Carnegie Mellon University

Alan S Waggoner

Ratnakar B Mujumdar

Bhalchandra M Karandikar

Jonathan Minden

Swati R Mujumdar

Marcel Pierre Bruchez

Lauren A Ernst

Brigitte F Schmidt

Susan Janet Fowler

Jonathan W Jarvik

Christopher S Szent-Gyorgyi

Peter B Berget

Subhasish K Chakraborty

D Lansing Taylor

Frederick Lanni

Mingrui Zhang

Philip L Southwick

Mark V Reddington

James Fitzpatrick

Chris Szent-Gyorgyi

Chris Szent-Gyorgi

Biosensor comprising an activatable acceptor fluorogen linked via a linker to a donor which transfers energy to the fluorogen on detecting an analyte wherein the fluorogen component reacts and a 100 fold increase in intensity results when the fluorogen interacts non-covalently with an activator e.g. fluorogen activator peptide.

Targeted probes of cellular physiology

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.

Optical biosensors

The present invention presents designs for high extinction quenched 'dyedrons' that can be activated by conversion of a single acceptor/quencher in the molecular assembly to a fluorescent state. The quencher is activated by noncovalent binding to a unique complementary expressible fluorogen activating peptide (FAP). In this way, the quencher serves as the homogeneous switch, receiving energy efficiently from each of the donor molecules of the dendronic antenna, and releasing it as fluorescence only when activated by binding. The sum of the extinction of the multiple dyes on the antenna will provide dramatic enhancements in the effective brightness of the probe in standard imaging systems. This approach provides a set of probes with exceptional brightness, specifically targeted to an expressed tag that activates the fluorescence of the dyedron.

Quenched dendrimeric dyes for florescence detection

Tissue slices and whole organisms offer substantial challenges to fluorescence imaging. Autofluorescence and absorption via intrinsic chromophores, such as flavins, melanin, and hemoglobins, confound and degrade output from all fluorescent tags. An 'optical window,' farther red than most autofluorescence sources and in a region of low hemoglobin and water absorbance, lies between 650 and 900 nm. This valley of relative optical clarity is an attractive target for fluorescence-based studies within tissues, intact organs, and living organisms. Novel fluorescent tags were developed herein, based upon a genetically targeted fluorogen activating protein and cognate fluorogenic dye that yields emission with a peak at 733 nm exclusively when complexed as a 'fluoromodule'. This tool improves substantially over previously described far-red/NIR fluorescent proteins in terms of brightness, wavelength, and flexibility by leveraging the flexibility of synthetic chemistry to produce novel chromophores.

Near infrared fluorogen and fluorescent activating proteins for in vivo imaging and live-cell biosensing

Disclosed are analogues of trimethine cyanine dyes, which are useful for importing fluorescent properties to target materials by covalent and non-covalent association.

Rigidized trimethine cyanine dyes

A process and a kit are provided for detecting differences in two or more samples of protein, including proteins bearing post-translational modifications and peptides. Proteins are prepared, for example, from each of a different group of cell samples or body fluid samples to be compared. Each protein extract is labeled with a different one of a luminescent dye from a matched set of dyes. The matched dyes have generally the same ionic and pH characteristics but emit light at different wavelengths to exhibit a different color upon luminescence detection. The labeled protein extracts are mixed together and separated together by electrophoresis or a chromatographic method. The separation is observed to detect proteins unique to one sample or present in a greater ratio in one sample than in the other. Those unique or excess proteins will fluoresce the color of one of the dyes used. Proteins common to each sample migrate together and fluoresce the same.

Difference detection methods using matched multiple dyes

The present invention pertains to luminescent dyes and methods for covalently attaching the dyes to a component or mixture of components so that the components may be detected and/or quantified by luminescence detection methods. The dyes are cyanine and cyanine-type dyes that contain or are derivatized to contain a reactive group. The reactive group is covalently reactive with amine, hydroxy and/or sulfhydryl groups on the component so that the dye can be covalently bound to the component. In addition, the dyes are preferably soluble in aqueous or other medium in which the component is contained. The components to be labeled can be either biological materials, such as antibodies, antigens, peptides, nucleotides, hormones, drugs, or non-biological materials, such as polymers, glass, or other surfaces. Any luminescent or light absorbing detecting step can be employed in the method of the invention.

Cyanine dyes as labeling reagents for detection of biological and other materials by luminescence methods

Disclosed are analogues of trimethine cyanine dyes which are useful for imparting fluorescent properties to target materials by covalent and non-covalent association. The compounds have general formula (2) optionally substituted by groups R -R wherein groups R , R , R and R are attached to the rings containing X and Y or, optionally are attached to atoms of the Z and Z ring structures and groups R -R are chosen to provide desired solubility, reactivity and spectralproperties to the fluorescent compounds; A is selected from O, S and NR where R is the substituted amino radical (a), where R&prime; is selected from hydrogen, a C alkyl and aryl and R&Prime; is selected from C alkyl, aryl, heteroaryl, an acyl radical having from 2-7 carbon atoms, and a thiocarbamoyl radical; Z and Z each represent a bond or the atoms necessary to complete one, two fused or three fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, non more than two oxygen, nitrogen and sulphur atoms.

The present invention provides low molecular weight fluorescent labeling complexes with large wavelength shifts between absorption of one dye in the complex and emission from another dye in the complex. These complexes can be used, for example, for multiparameter fluorescence cell analysis using a single excitation wavelength. The low molecular weight of the complex permits materials labeled with the complex to penetrate cell structures for use as probes. The labeling complexes are synthesized by covalently attaching through linkers at least one cyanine fluorochrome to another low molecular weight fluorochrome to form energy donor-acceptor complexes. Resonance energy transfer from an excited donor to fluorescent acceptor provides wavelength shifts up to 300 nm. The fluorescent labeling complexes preferably contain reactive groups for the labeling of functional groups on target compounds, such as derivatized oxy and deoxy polynucleic acids, antibodies, enzymes, proteins and other materials. The complexes may also contain functional groups permitting covalent reaction with materials containing reactive groups.

Fluorescent labeling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer

Fluorochromes labelling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer

Growing community of inventors

Pittsburgh, PA, United States of America

Alan S Waggoner