Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-10-06
2003-03-25
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S968000, C435S007200
Reexamination Certificate
active
06537771
ABSTRACT:
COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The present invention is in the field of transmembrane potential measurement using Nernstian dyes, e.g., in microfluidic systems.
BACKGROUND OF THE INVENTION
Cell-based assays are often preferred for an initial screening of biologically active compounds, due to the approximation of in vivo systems by cells, combined with their capability to be rapidly screened. A variety of cell responses to stimuli can be detected, including cell death, transporter function and response to chemical stimuli.
The distribution of a permeable ion between the inside and outside of a cell or vesicle depends on the transmembrane potential of the cell membrane. In particular, for ions separated by a semi permeable membrane, the electrochemical potential difference (&Dgr;&mgr;
j
) which exists across the membrane, is given by &Dgr;&mgr;
j
=2.3 RT log [j
I
]/[j
o
]+zE
R
F, where R is the universal gas constant, T is an absolute temperature of the composition, F is Faraday's constant in coulombs, [j
I
] is the concentration of an ion (j) on an internal or intracellular side of the at least one membrane, [j
o
] is the concentration of j on an external or extracellular side of the at least one membrane, z is a valence of j and E
R
is a measured transmembrane potential. Thus, the calculated equilibrium potential difference (E
j
) for ion j=−2.3RT(zF)
−1
log[j
I
]/[j
o
] (this is often referred to as the Nernst equation). See, Selkurt, ed. (1984)
Physiology
5
th
Edition
, Chapters 1 and 2, Little, Brown, Boston, Mass. (ISBN 0-316-78038-3); Stryer (1995)
Biochemistry
4th edition Chapters 11 and 12, W.H. Freeman and Company, N.Y. (ISBN 0-7167-2009-4); Haugland (1996)
Handbook of Fluorescent Probes and Research Chemicals Sixth Edition
by Molecular Probes, Inc. (Eugene Oreg.) Chapter 25 (Molecular Probes, 1996) and http://www.probes.com/handbook/sections/2300.html (Chapter 23 of the on-line 1999 version of the
Handbook of Fluorescent Probes and Research Chemicals Sixth Edition
by Molecular Probes, Inc.) (Molecular Probes, 1999) and Hille (1992)
Ionic Channels of Excitable Membranes
, second edition, Sinauer Associates Inc. Sunderland, Mass. (ISBN 0-87893-323-9) (Hille), for an introduction to transmembrane potential and the application of the Nernst equation to transmembrane potential. In addition to the Nernst equation, various calculations which factor in the membrane permeability of an ion, as well as Ohm's law, can be used to further refine the model of transmembrane potential difference, such as the “Goldman” or “constant field” equation and Gibbs-Donnan equilibrium. See Selkurt, ed. (1984)
Physiology
5
th
Edition
, Chapter 1, Little, Brown, Boston, Mass. (ISBN 0-316-78038-3) and Hille at e.g., chapters 10-13.
Increases and decreases in resting transmembrane potential—referred to as membrane depolarization and hyperpolarization, respectively—play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signaling and ion-channel gating. Potentiometric optical probes (typically potentiometric dyes) provide a tool for measuring transmembrane potential and changes in transmembrane potential over time (e.g., transmembrane potential responses following the addition of a composition which affects transmembrane potential) in membrane containing structures such as organelles (including mitochondria and chloroplasts), cells and in vitro membrane preparations. In conjunction with probe imaging techniques (e.g., visualization of the relevant dyes), these probes are employed to map variations in transmembrane potential across excitable cells and perfused organs.
For example, the plasma membrane of a cell at rest typically has a transmembrane potential of approximately −20 to −70 mV (negative inside) as a consequence of K
+
, NA
+
and Cl
−
concentration gradients (and, to a lesser extent, H
+
, Ca
2+
, and HCO
3
−
) that are maintained by active transport processes. Potentiometric probes are important tools for studying these processes, as well as for visualizing, e.g., mitochondria (which exhibit a large transmembrane potential of approximately −150 mV, negative inside matrix), and for cell viability assessment. See, Molecular Probes (1996) chapter 25 and the references cited therein.
Potentiometric probes include cationic or zwitterionic styryl dyes, cationic rhodamines, anionic oxonols, hybrid oxonols and merocyanine 540. The class of dye determines factors such as accumulation in cells, response mechanism and cell toxicity. See, Molecular Probes 1999 and the reference cited therein; Plasek et al. (1996) “Indicators of Transmembrane potential: a Survey of Different Approaches to Probe Response Analysis.”
J Photochem Photobiol
: Loew (1994) “Characterization of Potentiometric Membrane Dyes.”
Adv Chem Ser
235, 151 (1994); Wu and Cohen (1993) “Fast Multisite Optical Measurement of Transmembrane potential”
Fluorescent and Luminescent Probes for Biological Activity
, Mason, Ed., pp. 389-404; Loew (1993) “Potentiometric Membrane Dyes.”
Fluorescent and Luminescent Probes for Biological Activity
, Mason, Ed., pp. 150-160; Smith (1990) “Potential-Sensitive Molecular Probes in Membranes of Bioenergetic Relevance.”
Biochim Biophys Acta
1016, 1; Gross and Loew (1989) “Fluorescent Indicators of Transmembrane potential: Microspectrofluorometry and Imaging.”
Meth Cell Biol
30, 193; Freedman and Novak (1989) “Optical Measurement of Transmembrane potential in Cells, Organelles, and Vesicles”
Meth Enzymol
172, 102 (1989); Wilson and Chused (1985) “Lymphocyte Transmembrane potential and Ca
+2
-Sensitive Potassium Channels Described by Oxonol Dye Fluorescence Measurements”
Journal of Cellular Physiology
125:72-81; Epps et al. (1993) “Characterization of the Steady State and Dynamic Fluorescence Properties of the Potential Sensitive dye bis-(1.3-dibutylbarbituric acid) trimethine oxonol (DiBAC
4
(3) in model systems and cells”
Chemistry of Physics and Lipids
69:137-150, and Tanner et al. (1993) “Flow Cytometric Analysis of Altered Mononuclear Cell Transmembrane potential Induced by Cyclosporin”
Cytometry
14:59-69.
Potentiometric dyes are typically divided into at least two categories based on their response mechanism. The first class of dyes, referred to as fast-response dyes (e.g., styrylpyridinium dyes; see, e.g., Molecular Probes (1999) at Section 23.2), operate by a change in the electronic structure of the dye, and consequently the fluorescence properties of the dye, i.e., in response to a change in an electric field which surrounds the dye. Optical response of these dyes is sufficiently fast to detect transient (millisecond) potential changes in excitable cells, e.g., isolated neurons, cardiac cells, and even intact brains. The magnitude of the potential-dependent fluorescence change is often small; fast-response probes typically show a 2-10% fluorescence change per 100 mV.
The second class of dyes, referred to as slow-response or Nemstian dyes (See, e.g., Molecular Probes, 1999 at Section 23.3), exhibit potential-dependent changes in membrane distribution that are accompanied by a fluorescence change. The magnitude of their optical responses is typically larger than that of fast-response probes. Slow-response probes, which include cationic carbocyanines, rhodamines and anionic oxonols, are suitable for detecting changes in a variety of transmembrane potentials of, e.g., nonexcitable cells caused by a variety of biological phenomena, such as respiratory activity,
Farinas Javier Anibal
Wada H. Garrett
Caliper Technologies Corp.
Filler Andrew L.
Leary Louise N.
Quine Jonathan Alan
Quine Intellectual Property Law Group P.C.
LandOfFree
Use of nernstein voltage sensitive dyes in measuring... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of nernstein voltage sensitive dyes in measuring..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of nernstein voltage sensitive dyes in measuring... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3038166