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Chemical probe compounds that become fluorescent upon reduction, and methods for their useRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Viable Micro-organism, Testing For Antimicrobial Activity Of A MaterialChemical probe compounds that become fluorescent upon reduction, and methods for their use description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060199242, Chemical probe compounds that become fluorescent upon reduction, and methods for their use. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Patent Application Ser. No. 60/657,944 filed Mar. 1, 2005, the contents of which are incorporated herein by reference. FIELD OF THE INVENTION [0002] The invention relates to quenched chemical stain compounds that can be reduced to a fluorescing form, and methods for their use. DESCRIPTION OF RELATED ART [0003] Oxidation and reduction are natural metabolic functions of living cells. Methods and materials for the detection of the oxidative states of cells have been desirable for some time, as they allow researchers to achieve a better understanding of the conditions within cells or cell compartments. Various methods have been used to date. [0004] One approach is to use a chemical probe that is not fluorescent outside of the cell, but which becomes oxidized to a fluorescent derivative once it enters the cell. Dihydrorhodamine and dihydroethidium are examples of such chemicals that oxidize inside a cell compartment (Haugland, Richard P.; Handbook of Fluorescent Probes and Research Products; ninth edition; 2002). These stains are used to gain entry into live cells, and are not used for measuring redox functions in cells. The stains can be readily oxidized by molecular oxygen. [0005] Derivatives of quinones, naphthaquinones, and anthraquinones have been reported to quench fluorescence when linked to a fluorophore (Kong, J. C. Y. and Loach, R. A.; J Heterocycl. Chem. 17: 737 (1978); Wasielewski, M. R. and Nienczyk, M. P.; J. Am. Chem. Soc. 106: 5040 (1984)). [0006] Israel Patent Application No. 156355 (filed Jun. 9, 2003; published Jan. 4, 2004) suggests the preparation of compounds containing a reversible quinone/hydroquinone redox site, a spacer, and a fluorophore. The compounds do not fluoresce when in the oxidized state, but become fluorescent once reduced. The compounds are suggested as being useful as fluorescent redox probes for chemical, biochemical, and biophysical investigations. [0007] U.S. Pat. No. 6,057,120 (issued May 2, 2000) describes the use of redox-active compounds for the determination of an analyte. The redox pairs have a benzoquinoxaline substructure. The compounds are suggested to be used to determine the reducing or oxidizing activities of cells and enzymes. [0008] 5-Cyano-2,3-ditolyl tetrazolium chloride ("CTC"; commercially available from Polysciences, Inc.; Warrington, PA) is a monotetrazolium redox dye which produces a fluorescent formazan ("CTF") upon reduction. According to the manufacturer, the dye can be used in flow cytometry with excitation using a 488 nm laser and detection in the red color region (Data Sheet #486; September 1999). [0009] Various species can effect the oxidation of dyes, complicating the interpretation of experimental results. Examples of such species include molecular oxygen, superoxide, and hydrogen peroxide. This oxidation is used in connection with compounds such as dihydrorhodamine and dihydroethidium, where the neutral reduced form can enter cells by diffusion, and be subsequently oxidized to the fluorescent dye. These materials are not generally used to measure the oxidative function of cells, as atmospheric oxygen can oxidize them as well. [0010] Despite advances made to date, there still exists a need for chemical probes and methods for the monitoring indicators of metabolic functions within cells, tissues, and other materials. SUMMARY OF THE INVENTION [0011] Chemical compound probes containing a fluorophore unit and a reducible quenching unit are disclosed. The probes can also contain a linker unit covalently bonded to the fluorophore unit and the reducible quenching unit. Upon reduction, the reducible quenching unit exhibits a diminished quenching ability, and fluorescence of the compound can be detected. The probes can be used in a variety of applications to monitor the conditions of cells (including bacteria), tissues, and other materials. Applications include monitoring of change in oxidation state of a material, change in electron transport chain function, and change in cellular vitality after treatment with antibiotics, inhibitors, or other chemical compounds. DESCRIPTION OF THE FIGURES [0012] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. [0013] FIG. 1 shows chemical intermediates and compounds that are illustrative of embodiments of the invention. [0014] FIG. 2 shows a titration curve for Gram-positive Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis). The x-axis represents the concentration of compound (18) in nM, while the y-axis represents FL1 GeoMean fluorescence. [0015] FIG. 3 shows a titration curve for Gram-negative Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). The x-axis represents the concentration of compound (18) in nM, while the y-axis represents FL1 GeoMean fluorescence. [0016] FIG. 4 shows growth curve data obtained during a time-course experiment. The x-axis represents time in hours, while the y-axis represents S. aureus cells per mL. [0017] FIG. 5 shows fluorescence data obtained during a time-course experiment. The x-axis represents time in hours, while the y-axis represents FL1 GeoMean fluorescence. [0018] FIG. 6 shows fluorescence data obtained during a time-course experiment. The x-axis represents time in hours, while the y-axis represents FL1 GeoMean fluorescence. 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