Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-08-21
2003-06-24
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S524000, C436S008000, C436S085000, C521S099000, C521S100000, C521S102000, C501S080000, C210S660000
Reexamination Certificate
active
06582971
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to molecularly imprinted polymer compositions, as well as methods of making and using the compositions. The invention further relates to methods of making and using molecularly imprinted polymer compositions for molecular recognition of various biomolecules, particularly high molecular weight biological macromolecules.
2. Background of the Related Art
Current biomolecular recognition elements for large proteins and other large molecular weight biological compounds rely on fragile biomolecules for recognition of the target analyte. The recognition elements typically include antibodies, nucleic acid probes, enzymes, and/or other receptors. These recognition elements are expensive to prepare, especially in large quantities, and are physically and chemically fragile. In addition, the utility of sensors and separation media that use these fragile recognition elements is limited because of their expense and fragility. A need exists for an inexpensive, synthetic, and rugged recognition element that can replace fragile antibodies, nucleic acids, enzymes, and other bioreceptors.
The highest specificity of binding between a biomolecule and a surface is currently achieved using affinity interaction between, for example, antibodies and antigen, receptor and ligand, etc. Both binding strength and specificity are important in specific binding reactions. Affinity-based systems often involve association constants in the range of 10
5
to 10
9
M
−1
. Exploiting naturally occurring biological binding systems currently produces surfaces capable of specific, high-affinity binding with a preselected macromolecule. These systems recognize their target molecule by exploiting a combination of specific electrostatic interactions, hydrophobic interactions, hydrogen bonding, and shape.
Molecularly imprinted polymers (MIPs) have only recently been recognized as rugged, artificial recognition elements. MIPs have been prepared for a variety of small molecules with the affinity and specificity of the MIP for the target molecule approaching that observed for monoclonal antibodies. However, the preparation of MIPs for proteins and other large biomolecules have had limited success.
Mosbach and coworkers, as exemplified by U.S. Pat. Nos. 5,110,833 and 5,461,175, have developed what they call “molecular imprinting”, which is a method of preparing polymers by polymerizing monomers around “print molecules”. Molecular imprinting of synthetic polymers is a process where functional and cross-linking monomers are copolymerized in the presence of the target analyte, which acts as a molecular template. Before polymerization, the functional monomers either form a complex with the template via non-covalent interactions, or are covalently coupled forming a polymerizable derivative of the template. After polymerization, the functional groups of the monomers are held in position by the highly cross-linked polymeric structure. Subsequent removal of the template by solvent extraction or chemical cleavage reveals binding sites that are complementary in size and shape to the analyte. In this way, a molecular memory is introduced in the polymer, which is now capable of rebinding the analyte with very high specificity.
Originally, MIPs were employed as stationary phases in HPLC, notably for chiral separation. Subsequently, their use has been extended to other analytical techniques such as thin layer chromatography, capillary electrochromatography, solid-phase extraction, and immunoassay type binding assays. The binding sites often have affinities and selectivities approaching those of antibody-antigen systems, and have been dubbed antibody-binding mimics. These mimics display some clear advantages over real antibodies for sensor technology. Because of their highly cross-linked nature, MIPs are intrinsically stable and robust, facilitating their application in extreme environments, such as in the presence of acids, bases, or metal ions, in organic solvents, or at high temperatures and pressures. Moreover, MIPs are cheap to produce and can be stored in a dry state at room temperature for long periods of time. It is, therefore, not surprising that there is progress towards the use of MIPs as recognition elements in biomimetic sensors, large-scale separations (i.e. preparative chromatography or cleanup), and in the area of analytical chemistry for small-scale separations.
To the present date, imprinted polymer technology has had limited success for large molecular weight proteins and biomolecules, such as proteins, nucleic acids, and carbohydrates. The native state of a protein is usually a single, folded, globular structure where the hydrophobic amino acid side chains are buried in the interior of the protein and sequestered from bulk water. The denatured state of a protein is comprised of many random, unfolded structures where the hydrophobic amino acid side chains are exposed to bulk water. Although many other forces contribute to the stability of proteins, the hydrophobic effect is perhaps the main contributor to protein stability. Hydrophobic groups tend to organize the water molecules about them in hydrogen-bond cages called clathrates. The water molecules in clathrates are more ordered than the water molecules in bulk water. The entropic penalty of ordered water around hydrophobic groups in the denatured state drives the protein to the folded state. This effect is so great that it overcomes the concomitant decrease in entropy associated with folding the protein from an unfolded, denatured state to an ordered, native state. The driving force associated with removing hydrophobic side chains from bulk water is minimized or even absent when proteins are exposed to organic solvents. Consequently, in the presence of organic solvents, the entropy gain associated with unfolding the native state dominates and the protein unfolds. Despite this fact, the present methods of molecularly imprinting polymers involves dissolving the print molecule and monomer(s) in an organic solvent, and polymerization is initiated to ultimately yield the imprinted polymer.
Since proteins are most likely unfolded in the presence of organic solvents, the imprint formed during the polymerization of the monomers will be toward a non-native state of the target protein. Therefore, the interactions between print molecule and monomers during the polymerization in organic solvent would be considerably different than the interactions between the protein and MIP in water. The low dielectric medium of the organic solvent will screen hydrophobic interactions and exaggerate electrostatic interactions between the print molecule and monomers during polymerization. However, binding experiments are typically performed in water, which is a high dielectric medium, where electrostatic interactions will be screened while hydrophobic interactions will be exacerbated.
Attempts have been made to overcome the problems associated with imprinting proteins by avoiding the use of organic solvents in the imprinting process. Paliwal, et al., as exemplified in U.S. Pat. No. 5,756,717, describes the preparation of imprinted gels for use as a chromatographic separation media. The imprinted gels are made of agarose, which is a water insoluble, commercially available polysaccharide. The agarose is derivatized to prepare both positively and negatively charged agarose. The polymers are then heated to solubilize the agarose and then cooled to produce a gel. To prepare the imprint, the gel is heated to approximately 45° C. to melt the gel. Print molecule is added to the melted gel solution and the solution is cooled to generate the imprinted gel. The last step is the removal of the print molecule from the gel by washing the gel with a concentrated sodium chloride solution. This solution causes the print molecule to lose its affinity for the gel by screening the electrostatic interaction between the print molecule and the gel. This is a common method to remove bound biomolecules from ion exchange and affinity chroma
Fyffe James
Gaskin David
Giletto Anthony
Singh Waheguru Pal
Counts Gary W.
Le Long V.
Lynntech Inc.
Streets Jeffrey L.
Streets & Steele
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