Fluorescent pH indicators for intracellular assays

Details Fluorescent pH indicators for intracellular assays Fluorescent pH indicators for intracellular assays

2002-03-27

2004-10-05

Owens, Amelia A. (Department: 1625)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C549S280000

Reexamination Certificate

active

06800765

ABSTRACT:

CROSS-REFERENCES TO ADDITIONAL MATERIALS
This application incorporates by reference in their entirety for all purposes the following U.S. Pat. No. 5,355,215, issued Oct. 11, 1994; U.S. Pat. No. 6,097,025, issued Aug. 1, 2000; and U.S. Pat. No. 6,159,425, issued Dec. 12, 2000.
This application incorporates by reference in their entirety for all purposes the following U.S. patent application Ser. No. 09/337,623, filed Jun. 21, 1999; Ser. No. 09/349,733, filed Jul. 8, 1999; Ser. No. 09/478,819, filed Jan. 5, 2000; Ser. No. 09/596,444, filed Jun. 19, 2000; Ser. No. 09/626,208, filed Jul. 26, 2000; Ser. No. 09/643,221, filed Aug. 18, 2000; Ser. No. 09/710,061, filed Nov. 10, 2000; Ser. No. 09/722,247, filed Nov. 24, 2000; Ser. No. 09/759,711, filed Jan. 12, 2001; Ser. No. 09/765,869, filed Jan. 19, 2001; Ser. No. 09/765,874, filed Jan. 19, 2001; Ser. No. 09/766,131, filed Jan. 19, 2001; Ser. No. 09/767,316, filed Jan. 22, 2001; Ser. No. 09/767,434, filed Jan. 22, 2001; Ser. No. 09/767,579, filed Jan. 22, 2001; Ser. No. 09/767,583, filed Jan. 22, 2001; Ser. No. 09/768,661, filed Jan. 23, 2001; Ser. No. 09/768,742, filed Jan. 23, 2001; Ser. No. 09/768,765, filed Jan. 23, 2001; Ser. No. 09/770,720, filed Jan. 25, 2001; Ser. No. 09/770,724, filed Jan. 25, 2001; Ser. No. 09/777,343, filed Feb. 5, 2001; Ser. No. 09/836,575, filed Apr. 16, 2001; Ser. No. 09/844,655, filed Apr. 27, 2001; Ser. No. 10/003,030, filed Oct. 29, 2001; Ser. No. 10/012,255, filed Nov. 12, 2001; Ser. No. 10/000,172, filed Nov. 30, 2001; and Ser. No. 10/061,416, filed Feb. 1, 2002.
This application incorporates by reference in their entirety for all purposes the following U.S. Provisional Patent Application Serial No. 60/223,642, filed Aug. 8, 2000; Serial No. 60/244,012, filed Oct. 27, 2000; Serial No. 60/250,683, filed Nov. 30, 2000; Serial No. 60/287,697, filed Apr. 30, 2001; Serial No. 60/316,704, filed Aug. 31, 2001; Serial No. 60/318,038, filed Sep. 7, 2001; and Serial No. 60/318,149, filed Sep. 7, 2001.
This application incorporates by reference in their entirety for all purposes the following publications: Richard P. Haugland,
Handbook of Fluorescent Probes and Research Chemicals
(6
th
ed. 1996); and Joseph R. Lakowicz,
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
(2
nd
Ed. 1999).
FIELD OF THE INVENTION
The invention relates to compositions and methods for measuring pH. More particularly, the invention relates to compositions and methods for measuring pH in cells, organelles, and other samples using fluorescent and fluorogenic 2′,7′-dialkylfluorescein derivatives.
BACKGROUND OF THE INVENTION
The measurement of the pH of biological fluids is an important aspect of a variety of assays. In particular, intracellular pH plays an important modulating role in many cellular events, including cell growth, calcium regulation, enzymatic activity, receptor-mediated signal transduction, ion transport, endocytosis, chemotaxis, cell adhesion, and other cellular processes. The use of fluorescence-based techniques may increase the sensitivity of measurements and allow the measurement of intracellular pH in single cells, for example, by flow cytometry or existing fluorescence-based automated high-throughput microplate assays. Imaging techniques that use fluorescent pH indicators also may allow researchers to investigate these processes with much greater spatial resolution and sampling density than can be achieved using other available technologies, such as microelectrodes. Fluorescence assays are typically able to utilize low concentrations of the pH indicator, potentially reducing toxicity and buffering effects.
In selecting a pH indicator, to allow the greatest sensitivity to small changes in pH of the medium, the equilibrium constant between the acidic and basic forms of the indicator for the dye (i.e., the pK
a
) should be near the pH of the selected assay medium. For physiological assays, such as blood and most intracellular fluid assays, the pH typically is in the range pH 6 to pH 8, and more typically in the range pH 6.5 to 7.6.
A variety of fluorescein-based pH indicators have been described, including fluorescein, carboxyfluorescein, fluorescein sulfonic acid, chloromethylfluorescein, carboxynaphthofluorescein, seminaphthorhodafluor derivatives, and seminaphthofluorescein derivatives. Typically, for intracellular assays, the dye is used as a cell-permeant diacetate derivative that is subsequently cleaved by nonspecific esterases within the cells of the sample, producing the active indicator.
Perhaps the most commonly utilized fluorescein-based pH indicator is 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, more commonly known as BCECF. BCECF has a pK
a
of 7.0, making it an ideal choice for intracellular assays. In addition, BCECF exhibits pH-dependent dual excitation and is excited efficiently by the 488-nm line of an argon-ion laser, as used in a variety of instruments. Ester derivatives of BCECF, most typically BCECF-AM, are nonfluorescent and membrane-permeant, and can be loaded into cells without disruption of the cell membranes. The conversion to the fluorescent version of the indicator can serve as an indicator of cell viability, as well as permitting subsequent pH measurements.
Unfortunately, for all its advantages, BCECF also possesses significant disadvantages. The synthesis of BCECF-AM typically produces a mixture of discrete compounds, as shown below in formulas I, II, and III.
This results in substantial variations in the ratios of active components and the profiles of impurities. Thus, different commercial sources of BCECF-AM commonly yield indicators of highly variable quality due to differences in their manufacturing processes. In fact, the quality often varies considerably from batch to batch even from a single manufacturer. The variation of component ratios has caused some difficulties in obtaining reproducible data due to the lack of consistent commercial material. In addition, the extremely weak excitation peak of BCECF at shorter wavelengths makes excitation-ratiometric measurements difficult, and it is impossible to perform emission-ratiometric pH measurements using BCECF due to the lack of pH-dependent dual emission. Thus, there is a need for improved fluorescent pH indicators, particularly for intracellular assays.
SUMMARY OF THE INVENTION
The invention provides systems including compositions and methods for measuring pH, particularly in cells, organelles, and other samples. The compositions include pH-sensitive fluorescent and fluorogenic 2′,7′-dialkylfluorescein derivatives and associated nonfluorescent precursor compounds. The compositions may allow ratiometric measurement in the excitation spectrum and the emission spectrum. The methods include adding a precursor compound to a sample cell, incubating the sample cell to release the free indicator, illuminating the sample cell, and detecting the fluorescence response of the free indicator.


REFERENCES:
patent: 0 025 912 (1981-04-01), None
The Fluorescent Probe BCECF Has a Heterogenous Distribution in Sea Urchin Eggs, Mozingo et al.,Cell Biology International Reports, vol. 14, No. 8, pp. 689-699, Aug. 1990.
A Novel In Vitro Screening Assay for Trypanocidal Activity Using the Fluorescent Dye BCECF-AM, Obexer e al.,Trop. Med. Parasitol, vol. 46, pp. 45-48, 1995.
Fluorescent Characteristics and Pharmacokinetic Profiles of the Fluorescent Probe BCECF in Various Tissues: The Role of Blood Content, Devoisselle et al.,Photochemistry and Photobiology, vol. 64, No. 6. pp. 906-910, 1996.
pH Indicators, Haugland,Handbook of Fluorescent Probes and Research Chemicals, pp. 551-570, ©1996.
Ratiometric Measurement of Intracellular pH of Cultured Cells With BCECF in a Fluorescence Multi-Well Pante Render, Grant et al.,In Vitro Cell Dev. Biol., vol. 33, pp. 256-260, Apr. 1997.
BCECF in Single Cultured Cells: Inhomogeneous Distribution but Homogeneous Response, Weinlich et al.,The Journal of Experimental Biology, vol. 201, pp. 57-62, 1998.
Analysis of the Uptake of the Fluorescent Marker 2′,7′-bis-(2-

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