Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-09-14
2002-10-22
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S968000, C435S004000, C435S173400, C436S063000, C436S172000
Reexamination Certificate
active
06468763
ABSTRACT:
BACKGROUND OF THE INVENTION
In biological and biochemical research, model systems are employed as screening tools to determine whether a given line of research will lead the investigator to the ultimately sought results, e.g., utility in medical, diagnostic or consumer applications, or the like. In order for these model systems to provide the most relevant information, it is often necessary for the model to come as close as possible to the ultimate application as is practical. In pharmaceutical research, the approximation of living systems has led to the substantial use of in vivo models, e.g., live animal testing, and pseudo-in vivo models, e.g., screening of living cell cultures in vitro, also termed cell-based assays.
Cell based assays are often preferred for initial screening assays due to their approximation of in vivo systems combined with their capability to be rapidly screened. Once a cellular system is selected as a model assay system, there is an additional problem of detecting the functioning of the cells in response to a particular stimulus. Specifically, most cellular responses of interest are not readily assayable by convenient means, e.g., using optical detection methods.
In order to address the detection problem, a number of solutions have been proposed. For example, one solution is to continually monitor the immediately surrounding environment of the cells for changes in pH, where the rate of pH change is related to the metabolic state of the particular cell. See, e.g., U.S. Pat. Nos. 5,278,048 and 5,496,697 to Parce et al. Alternative methods employ intracellular dyes which indicate levels of intracellular components, e.g., calcium, pH, and the like, and use those measurements as an indication of cellular functioning.
Another class of assays simply employs dyes that label internal components of the cells, e.g., nucleic acids. Upon cellular death, the integrity of the cellular membrane is compromised, leading to a leaking of the nucleic acids and dye. Thus, the presence of the dye within the cell is used as a measure of cellular viability.
In still another class of assays, the electrical potential gradient between the inside and the outside of a cell or of a cell compartment is used as an indication of the ion transport functions of cellular components. A variety of methods have been previously described for detecting transmembrane potentials in vesicles. Many of these methods, e.g., Biophys. Biochim. Acta 1237:121-126 (1995), Biophys. J. 71:2680-2691 (1996), and Ann. Rev. Biophys. Bioeng. 10:217-244 (1981), are not suitable for use with live cells in cell based assays because of nonspecific staining, toxicity, slow response time, or lack of sensitivity. Some methods have been described for detecting transmembrane potential in living cells. See, e.g., U.S. Pat. No. 5,661,035, and references cited therein, Biophys. J. 69:1272-1280 (1995), Chem Biol. 4:269-277 (1997), J. Biomol. Screen. 1:75-80 (1999), Cytometry 14:59-69 (1993) and J. Memb. Biol. 130:1-10 (1992).
Despite the availability of membrane potential sensor compositions and assays, there still exists a need for rapid detection of transmembrane potential in a readily automatable, high-throughput format. The present invention generally meets these and a variety of other needs.
SUMMARY OF THE INVENTION
The present invention provides compositions that are used in the detection of transmembrane potentials in living cells, methods of using these compositions in assaying biological function and in screening for effectors of that function, systems and microfluidic devices that use such compositions in performing these biological assays, and kits including these compositions. The compositions include first and second membrane associated components which produce or quench a fluorescent signal when adjacent to each other. The first component typically comprises a non-fluorescent anionic or cationic fluorescence quenching compound or a cationic fluorophore that translocates from one leaflet of the membrane to the other leaflet, in response to a change in transmembrane potential. The second component is disposed adjacent to or within one leaflet of the membrane and is selected from a fluorophore or a quencher, depending upon the nature of the first component. As a result, changes in transmembrane potential produce an increase or a decrease in the level of fluorophore quenching (or a decrease or increase in the amount of emitted fluorescence, respectively).
The present invention also provides methods of detecting transmembrane potential changes in a cell, kits for detecting transmembrane potential changes, and microfluidic devices and systems in which these methods may be carried out.
REFERENCES:
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patent: 5661035 (1997-08-01), Tsien et al.
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Cafiso, D.S. et al., “EPR Determination of Membrane Potentials,”Ann. Rev. Biophys. Bioeng.(1981) 10:217-244.
Gonazlez, J.E. et al., “Voltage Sensing by Fluorescence Resonance Energy Transfer in Single Cells,”Biophys. J.(1995) 69:1272-1280.
Gonzalez, J.E. et al., “Improved Indicators of Cell Membrane Potential that use Fluorescence Resonance Energy Transfer,”Chemistry&Biology(1997) 4:269-277.
Jankowski, A. et al., “A noninvasive fluorimetric procedure for measurement of membrane potential,”J. Biol. Chem.(1999) 26098-26104.
Leenhouts, J.M. et al., “Membrane potential-driven translocation of a lipid-conjugated rhodamine,”Biophys. Biochim. Acta(1995) 1237:121-126.
Loew, L.M. et al., “A Naphthyl Analog of the Aminostyryl Pryidinium Class of Potentiometric Membrane Dyes Shows Consistent Sensitivity in a Variety of Tissue, Cell, and Model Membrane Preparations,”J. membrane Biol.(1992) 130:1-10.
Matko, J. et al., “Luminescence Quenching by Nitroxide Spin Labels in Aqueous Solution: Studies on the Mechanism of Quenching,”Biochem(1992) 31:703-711.
Schroeder, K.S. et al., “FLIPR: A New Instrument for Accurate, High Throughout Optical Screening,”J. Biomol. Screen(1999) 1:75-80.
Tanner, M.K. et al., “Flow Cytometric Analysis of Altered Mononuclear Cell Transmembrane Potential Induced by Cyclosporin,”Cytometry(1993) 14:59-69.
Yguerabide, J., “Theory for Establishing Proximity Relations in Biological Membranes by Excitation Energy Transfer Measurements,”Biophys.J.(1994) 66:683-693.
Caliper Technologies Corp.
Leary Louise N.
Murphy Matthew B.
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