Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
2003-04-16
2004-11-30
Foelak, Morton (Department: 1711)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C106S122000, C424S145100, C424S146100, C521S084100
Reexamination Certificate
active
06824776
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The disclosed invention relates to aerogels and specifically to silica aerogels having three-dimensional nanoarchitecture with colloidal gold~protein superstructures nanoglued therein. The disclosure describes methods for making the composite bioaerogels and their physical and chemical characteristics.
2. Description of Background Art
Much attention has been focused on immobilization of biomolecules in silicate glass formed by the sol-gel method, Eggers et al.,
Protein Sci.
2001, 10, 250-261. The process involves hydrolyzing an alkoxide to produce a sol, which then undergoes polycondensation to form a gel. Biomolecules are immobilized by being entrapped in the gel during the sol-to-gel transition. The sol-gel materials offer advantages over more traditional organic polymers for biomolecule entrapment in that these materials have increased mechanical strength, chemical stability, biocompatibility, and resistance to microbial attack.
While one can encapsulate a variety of biomolecules (enzymes, proteins, antibodies, cells) in sol-gel-derived matrices, the earliest reported bio/silicate-gels had only 30% activity as prepared using the conventional, alcohol-rich sol-gel preparation. Bioactivity of caged biomolecules rose to 75-95% upon the advent of the Dunn procedure, Dunn et al.,
Acta Mater.
1998, 46, 737-741, which uses less alcohol and provides better buffering of the sol. Traditionally when biomolecules have been incorporated into sol-gel-derived materials, the resultant gels are either kept wet (forming hydrogels) or are dried from aqueous conditions (forming xerogels) resulting in pore collapse of the material and long-sensing response times. The hydrogels are not ideal for real-world sensing either, in that they must be kept wet, stored at 4° C., and the long-term stability of the encapsulated biomolecule has not been investigated. The longest reported lifetime of these materials is approximately a month when stored at 4° C.
Heme proteins, such as horseradish peroxidase, Bhatia et al.,
Chem. Mater.
2000, 12, 2434-2441, cytochrome c (cyt. c) Lloyd et al.,
langmuir
2000, 16, 9092-9094 and myoglobin, Ellerby et al.,
Science
1992, 257, 1113-1116, have been extensively studied in sol-gel encapsulation. These proteins retain their spectroscopic properties and chemical functions of oxidation and reduction, ligand binding, or biocatalysis upon encapsulation. In one case, cyt. c was encapsulated into a sol-gel and absorbance-based spectral shifts were used to monitor binding of nitric oxide. Unfortunately, the sensor reaction is reported to have taken two hours to reverse, making dynamic measurements impractical, Aylott et al.,
Chem. Mater.
1997, 9, 2261-2263.
BRIEF SUMMARY OF THE INVENTION
We have encapsulated heme protein into a silica framework, facilitated by formation of a protein—protein superstructure nucleated in the liquid phase by colloidal gold. In solution, the protein is known to specifically adsorb via surface lysine residues to the surface of the Au with the heme pocket toward the metal. We posit that this adsorption-induced presentation of the back face of the protein to the protein-buffer medium leads to “tail—tail”-directed protein-protein assembly, alternating with “head—head” protein-protein association, to leave essentially no unassociated protein in the buffered medium. The ‘outer skin’ of protein acts as a protective barrier and stabilizes the proteins within the superstructure against denaturants, including those arising during synthesis and processing of the silica nanoarchitecture. The gold~protein-protein superstructure of the invention is nanoglued into the silica framework during the sol-to-gel transition. The wet gel is dried from supercritical fluid (SCF), forming a mesoporous aerogel, which permits true gasphase sensing with facile molecular transport into the biomolecule-modified three-dimensional nanoarchitecture of the aerogel. Although gold is described specifically throughout this disclosure, it is to be understood that silver, platinum, palladium, copper, and nickel may take the place of gold in this invention.
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B. Dunn et al., “Strategies for Encapsulating Biomolecules in Sol-Gel Matrices,” Acta Mater., 1998, 46, 737-741.
Rimple B. Bhatia et al., “Aqueous Sol-Gel Process for Protein Encapsulation,” Chem. Mater., 2000, 12, 2434-2441.
Christopher R. Lloyd et al., “Protecting Heme Enzyme Peroxidase Activity from H2O2 Inactivation by Sol-Gel Encapsulation,” Langmuir, 2000, 16, 9092-9094.
Lisa M. Ellerby et al., “Encapsulation of Proteins in Transparent Porous Silicate Glasses Prepared by the Sol-Gel Method,” Science, 1992, 225, 1113-1115.
Jonathan W. Aylott et al., “Optical Biosensing of Gaseous Nitric Oxide Using Spin-Coated Sol-Gel Thin Films,” Chem. Mater., 1997, 9, 2261-2263.
Catherine A. Morris et al., “Silica Sol as a Nanoglue: Flexible Synthesis of Composite Aerogels,” Science, 1999, 284, 622-624.
Nicholas Leventis et al., “Durable Modification of Silica Aerogel Monoliths with Fluorescent 2,7-Diazapyrenium Moieties. Sensing Oxygen near the Speed of Open-Air Diffusion,” Chem. Mater., 1999, 11, 2837-2845.
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Aimin Yu et al., “Nanostructured Electrochemical Sensor Based on Dense Gold Nanoparticle Films,” Nano Lett., 2003, 3, 1203-1207.
Pietron Jeremy J.
Rice Jane K.
Rolison Debra R.
Stroud Rhonda M.
Wallace Jean M.
Foelak Morton
Forman Rebecca L.
Karasek John J.
The United States of America as represented by the Secretary of
LandOfFree
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