WO2016164488A1

WO2016164488A1 - Flow cytometry based assay for bioavailable zinc

Info

Publication number: WO2016164488A1
Application number: PCT/US2016/026255
Authority: WO
Inventors: Chad ROBINSON; George BABCOCK
Original assignee: Shriners Hospital For Children
Priority date: 2015-04-06
Filing date: 2016-04-06
Publication date: 2016-10-13

Abstract

Complexes and methods for detecting bioavailable zinc are provided. In particular, a zinc-binding complex can include a biotinylated zinc-binding protein and a polymeric microsphere. Methods can include contacting the zinc-binding complex with a sample, adding a fluorophore, and detecting and measuring a fluorescence signal emitted by the fluorophore. The methods can be used in a wide range of analytical assays.

Description

FLOW CYTOMETRY BASED ASSAY FOR BIOAVAILABLE ZINC

PRIORITY TO RELATED APPLICATIONS

This application claims priority to United States Provisional Application No. 62/143,599, filed April 6, 2015, which is incorporated by reference herein in its entirety.

FIELD

The presently disclosed subject matter relates to complexes and methods for detecting bioavailable zinc.

BACKGROUND

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. The broad scope of zinc's effects demands sensitive and accurate techniques for binding, detecting, and quantifying zinc ions in a sample.

One method for sensitively detecting zinc is through inductively coupled plasma mass spectrometry (ICP-MS). During this method, 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, however, 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. For example, 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. However, 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.

Additional methods for detecting zinc are known in the art. For example, Malavolta et al., Cytometry A. 2006 Oct l;69(10): 1043-53 discloses a method for detecting zinc ions within biological cells using Zynpyr-1 as a capture protein. Similarly to plate assays, the sample must be processed to isolate and wash cells. Further, this method can only be used to measure intracellular zinc. However, both intracellular and extracellular zinc are important in various cellular functions and it is desirable to measure the total zinc concentration, particularly, the zinc concentration in serum.

Complexes and methods for detecting zinc are known in the art. For example, 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.

However, there remains a need in the art for complexes and methods for more accurately, sensitively, and quickly detecting bioavailable zinc ions that can be used in a wide range of analytical assays with fewer confounding signals, such as protein bound zinc. The presently disclosed subject matter provides such significant advantages over currently available systems. SUMMARY

The presently disclosed subject matter provides zinc-binding complexes and methods for detecting bioavailable zinc. In particular, 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.

In certain embodiments, 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. In specific embodiments, the biotinylated zinc-binding protein includes Metallothionein-2.

In certain embodiments, 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. In specific embodiments, 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. In specific embodiments, the polymeric microsphere includes streptavidin. In particular embodiments, the polymeric microsphere includes streptavidin coated polystyrene beads. The polymeric microsphere can have a diameter of from about 1 μιη to about 20 μιη.

In certain embodiments, 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.

In certain embodiments, the biotinylated zinc-binding protein can be attached to the polymeric microsphere. The biotinylated zinc -binding protein can include Metallothionein-2. In other embodiments, 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.

In certain embodiments, 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- (ethyloxycarbonylmethyloxy) quinoline (Zinquin ethyl ester), 6-Methoxy-(8-p- toluenesulfonamido)quinoline (TSQ), rhodafluor dyes (e.g., (l-[9'-(0- carboxyphenyl)-6'-amino-2'-chloro-3'-xanthanone]-4,10-(diethyl)-7-(2- pyridylmethyl)-l,4,7,10-tetraazacyclododecane), 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 a combination thereof. In specific embodiments, the fluorophore includes 4',5'-Bis[bis(2-pyridylmethyl)aminomethyl]-2',7 '-dichlorofluorescein.

In specific embodiments, the step of detecting a fluorescence signal in the method is performed using flow cytometry. In certain embodiments, the intensity of the fluorescence signal indicates zinc ion levels in the concentration range from about 0.01 μΜ to about 50 μΜ.

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.

In certain embodiments, the fluorophore includes 4',5'-Bis[bis(2- pyridylmethyl)aminomethyl]-2',7 '-dichlorofluorescein. BRIEF DESCRIPTION OF THE FIGURES

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.

DETAILED DESCRIPTION

The presently disclosed subject matter relates to complexes and methods for detecting bioavailable zinc. In particular, the presently disclosed subject matter provides zinc -binding complexes and methods for detecting one or more zinc ions in a sample.

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measure or determine, e.g. , the limitations of the measurement system. For example, "about" can mean a range of up to 20%, up to 10%, up to 5%, and or up to 1% of a given value.

ZINC BINDING COMPLEX

The zinc binding complex of the presently disclosed subject matter includes zinc-binding proteins, polymeric microspheres, and fluorophores.

Zinc-Binding Proteins

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.

In certain embodiments, the zinc -binding protein includes a zinc -binding group. Additionally or alternatively, 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. For example, but not by way of limitation, 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.

In certain embodiments, 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. For example, but not by way of limitation, 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.

Polymeric Microspheres

The zinc-binding complex of the presently disclosed subject matter further includes a polymeric microsphere. In certain embodiments, 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, for example, 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. For example, but not by way of limitation, the microsphere can have a diameter from about 1 μιη to about 20 μιη, or from about 1 μιη to about 10 μιη. In certain embodiments, the microsphere has a diameter of about 5 μιη or about 10 μιη. In addition, the microsphere can have a shape that is predominantly spherical in form. In certain embodiments, the diameter of the microsphere can be chosen to differentiate the microsphere from one or more cell sizes in a sample.

In specific embodiments, the microsphere is composed of a particulate material. For example, but not by way of limitation, the particulate material can include silica gel, glass, nylon, latex, polystyrene, Sephadex, Sepharose, cellulose, metal, polyethylene, or polypropylene. In addition, the microsphere can be swellable (e.g. , Wang resin bead) or non-swellable (e.g. , controlled-pore glass).

Fluorophores

In certain embodiments, the zinc-binding complex of the presently disclosed subject matter includes a fluorophore. In certain embodiments, 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. , Ν,Ν,Ν',Ν'- tetramethyl-6-carboxyrhodamine, 6-carboxy-X-rhodamine, 5-carboxyrhodamine-6G, 6-carboxyrhodamine-6G, and rhodamine 110), cyanine (e.g., Cy3, Cy5, Cy7), alexa (e.g., Alexa Fluor 555, Alexa Fluor 594), dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, cascade blue, coumarin derivatives (e.g. , umbelliferone), benzimide (e.g. , Hoechst 33258), phenanthridine (e.g., Texas red), ethidium, acridine, carbazole, phenoxazine, porphyrin, polymethine, boron- dipyrromethene (BODIPY), quinoline, pyrene derivatives, pyridyloxazole derivatives, 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 fluorescamine.

Attachment Means

In certain embodiments, 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. In certain embodiments, biotin is any suitable biotin derivative including, but not limited to biotin, biotin sulfone, caprolyamidobiotin, biocytin, desthiobiotin, desthiobiocytin, iminobiotin, or analogs and derivatives thereof. For example, but not by way of limitation, 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. Non-limiting examples of biotin-binding proteins include, but are not limited to streptavidin, avidin, ferritin avidin, nitroavidin, nitrostreptavidin, neutravidin, captavidin, or analogs and derivatives thereof.

In certain embodiments, 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. For example, but not by way of limitation, 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.

METHODS FOR DETECTING Z/NC IONS

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. Furthermore, 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. Non-limiting examples of 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. In certain embodiments, 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. Additionally, 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. In certain embodiments, 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.

Additionally, 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 μΐ_^ undiluted sample. For example, but by way of limitation, 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. 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. , 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- (ethyloxycarbonylmethyloxy) quinoline (Zinquin ethyl ester), 6-Methoxy-(8-p- toluenesulfonamido)quinoline (TSQ), rhodafluor dyes (e.g. , (l-[9'-(0- carboxyphenyl)-6'-amino-2'-chloro-3'-xanthanone]-4,10-(diethyl)-7-(2- pyridylmethyl)- l,4,7,10-tetraazacyclododecane), 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. For example, but not by way of limitation, U.S. Patent Pub. No. 2005/0142067 and Landero-Figueroa et al., 2014, Selectivity and Specificity of Small Molecule Fluorescent Dyes/Probes Used for the Detection of Zn and Ca in Cells, Metallomics, 6(2):301-305 discloses zinc -binding fluorophores, which are incorporated herein by reference in their entirety.

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. For example, but not by way of limitation, 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.

In certain embodiments, 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). For example, but not by way of limitation, U.S. Patent Pub. No. 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. In certain embodiments, the fluorescence signal can indicate zinc ion levels from about 0.01 μΜ to about 50 μΜ. For example, but not by way of limitation, the fluorescence signal indicates zinc ion levels from about 0.01 μΜ to about 0.05 μΜ, about 0.01 μΜ to about 0.1 μΜ, about 0.01 μΜ to about 0.5 μΜ, about 0.01 μΜ to about 1 μΜ, about 0.01 μΜ to about 3 μΜ, about 0.01 μΜ to about 5 μΜ, about 0.01 μΜ to about 10 μΜ, about 0.01 μΜ to about 20 μΜ, about 0.01 μΜ to about 30 μΜ, about 0.01 μΜ to about 40 μΜ, about 0.01 μΜ to about 50 μΜ, about 0.05 μΜ to about 50 μΜ, about 0.1 μΜ to about 10 μΜ, about 0.1 μΜ to about 50 μΜ, about 0.5 μΜ to about 50 μΜ, about 1 μΜ to about 50 μΜ, about 10 μΜ to about 50 μΜ, about 20 μΜ to about 50 μΜ, about 30 μΜ to about 50 μΜ, or about 40 μΜ to about 50 μΜ. Thus, the disclosed method can be used over a wider dynamic range than certain known methods such as plate assays. As a result, the disclosed method can be used to measure low concentrations of zinc, e.g., less than about 1 μΜ, without requiring multiple dilution steps to bring samples with high concentrations of zinc, e.g., greater than about 3 μΜ, within the range.

In certain embodiments, the steps of detecting and measuring a fluorescence signal can be performed using an epifluorescent microscope, laser scanning confocal microscope, or fluorsecent plate reader.

In certain embodiments, 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.

As described above, the disclosed method can be used in a wide range of analytical assays. In certain embodiments, the disclosed method can be used in any suitable existing flow multiparameter based evaluation. For example, the method can include the simultaneous measurement of two or more parameters using one or more parallel assays. For example, gating can be employed to target populations including the zinc-binding complex. In this manner, 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, Alternatively or additionally, zinc measurement can be combined with a functional assay, for example, to analyze phagocytosis, oxidative burst, and the like. In embodiments where a second assay is used, 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.

In certain embodiments where the method is used for a sample from a human or animal subject, the methods can be performed in vitro. For example, 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. Alternatively, the methods can be performed in vivo. For example, 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 Z/NC IONS

The presently disclosed subject matter further provides a kit for detecting zinc ions in a sample. For example, but not by way of limitation, the kit 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.

In certain embodiments, the zinc -binding complex can be stored and utilized in a plastic container. For example, but not by way of limitation, 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.

APPLICATIONS

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. Non-limiting examples of 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. For example, but by way of limitation, 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. In certain embodiments, 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.

EXAMPLE

The following example is merely illustrative of the presently disclosed subject matter and it should not be considered as limiting the scope of the invention in any way.

EXAMPLE 1: Metallothionein-2 Bound Microspheres for Zinc-Binding Fluorescence Analysis

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-Link™ 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. Upon finish of incubation, the Metallothionein-2 and NHS-PEG4- Biotin solution was applied directly onto the center of the resin bed of the Zeba™ 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.

The biotinylated Metallothionein-2 was then attached to polymeric microspheres. The biotinylated Metallothionein-2 (200 μg) was first added to 10 mg of washed SuperAvidin™ Coated Microspheres (9.4 μιη, 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 μΐ_^ buffer provided in the microsphere kit. The resulting Metallothionein-2 bound microspheres were then re-suspended in the same 100 μΐ_^ 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). After this addition, 1-1.25 μΜ of 4',5'-Bis[bis(2-pyridylmethyl)aminomethyl]-2',7'-dichlorofluorescein (Zinpyr- 1) (sc-21382) from Santa Cruz Biotechnology, Inc. was maintained in 100-500 μΐ_^ of the zinc solution at a temperature range of 4-25° C.

Fluorescence of the Metallothionein-2 bound microspheres with Zinpyr- 1 was then captured with a BD™ 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). For further comparison, 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). Unlike TPEN, 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 μΜ and 10 μΜ) (Figure 4). Thus, 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.

* * *

Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosed subject matter as defined by the appended claims. Moreover, the scope of the disclosed subject matter is not intended to be limited to the particular embodiments described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the disclosed subject matter, alternatives presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such alternatives. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

Various publications, patents, and patent applications are cited herein, the contents of which are hereby incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A zinc-binding complex comprising a biotinylated zinc-binding protein and a polymeric microsphere.

2. The zinc-binding complex of claim 1, wherein the biotinylated zinc-binding protein is attached to the polymeric microsphere.

3. The zinc-binding complex of claim 1, wherein the biotinylated zinc-binding protein is selected from the group consisting of Metallothionein-1, Metallothionein-2, Metallothionein-3, Metallothionein-4, alpha-2-Macroglobulin, S 100 proteins, albumin, and combinations thereof.

4. The zinc-binding complex of claim 3, wherein the biotinylated zinc-binding protein comprises Metallothionein-2.

5. The zinc -binding complex of claim 1, wherein the polymeric microsphere comprises a particulate material selected from the group consisting of silica gel, glass, nylon, latex, polystyrene, Sephadex, Sepharose, cellulose, metal, polyethylene, polypropylene, and combinations thereof.

6. The zinc -binding complex of claim 5, wherein the polymeric microsphere comprises polystyrene.

7. The zinc -binding complex of claim 1, wherein the polymeric microsphere comprises a biotin-binding protein selected from the group consisting of streptavidin, avidin, ferritin avidin, nitroavidin, nitrostreptavidin, neutravidin, captavidin, and analogs and derivatives thereof.

8. The zinc -binding complex of claim 7, wherein the polymeric microsphere comprises streptavidin.

9. The zinc -binding complex of claim 1, wherein the polymeric microsphere comprises streptavidin coated polystyrene beads.

10. The zinc -binding complex of claim 1, wherein the polymeric microsphere has a diameter of from about 1 μιη to about 20 μιη.

11. The zinc -binding complex of claim 1, further comprising a fluorophore selected from the group consisting of fluoresceine derivatives, rhodamine derivatives, cyanine, alexa, phenanthridine, ethidium, acridine, carbazole, phenoxazine, porphyrin, polymethine, boron-dip yrromethene, quinoline, pyrene derivatives, pyridyloxazole derivatives, indicators based on the N-(2-methoxyphenyl) iminodiacetate chelator, BAPTA-based indicators, fluorescamine, and combinations thereof.

12. The zinc-binding complex of claim 8, wherein the fluorophore is modified with a reactive group selected from the group consisting of dichlorotriainyl, isothiocyanate, succinimidyl ester, and sulfonyl chloride.

13. A method for detecting zinc ions in a sample, comprising:

(a) providing a zinc -binding complex comprising a biotinylated zinc- binding protein and a polymeric microsphere;

(b) contacting the sample with the zinc -binding complex;

(c) adding a fluorophore to the sample;

(d) detecting a fluorescence signal emitted by the fluorophore; and

(e) measuring the fluorescence signal, wherein intensity of the fluorescence signal indicates zinc ion levels.

14. The method of claim 13, wherein the biotinylated zinc -binding protein is attached to the polymeric microsphere.

15. The method of claim 13, wherein the biotinylated zinc -binding protein comprises Metallothionein-2.

16. The method of claim 13, wherein the polymeric microsphere comprises streptavidin coated polystyrene beads.

17. The method of claim 13, wherein the zinc -binding complex is present in a concentration of from about 100 complexes/mL to about 100,000 complexes/mL.

18. The method of claim 13, wherein the fluorophore is selected from the group consisting of 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-(ethyloxycarbonylmethyloxy) quinoline (Zinquin ethyl ester), 6-Methoxy-(8-p-toluenesulfonamido)quinoline (TSQ), rhodafluor dyes (e.g. , (l-[9'-(O-carboxyphenyl)-6'-amino-2'-chloro-3'-xanthanone]-4, 10-(diethyl)-7- (2-pyridylmethyl)-l,4,7, 10-tetraazacyclododecane), 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), and combinations thereof

19. The method of claim 18, wherein the fluorophore comprises 4',5'-Bis[bis(2- pyridylmethyl)aminomethyl]-2',7 '-dichlorofluorescein.

20. The method of claim 13, wherein the step of detecting a fluorescence signal is performed using flow cytometry.

21. The method of claim 13, wherein the intensity of the fluorescence signal indicates zinc ion levels in a concentration of from about 0.01 μΜ to about 50 μΜ.

22 A kit for detecting zinc ions in a sample, comprising a zinc -binding complex according to claim 1 and a fluorophore, packaged in one or more containers with one or more further reagents.

23. The kit of claim 22, wherein the fluorophore comprises 4',5'-Bis[bis(2- pyridylmethyl)aminomethyl]-2',7'-dichlorofluorescein.