Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-12-11
2003-12-09
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C436S501000, C436S528000, C436S531000, C436S527000
Reexamination Certificate
active
06660484
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and compositions for the direct detection of analytes using observable spectral changes in biopolymeric systems. In particular, the present invention allows for the direct colorimetric detection of analytes using color changes that occur in glycopolythiophene polymer systems in response to selective binding of analytes.
BACKGROUND OF THE INVENTION
A major goal of analyte detection research is to develop inexpensive, fast, reliable, and sensitive detectors. Unfortunately, the technologies developed to date have only met some of these goals, and no single device has sufficiently attained a majority of them.
Classical detection methods such as liquid chromatography (LC), gas chromatography (GC), and supercritical fluid chromatography (SFC), in combination with mass spectrometry, are widely used and provide accurate identification of analytes and quantitative data. However, these techniques are time consuming, extremely expensive, require sample preconcentration, and are difficult or impossible to adapt to field use.
Biosensors (i.e., devices containing biological material linked to a transducing apparatus) have been developed to overcome some of the shortcomings of the classical analyte detection techniques. Many currently used biosensors are associated with transducer devices that use photometry, fluorimetry, and chemiluminescence; fiber optics and direct optical sensing (e.g., grating coupler); surface plasmon resonance; potentiometric and amperometric electrodes; field effect transistors; piezoelectric sensing; and surface acoustic wave (Krämer, J. AOAC Intern. 79: 1245 [1996]). However, there are major drawbacks to these devices, including their dependence on a transducing device, which prevents miniaturization and requires a power source. These disadvantages make such devices too complex, expensive, or unmanageable for many routine analyte detection applications such as field work or home use. Additionally, many of these devices are limited by the lack of stability and availability of the biological materials (e.g., proteins, antibodies, cells, and organelles).
The art remains in need of analyte detectors that provide the specificity of biosensors but also provide the cost-efficiency, stability, accuracy, reliability, reproducibility, and robustness that is lacking from available technologies. In particular, development of devices that can be miniaturized, that allow the detection of multiple analyte types, and that do not rely on an energy source would also be very beneficial, particularly for routine field work and home use.
SUMMARY OF THE INVENTION
The present invention relates to methods and compositions for the direct detection of analytes using observable spectral changes in biopolymeric systems. In particular, the present invention allows for the direct colorimetric detection of analytes using color changes that occur in glycopolythiophene polymer systems in response to selective binding of analytes.
The present invention provides biopolymeric materials comprising a plurality of polymerized monomers and one or more ligands, wherein the biopolymeric materials change color in the presence of analyte. In some embodiments, the ligands are selected from the group consisting of peptides, proteins, antibodies, receptors, channels, and combinations thereof, although the present invention contemplates all protein ligands (i.e., with protein being defined in its broadest sense). In other embodiments, the ligands are non-proteins (e.g., lectins, carbohydrates, glycolipids, phospholipids, and the like). However, the present invention is not limited to any particular ligand-analyte binding partners.
In particularly preferred embodiments, the biopolymeric materials comprise water-soluble glycopolythiophenes (e.g., containing sialic acid or mannose ligands) such materials have been synthesized by oxidative co-polymerization of methyl thiopheneacetate and thiophene-carbohydrate monomers.
In some embodiments, the inventive biopolymeric materials comprise three portions: a polymer, a spacer, and a ligand. Because of the adaptability of these assemblies, modifications may be made which give it great variety in application and design. The present invention is not limited to the following variations in some embodiments. Some preferred embodiments of the present invention employ variation in the polymer backbone, thereby producing different shapes of conjugation. This is accomplished through the addition of aromatic and or non-aromatic units (e.g., thiazole, pyrrole, selenophene phenyl unit, phenylene vinylene unit and diacetylene) as co-monomers without losing conjugation. Alternatively, the polymer backbone is altered by heterocyclic atoms used in place of carbon (e.g., N, O, and Se). Other embodiments of the present invention vary the length or composition of the spacer element. For example, almost any length spacer (e.g., one or more carbon atoms) having hydrophilic/lipophilic properties is permitted. The alteration of spacer length and composition is directed by observing the colorimetric responses obtained such that a desired response is reached. It is contemplated that the ability to vary spacer length and composition allows the polymer assemblies greater access to high molecular weight molecules (e.g., viruses, bacteria, and parasites). In preferred embodiments, neither the ligand, dopant, spacer, or polymer assembly comprises a lipid.
In still other embodiments, the ligand is varied according to the analyte to be detected. For example, in some embodiments, the ligand(s) employed include sugars, altered or naturally occurring polynucleotides (DNA, RNA, etc.), polypeptides, and other organic molecules capable of specifically binding to a receptor (e.g., cyclosporin, benzadiazapam, or serotonin uptake transporters, ACE), metal-complexes, and inorganic materials such as transition and lanthanide series metals.
It is not intended that the present invention be limited to one particular type of ligand molecule. A variety of ligand are contemplated. For example, the present invention provides for both protein and non-protein ligands. In some embodiments, protein ligands comprise antibodies or portions of antibodies, proteins, or polypeptides, and the like. In other embodiments that employ non-protein ligands, a number of non-protein molecules are contemplated (e.g., carbohydrates, nucleic acids, drugs, chromophores, antigens, chelating compounds, molecular recognition complexes, ionic groups, polymerizable groups, linker groups, electron donors, electron acceptor groups, hydrophobic groups, hydrophilic groups, receptor binding groups, polysacchrides (e.g., trisaccharides, tetrasaccharides, etc.) ganglioside G
M1
, ganglioside G
T1b
, sialic acid, and combinations thereof).
In alternative embodiments of the present invention, the portions of the ligand or monomer assemblies are manipulated to alter their shape and electronic conformation of the composition. For example, in some embodiments of the present invention, a carbohydrate ligand was placed next to the phenyl group of the glycopolythiophene assemblies to prevent neuraminidase cleavage of the O-linked glycosides of sialic acid and to provide tighter binding to
Escherichia coli.
Certain embodiments of the present invention were modified in this manner without losing binding properties.
Likewise, it is not intended that the present invention be limited to detecting any particular analyte. A variety of analytes are contemplated. In some embodiments, the analyte is selected from the group consisting of pathogens, drugs, receptor ligands, antigens, ions, hormones, blood components, disease indicators, cell components, antibodies, lectins, enzymes, organic solvents, volatile organic compounds, pollutants, and genetic material. Other embodiments are directed to pathogenic analytes, for example, viruses, bacteria, parasites, and fungi. In particular embodiments of the present invention, viral analytes are selected from the group consisting of influenza, rubella, var
Baek Myung-Gi
Charych Deborah J.
Brown Stacy S.
Hedlen & Carroll, LLP
Housel James
Regents of the University of California
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