Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54346—Nanoparticles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
  • B01J20/28004—Sorbent size or size distribution, e.g. particle size
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
  • B01J20/28009—Magnetic properties
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
  • B01J20/28016—Particle form
  • B01J20/28019—Spherical, ellipsoidal or cylindrical
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
  • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
  • B01J20/28023—Fibres or filaments
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
  • B01J20/3204—Inorganic carriers, supports or substrates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
  • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
  • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
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  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
  • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
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  • B01J20/3257—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J20/3259—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulfur with at least one silicon atom
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J20/3261—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
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  • C07K—PEPTIDES
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
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  • C09C—TREATMENT OF INORGANIC MATERIALS, OTHER THAN FIBROUS FILLERS, TO ENHANCE THEIR PIGMENTING OR FILLING PROPERTIES ; PREPARATION OF CARBON BLACK  ; PREPARATION OF INORGANIC MATERIALS WHICH ARE NO SINGLE CHEMICAL COMPOUNDS AND WHICH ARE MAINLY USED AS PIGMENTS OR FILLERS
  • C09C1/00—Treatment of specific inorganic materials other than fibrous fillers; Preparation of carbon black
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  • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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Definitions

• the invention relates broadly to graphitic nanotubes, which includes tubular fullerenes (commonly called "buckytubes") and fibrils, which are functionalized by chemical substitution or by adsorption of functional moieties. More specifically the invention relates to graphitic nanotubes which are uniformly or non-uniformly substituted with chemical moieties or upon which certain cyclic compounds are adsorbed and to complex structures comprised of such functionalized fibrils linked to one another. The invention also relates to methods of introducing functional groups onto the surface of such fibrils. BACKGROUND OF THE INVENTION
• This invention lies in the field of submicron graphitic fibrils, sometimes called vapor grown carbon fibers.
• Carbon fibrils are vermicular carbon deposits having diameters less than l.O ⁇ , preferably less than 0.5 ⁇ , and even more preferably less than 0.2 ⁇ . They exist in a variety of forms and have been prepared through the catalytic decomposition of various carbon- containing gases at metal surfaces. Such vermicular carbon deposits have been observed almost since the advent of electron microscopy.
• a good early survey and reference is found in Baker and Harris, Chemistry and Physics of Carbon. Walker and Thrower ed. , Vol. 14, 1978, p. 83, hereby incorporated by reference. See also, Rodriguez, N. , J. Mater. Research. Vol. 8, p. 3233 (1993) , hereby incorporated by reference.
• Tennent U.S. Patent No. 4,663,230, hereby incorporated by reference, succeeded in growing cylindrical ordered graphite cores, uncontaminated with pyrolytic carbon.
• the Tennent invention provided access to smaller diameter fibrils, typically 35 to 700 A (0.0035 to 0.070 ⁇ ) and to an ordered, "as grown" graphitic surface.
• Fibrillar carbons of less perfect structure, but also without a pyrolytic carbon outer layer have also been grown.
• the fibrils, buckytubes and nanofibers that are functionalized in this application are distinguishable from continuous carbon fibers commercially available as reinforcement materials.
• continuous carbon fibers In contrast to fibrils, which have, desirably large, but unavoidably finite aspect ratios, continuous carbon fibers have aspect ratios (L/D) of at least 10 4 and often 10 6 or more.
• the diameter of continuous fibers is also far larger than that of fibrils, being always >1.0 ⁇ and typically 5 to 7 ⁇ .
• Continuous carbon fibers are made by the pyrolysis of organic precursor fibers, usually rayon, polyacrylonitrile (PAN) and pitch. Thus, they may include heteroatoms within their structure.
• PAN polyacrylonitrile
• the graphitic nature of "as made" continuous carbon fibers varies, but they may be subjected to a subsequent graphitization step. Differences in degree of graphitization, orientation and crystallinity of graphite planes, if they are present, the potential presence of heteroatoms and even the absolute difference in substrate diameter make experience with continuous fibers poor predictors of nanofiber chemistry.
• U.S. Patent No. 4,663,230 describes carbon fibrils that are free of a continuous thermal carbon overcoat and have multiple graphitic outer layers that are substantially parallel to the fibril axis. As such they may be characterized as having their c-axes, the axes which are perpendicular to the tangents of the curved layers of graphite, substantially perpendicular to their cylindrical axes. They generally have diameters no greater than 0.1 ⁇ and length to diameter ratios of at least 5. Desirably they are substantially free of a continuous thermal carbon overcoat, i.e., pyrolytically deposited carbon resulting from thermal cracking of the gas feed used to prepare them.
• the carbon planes of the graphitic nanofiber take on a herring bone appearance.
• These are termed fishbone fibrils.
• Carbon nanotubes of a morphology similar to the catalytically grown fibrils described above have been grown in a high temperature carbon arc (Iijima, Nature 354 56 1991) . It is now generally accepted (Weaver, Science 265 1994) that these arc-grown nanofibers have the same morphology as the earlier catalytically grown fibrils of Tennent. Arc grown carbon nanofibers are also useful in the invention.
• Fibrils have also been oxidized non-uniformly by treatment with nitric acid.
• International Application PCT/US94/10168 discloses the formation of oxidized fibrils containing a mixture of functional groups.
• Hoogenvaad, M.S., et al. Metal Catalysts supported on a Novel Carbon Support", Presented at Sixth International Conference on Scientific Basis for the Preparation of Heterogeneous Catalysts, Brussels, Belgium, September 1994
• Hoogenvaad, M.S., et al. Metal Catalysts supported on a Novel Carbon Support
• it beneficial in the preparation of fibril-supported precious metals to first oxidize the fibril surface with nitric acid.
• Such pretreatment with acid is a standard step in the preparation of carbon-supported noble metal catalysts, where, given the usual sources of such carbon, it serves as much to clean the surface of undesirable materials as to functionalize it.
• McCarthy and Bening Polymer Preprints ACS Div. of Polymer Chem. 3_0 (1)420(1990) prepared derivatives of oxidized fibrils in order to demonstrate that the surface comprised a variety of oxidized groups.
• the compounds they prepared, phenylhydrazones, haloaromaticesters, thallous salts, etc. were selected because of their analytical utility, being, for example, brightly colored, or exhibiting some other strong and easily identified and differentiated signal. These compounds were not isolated and are, unlike the derivatives described herein, of no practical significance.
• Fig. 1 is a graphical representation of an assay of BSA binding to plain fibrils, carboxy fibrils, and PEG-modified fibrils.
• Fig. 2 is a graphical representation of an assay of ⁇ -lactoglobulin binding to carboxy fibrils and PEG-modified fibrils prepared by two different methods.
• Fig. 3 is a graphical representation of the elution profile of bovine serum albumin (BSA) on a tertiary amine fibril column.
• BSA bovine serum albumin
• Fig. 4 is a graphical representation of the elution profile of BSA on a quaternary amine fibril column.
• Fig. 5 is the reaction sequence for the preparation of lysine-based dendrimeric fibrils.
• Fig. 6 is a graphical representation of cyclic voltammograms demonstrating the use of iron phthalocyanine modified fibrils in a flow cell.
• Fig. 7 is the reaction sequence for the preparation of bifunctional fibrils by the addition of N £ -(tert-butoxycarbonyl)-L-lysine.
• Fig. 8 is a graphical representation of the results of the synthesis of ethyl butyrate using fibril- immobilized lipase.
• Fig. 9 is a graphical representation of the results of separation of alkaline phosphatase (AP) from a mixture of AP and 0-galactosidase (BG) using AP inhibitor-modified fibrils.
• AP alkaline phosphatase
• BG 0-galactosidase
• Fig. 10 is a graphical representation of the results of separation of BG from a mixture of AP and BG using BG-modified fibrils.
• compositions which broadly have the formula [CnH ⁇ P ⁇ where n is an integer, L is a number less than O.ln, m is a number less than 0.5n, each of R is the same and is selected from S0 3 H, COOH, NH 2 , OH, R'CHOH, CHO, CN, COC1, halide, COSH, SH, COOR' , SR', SiR' 3 , Si+OR' y R' 3 _ y , Si-fO-SiR' 2 -K>R' / R" , Li, A1R' 2 , Hg-X, T1Z 2 and Mg-X, y is an integer equal to or less than 3,
• R' is hydrogen, alkyl, aryl, cycloalkyl, or aralkyl, cycloaryl, or poly(alkylether)
• R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl, fluoroaralkyl or cycloaryl
• X is halide
• Z is carboxylate or trifluoroacetate.
• the carbon atoms, C n are surface carbons of a substantially cylindrical, graphitic nanotube of substantially constant diameter.
• the nanotubes include those having a length to diameter ratio of greater than 5 and a diameter of less than 0.5 ⁇ , preferably less than O.l ⁇ .
• the nanotubes can also be substantially cylindrical, graphitic nanotubes which are substantially free of pyrolytically deposited carbon, more preferably those characterized by having a projection of the graphite layers on the fibril axis which extends for a distance of at least two fibril diameters and/or those having cylindrical graphitic sheets whose c-axes are substantially perpendicular to their cylindrical axis. These compositions are uniform in that each of R is the same.
• Non-uniformly substituted nanotubes are also prepared. These include compositions of the formula
• Functionalized nanotubes having the formula [C n H L ⁇ R, where n, L, m, R and R' have the same meaning as above and the carbon atoms are surface carbon atoms of a fishbone fibril having a length to diameter ratio greater than 5, are also included within the invention. These may be uniformly or non-uniformly substituted.
• the nanotubes are free of thermal overcoat and have diameters less than 0.5 ⁇ .
• functionalized nanotubes having the formula
• the carbon atoms, C n are surface carbons of a substantially cylindrical, graphitic nanotube of substantially constant diameter.
• the nanotubes have a length to diameter ratio of greater than 5 and a diameter of less than 0.5 ⁇ , preferably less than O.l ⁇ .
• the nanotubes may be nanotubes which are substantially free of pyrolytically deposited carbon. More preferably, the nanotubes are those in which the projection of the graphite layers on the fibril axes extends for a distance of at least two fibril diameters and/or those having cylindrical graphitic sheets whose c-axes are substantially perpendicular to their cylindrical axis.
• the surface atoms C n are reacted.
• edge or basal plane carbons of lower, interior layers of the nanotube may be exposed.
• surface carbon includes all the carbons, basal plane and edge, of the outermost layer of the nanotube, as well as carbons, both basal plane and/or edge, of lower layers that may be exposed at defect sites of the outermost layer.
• edge carbons are reactive and must contain some heteroatom or group to satisfy carbon valency.
• Y is an appropriate functional group of a protein, a peptide, an amino acid, an enzyme, an antibody, a nucleotide, an oligonucleotide, an antigen, or an enzyme substrate, enzyme inhibitor or the transition state analog of an enzyme substrate or is selected from R'-OH, R'-NR' 2 , R'SH, R'CHO, R'CN, R'X, R'N + (R') 3 X ⁇ , R'SiR' 3 , R'Si-fOR'-)- y R' 3 _ y , R'Si-fO-SiR' 2 -K>R' ,
• w is an integer greater than one and less than 200.
• the carbon atoms, C n are surface carbons of a substantially cylindrical, graphitic nanotube of substantially constant diameter.
• the nanotubes include those having a length to diameter ratio of greater than 5 and a diameter of less than 0.l ⁇ , preferably less than 0.05 ⁇ .
• the nanotubes can also be substantially cylindrical, graphitic nanotubes which are substantially free of pyrolytically deposited carbon. More preferably they are characterized by having a projection of the graphite layers on the fibril axes which extends for a distance of at least two fibril diameters and/or they are comprised of cylindrical graphitic sheets whose c-axes are substantially perpendicular to their cylindrical axes.
• the nanotubes are free of thermal overcoat and have diameters less than 0.5 ⁇ .
• the functional nanotubes of structure [C n H L ⁇ [R'-R] m may also be functionalized to produce compositions having the formula [C n H L [R'-A] m where n, L, m, R' and A are as defined above.
• the carbon atoms, C n are surface carbons of a substantially cylindrical, graphitic nanotube of substantially constant diameter.
• the nanotubes include those having a length to diameter ratio of greater than 5 and a diameter of less than 0.5 ⁇ , preferably less than O.l ⁇ .
• the nanotubes can also be substantially cylindrical, graphitic nanotubes which are substantially free of pyrolytically deposited carbon.
• compositions of the invention also include nanotubes upon which certain cyclic compounds are adsorbed. These include compositions of matter of the formula
• C n H L ⁇ [X-R a ] ra where n is an integer, L is a number less than O.ln, m is less than 0.5n, a is zero or a number less than 10, X is a polynuclear aromatic, polyheteronuclear aromatic or metallopolyheteronuclear aromatic moiety and R is as recited above.
• the carbon atoms, C n are surface carbons of a substantially cylindrical, graphitic nanotube of substantially constant diameter.
• the nanotubes include those having a length to diameter ratio of greater than 5 and a diameter of less than 0.5 ⁇ , preferably less than O.l ⁇ .
• the nanotubes can also be substantially cylindrical, graphitic nanotubes which are substantially free of pyrolytically deposited carbon and more preferably those characterized by having a projection of the graphite layers on said fibril axes which extend for a distance of at least two fibril diameters and/or those having cylindrical graphitic sheets whose c-axes are substantially perpendicular to their cylindrical axes.
• the nanotubes are free of thermal overcoat and have diameters less than 0.5 ⁇ .
• Preferred cyclic compounds are planar macrocycles as described on p. 76 of Cotton and
• More preferred cyclic compounds for adsorption are porphyrins and phthalocyanines.
• compositions include compounds of the formula
• the matrix is an organic polymer (e.g., a thermoset resin such as epoxy, bismaleimide, polyamide, or polyester resin; a thermoplastic resin; a reaction injection molded resin; or an elastomer such as natural rubber, styrene- butadiene rubber, or cis-l,4-polybutadiene) ; an inorganic polymer (e.g., a polymeric inorganic oxide such as glass), a metal (e.g., lead or copper), or a ceramic material (e.g., Portland cement).
• Beads may be formed from the matrix into which the fibrils have been incorporated. Alternately, functionalized fibrils can be attached to the outer surface of functionalized beads.
• the functionalized fibrils are better dispersed into polymer systems because the modified surface properties are more compatible with the polymer, or, because the modified functional groups (particularly hydroxyl or amine groups) are bonded directly to the polymer as terminal groups.
• polymer systems such as polycarbonates, polyurethanes, polyesters or polyamides/imides bond directly to the fibrils making the fibrils easier to disperse with improved adherence.
• the invention is also in methods of introducing functional groups onto the surface of carbon fibrils by contacting carbon fibrils with a strong oxidizing agent for a period of time sufficient to oxidize the surface of said fibrils and further contacting said fibrils with a reactant suitable for adding a functional group to the oxidized surface.
• the oxidizing agent is comprised of a solution of an alkali metal chlorate in a strong acid.
• the alkali metal chlorate is sodium chlorate or potassium chlorate.
• the strong acid used is sulfuric acid. Periods of time sufficient for oxidation are from about 0.5 hours to about 24 hours.
• a composition having the formula [C n H L -H-CH(R')OH] m is formed by reacting R'CH 2 OH with the surface carbons of a nanotube in the presence of a free radical initiator such as benzoyl peroxide.
• the invention is also in a method for linking proteins to nanotubes modified by an NHS ester, by forming a covalent bond between the NHS ester and the amino group of the protein.
• the invention is also in methods for producing a network of carbon fibrils comprising contacting carbon fibrils with an oxidizing agent for a period of time sufficient to oxidize the surface of the carbon fibrils, contacting the surface-oxidized carbon fibrils with reactant suitable for adding a functional group to the surface of the carbon fibrils, and further contacting the surface-functionalized fibrils with a cross-linking agent effective for producing a network of carbon fibrils.
• a preferred cross-linking agent is a polyol, polyamine or polycarboxylic acid.
• Functionalized fibrils also are useful for preparing rigid networks of fibrils.
• a well-dispersed, three-dimensional network of acid-functionalized fibrils may, for example, be stabilized by cross-linking the acid groups (inter-fibril) with polyols or polyamines to form a rigid network.
• the invention also includes three-dimensional networks formed by linking functionalized fibrils of the invention. These complexes include at least two functionalized fibrils linked by one or more linkers comprising a direct bond or chemical moiety. These networks comprise porous media of remarkably uniform equivalent pore size. They are useful as adsorbents, catalyst supports and separation media.
• interstices between these fibrils are irregular in both size and shape, they can be thought of as pores and characterized by the methods used to characterize porous media.
• the size of the interstices in such networks can be controlled by the concentration and level of dispersion of fibrils, and the concentration and chain lengths of the cross-linking agents.
• Such materials can act as structured catalyst supports and may be tailored to exclude or include molecules of a certain size. Aside from conventional industrial catalysis, they have special applications as large pore supports for biocatalysts.
• the rigid networks can also serve as the backbone in biomimetic systems for molecular recognition. Such systems have been described in US Patent No. 5,110,833 and International Patent Publication No. W093/19844.
• the appropriate choices for cross-linkers and complexing agents allow for stabilization of specific molecular frameworks.
• the uniformly functionalized fibrils of the invention can be directly prepared by sulfonation, electrophilic addition to deoxygenated fibril surfaces or metallation.
• arc grown nanofibers When arc grown nanofibers are used, they may require extensive purification prior to functionalization. Ebbesen et al. (Nature 367 519 (1994)) give a procedure for such purification.
• the carbon fibrils are processed prior to contacting them with the functionalizing agent.
• processing may include dispersing the fibrils in a solvent.
• the carbon fibrils may then be filtered and dried prior to further contact.
• Activated C-H (including aromatic C-H) bonds can be sulfonated using fuming sulfuric acid (oleum) , which is a solution of cone, sulfuric acid containing up to 20% S0 3 .
• the conventional method is via liquid phase at T-80°C using oleum; however, activated C-H bonds can also be sulfonated using S0 3 in inert, aprotic solvents, or S0 3 in the vapor phase.
• the reaction is:
• Reactions were carried out in the gas phase and in solution without any significant difference in results.
• the vapor phase reaction was carried out in a horizontal quartz tube reactor heated by a Lindberg furnace.
• H S0 fitted with gas inlet/outlet tubes was used as the S0 3 source.
• a weighed sample of fibrils (BN or CC) in a porcelain boat was placed in the 1" tube fitted with a gas inlet; the outlet was connected to a cone.
• H 2 S0 4 bubbler trap Argon was flushed through the reactor for 20 min to remove all air, and the sample was heated to 300°C for 1 hour to remove residual moisture. After drying, the temperature was adjusted to reaction temperature under argon.
• the S0 3 source was connected to the reactor tube and an argon stream was used to carry S0 3 vapors into the quartz tube reactor. Reaction was carried out for the desired time at the desired temperature, after which the reactor was cooled under flowing argon. The fibrils were then dried at 90°C at 5" Hg vacuum to obtain the dry weight gain. Sulfonic acid (-S0 3 H) content was determined by reaction with 0.100N NaOH and back-titration with 0.100N HC1 using pH 6.0 as the end point.
• the liquid phase reaction was carried out in cone, sulfuric acid containing 20% S0 3 in a multi-neck 100 cc flask fitted with a thermometer/temperature controller and a magnetic stirrer. A fibril slurry in cone. H 2 S0 4 (50) was placed in the flask. The oleum solution (20 cc) was preheated to -60°C before addition to the reactor. After reaction, the acid slurry was poured onto cracked ice, and diluted immediately with 1 1 DI water. The solids were filtered and washed exhaustively with DI water until there was no change in pH of the wash effluent. Fibrils were dried at 100°C at 5" Hg vacuum. Due to transfer losses on filtration, accurate weight gains could not be obtained. Results are listed in Table 1. TABLE I
• the surface carbons in fibrils behave like graphite, i.e., they are arranged in hexagonal sheets containing both basal plane and edge carbons. While basal plane carbons are relatively inert to chemical attack, edge carbons are reactive and must contain some heteroatom or group to satisfy carbon valency. Fibrils also have surface defect sites which are basically edge carbons and contain heteroatoms or groups.
• the most common heteroatoms attached to surface carbons of fibrils are hydrogen, the predominant gaseous component during manufacture; oxygen, due to its high reactivity and because traces of it are very difficult to avoid; and H 2 0, which is always present due to the catalyst.
• Pyrolysis at ⁇ 1000°C in a vacuum will deoxygenate the surface in a complex reaction with unknown mechanism, but with known stoichiometry.
• the products are CO and C0 2 , in a 2:1 ratio.
• the resulting fibril surface contains radicals in a C- ⁇ -C, ⁇ alignment which are very reactive to activated olefins.
• the surface is stable in a vacuum or in the presence of an inert gas, but retains its high reactivity until exposed to a reactive gas.
• fibrils can be pyrolized at
• RFS + CH 2 CH-CN > Fibril-R'CN where R' is a hydrocarbon radical (alkyl, cycloalkyl, etc.)
• EXAMPLE 2 Preparation of Functionalized Fibrils by Reacting Acrylic Acid with Oxide-Free Fibril Surfaces
• BN fibrils in a porcelain boat is placed in a horizontal 1" quartz tube fitted with a thermocouple and situated in a Lindberg tube furnace. The ends are fitted with a gas inlet/outlets.
• the tube is purged with dry, deoxygenated argon for 10 minutes, after which the temperature of the furnace is raised to 300°C and held for 30 minutes. Thereafter, under a continued flow of argon, the temperature is raised in 100°C increments to 1000°C, and held there for 16 hours. At the end of that time, the tube is cooled to room temperature (RT) under flowing argon.
• RT room temperature
• the flow of argon is then shunted to pass through a multi-neck flask containing neat purified acrylic acid at 50°C and fitted with gas inlet/outlets.
• the flow of acrylic acid/argon vapors is continued at RT for 6 hours.
• residual unreacted acrylic acid is removed, first by purging with argon, then by vacuum drying at 100°C at ⁇ 5" vacuum.
• the carboxylie acid content is determined by reaction with excess 0.100N NaOH and back- titrating with 0.100N HC1 to an endpoint at pH 7.5.
• the resulting surface is very reactive and activated olefins such as acrylic acid, acryloyl chloride, acryla ide, acrolein, maleic anhydride, allyl amine, allyl alcohol or allyl halides will react even at room temperature to form clean products containing only that functionality bonded to the activated olefin.
• olefins such as acrylic acid, acryloyl chloride, acryla ide, acrolein, maleic anhydride, allyl amine, allyl alcohol or allyl halides
• Aromatic C-H bonds can be metallated with a variety of organometallic reagents to produce carbon- metal bonds (C-M) .
• M is usually Li, Be, Mg, Al, or Tl; however, other metals can also be used.
• the simplest reaction is by direct displacement of hydrogen in activated aromatics:
• the reaction may require additionally, a strong base, such as potassium t-butoxide or chelating diamines.
• Aprotic solvents are necessary (paraffins, benzene) .
• the metallated derivatives are examples of primary singly-functionalized fibrils. However, they can be reacted further to give other primary singly- functionalized fibrils. Some reactions can be carried out sequentially in the same apparatus without isolation of intermediates.
• CC fibrils One gram of CC fibrils is placed in a porcelain boat and inserted into a 1" quartz tube reactor which is enclosed in a Lindberg tube furnace. The ends of the tube are fitted with gas inlet/outlets. Under continuous flow of H 2 , the fibrils are heated to 700°C for 2 hours to convert any surface oxygenates to C-H bonds. The reactor is then cooled to RT under flowing H 2 .
• the hydrogenated fibrils are transferred with dry, de-oxygenated heptane (with LiAlH 4 ) to a 1 liter multi-neck round bottom flask equipped with a purified argon purging system to remove all air and maintain an inert atmosphere, a condenser, a magnetic stirrer and rubber septum through which liquids can be added by a syringe. Under an argon atmosphere, a 2% solution containing 5 mmol butyllithium in heptane is added by syringe and the slurry stirred under gentle reflux for 4 hours.
• the fibrils are separated by gravity filtration in an argon atmosphere glove box and washed several times on the filter with dry, deoxygenated heptane. Fibrils are transferred to a 50 cc r.b. flask fitted with a stopcock and dried under 10 "4 torr vacuum at 50°C. The lithium concentration is determined by reaction of a sample of fibrils with excess O.IOON HCl in DI water and back-titration with O.IOON NaOH to an endpoint at pH 5.0.
• Ex. 6 are transferred with dry, deoxygenated heptane in an argon-atmosphere glove bag to a 50 cc single neck flask fitted with a stopcock and magnetic stirring bar. The flask is removed from the glove bag and stirred on a magnetic stirrer. The stopcock is then opened to the air and the slurry stirred for 24 hours. At the end of that time, the fibrils are separated by filtration and washed with aqueous MeOH, and dried at 50°C at 5" vacuum.
• the concentration of OH groups is determined by reaction with a standardized solution of acetic anhydride in dioxane (0.252 M) at 80°C to convert the OH groups to acetate esters, in so doing, releasing 1 equivalent of acetic acid/mole of anhydride reacted.
• the total acid content, free acetic acid and unreacted acetic anhydride, is determined by titration with O.IOON NaOH to an endpoint at pH 7.5.
• phthalocyanine derivative fibrils for protein immobilization has significant advantages over the prior art methods of protein immobilization. In particular, it is simpler than covalent modifications. In addition, the phthalocyanine derivative fibrils have high surface area and are stable in almost any kind of solvent over a wide range of temperature and pH.
• Porphyrins and Phthalocyanines Adsorption of Porphyrins and Phthalocyanines onto Fibrils
• the preferred compounds for physical adsorption on fibrils are derivatized porphyrins or phthalocyanines which are known to adsorb strongly on graphite or carbon blacks.
• Several compounds are available, e.g., a tetracarboxylic acid porphyrin, cobalt (II) phthalocyanine or dilithium phthalocyanine. The latter two can be derivatized to a carboxylic acid form.
• Dilithium phthalocyanine In general, the two Li + ions are displaced from the phthalocyanine (Pc) group by most metal (particularly multi-valent) complexes.
• Cobalt (II) complexes are particularly suited for this.
• Co ++ ion can be substituted for the two Li + ions to form a very stable chelate.
• the Co ++ ion can then be coordinated to a ligand such as nicotinic acid, which contains a pyridine ring with a pendant carboxylic acid group and which is known to bond preferentially to the pyridine group.
• a ligand such as nicotinic acid, which contains a pyridine ring with a pendant carboxylic acid group and which is known to bond preferentially to the pyridine group.
• Co(II)Pc can be electrochemically oxidized to Co(III)Pc, forming a non-labile complex with the pyridine moiety of nicotinic acid.
• the free carboxylic acid group of the nicotinic acid ligand is firmly attached to the fibril surface.
• Suitable ligands are the aminopyridines or ethylenedia ine (pendant NH 2 ) , mercaptopyridine (SH) , or other polyfunctional ligands containing either an amino- or pyridyl- moiety on one end, and any desirable function on the other.
• the loading capacity of the porphyrin or phthalocyanines can be determined by decoloration of solutions when they are added incrementally.
• the deep colors of the solutions deep pink for the tetracarboxylic acid porphyrin in MeOH, dark blue-green for the Co(II) or the dilithium phthalocyanine in acetone or pyridine
• Loading capacities were estimated by this method and the footprints of the derivatives were calculated from their approximate measurements (-140 sq. Angstroms) . For an average surface area for fibrils of 250 m 2 /g, maximum loading will be -0.3 mmol/g.
• the tetracarboxylic acid porphyrin was analyzed by titration. The integrity of the adsorption was tested by color release in aqueous systems at ambient and elevated temperatures.
• the fibril slurries were initially mixed (Waring blender) and stirred during loading. Some of the slurries were ultra-sounded after color was no longer discharged, but with no effect.
• Runs 169-11, -12, -14 and -19-1 were washed in the same solvent to remove occluded pigment. All gave a continuous faint tint in the wash effluent, so it was difficult to determine the saturation point precisely.
• Runs 168-18 and -19-2 used the calculated amounts of pigment for loading and were washed only very lightly after loading.
• the tetracarboxylic acid porphyrin (from acetone) and the Co phthalocyanine (from pyridine) were loaded onto fibrils for further characterization (Runs 169-18 and —19-2, respectively). Analysis of Tetracarboxylic Acid Porphyrin Addition of excess base (pH 11-12) caused an immediate pink coloration in the titrating slurry.
• a number of substituted polynuclear aromatic or polyheteronuclear aromatic compounds were adsorbed on fibril surfaces.
• the number of aromatic rings should be greater than two per rings/pendant functional group.
• substituted anthracenes, phenanthrenes, etc. containing three fused rings, or polyfunctional derivatives containing four or more fused rings can be used in place of the porphyrin or phthalocayanine derivatives.
• substituted aromatic heterocycles such as the quinolines, or multiply substituted heteroaromatics containing four or more rings can be used.
• Table II summarizes the results of the loading experiments for the three porphyrin/phthalocyanine derivatives.
• TCAPor ⁇ h Tetracarboxylic Acid Porphyrin (cal)-calculated
• Examples 11 and 12 illustrate methods for the adsorption of two different phthalocyanine derivatives on carbon nanotubes.
• thermolysin The amount of thermolysin on these fibrils was determined by measuring the enzyme activity of the fibrils.
• Thermolysin can react with substrate FAGLA (N- (3-[2-furyl]acryloyl)-gly-leuamide) and produce a compound that causes absorbance decrease at 345 nm with extinction coefficient of -310 M ⁇ -cm -1 .
• the assay buffer condition for this reaction was 40mM Tris, lOmM CaCl 2 and 1.75 M NaCl at pH 7.5.
• the reaction was performed in 1 ml cuvette by mixing 5 ⁇ l of FAGLA stock solution (25.5 mM in 30% DMF in dH 2 0) and lO ⁇ g of thermolysin fibrils in 1 ml of assay buffer.
• the absorbance decrease at 345 nm was monitored by time scan over 10 minutes.
• the enzyme activity ( ⁇ M/min) was then calculated from the initial slope using the extinction coefficient -310 M ⁇ cm -1 .
• the amount of active thermolysin per gram of fibril was 0.61 ⁇ moles.
• Thermolysin was immobilized on these phthalocyanine derivative fibrils by adsorption according to the method of Example 34.
• the amount of active thermolysin per gram of fibrils was 0.70 ⁇ moles.
• Phthalocyanine derivative fibrils on which thermolysin has been immobilized can be used to catalyze the synthesis of a precursor of the artificial sweetener aspartame.
• the reaction is carried out by mixing 80 mM L-Z-Asp and 220 mM L-PheOMe in ethyl acetate with 10 ⁇ M fibril immobilized thermolysin.
• the product Z-Asp-PheOMe is monitored by HPLC to determine the yield.
• the filter cake was then transferred to a Soxhlet thimble and washed in a Soxhlet extractor with DI water, exchanging fresh water every several hours. Washing was continued until a sample of fibrils, when added to fresh DI water, did not change the pH of the water. The fibrils were then separated by filtration and dried at 100°C at 5" vacuum overnight.
• the carboxylic acid content was determined by reacting a sample with excess O.IOON NaOH and back- titrating with 0.l00 n HCl to an endpoint at pH 7.5. The results are listed in the Table.
• AMINO FUNCTIONALIZATION OF FIBRILS can be introduced directly onto graphitic fibrils by treating the fibrils with nitric acid and sulfuric acid to get nitrated fibrils, then reducing the nitrated form with a reducing agent such as sodium dithionite to get amino-functionalized fibrils according to the following formula: Fib 0 3 -/H 2 5S0 £ 4 F hinder ⁇ .b. -N w 0 2 NaS, ⁇ _0 4 4 «.Fib-NH 2
• the resulting fibrils have many utilities, including the immobilization of proteins (e.g., enzymes and antibodies) , and affinity and ion exchange chromatography.
• proteins e.g., enzymes and antibodies
• affinity and ion exchange chromatography e.g., affinity and ion exchange chromatography
• the reaction was stopped and centrifuged. The aqueous layer was removed and the fibrils washed with water (X5) . The residue was treated with 10% sodium hydroxide (X3) , and washed with water (X5) to furnish nitrated fibrils.
• the fibrils were coupled with horseradish peroxidaese.
• the HRP-coupled amino fibrils were then extensively dialyzed. Following dialysis, the fibrils were washed 15 times over the following week.
• the enzyme-modified fibrils were assayed as follows:
• RADICAL INITIATOR The high degree of stability of carbon nanotubes, while allowing them to be used in harsh environments, makes them difficult to activate for further modification. Previous methods have involved the use of harsh oxidants and acids. It has now been surprisingly found that terminal alcohols can be attached to carbon nanotubes using a free radical initiator such as benzoyl peroxide (BPO) . Carbon nanotubes are added to an alcohol having the formula RCH 2 OH, wherein R is hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cycloaryl, or poly(alkylether) along with a free radical initiator and heated to from about 60°C to about 90°C. Preferred alcohols include ethanol and methanol.
• the reaction mixture is filtered and the carbon nanotube material is washed and dried, yielding modified nanotubes of the formula Nanotube-CH(R)OH.
• This method can also be used to couple bifunctional alcohols. This allows one end to be linked to the carbon nanotube and the other to be used for the indirect linkage of another material to the surface.
• Non-specific binding to high surface area carbon material is ubiquitous. It has been found that attaching hydrophilic oligomers such as PEG to carbon nanotubes can reduce non-specific binding. Further, it has been found that by attaching one end of chain-like molecules such as PEG to the surface of the nanotubes the free end can contain a functional group that can be used for attachment of other materials of interest while still retaining the properties of the PEG (or other material) layer to reduce non-specific binding. Reduction of Non-specific Binding of Bovine Serum Albumen with PEG-modified Fibrils
• Stock dispersions of unmodified fibrils, chlorate oxidized fibrils and PEG modified fibrils at 0.1 mg/ml in 50 mM potassium phosphate buffer at pH 7.0 were prepared by dispersing 1.0 mg of each in 10 mis of buffer with sonication. 2 mis of 2-fold serial dilutions of each were placed in each of 9 polypropylene tubes. 100 ⁇ l of a 0.2 mg/ml solution of bovine serum albumin (BSA) in the same buffer was added to each tube and to three buffer blanks. Three buffer tubes without protein were also prepared. All tubes were mixed on a vortex mixer and allowed to incubate for 30 minutes with 30 seconds of vortexing every 10 minutes.
• BSA bovine serum albumin
• the number of secondary derivatives which can be prepared from just carboxylic acid is essentially limitless. Alcohols or amines are easily linked to acid to give stable esters or amides. If the alcohol or amine is part of a di- or bifunctional poly-functional molecule, then linkage through the O- or NH- leaves the other functionalities as pendant groups.
• Typical examples of secondary reagents are:
• R alkyl, aralkyl, R- Methanol, phenol, tri- aryl, fluoroethanol, fluorocarbon, OH-terminated polymer, SiR' 3 Polyester, silanols
• H 2 N-R R same as above R- Amines, anilines, fluorinated amines, silylamines, amine terminated polyamides, proteins
• R alkyl
• H0- Ethyleneglycol PEG
• Penta- aralkyl CH 2 0- erythritol, bis-Phenol
• the reactions can be carried out using any of the methods developed for esterifying or arainating carboxylic acids with alcohols or amines.
• the methods of H.A. Staab, Angew. Chem. Internat. Edit.. (1), 351 (1962) using N,N , -carbonyl diimidazole (CDI) as the acylating agent for esters or amides and of G.W. Anderson, et al., J. Amer. Chem. Soc. 86, 1839 (1964), using N-hydroxysuccinimide (NHS) to activate carboxylic acids for amidation were used.
• CDI N,N , -carbonyl diimidazole
• NHS N-hydroxysuccinimide
• Amidation of amines occurs uncatalyzed at RT.
• the first step in the procedure is the same. After evolution of C0 2 , a stoichiometric amount of amine is added at RT and reacted for 1-2 hours. The reaction is quantitative.
• the reaction is:
• Trialkylsilylchlorides or trialkylsilanols react immediately with acidic H according to: R-COOH + Cl-SiR' 3 > R-CO-SiR' 3 + HCl
• DABCO DABCO
• Suitable solvents are dioxane and toluene.
• Aryl sulfonic acids, as prepared in Example 1, can be further reacted to yield secondary derivatives.
• Sulfonic acids can be reduced to mercaptans by LiAlH 4 or the combination of triphenyl phosphine and iodine (March,
• R-CONHS + R'NH 2 > R-CO-NHR' This method is particularly useful for the covalent attachment of protein to graphitic fibrils via the free NH 2 on the protein's side chain.
• proteins which can be immobilized on fibrils by this method include trypsin, streptavidin and avidin.
• streptavidin (or avidin) fibrils provide a solid carrier for any biotinylated substance
• Trypsin fibrils were prepared by mixing 1.1 mg NHS-ester fibrils (treated as in avidin fibrils) and 200 ⁇ l of 1.06 mM trypsin solution made in 5 mM sodium phosphate buffer > (pH 7.1) and rotating at room temperature for 6.5 hours. The trypsin fibrils were then washed by 1 ml of 5 mM sodium phosphate buffer (pH 7.1) three times and suspended in 400 ⁇ l of the same buffer for storage.
• Trypsin can react with substrate L-BAPNA (N ⁇ - benzoyl-L-arginine p-nitroanilide) and release a colored compound that absorbs light at 410 nm.
• the assay buffer for this reaction was 0.05 M Tris, 0.02 M CaCl 2 , pH 8.2.
• the reaction was performed in 1 ml cuvette by mixing 5 ⁇ l of L-BAPNA stock solution (50 mM in 37% DMSO in H 2 0) and 10-25 ⁇ g of trypsin fibrils in a 1 ml of assay buffer.
• the absorbance increase at 410 nm was monitored over 10 minutes.
• the enzyme activity ( ⁇ M/min) was then calculated from the initial slope.
• the activity was 5.24 ⁇ M/min per 13 ⁇ g fibrils. This result can be converted to the amount of active trypsin on fibrils by dividing the activity of a known concentration of trypsin solution, which was measured to be 46 ⁇ M/min per 1 ⁇ M trypsin under the same assay conditions. Therefore the amount of active trypsin per gram of fibrils was 8.3 ⁇ moles (or 195 mg) .
• the resulting material was filtered onto a polycarbonate membrane filter, washed 2X with buffer, IX with DI water and 2X with absolute EtOH, all under an argon blanket.
• Gold foil Alfa/Aesar
• 2 cm x 0.8 cm was cleaned with a solution of 1 part 30% H 2 0 2 and 3 parts concentrated H 2 S0 for 10 minutes and rinsed with DI water.
• the foil piece was connected to an Au wire lead and cycled electrochemically between -0.35 V vs. Ag/AgCl and 1.45 V vs. Ag/AgCl in 1 M H 2 S0 4 at 50 mv/sec until the cyclic voltammograms were unchanged, approx. 10 minutes. It was then rinsed with DI water and dried.
• the large piece was cut into four strips 0.5 cm x 0.8 cm.
• the Au foil samples exposed to the CN/ethylenediamine and CN/SH were examined by scanning electron microscopy (SEM) to detect the presence or absence of CN on the surface. Examination at 40,000X revealed the presence of CN distributed over the surface exposed to CN/SH but no CN were observed on the Au foil sample exposed to CN/ethylenediamine.
• SEM scanning electron microscopy
• EXAMPLE 25 Preparation of Maleimide Fibrils From Amino Fibrils
• Amino fibrils were prepared according to Example 13. The amino fibrils (62.2 mg) were then sonicated in sodium phosphate buffer (5 ml, 5 mM at pH 7.2) . Sulfosuccinmidyl-4-(N-maleimidomethyl)cyclohexane- 1-carboxylate (SMCC; 28.8 mg, 0.66 mmols; Pierce, Cat. No.22360) was added to the fibril suspension. The reaction mixture was stirred overnight at room temperature. The fibrils were washed with water and methanol, and the product fibrils were dried under vacuum. Antibody immobilization on the product confirmed the presence of maleimide fibrils.
• SMCC Sulfosuccinmidyl-4-(N-maleimidomethyl)cyclohexane- 1-carboxylate
• maleimides with different linkers e.g., sulfo-SMCC, succinimidyl 4-[p- maleimidophenyl]butyrate [SMPB], sulfo-SMPB, m- maleimidobenzyl-N-hydroxysuccinimide ester [MBS], sulfo- MBS etc.
• linkers e.g., succinimidyl 4-[p- maleimidophenyl]butyrate [SMPB], sulfo-SMPB, m- maleimidobenzyl-N-hydroxysuccinimide ester [MBS], sulfo- MBS etc.
• the resulting maleimide fibrils can be used as a solid support for the covalent immobilization of proteins, e.g. antibodies and enzymes.
• Antibodies were covalently immobilized on malemide activated fibrils. The capacity of antibody was 1.84 milligrams per gram of fibrils when amino fibrils obtained from nitration/reduction method (Example 13) were used and 0.875 milligrams per gram of fibrils when amino fibrils derivatized from carboxyl fibrils were used.
• the carboxylic acid functionalized fibrils were prepared as in Example 14.
• the carboxylic acid content was 0.75 meq/g.
• Fibrils were reacted with a stoichiometric amount of CDI in an inert atmosphere with toluene as solvent at R.T. until C0 2 evolution ceased. Thereafter, the slurry was reacted at 80 °C with a 10- fold molar excess of polyethyleneglycol (MW 600) and a small amount of NaOEt as catalyst. After two hours reaction, the fibrils were separated by filtration, washed with toluene and dried at 100 °C.
• ESCA was carried out to quantify the amount of N present on the aminated fibrils (GF/NH ) .
• ESCA analysis of 177-046-1 showed 0.90 at% N (177-059).
• a derivative was made by the gas phase reaction with pentafluorobenzaldehyde to produce the corresponding Schiff Base linkages with available primary amine groups.
• ESCA analysis still showed the 0.91 at% N, as expected, and 1.68 at%F. This translates into a 0.34 at% of N present as reactive primary amine on the aminated fibrils (5 F per pentafluorobenzaldehyde molecule) .
• a level of 0.45 at% N would be expected assuming complete reaction with the free ends of each N.
• the observed level indicates a very high yield from the reaction of N with NHS-activated fibril and confirms the reactivity of the available free amine groups.
• Carboxy1 fibrils were also converted to amino fibrils using mono-protected 1,6-diaminohexane (a six- carbon linker) , rather than ethylenediamine (a two-carbon linker) .
• EXAMPLE 28
• Carboxyl groups on fibrils can be modified by reacting the carboxyl groups with one amino group of a compound having two or more amino groups (at least one of which is unprotected by groups such as t-Boc or CBZ) .
• the fibrils so generated are amide derivatives in which the amide carbonyl is derived from the fibril carboxyl group and the amide nitrogen is substituted with a group (such as an alkyl group) containing one or more primary amines.
• the amino groups are then available for use or further modification.
• One gram of carbon fibrils was placed in a dry scintered glass filter tunnel, the outlet of which was tightly stoppered with a rubber serum septum, and anhydrous dichloromethane was added to cover.
• N- Methylmorpholine (758 ⁇ L, 7 mmol) was added, the suspension was mixed with the aid of a spatula. Then isobutyl chloroformate (915 ⁇ L, 7 mmol) was added, and the suspension mixed periodically for one hour. The mixture was protected from atmospheric moisture by a cover of Parafilm as much as was practical. Meanwhile, N-boc-l,6-diaminohexane hydrochloride (1.94 g, 7.7 mmol) was partitioned between dichloromethane (10 mL) and 1 M NaOH (10 mL) . The lower, organic phase was dried over anhydrous potassium carbonate and filtered through a disposable Pasteur pipette containing a cotton plug, and N-methylmorpholine (758 ⁇ L, 7 mmol) was added.
• the serum septum was removed from the filter funnel, the reagents were removed from the fibrils by vacuum filtration, and the fibrils were washed with anhydrous dichloromethane.
• the serum septum was replaced, and the mixture of N-methylmorpholine and monoprotected diaminohexane was added to the fibrils. The mixture was stirred periodically for one hour.
• the reagents were removed by filtration, and the fibrils were washed successively with dichloromethane, methanol, water, methanol, and dichloromethane.
• a 50% mixture of trifluoric acid and dichloromethane was added to the fibrils and the mixture stirred periodically for 20 minutes.
• the solvents were removed by filtration, and the fibrils were washed successively with dichloromethane, methanol, water, 0.1 M NaOH, and water.
• HRP horseradish peroxidase
• Table VI summarizes the secondary derivative preparations. Products are analyzed by ESCA. The analysis confirms the incorporation of the desired pendant groups. The products are analyzed by ESCA for C, 0, N, Si and F surface contents. TABLE VI
• Tertiary and quaternary amine functional groups can be attached to the surface of carbon nanotubes via an amide or ester bond via a carboxyl group on the nanotube and either an amine or hydroxyl group of the tertiary or quaternary amine precursor.
• Such tertiary or quaternary amine fibrils are useful as chromatographic matrices for the separation of biomolecules.
• the tertiary or quaternary amine fibrils can be fabricated into disk- shaped mats or mixed with conventional chromatographic media (such as agarose) for separation purposes.
• the resulting fibrils were washed three times with 20 ml dimethylformamide, three times with 20 ml methylene chloride, three times with 20 ml methanol and finally three times with de-ionized water.
• the product was dried under vacuum. Results from an elemental analysis of nitrogen showed that about 50% of the carboxyl groups on the fibril had reacted with the primary amino group in the quaternary amine moiety.
• BSA Bovine Serum Albumin
• the column was eluted with 5 mM sodium phosphate at a flow rate of 0.2 ml/min and 0.6ml fractions were collected.
• the elution profile was monitored using a UV- visible detector, and is shown in Fig 3. Once the detector indicated that no more protein was eluting from the column, bound BSA was eluted by adding 1 M KC1 in 5 mM sodium phosphate (pH 7.3). The presence of the protein in each fraction was identified by micro BCA assay (Pierce, Rockford, II) .
• the elution profile was monitored using a UV-visible detector (Fig. 4) . Once the detector indicated that protein was no longer being eluted with 5 mM sodium phosphate buffer, the solvent was changed to 1 M KC1 in 5 mM sodium phosphate (pH 7.3). The presence of the protein in each fraction was identified by micro BCA assay (Pierce, Rockford, II) .
• Aqueous suspensions of solid graphitic carbon are made containing one or more enzymes that are capable of accepting the graphitic carbon as a substrate and performing a chemical reaction resulting in chemically- modified graphitic carbon.
• the aqueous suspension is maintained at conditions acceptable for the enzyme(s) to carry out the reaction (temperature, pH, salt concentration, etc.) for a time sufficient for the enzyme(s) to catalytically modify the surface of the graphitic carbon.
• the suspension is continually mixed to allow the enzyme(s) access to the surface of the graphitic carbon.
• the enzyme is removed from the carbon by filtration washing.
• cytochrome p450 enzymes and peroxidase enzymes.
• the types of enzymes have been well-studied, they accept aromatic type substrates, and their optimal reaction conditions have been worked out. Both enzyme types introduce hydroxyl groups into their substrates and may introduce hydroxyl groups into graphitic carbon.
• other biocatalysts such as ribozymes and catalytic antibodies, or non-biological mimics of enzymes, could be designed to catalytically functionalize carbon nanotubes.
• EXAMPLE 34 Enzymatic Functionalization Using Rat Liver Microsomes Cytochrome p450 enzymes are generally believed to function in the liver as detoxifying agents (F. Peter Guengerich, American Scientist.
• cytochrome p450 enzymes Two rats (“experimental” rats) were administered phenobarbital (lg/L, pH 7.0) in their drinking water for one week to induce expression of cytochrome p450 enzymes. Two other rats (“control” rats) were given water without phenobarbital. The rats were then sacrificed and cytochrome p450-containing microsomes were prepared from their livers by standard procedures (see for example. Methods in Enzymology, Vol. 206) . The microsomes were mixed with carbon nanotubes
• fibrils both "plain” or nonfunctionalized and “COOH” or oxidized fibrils
• microsomes both experimental and control microsomes
• NADPH was included as a co-substrate for cytochrome p450s and glucose-6-phosphate, glucose-6- phosphate dehydrogenase were added to regenerate NADPH from NADP + (if NADP + is generated by cytochrome p450s) .
• the mixtures were rotated at room temperature for about 1.5 days in microcentrifuge tubes. Following the incubation, the fibrils were washed extensively in deionized water, 1 M HCl, 1 M NaOH, 0.05% Triton X-100, 0.05% Tween, methanol, and 1 M NaCl.
• cytochrome p450 enzymes were purchased (GENTEST, Woburn, MA) . Because cytochrome p450 enzymes are only active in association with membranes, these enzymes are supplied as microsomal preparations.
• cytochrome p450s CYP1A1 (cat.# Mlllb) , CYP1A2 (cat.# M103C) , CYP2B6 (cat.# 110a), CYP3A4 (with reductase, cat.# 107r) .
• MgCl 2 (0.67 mg/mL) was also included in the reaction solution. In this experiment, fibrils were washed with the aid of a Soxhlet apparatus.
• DNBA 3,5- dinitrobenzoic acid
• fibrils 11 mg were mixed in a solution containing 50 mM sodium acetate (1.25 mL, pH 5.0), horseradish peroxidase (200 nM) , and dihydroxyfumaric acid (15 mg) was added 5 mg at a time for the first 3 hours of the reaction. The reaction was carried out for a total of 5 hours at 4° C with intermittent bubbling of gaseous oxygen. Following the reaction, the fibrils were washed with water, 1 N NaOH, methanol, and methylene chloride (200 mL of each) . A control reaction was carried out using peroxidase that had been heat inactivated (100° C for 5 minutes) .
• the primary products obtainable by addition of activated electrophiles to oxygen-free fibril surfaces have pendant -COOH, -C0C1, -CN, -CH 2 NH 2 , -CH 2 OH, -CH 2 -
• Fibril-CH 2 OH + HOOC-R-Y > F-CH 2 OCOR-Y Fibril-CH 2 -Halogen + Y " > F-CH 2 -Y + X " Y " NCO", -
• the concentration of functional groups on the surface of nanotubes can be increased by modifying the nanotubes with a series of generations of a polyfunctional reagent that results in the number of the specific functional groups increasing with each generation to form a dendrimer-like structure.
• the resulting dendrimeric nanotubes are particularly useful as a solid support upon which to covalently immobilize proteins, because they increase the density of protein immobilized on the nanotube surface.
• the present invention demonstrates that high densities of a specific chemical functionality can be imparted to the surface of high surface area particulate carbon, which has been difficult with previous high surface area carbons.
• the reaction sequence is shown in Fig. 5.
• a suspension of amino fibrils (90 mg) in sodium bicarbonate (5 ml, 0.2 M, pH 8.6) was added a solution of N ⁇ ,Ne-di-t-boc-L-lysine N-hydroxysuccinimide ester (120 mg, 0.27 mmol) in diosane (5 ml).
• the reaction mixture was stirred overnight at room temperature.
• the tert-butoxycarbonyl protected lysine fibrils were extensively washed with water, methanol and methylene chloride and dried under vacuum.
• the tert- butoxycarbonyl protected lysine fibrils were then treated with trifloroacetic acid (5 ml) in methylene chloride (5 ml) for 2 hours at room temperature.
• the product amino lysine fibrils were extensively washed with methylene chloride, methanol and water and dried under vacuum.
• Preparation of the second and the third generation lysine fibrils followed the same procedure.
• the amino acid analysis data showed that the first generation lysine fibrils contained 0.6 ⁇ ols lysine per gram of fibrils, the second generation lysine fibrils contained 1.8 ⁇ mols per gram of fibrils, and the third generation lysine had 3.6 ⁇ mols lysine per gram of fibrils.
• Carboxyl dendrimeric fibrils can be prepared by the same method by using aspartic or glutamic acid with carboxyl fibrils.
• Carboxylate terminated dendrimers with a carbon nanotube (CN) core are produced by successive, sequential couplings of aminobuty-nitrilotriacetic acid (NTA) and beginning with the NHS ester of chlorate oxidized carbon nanotubes. Preparation of NTA
• CN/NHS were prepared according to the method of Example 20. Preparation of CN/NTA 0.4 g of NTA»HC1 was dissolved in 25 mis of
• CN/NTA was first converted to the NHS active ester. 0.396 grams of CN/NTA was dried in an oven at 90°C for 30 minutes and then placed in a 100 ml RB flask with 30 mis of anhydrous dioxane and purged with argon. 0.4 g of N-hydroxysuccinimide added with stirring followed by 0.67 grams of EDC with continued stirring for an additional hour. The CN tended to agglomerate together during this time. The dioxane was decanted off and the solids were washed 2X with 20 mis of anhydrous dioxane. The solids were washed with 20 mis of anhydrous MeOH during which the agglomerates broke up.
• HRP horseradish peroxidase
• Plain fibrils (0.49 mg) , amino fibrils (0.32 mg) , first generation lysine fibrils (0.82 mg) , second generation lysine fibrils and third generation lysine fibrils were sonicated with sodium bicarbonate conjugate buffer (600 ⁇ l, 0.1 M, containing 0.9% NaCl) for 15 minutes at room temperature. Then they were incubated with HRP solution in sodium bicarbonate conjugate buffer (490 ml, enzyme stock solution of 5.6 mg/ml) for 19 hours at room temperature.
• the HRP immobilized fibrils were washed with the following buffer (1 ml) : 10 mM NaHC0 3 buffer containing 0.9% NaCl at pH 9.5 (IX washing buffer) seven times, 0.1% Triton X-100 in IX washing buffer five times, 50% ethylene glycol in IX washing buffer three times.
• the activity of HRP was assayed with hydrogen peroxide solution (10 ⁇ l, 10 mM stock solution) and 2,2-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid diammoniu salt (ABTS, 3 ⁇ l, mM stock solution) in glycine assay buffer (50 mM, pH 4.4) at 414 nm.
• ABTS 2,2-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid diammoniu salt
• N ⁇ -CBZ-N e - (tert-butoxycarbony1)-L-lysine was treated with 0.2 M calcium carbonate (4 ml) and the aqueous layer was removed to obtain a white solid.
• the solid was resuspended in N,N- dimethylformamide(40 ml) and benzyl bromide (1.16 ml). The reaction mixture was stirred overnight at room temperature. The reaction mixture was worked up with ethyl acetate and water, and the organic layer was dried over magnesium sulphate.
• N ⁇ -CBZ-L-lysine benzyl ester fibrils 113 mg
• sodium hydroxide I N, 4 ml
• the product N ⁇ -CBZ-L-lysine fibrils was extensively washed with water and methanol and the fibrils were dried under vacuum.
• the final bifunctional fibrils were extensively washed with water, methanol, 0.5 N sodium hydroxide, acetonitrile and methylene chloride. Amino acid analysis showed 0.3 ⁇ mols lysine per gram of fibrils.
• Hydroxyl and carboxyl (or amino) bifunctional fibrils can be made by a similar method to that described here by using serine, threonine, or tyrosine.
• Thiolated and carboxyl (or amino) bifunctional fibrils can be made using cysteine.
• Carboxyl and amino bifunctional fibrils can be made using aspartic or glutamic acid.
• Functionalized graphitic nanotubes are useful as solid supports in many biotechnology applications due to their high porosity, chemical and thermal stability and high surface area. They have been found to be compatible with harsh chemical and thermal treatments and very amenable to chemical functionalization.
• an enzyme can be covalently immobilized on a modified nanotube while retaining its biological activity.
• nanotubes are also suitable for use as affinity chromatographic supports in biomolecular separations.
• enzyme inhibitors have been prepared on nanotubes in multi-step syntheses such that the immobilized inhibitors were accessible to macromolecules, and reversible specific biological recognition occurred between proteins and modified fibrils.
• the hydrophobicity of the nanotube surface is not enough to immobilize high densities of proteins by adsorption.
• alkyl chains of varying lengths have been coupled to the nanotube surface.
• Proteins that have been immobilized on alkyl nanotubes by adsorption include trypsin, alkaline phosphatase, lipase and avidin.
• the enzyme activities of these immobilized proteins are comparable with those of the free enzymes, proven by the catalytic efficiencies toward the hydrolysis of their substrates in aqueous solutions.
• phenyl-alkyl nanotubes which are alkyl nanotubes with the addition of a phenyl group on the end of the alkyl chain, have also been prepared.
• This modification introduced an aromatic structure that interacts with the amino acids phenylalanine, tyrosine, and tryptophan in proteins through ⁇ - ⁇ interactions.
• the adsorption of alkaline phosphatase and lipase on phenyl- alkyl nanotubes was comparable to the adsorption on C 8 - alkyl nanotubes.
• Alkyl fibrils were prepared by reacting lOmg of carboxyl fibrils, which contained approximately 0.007 mmoles of
• the enzymes lipase, trypsin, alkaline phosphatase and avidin were immobilized on the alkyl fibrils of this example by adsorption.
• the alkyl fibrils and enzyme were mixed at room temperature for three to four hours, followed by washing two to four times with 5mM sodium phosphate (pH 7.1).
• Alkaline phosphatase was immobilized on C 8 -fibrils and C 6 OH-fibrils; trypsin on C 6 - , C 8 -, C 10 - and C 18 -fibrils, lipase on C 6 OH-, C 8 -, C 10 - and C 18 -fibrils, and avidin on C 8 -fibrils.
• the results are shown in the following table: 1 Enzyme ⁇ mol/g fibril mg/g fibril lipase 6.8 816 trypsin 1.7 40 alkaline phosphatase 0.66 56
• Phenyl-alkyl fibrils were prepared by two different reactions.
• Reaction 1 mixed 20 mg carboxyl fibrils (containing approximately 0.014 mmoles of -COOH group) with 0.28 mmoles of 4-phenylbutylamine, 0.28 mmoles EDC and 0.28 mmoles DMAP (4-dimethylaminopyridine) in 1.5 ml of DMF (N,N-dimethylformamide) .
• Reaction 2 mixed 20 mg carboxyl fibrils with 0.28 mmoles of 6- phenyl-1-hexanol, 0.28 mmoles DCC (1,3- dicyclohexylcarbodiimide) and 0.28 mmoles DMAP in 1.5 ml of DMF.
• the reactions were performed at room temperature with stirring overnight.
• the fibrils were then washed rigorously with 3 x 25 ml CH 2 C1 2 , 3 x 25 ml MeOH, and 3 x 25 ml dH 2 0.
• Alkaline phosphatase reacts with substrate p- nitrophenyl phosphate and releases a color compound that absorbs light at 405 nm with extinction coefficient of 18,200 M ⁇ 1 cm "1 .
• the reaction was performed in 1 ml cuvette by mixing 5 ⁇ l of p-nitrophenyl phosphate stock solution (0.5 M in 33% DMSO in assay buffer) and 13 ⁇ g of alkaline phosphatase fibrils in 1 ml of assay buffer.
• the absorbance increase of 405 nm was monitored by time scan over 0 minutes.
• the enzyme activity ( ⁇ M/min) was then calculated from the initial slope using the extinction coefficient 18200 M ⁇ cm" 1 .
• the activity was 6.95 ⁇ M/min per 13 ⁇ g fibrils.
• the activity was 2.58 ⁇ M/min per 13 ⁇ g fibrils.
• phenyl-alkyl fibrils were suspended in 50 ⁇ l of 5 mM sodium phosphate buffer (pH 7.1) and sonicated for 20 minutes.
• 5 mM sodium phosphate buffer pH 7.1
• lipase solution 0.2 mM in 5 mM sodium phosphate buffer, pH 7.1
• the fibrils were then washed with 600 ⁇ l of 5 mM sodium phosphate buffer (pH 7.1) three times and suspended in 200 ⁇ l of the same buffer.
• Lipase can react with the substrate l,2-o- dilauryl-rac-glycero-3-glutaric acid-resorufin ester (Boehringer Mannheim, 1179943) and produce a color compound that absorbs light at 572 nm with extinction coefficient of 60,000 M ⁇ cm "1 .
• the reaction was performed in 1 ml cuvette by mixing 5 ⁇ l of substrate stock solution (7.6 mM in 50% dioxane in
• a 10.0 g sample of graphitic fibrils was slurried in 450 mL concentrated H 2 S0 4 by mixing with a spatula, then transferred to a reactor flask fitted with inlet/outlets and an overhead stirrer. With stirring and under a slow flow of argon, a charge of 8.68 g of NaCl ⁇ 3 was added in portions at room temperature over a 24 hour period. Chlorine vapors, which were generated during the entire course of the run, were swept out of the reactor into an aqueous NaOH trap. At the end of the run, the fibril slurry was poured over cracked ice and vacuum filtered.
• the fibrils were then washed twice with conjugation buffer in an Eppendorf tube and suspended 430 ⁇ L conjugation buffer. A 50- ⁇ L aliquot of the suspension (0.14 mg fibrils) was mixed with 4.0 mg activated HRP (Pierce, Rockford, IL) dissolved in 50 ⁇ L deionized water and the resulting suspension was rotated overnight at 4°C. The HRP- conjugated fibrils were washed extensively in an Eppendorf tube and suspended 430 ⁇ L conjugation buffer. A 50- ⁇ L aliquot of the suspension (0.14 mg fibrils) was mixed with 4.0 mg activated HRP (Pierce, Rockford, IL) dissolved in 50 ⁇ L deionized water and the resulting suspension was rotated overnight at 4°C. The HRP- conjugated fibrils were washed extensively in an HRP (Pierce, Rockford, IL) dissolved in 50 ⁇ L deionized water and the resulting suspension was rotated overnight at 4°C. The HRP- conjugated fibrils were was
• Carboxylated fibrils were used to prepare NHS ester fibrils as described in Example 50 above.
• NHS ester fibrils 114 mg
• 10 equivalents based on the estimation of 0.7 meq NHS ester per gram of fibrils
• Dry triethylamine (10 equiv.) was added and the mixture was stirred for 3 hours at room temperature.
• the tyraminyl fibrils were washed under vacuum in a scintered glass funnel first with acetone, then extensively with deionized water.
• 4-(p-Aminophenylazo)-phenylarsonic acid (66 mg) was suspended in 4 mL of 1 N HCl. The suspension was cooled to 4°C and mixed slowly with 0.36 mL of 0.5 M NaN0 2 . After 15 minutes, the arsonic acid/NaN0 2 mixture was added to the tyraminyl fibrils, which were suspended in 10 mL of 0.1 M NaC0 3 (pH 10.0). The reaction mixture (pH « 10) was stirred overnight at 4°C. The fibrils were then treated with successive washes of 0.1 M Na 2 C0 3 (pH 10.0), 8 M guanidine HCl, 25 mM NaOH, and water until the effluent became clear.
• Atomic absorption analysis of arsenic in the AP-inhibitor fibrils was carried out by Galbraith Laboratories (Knoxville, TN) .
• AP-inhibitor fibrils which contain sidechains containing one atom of arsenic were found by atomic absorption analysis to have any arsenic content of 0.4%. This indicates that roughly 10% of the estimated initial COOH groups were converted to AP-inhibitors in this multi-step synthesis. Based on the surface area of fibrils, this means that there would be one inhibitor molecule (enzyme binding site) for every 50 ⁇ A 2 of surface area.
• TPEG B-Galactosidase-Inhibitor Fibrils
• p-Amino-phenyl-B-D-thiogalactoside (TPEG) derivatized fibrils were prepared based on the method of Ullman, (1984) Gene. 29:27-31.
• To 8 mg of carboxylated fibrils in 0.2 mL deionized water was added 2.24 mg TPEG.
• the pH of the suspension was adjusted to 4.0 with 0.1 M HCl and 15 mg EDAC was added.
• the mixture was stirred for 3 hours at pH 4.0 and room temperature. The reaction was stopped by rapid centrifugation in an Eppendorf tube and removal of the liquid.
• the B-galactosidase-inhibitor fibrils were washed five times by repeated resuspension in deionized water and centrifugation.
• alkaline phosphatase from E. coli , Type III; Sigma Chemical Co., St. Louis, MO
• BG B- galactosidase
• B-Galactosidase was assayed by spectrophotometrically monitoring the enzyme's ability to hydrolyze 2-nitro-galacto-B-D-pyranoside (ONPG) .
• a mixture of AP and BG were added.
• the concentrations of added enzymes were in large excess of the immobilized inhibitor concentrations.
• 0.550 ⁇ mol AP/g fibrils was bound (as opposed to non-specific binding of 0.020 ⁇ mol BG/g fibrils) .
• the capacity was determined to be 0.093 ⁇ mol BG/g fibrils (in contrast with non-specific binding of 0.012 ⁇ mol AP/g fibrils).
• the results of the affinity chromatography experiments are shown in Figs. 9 and 10.
• AP-inhibitor fibrils did not appreciably bind BG, but bound AP, which specifically eluted when 40 mM phosphate, a competing inhibitor, was added to the buffer (Fig. 9) .
• Fibrils derivatized with BG did not bind substantial amounts of AP, but bound BG, which specifically eluted when the pH was raised to weaken the enzyme-inhibitor association (Fig. 10) .
• antibodies can be immobilized on functionalized nanotubes, and that such antibody nanotubes have unique advantages for many applications due to their high surface area per weight, electrical conductivity, and chemical and physical stability.
• antibody nanotubes can be used as affinity reagents for molecular separations.
• Antibody nanotubes are also useful for analytical applications, including diagnostic immunoassays such as ECL-based immunoassays.
• Antibodies can be immobilized either by covalent binding or non-covalent adsorption.
• Covalent immobilization was accomplished by various methods; including reductive amination of antibody carbohydrate groups, NHS ester activation of carboxylated fibrils (see Example 27, supra), and reaction of thiolated or maleimido fibrils with reduced or maleimido-modified antibodies (see Examples 23 and 25 supra) .
• the best method for attaching antibodies to nanotubes will depend on the application they are to be used in.
• the preferred method may be non-covalent adsorption because the capacity of protein binding seems to be the highest for this method.
• covalent methods may be preferred (the alkyl appendages are weak electrical conductors and can be expected to insulate the fibrils) .
• Reductive amination may be the best way to covalently attach antibodies to fibrils because, by using this method, the antibodies are correctly oriented so that their binding sites are pointing outward (away from the fibrils) .
• NAD + fibrils have been used as a solid support for the purification of dehydrogenases.
• the main advantage of using fibrils is their large amount of accessible surface area.
• An affinity matrix with high surface area is desirable because of the high potential capacity.
• the fibrils may either be a loose dispersion or fixed into a column or mat.
• Fibrils were oxidized to introduce carboxyl groups according to Examples 14 and 15.
• sodium bicarbonate solution 3ml, 0.2 M, pH 8.6
• N 6 - [aminohexyl]carbamoylmethyl)-nicotinamide adenine dinucleotide lithium salt solution 25 mg from Sigma in 5 ml sodium bicarbonate solution.
• the reaction mixture was stirred overnight at room temperature.
• the product fibrils were extensively washed with water, N,N- dimethylformamide, and methanol.
• the elemental analysis data showed that the product fibrils contained 130 mmols of NAD molecules per gram of fibrils by nitrogen analysis and 147 mmols of NAD molecules per gram of fibrils by phosphorus analysis.
• Other NAD + analogs having linkers terminating in an amino group can be used to prepare NAD + fibrils.
• the NAD + immobilized fibrils (0.26 mg) and plain fibrils (0.37 mg) were sonicated with 0.1% polyethylene glycol (PEG, MW 1000) in sodium phosphate (1 ml, 0.1 M, at pH 7.1) for 30 minutes at 40°C, then incubated for 30 minutes at 40°c.
• the fibril suspension was centrifuged and the supernatant were removed.
• the fibrils were incubated with the mixture of L-lactate dehydrogenase (LDH) in 0.1% PEG (1000) sodium phosphate buffer (250 ⁇ l, the ratio of the LDH solution and the 0.1% PEG buffer was 1:1) for 90 minutes at 4 ⁇ C. Then the mixtures were equilibrated for 30 minutes at room temperature.
• LDH L-lactate dehydrogenase
• the fibrils were washed with 0.1% PEG (1000) in sodium phosphate buffer (5 X 1000 ⁇ l) and every washing took 15 minutes with rotation.
• the LDH was eluted with a 5 mM solution of NADH in 0.1% PEG (1000) sodium phosphate buffer (5 mM 3X1000 ⁇ l) .
• the LDH activity in the eluents was assayed by measuring the absorbance change at 340 nm during reduction of pyruvate.
• the assay mixture contained 0.1% PEG (1000) in sodium phosphate buffer (980 ⁇ l) , pyruvate (3.3 ⁇ l, 100 mM stock solution), and each elution fraction (16.7 ⁇ l) .
• the enzyme reaction is shown below:
• N,N-dimethylformamide N,N-dimethylformamide (DMF, 2 ml) and methylene chloride (8 ml) were added N-(9- fluorenylmethoxycarbonyl)-0-butyl-L-serine (215 mg, 0.56 mmol), 1,3-dicyclohexylcarbodiimide (DCC, 115 mg, 0.56 mmol) and 4 dimethylaminopyridine (DMAP, 3.4 mg, 0.028 mmol) .
• DMF 1,3-dicyclohexylcarbodiimide
• DMAP dimethylaminopyridine
• fibril surfaces can be functionalized by biotinylation or by both alkylation and biotinylation.
• the fibrils containing such modifications can then bind any streptavidin conjugated substances such as streptavidin beads and streptavidin enzymes.
• Fibrils offer great advantages as solid carriers because of their high surface area. Beads, which can be made strongly magnetic, are extremely useful in separation assays.
• the biotinylated fibrils described herein combine the advantages of both the fibrils and the beads.
• the biotinylated alkyl fibrils are an extension of the same concept but exhibit the additional protein adsorption property of alkyl fibrils.
• streptavidin- and biotin-coated fibrils can be used in diagnostics and can be used as capture agents for assays such as electrochemiluminescence assays.
• a novel feature of this invention is the combination of two solid carriers on one fibril to create a bifunctional fibril. Moreover, the disclosed process increases the surface area for beads and magnifies fibril magnetization.
• Biotinylated fibrils were prepared by mixing 2.4 mg of amino fibrils prepared as described in Example 16 and 9 mg of NHS ester long chain biotin in buffer 0.2 M NaHC0 3 at a pH of 8.15. The mixture was rotated at room temperature for four hours and washed with the same buffer twice.
• Biotinylated alkyl fibrils were prepared by a two step reaction. First, 4.25 mg of bifunctional fibrils (containing both amino and carboxyl) and 25 mg of NHS ester long chain biotin were mixed. The fibrils were washed and dried under vacuum.
• the second reaction was carried out by mixing 4 mg of biotinylated bifunctional fibrils with 11 mg of EDC (l-ethyl-3-3-dimethylaminopropyl)carbodiimide) , 7.5 mg of DMAP (4-dimethylaminopyridine) and 10 ⁇ l of NH 2 (CH 2 ) 7 CH 3 in 0.5 ml of DMF. The mixture was stirred at room temperature overnight. The final biotinylated alkyl fibrils were washed by CH 2 C1 2 , MeOH, and dH 2 0.
• Biotinylated fibrils can be used in assays involving formats that require streptavidin-biotin or avidin-biotin interactions. Biotinylated fibrils could, for example, be further derivatized with streptavidin. Biotin covalently linked to fibrils (see Example 50) could form strong non-covalent binding interactions with streptavidin. Because streptavidin is a tetrameric protein with four equivalent binding sites, streptavidin bound to biotinylated fibrils would almost certainly have unoccupied binding sites to which additional biotinylated reagents could bind. Thus, biotinylated fibrils would be converted to streptavidin-coated fibrils.
• FBS fibril-biotin-streptavidin
• a biotinylated anti-analyte antibody could be captured on the FBS support (either before or after the antibody has complexed to an analyte) .
• Assays using biotinylated anti-analyte antibodies are well established. Such assays include competitive assays where the analyte of interest competes with a labeled analyte for binding to the anti-analyte antibody. Free (unbound) analyte and free (unbound) labeled analyte can be washed from the fibril immobilized antibody. The washing step depends on the fibrils being physically separated from the solution phase by common practices involving centrifugation, filtration, or by attraction to a magnet.
• Sandwich immunoassays are well known in the field of diagnostics. Such assays involve an analyte being bound simultaneously by two antibodies; a first "primary” antibody which is captured on a solid surface by for example being labeled with biotin, and a "secondary” antibody which is not captured by a solid surface but is labeled with a reporter group.
• a sandwich assay could be carried out using fibrils as a solid capture support whereby the fibrils are captured as described in the previous paragraph.
• the fibril would have covalently linked to it biotin, which would be bound to streptavidin, which would in turn be bound to a biotinylated primary antibody, which would be bound to analyte (if present) , which would be bound to a labeled secondary antibody.
• DNA probe assays could be carried out using FBS supports. Biotinylated single stranded DNA can be bound to FBS supports and competitive hybridization can occur between complementary single stranded analyte DNA molecules and complementary labeled oligonucleotides.
• biotinylated fibrils can be used in immunoassays and DNA probe assays.
• bifunctional fibrils can be modified by covalent attachment of biotin to one type of functional group and alkyl chains to the other type of functional group.
• the resultant alkylated, biotinylated fibrils can be used both in specific association with streptavidin or avidin (via biotin) and also for adsorption of proteins (via the alkyl chains) .
• Alkyl fibrils could be used in conjunction with other solid supports, such as streptavidin-coated magnetic beads.
• One advantage of fibrils over such beads is that they have a much higher surface area (per unit weight) .
• fibrils could be attached to the outside surface of the magnetic beads, this would dramatically improve the surface area and hence the binding capacity of the beads.
• alkylated, biotinylated fibrils could be mixed with streptavidin-coated beads resulting in high affinity streptavidin(bead)-biotin(fibril) interactions and hence fibril-coated beads with an extremely high surface area.
• fibril-coated beads could be further derivatized with adsorbed proteins including streptavidin and antibodies.
• streptavidin or antibody coated fibrils can be used in immunoassays and DNA probe assays.
• fibril-coated beads could improve the properties of the beads by dramatically increasing their surface area such that fewer beads would be required in a given assay to give the same result.
• 3-DIMENSIONAL STRUCTURES The oxidized fibrils are more easily dispersed in aqueous media than unoxidized fibrils. Stable, porous 3-dimensional structures with meso- and macropores (pores >2 nm) are very useful as catalysts or chromatography supports.
• fibrils can be dispersed on an individualized basis, a well-dispersed sample which is stabilized by cross-links allows one to construct such a support.
• Functionalized fibrils are ideal for this application since they are easily dispersed in aqueous or polar media and the functionality provides cross-link points. Additionally, the functionality provides points to support the catalytic or chromatographic sites. The end result is a rigid, 3-dimensional structure with its total surface area accessible with functional sites on which to support the active agent.
• Typical applications for these supports in catalysis include their use as a highly porous support for metal catalysts laid down by impregnation, e.g. , precious metal hydrogenation catalysts.
• the ability to anchor molecular catalysts by tether to the support via the functionality combined with the very high porosity of the structure allows one to carry out homogeneous reactions in a heterogeneous manner.
• the tethered molecular catalyst is essentially dangling in a continuous liquid phase, similar to a homogeneous reactor, in which it can make use of the advantages in selectivities and rates that go along with homogeneous reactions.
• being tethered to the solid support allows easy separation and recovery of the active, and in many cases, very expensive catalyst.
• the 3-dimensional structure of functionalized fibrils is an electrode, or part of an electrode, and the functionalization has resulted from adsorption of Co(II)Pc
• electrochemical oxidation of Co(II) to Co(III) in the presence of nicotinic acid will produce a non- labile Co(III)-pyridyl complex with a carboxylic acid as the pendent group.
• Attaching a suitable antigen, antibody, catalytic antibody, or other site-specific trapping agent will permit selective separations of molecules (affinity chromatography) which are otherwise very difficult to achieve.
• the Co(III) complex containing the target molecule can be electrochemically reduced to recover the labile Co(II) complex.
• the ligand on Co(II) containing the target molecule can then be recovered by mass action substitution of the labile Co(II) ligand, thereby effecting a separation and recovery of molecules which are otherwise very difficult or expensive to perform (e.g., chiral drugs).
• the pores within the functionalized carbon fibril mats were too small to allow significant flow and thus would not be useful as flow through electrodes.
• particulate carbon or other carbon based materials such as Reticulated Vitreous Carbon (RVC)
• RVC Reticulated Vitreous Carbon
• the porous electrode materials could not be formed in situ, packed too densely and formed voids or channels, were subject to dimensional instability during changes in solvent and flow conditions, and were unable to form very thin electrodes.
• the use of functionalized carbon fibrils as electrodes in a flow cell solved such problems.
• the functionalized carbon fibrils used as electrodes in a flow cell can be modified by surface treatment with electroactive agents.
• the fibrils can also be modified with non-electroactive materials that may serve a catalytic or electrocatalytic function or serve to inhibit unwanted reactions or adsorption of materials from the flowing stream.
• Graphitic fibrils were modified by adsorbing Iron(III)phthalocyanine-bis-pyridine (FePc-2Py) (Aldrich 41,016-0). 0.403 grams of fibrils and 0.130 grams of FePc-2Py were added to 150 mis of absolute EtOH and sonicated with a 450 Watt Branson probe sonicator for 5 in. The resulting slurry was filtered onto a 0.45 ⁇ ro MSI nylon filter in a 47 mm Millipore membrane vacuum filter manifold, rinsed with water and dried in a vacuum oven overnight at 35°C. The final weight was 0.528 grams, indicating substantial adsorption. A spectrophotometric analysis of the filtrate accounted for the remaining FeP-2Py
• a electrochemical flow cell was constructed from a 13 mm, plastic, Swinney type membrane filter holder by placing a 13 mm diameter disk of gold mesh (400 mesh, Ladd Industries) on top of the membrane support and making electrical contact to the screen with a platinum wire, insulated with Teflon® heat shrink tubing that was fed through the wall of the filter holder for external connection as the working electrode of a three electrode potentiostat circuit.
• the gold mesh was fixed in place with a minimal amount of epoxy around the outer edge.
• a strip of gold foil was fashioned into a ring and placed in the bottom, down stream section of the filter holder and connected with an insulated Pt wire lead for connection as the counter electrode of a three electrode potentiostat circuit.
• a ring of 0.5mm diameter silver wire, electrochemically oxidized in 1M HCl, was placed in the top section of the filter holder with an insulated lead for connection as the reference electrode.
• the 0.5 inch diameter disk of FePc-2Py modified CN was placed in the flow cell, which was then connected to the appropriate leads of an EG&G PAR 273 potentiostat.
• the flow cell was connected to a Sage syringe pump filled with 0.1M KC1 in 0.1M potassium phosphate buffer at pH 7.0.
• Cyclic voltammograms (CVs) were recorded under no flow (static) and flow (0.4 mls/min.) at a potential scan rate of 20 mv/sec. (see Fig. 6) .
• the CVs were nearly identical with and without flow and showed two persistent, reversible oxidation and reduction waves consistent with surface confined FePc-2Py.
• the persistence of the redox peaks under fluid flow conditions demonstrates that the FePc-2Py is strongly bound to the carbon fibrils and that the use of iron phthalocyanine modified fibrils function well as a flow through electrode material.
• Alumina-Fibril Composites (185-02-01)
• One g of nitric acid oxidized fibrils (185-01- 02) was highly dispersed in 100 cc DI water using and U/S disintegrator.
• the fibril slurry was heated to 90°C and a solution of 0.04 mol aluminum tributoxide dissolved in 20 cc propanol was slowly added. Reflux was continued for 4 hr, after which the condenser was removed to drive out the alcohol. After 30 in the condenser was put back and the slurry refluxed at 100°C overnight.
• a black sol with uniform appearance was obtained.
• the sol was cooled to RT and after one week, a black gel with a smooth surface was formed.
• the gel was heated at 300°C in air for 12 hr.
• the alumina-fibril composites were examined by SEM. Micrographs of cracked surfaces showed a homogeneous dispersion of fibrils in the gel.
• nitric acid oxidized fibrils (173-83- 03) were highly dispersed on 200 cc ethanol using ultrasonification.
• a solution of 0.1 mol tetraethoxysilane dissolved in 50 cc ethanol was slowly added to the slurry at RT, followed by 3 cc cone. HCL.
• the mixture was heated to 85°C and maintained at that temperature until the volume was reduced to 100 cc.
• the mixture was cooled and set aside until it formed a black solid gel. The gel was heated at 300°C in air.
• silica-fibril composites were examined by SEM. Micrographs of cracked surfaces showed a homogeneous dispersion of fibrils in the gel. Similar preparations with other ceramics, such as zirconia, titania, rare earth oxides as well as ternary oxides can be prepared.
• Polymer beads especially magnetic polymer beads containing an Fe 3 0 4 core, such as those manufactured by Dynal and others, have many uses in diagnostics. These beads suffer, however, from having a low surface area compared to that available from nanotubes.
• Functionalized fibrils can be incorporated onto the surface of beads, which allows the polymer/fibril composites to be used as solid supports for separations or analytical application (e.g., electrochemiluminescence assays, enzyme immobilization) .
• the invention has application in the formulation of a wide variety of functionalized nanotubes and uses therefor.

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