-3-xanthanone, 9-(0-carboxy-phenyl)-4,5-bis[bis(2- pyridylmethyl)-aminomethyl]-6-hydroxy-3-xanthanone, 9-(0-carboxy-phenyl)-2- chloro-5-[2- ⁇ bis(2-pyridylmethyl)aminomethyl ⁇ -N-methylaniline]-6-hydroxy-3- xanthanone), 2-Methyl-8-[(4-methylphenyl)sulfonylamino]-6- (e
  • the step of detecting a fluorescence signal in the method is performed using flow cytometry.
  • the intensity of the fluorescence signal indicates zinc ion levels in the concentration range from about 0.01 ⁇ to about 50 ⁇ .
  • the presently disclosed subject matter further provides a kit for detecting zinc ions in a sample including the zinc -binding complex and a fluorophore, packaged in one or more containers with one or more further reagents.
  • the fluorophore includes 4',5'-Bis[bis(2- pyridylmethyl)aminomethyl]-2',7 '-dichlorofluorescein.
  • Figure 1 depicts a method for biotinylating Metallothionein-2 according to one exemplary embodiment of the disclosed subject matter.
  • Figure 2 depicts a method for detecting zinc ions in a sample according to one exemplary embodiment of the disclosed subject matter.
  • Figure 3 depicts labeling of a zinc -binding complex with a fluorophore and its response to zinc ions in a sample according to one exemplary embodiment of the disclosed subject matter.
  • Figure 4 depicts fluorescence levels of a zinc -binding complex (represented by solid lines) and a metal chelator with a high affinity for zinc (represented by dashed lines) with a fluorophore across increasing concentrations of zinc ions.
  • the presently disclosed subject matter relates to complexes and methods for detecting bioavailable zinc.
  • the presently disclosed subject matter provides zinc -binding complexes and methods for detecting one or more zinc ions in a sample.
  • the zinc binding complex of the presently disclosed subject matter includes zinc-binding proteins, polymeric microspheres, and fluorophores.
  • the zinc-binding complex of the presently disclosed subject matter includes at least one zinc -binding protein.
  • the zinc -binding protein includes any protein known to one of ordinary skill in the art that is capable of binding, coordinating, and/or chelating one or more zinc ions.
  • Non-limiting examples of the zinc -binding protein include, but are not limited to Metallothionein-1, Metallothionein-2, Metallothionein- 3, Metallothionein-4, alpha-2-Macroglobulin, S 100 proteins, albumin, or combinations thereof.
  • the zinc -binding protein includes a zinc -binding group.
  • the zinc-binding group can be any suitable functional group known to one of ordinary skill in the art that is capable of binding, coordinating, and/or chelating one or more zinc ions.
  • the functional group can include a polyalkylene oxide, hydroxylated group; or a group having at least one amine, ammonium salt, carboxylate, sulfanyl, sulfinyl, sulfonyl, phosphate, phosphonate, phosphate, tertiary amine, pyridyl group, or combinations thereof.
  • the zinc-binding group can be any suitable chemical moiety known to one of ordinary skill in the art that is capable of binding, coordinating, and/or chelating with one of more zinc ions.
  • the chemical moiety can include ethylenediaminetetra-acetic acid (EDTA); diethyldithiocarbamate (DEDTC); histidine-containing compounds; sulfonamide-containing compounds; 1, 10-phenathroline, pyridyl-containing compounds; or amine-containing compounds.
  • Additional non-limiting examples of the chemical moiety include, but are not limited to analogs of l,2-bis(2- aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA) disclosed in U.S. Patent No. 8,445,702, which is incorporated herein by reference in its entirety.
  • BAPTA l,2-bis(2- aminophenoxy)ethane-N,N,N',N',-tetraacetic acid
  • the zinc-binding complex of the presently disclosed subject matter further includes a polymeric microsphere.
  • the polymeric microsphere includes any suitable microsphere known to one of ordinary skill in the art that is capable of acting as a solid phase or object.
  • Such microspheres are available through various sources including Bangs Laboratories, Inc., Polysciences, Inc., 3M Scotchlite Glass Bubbles, Biosphere Medical, Luminex microspheres, Spherotech, Inc., and Structure Probe, Inc.
  • the microsphere can have any shape and dimension.
  • the microsphere can have a diameter from about 1 ⁇ to about 20 ⁇ , or from about 1 ⁇ to about 10 ⁇ .
  • the microsphere has a diameter of about 5 ⁇ or about 10 ⁇ .
  • the microsphere can have a shape that is predominantly spherical in form.
  • the diameter of the microsphere can be chosen to differentiate the microsphere from one or more cell sizes in a sample.
  • the microsphere is composed of a particulate material.
  • the particulate material can include silica gel, glass, nylon, latex, polystyrene, Sephadex, Sepharose, cellulose, metal, polyethylene, or polypropylene.
  • the microsphere can be swellable (e.g. , Wang resin bead) or non-swellable (e.g. , controlled-pore glass).
  • the zinc-binding complex of the presently disclosed subject matter includes a fluorophore.
  • the fluorophore includes any suitable fluorophore known to one of ordinary skill in the art that is capable of generating fluorescence in response to a binding of one or more zinc ions to the zinc-binding complex and is capable of being detected following application of a suitable excitatory light.
  • Non-limiting examples of the fluorophore include, but are not limited to fluoresceine derivatives (e.g.
  • fluorescein isothiocyanate 6- carboxyfluorescein, 6-carboxy-2',4',7',4,7-hexachlorofluorescein, 6-carboxy-4',5'- dichloro-2',7'-dimethoxyfluorescein), rhodamine derivatives (e.g.
  • cyanine e.g., Cy3, Cy5, Cy7
  • alexa e.g., Alexa Fluor 555, Alexa Fluor 594
  • dansyl e.g., Alexa Fluor 555, Alexa Fluor 594
  • dansyl e.g., Alexa Fluor 555, Alexa Fluor 594
  • dansyl e.g., dansyl
  • phycoerythrin e.g., Alexa Fluor 594
  • dansyl e.g., Alexa Fluor 555, Alexa Fluor 594
  • dansyl e.g., Alexa Fluor 555, Alexa Fluor 594
  • dansyl e.g., Alexa Fluor 555, Alexa Fluor 594
  • dansyl e.g., Alexa Fluor 555, Alexa Fluor 594
  • dansyl e.g., Alexa Fluor 555, Alexa Fluor 594
  • dansyl e.g., Alex
  • phenanthridine e.g., Texas red
  • ethidium e.g., acridine
  • carbazole e.g., phenoxazine
  • porphyrin polymethine
  • BODIPY boron- dipyrromethene
  • quinoline e.g. , FluoZin- 1, 2 and 3, RhodZin-3, Newport Green DCF and PDF
  • BAPTA-based indicators e.g., fura-2, fluo-3 and 4
  • the zinc-binding protein is attached to the polymeric microsphere.
  • the zinc -binding protein can be attached to the polymeric microsphere using any suitable linker for covalent and/or non-covalent binding or interaction known to one of ordinary skill in the art including, but not limited to strep tavidin- biotin, avidin-biotin, thiol-malemide, azide-phosphine, carboxylate-amine, glutaraldehyde, polyglutaraldehyde, carbodiimide, or diethylpyrocarbonate (DEPC)- carbodiimide.
  • linker for covalent and/or non-covalent binding or interaction known to one of ordinary skill in the art including, but not limited to strep tavidin- biotin, avidin-biotin, thiol-malemide, azide-phosphine, carboxylate-amine, glutaraldehyde, polyglutaraldehyde, carbodiimide, or diethy
  • biotin is any suitable biotin derivative including, but not limited to biotin, biotin sulfone, caprolyamidobiotin, biocytin, desthiobiotin, desthiobiocytin, iminobiotin, or analogs and derivatives thereof.
  • the zinc-binding protein 101 can be biotinylated 102 using methods known to one of ordinary skill in the art (e.g., NHS- PEG4-Biotin reaction) and then reacted 201 with the polymeric microsphere 202 that can be coated with a biotin-binding protein.
  • biotin-binding proteins include, but are not limited to streptavidin, avidin, ferritin avidin, nitroavidin, nitrostreptavidin, neutravidin, captavidin, or analogs and derivatives thereof.
  • the fluorophore is attached to the zinc-binding protein.
  • the fluorophore can be attached to the zinc-binding protein using any suitable linker for covalent and/or non-covalent binding or interaction known to one of ordinary skill in the art including, but not limited to streptavidin-biotin, avidin- biotin, alkyl groups, ether, polyether, alkyl amide, peptide, modified peptide, Poly(ethylene glycol) (PEG), polyaminoacids (e.g., polylysine), functionalized PEG, polysaccharides, glycosaminoglycans, dendritic polymers, PEG-chelant polymers, oligonucleotide, phospholipid, alkenyl chains, alkynyl chains, disulfide, glutaraldehyde, polyglutaraldehyde, carbodiimide, or diethylpyrocarbonate (DEPC)- carbodiimide.
  • PEG poly(ethylene
  • the zinc -binding protein can be attached to an amine-containing linker and then reacted with the fluorophore that can be modified with a reactive group including, but not limited to dichlorotriainyl, isothiocyanate, succinimidyl ester, and sulfonyl chloride.
  • the presently disclosed subject matter provides a method for detecting zinc ions in a sample.
  • the methods disclosed herein provide the benefit and ability to measure single samples.
  • the methods disclosed herein can be used for direct measurement of a sample, i.e., without deproteinating, isolating, washing, or otherwise processing the sample.
  • the method includes providing the zinc -binding complex as described herein.
  • the zinc -binding complex can be used as an indicator of zinc ion levels in a suitable sample.
  • a suitable sample include, but are not limited to human blood serum, goat blood serum, bovine blood serum, semen, urine, feces, lavage fluid, cell culture media, cell lysates (e.g., bacteria and organ homogenates), soil, or water.
  • the method can further include contacting 203 the sample with the zinc- binding complex.
  • the step of contacting the sample can be performed at any suitable temperature including, but not limited to a temperature from about 4° C to about 25° C.
  • the step of contacting the sample can be performed at any suitable pH including, but not limited to a pH from about 6.0 to about 9.0.
  • the step of contacting the sample can be performed using any suitable buffer including, but not limited to nitriloacetic acid, PBS, HBSS, HEPES, monopotassium phosphate, disodium phosphate, MOPS, or citrate.
  • the step of contacting the sample can be performed using any suitable concentration of zinc -binding complexes including, but not limited to a concentration from about 100 complexes/mL to about 100,000 complexes/mL in an approximately 300 ⁇ ⁇ undiluted sample.
  • the step of contacting the sample can be performed using a concentration of zinc-binding complexes from about 100 complexes/mL to about 1,000 complexes/mL, about 100 complexes/mL to about 5,000 complexes/mL, about 100 complexes/mL to about 10,000 complexes/mL, about 100 complexes/mL to about 50,000 complexes/mL, about 100 complexes/mL to about 100,000 complexes/mL, about 1,000 complexes/mL to about 100,000 complexes/mL, about 5,000 complexes/mL to about 100,000 complexes/mL, about 10,000 complexes/mL to about 100,000 complexes/mL, or about 50,000 complexes/mL to about 100,000 complexes/mL.
  • the method can further include adding 204 a fluorophore to the sample.
  • a fluorophore Any suitable zinc -binding fluorophore known to one of ordinary skill in the art can be used as long as it allows for zinc to be co-bound by protein.
  • Non-limiting examples of a suitable zinc-binding fluorophore include, but are not limited to 4',5'-Bis[bis(2- pyridylmethyl)aminomethyl]-2',7'-dichlorofluorescein (Zinpyr-1 or ZP- 1), zinpyr dyes (e.g.
  • TFLZn potassium salt indicators based on the N-(2-methoxyphenyl) iminodiacetate chelator (e.g., FluoZin- 1, 2 and 3, RhodZin-3, Newport Green DCF and PDF), BAPTA-based indicators (e.g. , fura-2, fluo-3 and 4), or combinations thereof.
  • N-(2-methoxyphenyl) iminodiacetate chelator e.g., FluoZin- 1, 2 and 3, RhodZin-3, Newport Green DCF and PDF
  • BAPTA-based indicators e.g. , fura-2, fluo-3 and 4
  • the method can further include detecting 205 a fluorescence signal emitted 206 by the fluorophore.
  • the fluorescence signal can be an optical response following application of a suitable excitatory light.
  • the optical response can result from changes including, but not limited to changes in wavelength distribution, intensity of absorbance or fluorescence, fluorescence polarization, fluorescence lifetime, or combinations thereof.
  • the changes can be caused by the zinc-binding complex binding, coordinating, and/or chelating one or more zinc ions in the sample.
  • the step of detecting a fluorescence signal emitted by the fluorophore is performed using assays including, but not limited to microscopy, fluorometry, optical scanning, UV- spectrophotometer, bioassays, ion chromatography, ion-selective electrodes or anodic stripping voltammetry, fluorescence spectroscopy, atomic absorption or emission spectroscopy, colorimeter, or inductively coupled plasma mass spectrometry (ICP-MS).
  • assays including, but not limited to microscopy, fluorometry, optical scanning, UV- spectrophotometer, bioassays, ion chromatography, ion-selective electrodes or anodic stripping voltammetry, fluorescence spectroscopy, atomic absorption or emission spectroscopy, colorimeter, or inductively coupled plasma mass spectrometry (ICP-MS).
  • assays including, but not limited to microscopy, fluorometry, optical scanning
  • 2014/0273038 discloses a bioluminescence assay for detecting fluorescence emitted from a zinc-bound fusion protein, which is incorporated herein by reference in its entirety.
  • An additional non-limiting example for performing the step of detecting a fluorescence signal emitted by the fluorophore includes, but is not limited to flow cytometry. Any suitable flow cytometric method known to one of ordinary skill in the art can be used. Non-limiting examples of suitable flow cytometric methods include, but are not limited to methods disclosed in Shapiro's Practical Flow Cytometry, Third Edition (Alan R. Liss, Inc. 1995) and U.S. Patent Pub. No. 2014/0093887, which are incorporated herein by reference in their entireties.
  • the method can further include measuring the fluorescence signal, wherein intensity of the fluorescence signal indicates zinc ion levels in the sample.
  • the polymeric microsphere of the zinc-binding complex can decrease background signals that can be caused by endogenous proteins in the sample and consequently can increase sensitivity and accuracy of fluorescence detection. Further, the amount of polymeric microspheres can be increased or decreased to modulate the dynamic range. By way of example, and not limitation, when used with samples having a low concentration of zinc, the amount of polymeric microspheres can be decreased to increase sensitivity. For further example, when used in samples having a high concentration of zinc, the amount of polymeric microspheres can be increased to provide adequate surface space for zinc binding.
  • the fluorescence signal can indicate zinc ion levels from about 0.01 ⁇ to about 50 ⁇ .
  • the fluorescence signal indicates zinc ion levels from about 0.01 ⁇ to about 0.05 ⁇ , about 0.01 ⁇ to about 0.1 ⁇ , about 0.01 ⁇ to about 0.5 ⁇ , about 0.01 ⁇ to about 1 ⁇ , about 0.01 ⁇ to about 3 ⁇ , about 0.01 ⁇ to about 5 ⁇ , about 0.01 ⁇ to about 10 ⁇ , about 0.01 ⁇ to about 20 ⁇ , about 0.01 ⁇ to about 30 ⁇ , about 0.01 ⁇ to about 40 ⁇ , about 0.01 ⁇ to about 50 ⁇ , about 0.05 ⁇ to about 50 ⁇ , about 0.1 ⁇ to about 10 ⁇ , about 0.1 ⁇ to about 50 ⁇ , about 0.5 ⁇ to about 50 ⁇ , about 1 ⁇ to about 50 ⁇ , about 10 ⁇ to about 50 ⁇ , about 20 ⁇ to about 50 ⁇ , about 30 ⁇ to about
  • the disclosed method can be used over a wider dynamic range than certain known methods such as plate assays.
  • the disclosed method can be used to measure low concentrations of zinc, e.g., less than about 1 ⁇ , without requiring multiple dilution steps to bring samples with high concentrations of zinc, e.g., greater than about 3 ⁇ , within the range.
  • the steps of detecting and measuring a fluorescence signal can be performed using an epifluorescent microscope, laser scanning confocal microscope, or fluorsecent plate reader.
  • intensity of the fluorescence signal can be analyzed using manual or computer means known to one of ordinary skill in the art including, but not limited to a look-up table or standard curve.
  • the disclosed method can be used in a wide range of analytical assays.
  • the disclosed method can be used in any suitable existing flow multiparameter based evaluation.
  • the method can include the simultaneous measurement of two or more parameters using one or more parallel assays.
  • gating can be employed to target populations including the zinc-binding complex.
  • the methods can be used to measure the amount of bioavailable zinc in addition to another parameter such as leukocyte (e.g., T cells, B cells, monocytes, and neutrophils) or other cell levels,
  • leukocyte e.g., T cells, B cells, monocytes, and neutrophils
  • zinc measurement can be combined with a functional assay, for example, to analyze phagocytosis, oxidative burst, and the like.
  • the parameters detected by the second assay can be differentiated by size and/or fluorescent wavelength from the zinc-binding complexes. Additionally, like the method for detecting zinc, the second assay can preferably be performed by direct measurement, i.e., without any processing of the sample.
  • the methods can be performed in vitro.
  • the sample can be obtained, e.g., withdrawn from the subject prior to contacting the sample with the zinc -binding complex and detecting a fluorescence signal.
  • the methods can be performed in vivo.
  • the zinc-binding complex can be introduced to the subject via injection or lavage, for example bronchial, peritoneal, or gastric lavage, and the sample can later be isolated from the subject and combined with a fluorophore for detecting a fluorescence signal.
  • kits for detecting zinc ions in a sample can include the zinc-binding complex as described herein and a fluorophore, packaged in one or more containers with one or more further reagents.
  • the one or more further reagents can include buffers and preservatives known to one of ordinary skill in the art that are appropriate for performing the method for detecting zinc ions in a sample as described herein.
  • Non-limiting examples of suitable buffers and preservatives include, but are not limited to 7% trichloracetic acid, zinc reagents (e.g., Zinc Reagent -1, and -2 of Bio Vision's Zinc Colorimetric Assay Kit; Reagent A, B, and C of Abnova's Zinc Assay Kit; Zinc Reagent 1 and 2 of Sigma- Aldrich's Zinc Assay Kit; and Reagent A, B, and C of QuantiChrom's Zinc Assay Kit), or combinations thereof.
  • zinc reagents e.g., Zinc Reagent -1, and -2 of Bio Vision's Zinc Colorimetric Assay Kit
  • Reagent A, B, and C of Abnova's Zinc Assay Kit Zinc Reagent 1 and 2 of Sigma- Aldrich's Zinc Assay Kit
  • Reagent A, B, and C of QuantiChrom's Zinc Assay Kit or combinations thereof.
  • the zinc -binding complex can be stored and utilized in a plastic container.
  • the plastic container can be a polypropylene container (e.g., BD Falcon tubes). Storing the zinc-binding complex in the plastic container can avoid contamination problems and consequent loss of fluorescence sensitivity.
  • the presently disclosed subject matter can be used to determine diseases or conditions associated with abnormal levels of zinc ions in a sample from a subject.
  • diseases and conditions which are associated with abnormal levels of zinc ions include, but are not limited to increased infection, weakened immune system, reduced growth in children, delayed wound healing, Alzheimer's disease, amyotropic lateral sclerosis (ALS), Parkinson's disease, epilepsy, ischemia, prostate cancer, and anemia.
  • ALS amyotropic lateral sclerosis
  • ischemia ischemia
  • prostate cancer prostate cancer
  • anemia anemia
  • U.S. Patent Pub. No. 2010/0099195 discloses methods for detecting zinc to determine the presence or elevated risk of developing prostate cancer in a subject, which is incorporated herein by reference in its entirety.
  • the subject can be a mammal including, but not limited to a human, goat, or bovine. Any suitable method known to one of ordinary skill in the art can be used to obtain the sample from the subject.
  • the present Example provides Metallothionein-2 bound microspheres for zinc-binding fluorescence analysis in accordance with the disclosed subject matter.
  • Metallothionein-2 (rabbit liver) (ALX-202-073-M001) from Enzo Life Sciences, Inc. was biotinylated using the EZ-LinkTM Micro NHS-PEG4-Biotinylation Kit (21955) from Thermo Fisher Scientific Inc.
  • Metallothionein-2 was first dissolved in 200-700 PBS.
  • NHS-PEG4-Biotin provided in the kit was then added to the Metallothionein-2 solution and incubated on ice for two hours or at room temperature for 30-60 minutes.
  • the Metallothionein-2 and NHS-PEG4- Biotin solution was applied directly onto the center of the resin bed of the ZebaTM Spin Desalting Column provided in the kit. The column was then centrifuged for 2 minutes and the flow-through solution was collected as biotinylated Metallothionein- 2.
  • biotinylated Metallothionein-2 was then attached to polymeric microspheres.
  • the biotinylated Metallothionein-2 (200 ⁇ g) was first added to 10 mg of washed SuperAvidinTM Coated Microspheres (9.4 ⁇ , 1.06 g/cm ) from Bangs Laboratories, Inc. The solution was incubated for 15 minutes at room temperature on a vortexer and then centrifuged. After the supernatant was decanted, any unbound biotinylated Metallothionein-2 was removed from the microspheres by washing two times in 100 ⁇ ⁇ buffer provided in the microsphere kit. The resulting Metallothionein-2 bound microspheres were then re-suspended in the same 100 ⁇ ⁇ buffer provided in the microsphere kit.
  • the Metallothionein-2 bound microspheres was then tested in a zinc solution.
  • Zinc sulfate concentrate (32047-1 Fluka) from Sigma- Aldrich was used as the source of zinc ions in solution.
  • N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (P4413-50MG) from Sigma-Aldrich was used as a metal-chelating reagent to provide a zinc ion free solution for negative control comparison to the Metallothionein-2 bound microspheres.
  • the Metallothionein-2 bound microspheres and TPEN were first added to the zinc solution. TPEN has also been added first to create a zinc free control, as well as after (not shown here).
  • Fluorescence of the Metallothionein-2 bound microspheres with Zinpyr- 1 was then captured with a BDTM LSR II (BD Biosciences) or Coulter Epics XL (Beckman Coulter) flow cytometer.
  • the forward-side scatter plot revealed a major population of events representing single beads ( Figure 3).
  • the gated population was then analyzed for fluorescence intensity and was shown to be positive by a shift in fluorescence between the negative and positive controls ( Figure 3).
  • the level of fluorescence was measured across increasing concentrations of zinc ions in response to the Metallothionein-2 bound microspheres and TPEN with Zinpyr- 1 ( Figure 4).
  • fluorescence from the Metallothionein-2 bound microspheres was able to be captured across every zinc concentration as tested ( Figure 4).
  • the fluorescence from the Metallothionein-2 bound microspheres even exceeded the fluorescence from TPEN at the higher zinc concentrations as tested (e.g. , 1 ⁇ and 10 ⁇ ) ( Figure 4).
  • flow cytometry based analysis of fluorescence from zinc-binding proteins (e.g., Metallothionein-2) bound to microspheres with fluorophores (e.g., Zinpyr- 1) are able to sensitively, accurately, more availably, and more quickly detect bioavailable zinc across a range of zinc concentrations in multiple sample sources in an at will manner.

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Abstract

L'invention concerne des complexes et des procédés de détection de zinc biodisponible. En particulier, un complexe de liaison au zinc peut comprendre une protéine de liaison au zinc biotinylée et une microsphère polymérique. Des procédés peuvent consister à mettre en contact le complexe de liaison au zinc avec un échantillon, à ajouter un fluorophore, et à détecter et à mesurer un signal de fluorescence émis par le fluorophore. Les procédés peuvent être utilisés dans une large plage d'études analytiques.
PCT/US2016/026255 2015-04-06 2016-04-06 Étude basée sur la cytométrie de flux pour zinc biodisponible WO2016164488A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5424440A (en) * 1988-07-08 1995-06-13 Jbl Scientific, Inc. Fluorescent benzothiazole derivatives
US5922302A (en) * 1993-05-17 1999-07-13 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin-metal chelating protein conjugates
US20050182253A1 (en) * 2004-01-09 2005-08-18 Shigenobu Yano Fluorescent zinc ion sensor
US20050250214A1 (en) * 2004-05-05 2005-11-10 Gee Kyle R Zinc binding compounds and their method of use
US20140335528A1 (en) * 2013-05-13 2014-11-13 Intelligent Bio-Systems, Inc Analyte Enrichment Methods And Compositions

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5424440A (en) * 1988-07-08 1995-06-13 Jbl Scientific, Inc. Fluorescent benzothiazole derivatives
US5922302A (en) * 1993-05-17 1999-07-13 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin-metal chelating protein conjugates
US20050182253A1 (en) * 2004-01-09 2005-08-18 Shigenobu Yano Fluorescent zinc ion sensor
US20050250214A1 (en) * 2004-05-05 2005-11-10 Gee Kyle R Zinc binding compounds and their method of use
US20140335528A1 (en) * 2013-05-13 2014-11-13 Intelligent Bio-Systems, Inc Analyte Enrichment Methods And Compositions

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