Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2001-05-08
2004-06-01
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C435S007100, C435S007920, C436S164000, C436S808000, C536S024310
Reexamination Certificate
active
06743640
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a fluorescent biosensor that functions by a novel Quencher-Tether-Ligand (QTL) mechanism. In particular, the polymer-QTL system provides for effective sensing of biological agents by observing fluorescence changes.
DISCUSSION OF THE BACKGROUND
The enzyme linked immunosorbant assay, ELISA, is the most widely used and accepted technique for identifying the presence and biological activity of a wide range of proteins, antibodies, cells, viruses, etc. It is a multi-step “sandwich assay” in which the analyte biomolecule is first bound to an antibody tethered to a surface. A second antibody then binds to the biomolecule. In some cases, the second antibody is tethered to a catalytic enzyme which subsequently “develops” an amplifying reaction. In other cases, this second antibody is biotinylated to bind a third protein (e.g., avidin or streptavidin). This protein is tethered either to an enzyme, which creates a chemical cascade for an amplified colorimetric change, or to a fluorophore, for fluorescent tagging.
Despite its wide use, there are many disadvantages to ELISA. For example, because the multi-step procedure requires both precise control over reagents and development time, it is time-consuming and prone to “false positives”. Further, careful washing is required to remove nonspecific adsorbed reagents. On the other hand, the polymer-QTL (Quencher-Tether-Ligand) approach of the present invention is a single-step, instantaneous, homogeneous assay where the amplification step is intrinsic to the fluorescent conjugated polymer. Furthermore, there are no reagents. Thus, the process is uniquely robust, simple, and accurate relative to ELISA or other sensor techniques.
Other technology advantages inherent in the polymer-QTL approach are similar to those used in fluorescence resonance energy transfer (FRET). FRET techniques have been applied to both polymerase chain reaction-based (PCT) gene sequencing and immunoassays. In particular, FRET uses homogeneous binding of an analyte biomolecule to activate the fluorescence of a dye that is quenched in the off-state. However, there are important limitations and differentiations to FRET relative to the polymer-QTL approach. In an example of FRET technology, a fluorescent dye is linked to an antibody (F-Ab), and this diad is bound to an antigen linked to a quencher (Ag-Q). The bound complex (F-Ab:Ag-Q) is quenched (i.e., non-fluorescent) by energy transfer. In the presence of identical analyte antigens which are untethered to Q (Ag), the Ag-Q diads are displaced quantitatively as determined by the equilibrium binding probability determined by the relative concentrations, [Ag-Q]/[Ag]. This limits the FRET technique to a quantitative assay where the antigen is already well-characterized, and the chemistry to link the antigen to Q must be worked out for each new case.
On the other hand, the QTL assay works by selective binding to the quencher-tether-ligand. The competition between the binding of QTL to the polymer vs. the analyte may be widely controlled by varying the polymer and QTL structure and charge density. Even more importantly, the QTL assay uses a fluorescent polymer rather than a small molecular fluorophore. This fundamental difference between FRET techniques and QTL assays allows the polymer-QTL technology to have significant amplification in quenching and detection sensitivity. In addition, the polymer-QTL approach is immediately applicable to powerful combinatorial techniques for generating new molecular recognition species for unknown and uncharacterized biomolecules. Due to the sensitivity of fluorescence quenching in the QTL technology relative to FRET, QTL eliminates the need for pre-concentration of the sample, thereby allowing the use of microliter quantities of reagents in capillary tubes. The polymer-QTL approach also avoids the elaborate preprocessing required by polymerase chain reaction (PCR), offering significant incentives to the further development of the polymer-QTL approach for detection of nucleic acids.
The ability to rapidly and accurately detect and quantify biologically relevant molecules with high sensitivity is a central issue for medical technology, national security, public safety, and civilian and military medical diagnostics. Many of the currently used approaches, including enzyme linked immunosorbant assays (ELISAs) and PCR are highly sensitive. However, they can be cumbersome and time-consuming, as discussed above. In order to address the problems in the art, the present inventors have developed a prototype for a new fluorescent biosensor which functions by a novel Quencher-Tether-Ligand (QTL) mechanism that provides a simple, rapid and highly-sensitive detection of biological molecules with structural specificity. The present invention may be adapted to a number of different formats ranging from a single or multiple species to medical diagnostics and fluorescent tags.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel chemical moiety formed of a quencher (Q), a tethering element (T), and a ligand (L).
It is another object of the present invention to provide a method of detecting target biological agents in a sample using the novel QTL molecule of the present invention and a fluorescent polymer.
It is a further object of the present invention to detect target biological agents in a sample by observing fluorescent changes.
It is a feature of the present invention that the change in fluorescence is indicative of the presence of the target biological agent.
It is another feature of the present invention that the fluorescent polymer can be tethered to the novel QTL molecule.
It is a further feature of the present invention that the fluorescent polymer used in detection can be anchored to a support.
It is an object of the present invention to rapidly and accurately detect and quantify target biological molecules in a sample.
It is a further feature of the invention that the fluorescent polymer comprises dye chromophores pendant as side chains of a polymeric backbone.
It is an advantage of the present invention that target biological molecules can be detected at near single molecule levels.
It is a further feature of the invention that detection of target molecules may be enhanced by application of electric fields.
It is a further advantage of the present invention that it is simple and requires no elaborate preprocessing.
These and other objects are met by a composition of matter comprising: a) a fluorescent polymer, and b) a chemical moiety QTL comprising a recognition element, which binds to a target biological agent, and a property-altering element which alters fluorescence emitted by the fluorescent polymer when complexed together to a distinguishable degree, bound together by a tethering element, the chemical moiety being adapted for complexation with the fluorescent polymer. In the presence of binding of the recognition element to said target biological agent, the fluorescence emitted by the polymer is altered from that emitted when binding between said recognition element and said target biological agent does not occur.
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Bergstedt Troy S.
Jones Robert
McBranch Duncan W.
Whitten David G.
Counts Gary W.
Kelber Steven B.
Le Long V.
Piper Rudnick LLP
QTL Biosystems LLC
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