Optical biosensors
AssigneeCarnegie Mellon University
InventorsBrigitte F. Schmidt, Christopher S. Szent-Gyorgyi, Alan S. Waggoner, Peter B. Berget, Marcel P. Bruchez, Jonathan W. Jarvik
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Abstract
Provided are biosensors, compositions comprising biosensors, and methods of using biosensors in living cells and organisms. The biosensors are able to be selectively targeted to certain regions or structures within a cell. The biosensors may provide a signal when the biosensor is targeted and/or in response to a property of the cell or organism such as membrane potential, ion concentration or enzyme activity.
Claims26
A ligand-dye complex, comprising a cognate ligand of a dye non covalently bound to the dye, wherein the cognate ligand comprises an scFv molecule consisting of a polypeptide sequence having at least 90% sequence identity to the polypeptide of SEQ ID NO:
- 3, wherein the dye comprises one of a thiazole orange or a thiazole orange analog that binds the scFv molecule, and wherein the bound dye and ligand exhibit an increase in detectable fluorescence signal at least ten times greater than the detectable fluorescence signal of the dye when not bound to the ligand.
A ligand-dye complex comprising:
- an scFv molecule having complementarity determining regions of SEQ ID NOs 76, 77 and 78 noncovalently bound to a dye comprising one of a thiazole orange or a thiazole orange analog that binds the scFv molecule;
- wherein the bound dye and ligand exhibit an increase in detectable fluorescence signal at least ten times greater than the detectable fluorescence signal of the dye when not bound to the ligand.
A method of forming a ligand-dye complex for use in detecting expression of a gene in a cell, the method comprising:
- adding a vector to a cell, wherein the vector comprises a nucleic acid gene encoding a scFv molecule consisting of a polypeptide sequence having at least 90% sequence identity to the polypeptide of SEQ ID NO:
- 3;
- causing expression of the gene to produce a gene product comprising the scFv molecule;
- adding dye to the cell to bind to the gene product, wherein the dye comprises one of thiazole orange or a thiazole orange analog comprising the following general structure:
- wherein R is selected from the group consisting of a moiety that controls water solubility and non-specific binding, a moiety that controls the ability of the dye to enter a cellular compartment, a group that comprises, optionally with a linker, biotin, a hapten, a His-tag, or a moiety to facilitate isolation of the ligand, a fluorescent label optionally comprising a linker, a photoreactive group, and a reactive group.
A method of forming and using a ligand-dye complex for detecting interaction of two proteins in a cell comprising:
- adding a vector to a cell, wherein the vector comprises a nucleic acid gene encoding a first protein fused to a first portion of a scFv polypeptide sequence having at least 90% sequence identity to the polypeptide of SEQ ID NO:
- 3;
- adding a vector to a cell, wherein the vector comprises a nucleic acid gene encoding a second protein fused to a second portion of the scFv polypeptide sequence;
- causing expression of the genes to produce fusion proteins comprised of the first and second proteins fused to first and second portions, respectively, of the scFv polypeptide;
- and
- adding dye to the cell, wherein the dye comprises one of thiazole orange or a thiazole orange analog comprising the following general structure:
- wherein R is selected from the group consisting of a moiety that controls water solubility and non-specific binding, a moiety that controls the ability of the dye to enter a cellular compartment, a group that comprises, optionally with a linker, biotin, a hapten, a His-tag, or a moiety to facilitate isolation of the ligand, a fluorescent label optionally comprising a linker, a photoreactive group, and a reactive group;
- and
- detecting a fluorescence signal indicative of the dye binding to the fusion proteins, wherein the fluorescence signal is at least ten times greater than the detectable fluorescence signal of the dye when not bound to the fusion proteins.
Dependencies are inferred from the claim text.