Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 11 to 14 amino acid residues in defined sequence
Reexamination Certificate
1997-08-29
2001-05-29
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
11 to 14 amino acid residues in defined sequence
C530S345000, C530S350000, C530S402000, C435S287100, C435S287900, C435S288700
Reexamination Certificate
active
06239255
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates in general to the field of biosensors, more particularly, the present invention is directed to a surface plasmon resonance (SPR) miniature integrated sensor using a gold-binding repeating polypeptide to which variable recognition elements are attached at the sensor surface.
BACKGROUND OF THE INVENTION
Without limiting the scope of the invention, its background is described in connection with surface plasmon resonance sensors, as an example.
Heretofore, in this field, the development of sensors for the detection of specific molecules within specimens or samples have required expensive equipment, highly trained personnel, large samples, and days to weeks to complete. While the detection of contaminants has been an active area of research, present systems are limited in the range of applications and are unable to detect specific compounds in real-time. Present systems for the analysis of specific binding phenomena have included antibody-antigen complex formation and receptor-ligand interactions for the detection of small molecules.
An active area of sensor development has been the use of an optical phenomenon known as surface plasmon resonance (SPR). Biosensors of this type, such as the BIAcore line from Biacore, Upsala, Sweden, are available for use in research and development. Two factors limiting the general use of SPR biosensors, however, are the relatively high cost of developing and using specific biosensors and the lack of mobility for field analysis.
The most difficult step in producing a surface plasmon resonance biosensor is the attachment of specific recognition elements to the gold surface of the sensor. The problems associated with attaching organic molecules to gold are manifold. One problem is the need to highly purify the specific recognition elements so as to avoid non-specific surface interactions by impurities in the preparation. Another problem is the difficulty in stably attaching the specific recognition element to a metal. Yet another problem is the need to prepare and treat the gold surface to prevent Non-Specific Binding (“NSB”).
The recognition elements of current SPR biosensor are attached to a gold sensor surface by forming a monolayer of long-chain alkanethiols with suitable reactive groups on one end of the molecule and a gold-complexing thiol on the other. The actual molecular recognition elements are attached directly to the alkanethiol monolayer or to a hydrogel layer. An example of hydrogel monolayer is carboxymethyl dextran, that is attached to the monolayer.
The purpose of providing an additional hydrogel layer over the alkanethiol monolayer is to favor normal protein interaction and function, which are disrupted by the thiol groups used for attaching proteins. The additional monolayer is also necessary to provide a more hydrophilic environment at the gold surface than is provided by the monolayer alone. The presence of a hydrogel is also necessary to reduce the non-specific binding of proteins on the gold surface and to stabilize the alkanethiol monolayer attachment to gold.
Under relatively stable and controlled conditions in the laboratory, the gold-sulfur association is relatively stable. Outside the laboratory, however, the association of molecules necessary to obtain a surface plasmon resonance reading are more unpredictable and less reliable. The decreased reliability is due in part to the presence of oxidants, other sulfur-containing compounds and acidic solutions, all of which limit the practical use of SPR biosensors constructed with alkanethiol monolayers. Consequently, present SPR biosensors have failed to approach their full potential of applications.
Brown recently cloned a novel Gold-Binding Protein (“GBP”) and characterized the specific recognition element. Stanley Brown,
Metal Recognition by Repeating Polypeptides
, Nature Biotechnology, Vol. 15, March 1997. He suggested that the GBP could be genetically engineered as a fusion protein with specific recognition elements for use in constructing biosensors. Brown did not, however, disclose a method of using a GBP to construct surface plasmon biosensors directed to a specific molecule or molecules. Nor does Brown discuss a method of employing protein-coupling chemistry to create chimeric proteins consisting of the GBP and several different recognition elements. Furthermore, Brown failed to disclose a method of using and making a biosensor using a GBP.
What is needed is a low cost, mobile sensor with more stable recognition elements. Also needed is an apparatus and method that enables the developer and user of Surface Plasmon Resonance-based biosensors (“SPR-biosensors”) to easily and reliably construct, evaluate, produce and use SPR-biosensors with specific recognition elements that attach to the gold surface.
SUMMARY OF THE INVENTION
The present invention disclosed herein comprises a method of producing a stable surface plasmon biosensor. More particulary, the present invention is directed to a method of improving the strength and stability of the molecular bonds between the gold surface of a surface plasmon resonance biosensor and the specific recognition element of the biosensor. The present invention is a method for immobilizing a molecule on a gold surface comprising, attaching a gold binding peptide to the gold surface and attaching the molecule to the gold binding peptide.
More particularly, the present invention is directed to a method of immobilizing a molecule on a gold surface in which the gold binding peptide is a naturally occurring polypeptide structure. Alternatively, the gold binding peptide may be a genetically engineered or synthetically constructed polypeptide structure. Even more particularly, the genetically engineered or synthetically constructed repeated polypeptide structure is the repeated amino acid sequence MHGKTQATSGTIQS (SEQ ID NO.: 1).
The present invention also encompasses a method of binding a recognition element to a gold surface comprising the steps of, attaching a Gold Binding Peptide-Alkaline Phosphotase chimera (“GBP-AP”) protein to the gold surface, digesting away the alkaline phosphatase domain of the protein with proteolytic enzymes leaving the GBP domain bound to the gold surface, and attaching a recognition element to the GBP domain. In one embodiment the proteolytic enzyme used for digesting away the alkaline phosphatase domain is trypsin. The method of the present invention may further comprise the step of cross linking the bound GBP domain to adjacent molecules.
Yet another embodiment of the present invention is a molecular specificity structure comprising, a gold surface, a gold binding peptide foundation layer coating the gold surface, and a sensing layer attached to said gold binding peptide layer, wherein the sensing layer is immobilized about the gold surface, the sensing layer having the characteristic quality of binding to a specific target analyte. The specificity structure may be further defined as comprising a specific recognition element, which may be for example, an antibody, a lectin, a hormone receptor, a nucleic acid, a carbohydrate, a lipid, or any antigen, hormone or other binding partner. The specificity layer of the present invention may be further defined as comprising an intermediary adaptive layer capable of reversibly binding a plurality of recognition elements with different specificities, and a specific recognition element bound to the intermediary adaptive layer.
REFERENCES:
Brown, S. ‘Metal Recognition by Repeating Polypeptides’, Nature of Biotechnology, vol. 15, pp. 269-272, Mar. 1997.*
Wang et al. ‘Simplified Purification and Testing of Colloidal Gold Probes’, Biochemistry, vol. 85, pp. 109-115, 1985.*
Ackerman et al. ‘Differential Surface Labeling and Internalizatio of Glugagon by Peripheral Leukocytes’, Journal of Histochemistry and Cytochemistry, vol. 31, No. 3, pp. 433-440, 1983.*
Wohlheuter et al. ‘Analysis of Binding of Monoclonal Antibody to Malarial Peptide by Surface Plasmon Resonace Biosensor and Integrated Rate Equation’, Journal of
Furlong Clement E.
Woodbury Richard
Flores Edwin S.
Gardere Wynne & Sewell LLP
Gupta Anish
Low Christopher S. F.
Warren, Jr. Sanford E.
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