Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
1999-06-22
2001-10-02
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C436S518000, C436S514000, C436S002000, C436S172000, C435S004000, C435S007100, C435S287100, C435S287200, C435S288700
Reexamination Certificate
active
06297059
ABSTRACT:
FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
Biological sensors are based on the immobilization of a recognition molecule at the surface of a transducer (a device that transforms the binding event between the target molecule and the recognition molecule into a measurable signal). In one prior approach, the transducer has been sensitive to any binding, specific or non-specific, that occurred at the transducer surface. Thus, for surface plasmon resonance or any other transduction that depended on a change in the index of refraction, such sensors have been sensitive to both specific and non-specific binding. Another prior approach has relied on a sandwich assay where, for example, the binding of an antigen by an antibody has been followed by the secondary binding of a fluorescently tagged antibody that is also in the solution along with the protein to be sensed. In this approach, any binding of the fluorescently tagged antibody will give rise to a change in the signal and, therefore, sandwich assay approaches have also been sensitive to specific as well as non-specific binding events. Thus, selectivity of many prior sensors has been a problem.
Another previous approach where signal transduction and amplification have been directly coupled to the recognition event is the gated ion channel sensor as described by Cornell et al., “A Biosensor That Uses Ion-Channel Switches”, Nature, vol. 387, Jun. 5, 1997. In that approach an electrical signal was generated for measurement. Besides electrical signals, optical biosensors have been described in U.S. Pat. No. 5,194,393 by Hugl et al. and U.S. Pat. No. 5,711,915 by Siegmund et al. In the later patent, fluorescent dyes were used in the detection of molecules.
Despite the recent progress in such signal transduction and amplification directly coupled to a recognition event, further improvements have been desired especially in the development of optical biosensors.
One object of the present invention is an optical biosensor using a transduction approach that amplifies specific binding events thereby amplifying both sensitivity and specificity.
Another object of the present invention is to provide an optical biosensor and process for use of optically tagged receptor molecules to trigger signal transduction and amplification by a recognition event.
Still another object of the present invention is to provide an optical biosensor and process for signal transduction based on aggregation of receptor molecules through either binding by a multivalent protein or binding of multiple receptor molecules to the same protein or protein clusters.
Yet another object of the present invention is an optical biosensor and process using optical signal transduction resulting from an internal reference by virtue of a simultaneous increase in a red fluorescence and a decrease in a blue fluorescence. This can be coupled with an isobestic point that can be used to reference the absolute intensity of fluorescence.
Another object of the present invention is the fabrication of biomimetic membranes (supported and hybrid phospholipid bilayers) containing the functionalized (optically tagged) receptor molecules such as glycolipid receptor molecules.
Another object of the present invention is to functionalize naturally occurring glycolipid receptors (e.g., Gal&bgr;1-3GalNAc&bgr;1-4Gal(3-2&agr;NeuAc)&bgr;1-4Glc&bgr;1-1Cer, (GM1) with an optical tag such as fluorescent dye molecules.
Another object of the present invention is an optical biosensor and process using donor and acceptor dye molecules each of which is covalently attached to the recognition element to effect energy transfer upon aggregation following binding by a multivalent protein.
Another object of the present invention is an optical biosensor and process using hydrophobic optical tags (dye molecules) and hydrophobic linker molecules that can attach the optical tags to, e.g., a glycolipid so that the dye molecule stably resides in the upper leaf (layer) of a bilayer, e.g., a phospholipid bilayer. This can minimize non-specific interactions of the dye molecule with interferents.
Another object of the present invention is to provide an optical biosensor and process using optical transduction triggered by a specific protein binding event which results in energy transfer yielding both a decrease in the fluorescence of one dye species (e.g., blue emission) with a concomitant increase in the fluorescence of another dye species (e.g., red emission).
SUMMARY OF THE INVENTION
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention provides an optical biosensor for the detection of a multivalent target biomolecule including a substrate having a fluid membrane thereon, recognition molecules situated at a surface of said fluid membrane, the recognition molecule capable of binding with the multivalent target biomolecule, the recognition molecule further characterized as including a fluorescence label thereon and as being movable at the surface; and, a means for measuring a change in fluorescence properties in response to binding between the two different recognition molecules and the multivalent target biomolecule.
The present invention also provides an optical biosensor for the detection of a multivalent target biomolecule including a substrate having a fluid membrane thereon, at least two different recognition molecules situated at a surface of the fluid membrane, the recognition molecules capable of binding with the multivalent target biomolecule wherein at least one recognition molecule includes a fluorescence donor label thereon and at least one recognition molecule includes a fluorescence acceptor label thereon; and, a means for measuring a change in fluorescence properties in response to binding between the recognition molecules and the multivalent target biomolecule.
The present invention also provides an optical biosensor for the detection of a multivalent target biomolecule including a recognition element capable of binding with the multivalent target biomolecule wherein the recognition molecule includes a fluorescence label from the group of a fluorescence donor label and a fluorescence acceptor label thereon; a supporting surface for the recognition molecule situated thereon, wherein said supporting surface includes a fluorescence label from the group of a fluorescence donor label and a fluorescence acceptor label thereon, the supporting surface including a fluorescence donor label when the recognition molecule includes a fluorescence acceptor label and the supporting surface including a fluorescence acceptor label when the recognition molecule includes a fluorescence donor label and, a means for measuring a change in fluorescence properties in response to binding between the recognition molecules and the multivalent target biomolecule.
The present invention also provides a method of detecting a multivalent target biomolecule including contacting a sample with a sensor including a substrate having a fluid membrane thereon, at least two different recognition molecules situated at the surface of the fluid membrane, the recognition molecules capable of multivalent binding with the multivalent target biomolecule wherein at least one recognition molecule includes a fluorescence donor label thereon and at least one recognition element includes a fluorescence acceptor label thereon; and measuring a change in fluorescence properties in response to binding between the recognition molecule and the multivalent target biomolecule.
The present invention also provides an optical biosensor for the detection of a target biomolecule including a substrate having a fluid membrane thereon, recognition molecules situated at a surface of said fluid membrane, said recognition molecule bound with a selected multivalent biomolecule, said recognition molecules further characterized as including a fluorescence label, and, a means for measuring a change in fluorescent properties in response to competitive binding with sai
Song Xuedong
Swanson Basil I.
Cottrell Bruce H.
Le Long V.
Pham Minh-Quan K.
The Regents of the University of California
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