KR20070030282A - Functionalized single walled carbon nanotubes - Google Patents

Abstract

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 single walled carbon nanotubes having diameters than 5 nanometers 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 nanotubes linked to one another. The invention also relates to methods for introducing functional groups onto the surface of such nanotubes. The invention further relates to uses for functionalized single walled carbon nanotubes. ® KIPO & WIPO 2007

Description

FUNCTIONALIZED SINGLE WALLED CARBON NANOTUBES}

Description of References to Related Applications

This application claims priority to Provisional Application No. 60 / 037,238, filed September 25, 1996, and is a sequential applicant of US application serial number 08 / 611,368, filed March 6, 1996, but now abandoned. US Patent Application Serial No. 08 / 812,856, filed on April 6, but now abandoned, is part of a serial application of US Patent Application Serial No. (attorney Docket No. 100647-3583), filed April 30, 2004. This application is also part of US Patent Application Serial No. 09 / 594,673, filed June 16, 2000, which is a divisional application of US Patent Application Serial No. 08 / 352,400 (currently US Patent 6,203,814), filed December 8, 1994. It is a continuous application. Each of the foregoing applications is incorporated herein by reference.

The present invention generally includes tubular fullerenes (commonly referred to as "bucky tubes") and fibrils, and functionalized graphite nanotubes by chemical substitution or adsorption of functional moieties. It's about (nanotube). More specifically, the present invention is a compound consisting of a single-walled carbon nanotube, which is uniformly or non-uniformly substituted with a chemical moiety, or a specific ring compound is adsorbed, and the functional group-attached fibrils bonded to each other. It is about a structure. The present invention also relates to a method of introducing functional groups on the surface of single-walled carbon nanotubes.

The present invention relates to the field of submicron graphite fibres, sometimes referred to as vapor grown carbon fibers. Carbon fibres are worm-shaped carbon deposits having a diameter of less than 1.0 μm, preferably less than 0.5 μm, and even more preferably less than 0.2 μm, which are present in a variety of forms and contain various carbon-containing gases at the metal surface. Prepared via catalytic cracking. Most of these worm-shaped carbon deposits have been observed since electron microscopy appeared. Beneficial initial investigations and explanations are described in Baker and Harris, Chemistry and Physics of Carbon , Walker and Thrower ed., Vol. 14, 1978, p. 83, incorporated herein by reference. See also, Rodriguez, N., J. Master . Research , Vol. 8, p. 3233 (1993), which is incorporated herein by reference. In 1976, Endo et al., Obelin, A., and Endo, M., J. of Crystal Growth , Vol. 32 (1976), pp. 335-349, incorporated herein by reference, revealed the basic mechanism by which such carbon fibrils grow. It was observed that they began to form from metal catalyst particles that began to supersaturate with carbon in the presence of a hydrocarbon containing gas. After the cylindrical regular graphite core is extruded, it immediately begins to be coated with the outer layer of pyrolytically deposited graphite according to the method of Endo et al. Fibrils having such pyrolysis overcoats generally have a diameter of more than 0.1 μ, more typically between 0.2 and 0.5 μ.

In 1983 Tennent succeeded in growing cylindrical regular graphite cores that were not contaminated with pyrolytic carbon in US Pat. No. 4,663,230, which is incorporated herein by reference. In other words, the tenant's invention gave access to smaller diameter fibres, fibres, typically 35 to 700 microns (0.0035 to 0.070 micron), and regular "as grown" graphite surfaces. In addition, fibrillar carbon, which has a less complete structure and no pyrolytic carbon outer layer, has also been grown.

The functional group-attached fibrils, buckytubes and nanofibers of the present application are different from continuous carbon fibers available on the market as reinforcing materials. A high aspect ratio is preferred, but in contrast to the inevitably limited fibrils, the aspect ratio (L / D) of the continuous carbon fibers is at least 10 ⁴ and often at least 10 ⁶ . In addition, the diameter of the continuous fibers is much greater than the diameter of the fibrils, always> 1.0μ and generally between 5-7μ.

Continuous carbon fibers are produced by pyrolysis of organic precursor fibers, generally rayon, polyacrylonitrile (PAN), and pitch. That is, the continuous carbon fiber may contain heteroatoms in its structure. Graphite types of "as made" continuous carbon fibers vary, but can be treated with subsequent graphitization steps. Differences in the degree of graphitization, orientation and crystallinity of the graphite plane, where present, the possibility of heteroatoms and even absolute differences in substrate diameters have made continuous fibers an unpredictable factor in nanofiber chemistry.

Tennant, in US Pat. No. 4,663,230, discloses carbon fibres with no continuous thermal carbon overcoat and having a plurality of graphite outer layers substantially parallel to the fibrous axis. This fibril is characterized by having a c-axis perpendicular to the tangent of the curved layer of graphite, which is itself substantially perpendicular to the cylindrical axis. In general, the diameter of the fibrils is 0.1 μm or less and a length to diameter ratio of at least 5. Preferably, the fibrils are substantially free of continuous thermal carbon overcoat, ie pyrolytically deposited carbon obtained by pyrolysis of the gaseous feedstock used in the production of these fibres.

Tennant et al., In US Pat. No. 5,171,560 (incorporated herein by reference), have a graphite layer that is substantially free of the fibrous axis and has no thermal overcoat, the protrusion of which is a distance of at least two fibrous diameters. Post the carbon fibre, which has been deployed. In general, such fibrils are substantially cylindrical graphite nanotubes of substantially constant diameter and contain a cylindrical graphite sheet having a c axis substantially perpendicular to this cylindrical axis. It is also substantially free of pyrolytically deposited carbon, having a diameter of less than 0.1 micron and having a length to diameter ratio of greater than five. Such fibrils are the main object of this invention.

Further details regarding the formation of fibrous fibrous aggregates are described in US patent application Ser. No. 149,573, filed Jan. 28, 1988 by Snyder et al., And PCT application No. US89 / 00322, filed Jan. 28, 1989. ("Carbon Fibrils") WO 89/07163, and US Patent Application Serial Nos. 413,837, filed September 28, 1989 by Moy et al. And PCT Application No. US90 / 05498, filed September 27, 1990 ( "Fibril Aggregates and Method of Making Same" can be found in the specification of WO 91/05089, all of which are assigned to the same assignee as the present invention and are incorporated herein by reference.

Moy et al. Are various macroscopic forms (injection forms) that are randomly entangled in USSN 07 / 887,307, filed May 22, 1992, incorporated herein by reference, to form entangled ball fibrils resembling a bird nest ("BN"). Fibrils prepared as an aggregate having the same as measured by an electron microscope; Or the relative orientation is substantially the same and the appearance of the combed yarn ("CY"), such as the longitudinal axis of each fibril (whether bent or twisted), extends in the same direction as the surrounding fibril of the bundle. An aggregate consisting of bundles of straight to slightly bent or twisted carbon fibrils held; Or fibrils made as aggregates of straight to slightly curved or twisted fibrils which are loosely entangled with one another to form an "open network" ("ON") structure. In an open network structure, the degree of fibrillar entanglement is greater than that observed in combed yarn-like aggregates (each fibre having substantially the same relative orientation) and less than that observed in bird nested fibres. CY and ON aggregates are more readily dispersed than BN, and are useful for preparing desirable composites with uniform properties throughout the structure.

When the protrusion of the graphite layer on the fibrous axis is developed by a distance less than two fibrous diameters, the carbon plane of the graphite nanofibers has a morphological appearance in cross section. Therefore, it is called golgol fibril. In US Pat. No. 4,855,091, which is incorporated herein by reference, Geus provides a method of making golgol fibril substantially free of pyrolytic overcoat. Such fibrils are also usefully used in the practice of the present invention.

Carbon nanotubes, similar in shape to the catalytically grown fibrils described above, were grown in hot carbon arcs (Iijima, Nature 354 56 1991). At present, such arc-grown nanofibers are generally accepted to have the same morphology as conventionally grown tenant-catalyzed fibrous fibers (Weaver, Science 265 1994). Arc-grown carbon nanofibers are also useful in the present invention.

Single-wall carbon nanotubes and methods for their preparation have been described since 1993 in "Single-shell carbon nanotubes of 1-nm diameter", S Iijima and T Ichihashi Nature , vol. 363, p. 603 (1993) and "Cobalt-catalysed growth of carbon nanotubes with single-atomic-layer walls", DS Bethune, CH Kiang, MS DeVries, G Gorman, R Savoy and R Beyers Nature , vol. 363, p .605 (1993), all of which are incorporated herein by reference.

Single-walled carbon nanotubes are also published in US Pat. No. 6,221,330 to Moy et al., Which is incorporated by reference in its entirety. Moi et al. In this document firstly have 1 to 6 carbon atoms and have only H, O, N, S or Cl as heteroatoms and optionally mixed with hydrogen under one or more gaseous carbon compounds, and under catalytic cracking reaction conditions. Forming a gaseous carbon starting stock gas mixture containing a gaseous metal containing compound that is unstable and forms a metal containing catalyst that acts as a decomposition catalyst under this reaction condition; A method of producing hollow single-walled carbon nanotubes is then disclosed by catalytic decomposition of one or more gaseous carbon compounds, in which the decomposition reaction is carried out under decomposition reaction conditions to yield nanotubes. This invention relates to a gas phase reaction in which a gaseous metal containing compound is also introduced into a reaction mixture containing a gaseous carbon source. The carbon source is typically a C ₁ to C ₆ compound having H, O, N, S or Cl as the heteroatom and optionally mixed with hydrogen. Carbon monoxide or carbon monoxide and hydrogen are preferred carbon feedstocks. Increased reaction zone temperatures of about 400 ° C. to 1300 ° C. and pressures between about 0 to about 100 p.sig are believed to cause decomposition of gaseous metal containing compounds to metal containing catalysts. Decomposition can be to an atomic metal or to a partially decomposed intermediate species. The metal containing catalyst catalyzes (1) CO decomposition and (2) SWNT formation. That is, the present invention relates to a method for forming SWNTs through catalytic decomposition of carbon compounds.

The invention of US Pat. No. 6,221,330 utilizes, in some embodiments, aerosol technology in which aerosols of metal containing catalysts are introduced into the reaction mixture. An advantage of the aerosol process for producing SWNTs is that efficient and continuous commercial or industrial production methods can produce catalyst particles of uniform size and scale. The previously discussed electric arc discharge and laser deposition methods have not been economically augmented for such commercial or industrial production. Examples of metal-containing compounds useful in this invention include metal carbonyl, metal acetyl acetonate, and other materials that can be introduced as vapors that decompose under decomposition conditions to form free-standing metal catalysts. Catalytically active metals are Fe, Co, Mn, Ni and Mo. Molybdenum carbonyl and iron carbonyl are preferred metal containing compounds that can decompose under reaction conditions to form a vapor phase catalyst. This solid form of metal carbonyl can be delivered to the pretreatment zone to be vaporized to become the vapor phase precursor of the catalyst. It has been found that there are two methods available for forming SWNTs on freestanding catalysts.

The first method is the direct injection of volatile catalysts. Direct injection methods are described in US Patent Application Serial No. 08 / 459,534, which is incorporated herein by reference. Direct injection of volatile catalyst precursors was observed to form SWNTs using molybdenum hexacarbonyl [Mo (CO) ₆ ] and dicobalt octacarbonyl [Co ₂ (CO) ₈ ] catalysts. Both materials are solid at room temperature but sublimate at or near room temperature. That is, the molybdenum compound is thermally stable up to at least 150 ° C., but the cobalt compound sublimates as it is decomposed [“Organic Syntheses via Metal Carbonyls”, Vol. 1, I. Wender and P. Pino, eds., Interscience Publishers, New York, 1968, p. 40].

The second method uses a vaporizer to introduce metal containing compounds (FIG. 12). According to one preferred aspect of this invention, the vaporizer 10 shown in FIG. 12 includes a quartz thermowell 20 having a seal 24 at a position about 1 "from the bottom to form a second zone. There are two 1/4 "holes 26 open in this zone that are exposed to the gaseous reactants. The catalyst is injected into this zone and then vaporized by the vaporizer furnace 32 at any desired temperature. This furnace is controlled by the first thermocouple 22. The metal containing compound, preferably the metal carbonyl, is vaporized at a temperature below its decomposition point and the gaseous reactant CO or CO / H ₂ is controlled by the reaction zone furnace 38 and the second thermocouple 42, respectively. Sweep the precursor with 34). While not intending to limit the operability to a particular theory, it is believed that at the reactor temperature the metal containing compound is partially decomposed into intermediate species or completely decomposed into metal atoms. These intermediate species and / or metal atoms coalesce into larger aggregated particles which are actually catalysts. The particles then grow to the exact size that catalyzes the decomposition of CO and promotes SWNT growth. In the apparatus of FIG. 11, catalyst particles and final carbon form are collected on the quartz wool plug 36. The growth rate of the particles depends on the concentration of the gaseous metal containing intermediate species. This concentration is determined by the vapor pressure of the vaporizer (and thus the temperature). If the concentration is too high, particle growth is too fast and structures other than SWNTs grow (eg, MWNTs, amorphous carbons, onions, etc.). The present invention is incorporated by reference in its entirety, including the examples of US Pat. No. 6,221,330.

US Patent 5,424,054 to Bethune et al., Incorporated herein by reference, describes a method of producing single-walled carbon nanotubes by contacting carbon vapor with a cobalt catalyst. Carbon vapor is produced by electric arc heating of solid carbon, which may be amorphous carbon, graphite, activated or decolorized carbon or mixtures thereof. Other carbon heating techniques are also discussed, such as laser heating, electric beam heating and RF induction heating.

Guo, T., Nikoleev, P., Thess, A., Colbert, D. T., and Smally, R. E., Chem. Phys. Lett. 243: 1-12 (1995), describes a method for producing single-walled carbon nanotubes in which graphite rods and transition metals are vaporized simultaneously by high temperature lasers.

See also Thess, A., Lee, R., Nikolaev, P., Dai, H., Petit, P., Robert, J., Xu, C., Lee, YH, Kim, In SG, Rinzler, AG, Colbert, DT, Scuseria, GE, Tonarek, D., Fischer, JE, and Smalley, RE, Science, 273: 483-487 (1996), Smoley et al. Are graphite containing small amounts of transition metals. A method for producing single-walled carbon nanotubes is described in which the rod is laser vaporized in an oven at about 1200 ° C. Single-walled nanotubes are reported to be produced in yields of over 70%.

Supported metal catalysts for the formation of SWNTs are also known. Dai., H., Rinzler, A.G., Nikolaev, P., Thess, A., Colbert, D. T., and Smalley, R. E., Chem. Phys. Lett. 260: 471-475 (1996), Smolley et al. Describe the supported Co, Ni and Mo catalysts for growing multi-walled nanotubes and single-walled nanotubes from CO, and the estimated mechanism of their formation.

McCarty et al. Disclose sulfuric acid (H ₂ SO) under reaction conditions (eg, time, temperature and pressure) sufficient to oxidize the surface of the fibrils in U.S. Patent Application Serial No. 351,967, incorporated herein by reference on May 15, 1989. ₄ ) and a method for oxidizing the surface of carbon fibrils, including contacting fibrils with an oxidant containing potassium chloride (KClO ₃ ) and fibrils. Fibrillars oxidized according to the McCarty et al. Method are oxidized heterogeneously, ie, the carbon atoms are substituted with a mixture of carboxy, aldehyde, ketone, phenol groups and other carbonyl groups.

In addition, the raw fiber was oxidized nonuniformly by nitric acid treatment. International application PCT / US94 / 10168 discloses the formation of oxidized fibrils containing a mixture of functional groups. Also, Hugenbad, M.S. ("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) nitrate fibrillar surfaces in the preparation of fibrillar-supported precious metals. First found that it was beneficial to oxidize. This acid pretreatment is a standard step in the preparation of carbon supported precious metal catalysts, provided that a common source of such carbon, if desired, removes as much undesirable material as possible to functionalize the surface.

In a published study, McCarthy and Benning (Polymer Preprints ACS Div. Of Polymer Chem. 30 (1) 420 (1990)) prepared derivatives of oxidized fibrils to demonstrate that the surface contains various oxidative groups. The prepared compounds, phenylhydrazone, haloaromatic esters, thallium salts, and the like, were chosen because of their analytical utility, such as bright, slightly different, strong, easily identified and distinct signals. Such compounds have not been isolated and have no practical value unlike the derivatives described herein.

While it has been found that carbon fibrils and aggregates of carbon fibrils have many uses, as described in the above-mentioned patents and patent applications, many other important uses may be developed if the fibrous surface is functionalized. Uniform or non-uniform functionalization allows various substrates and functionally attached fibrils to interact to form unique composites of materials with unique properties, based on the bonds between the functional sites present on the fibrous surface. Allow the fibrous structure to be formed.

Purpose of the Invention

Accordingly, a first object of the present invention is to provide a single-walled carbon nanotube with a functional group attached less than 5 nanometers in diameter, i.e. a single-walled carbon nanotube whose surface is uniformly or non-uniformly modified to bond functional chemical moieties. To provide.

Yet another object of the present invention is to provide single-walled carbon nanotubes having a diameter of less than 5 nanometers and having functional groups attached to the surface by reaction with an oxidation medium or other chemical medium.

Another third object of the present invention is to provide single-walled carbon nanotubes whose diameter is less than 5 nanometers and whose surface is uniformly modified by chemical reactions or by physical adsorption of species with their own chemical reactivity.

Another fourth object of the present invention is to provide single-walled carbon nanotubes whose diameter is less than 5 nanometers and whose surface is modified by oxidation or the like and then reacted with functional groups.

Another fifth object of the present invention is to provide single-walled carbon nanotubes whose diameter is less than 5 nanometers and whose surface has been modified with a wide range of functional groups to physically bind or chemically react with chemical groups present in various substrates. .

Another sixth object of the present invention is to provide a composite structure of single-walled carbon nanotubes in which functional groups on single-walled carbon nanotubes of less than 5 nanometers in diameter are bonded to one another via various linker chemistries.

Another seventh object of the present invention is a method of chemically modifying a fibrous surface in order to bind the functional moiety to the surface of the fibrous fiber in each case and physically attaching the species to the surface of single-walled carbon nanotubes of less than 5 nanometers in diameter It provides a method of adsorbing.

Another eighth object of the present invention is to provide a novel composite of a material based on functionally attached single-walled carbon nanotubes of less than 5 nanometers in diameter.

1 is a graph showing the results of analyzing the BSA binding to the basic fibrils, carboxy fibrils and PEG modified fibrils.

Figure 2 is a graph showing the results of the analysis of β-lactoglobulin binding to carboxy fibrils and PEG modified fibrils prepared by two different methods.

3 is a graph depicting the elution profile of bovine serum albumin (BSA) in tertiary amine fibril columns.

4 is a graph depicting the elution profile of BSA in a quaternary amine fibril column.

5 is a reaction sequence for preparing a lysine dendritic fibril.

6 is a graph of cyclic voltammetry demonstrating the use of iron phthalocyanine modified fibril in flow cells.

FIG. 7 shows a reaction sequence for preparing bifunctional fibril by adding N _ε- (tert-butoxycarbonyl) -L-lysine.

8 is a graph illustrating the results of synthesizing ethyl butyrate using lipase immobilized on fibril.

9 is a graph showing the results of the separation of alkaline phosphatase (AP) from a mixture of AP and β-galactosidase (βG) using AP inhibitor modified fibrils.

10 is a graph showing the results of separating βG from a mixture of AP and βG using βG-modified fibril.

11 illustrates a reactor capable of producing single wall carbon nanotubes.

FIG. 12 shows the vaporizer components of the reactor shown in FIG. 11.

Detailed description of the invention

The present invention relates generally to composites represented by the formula:

[C _n H _L ] -R _m

Wherein n is an integer, L is a number less than 0.1 n, m is a number less than 0.5 n,

R are the same each, SO ₃ H, COOH, NH ₂ , OH, R'CHOH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR') _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR' ₂ , Hg-X, TlZ ₂ and Mg-X,

y is an integer of 3 or less,

R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cycloaryl or poly (alkylether),

R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl, fluoroaralkyl or cycloaryl,

X is a halide,

Z is a carboxylate or trifluoroacetate.

The carbon atom, C _n, is the surface carbon of the substantially cylindrical graphite nanotubes of substantially constant diameter. Such nanotubes have a length to diameter ratio of greater than 5 and a diameter of less than 0.5 microns, preferably less than 0.1 microns. In addition, the nanotubes may be substantially cylindrical graphite nanotubes, substantially free of pyrolytically deposited carbon, more preferably having graphite layer protrusions on the fibrous axis that are deployed by at least two fibrillar diameter distances. Or (c) nanotubes, characterized in that the c-axis holds a cylindrical graphite sheet substantially perpendicular to the cylindrical axis. In a preferred embodiment, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube having a substantially constant diameter or less than 5 nanometers in diameter. Most preferably, carbon atoms C _n are surface atoms of single-walled carbon nanotubes less than 5 nanometers in diameter. This composite is uniform in that each R is identical.

Heterogeneously substituted nanotubes are also prepared. Such nanotubes include compounds represented by the formula:

[C _n H _L ] -R _m

In this formula, n, L, m, R and the nanotubes themselves are as described above, provided that each R does not contain oxygen or that each R is an oxygen containing group and not COOH.

Also included in the present invention are functionally attached nanotubes represented by the formula:

[C _n H _L ] -R _m

Where n, L, m and R are as defined above and the carbon atoms are surface carbon atoms of fishbone fibrils having a length-to-diameter ratio greater than 5 or single-walled carbons of less than 5 nanometers in diameter It is a carbon atom of nanotubes. Such nanotubes may be substituted uniformly or heterogeneously. The nanotubes preferably have no thermal overcoat and are less than 0.5 microns in diameter.

Also included in the present invention are functionally attached nanotubes represented by the formula:

[C _n H _L ]-[R'-R] _m

In this formula, n, L, m, R 'and R have the same meaning as described above. The carbon atom C _n is the surface carbon of the substantially cylindrical graphite nanotubes having a substantially constant diameter. This nanotube has a length to diameter ratio of greater than 5 and a diameter of less than 0.5 microns, preferably less than 0.1 microns. The nanotubes can be nanotubes substantially free of pyrolytically deposited carbon. The nanotubes also have projections of the graphite layer developed at least two fibrous diameter distances on the fibrous axis and / or have cylindrical graphite sheets whose c axis is substantially perpendicular to the cylindrical axis. In a preferred embodiment, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube having a substantially constant diameter or less than 5 nanometers in diameter. Most preferably, the carbon atoms C _n are surface atoms of single-walled carbon nanotubes less than 5 nanometers in diameter.

In uniformly and heterogeneously substituted nanotubes, the surface atoms C _n are reactive. Most of the carbon atoms present in the surface layer of graphite fibrils are basal plane carbon, as in graphite. Basal planar carbon is relatively inert to chemical attack. Defective sites, such as those where the fibrous perimeter is not completely enclosed by the graphite plane, have carbon atoms similar to the edge carbon atoms of the graphite plane (see Urry, Elementary Equilibrium Chemistry of Carbon, Wiley , New York, 1989).

Defect sites may expose the edges or underlying planar carbon of the lower inner layer of the nanotubes. Thus, the term surface carbon includes the base plane and / or the edge carbon of the underlying layer, which may be exposed at defect sites of the outermost layer, as well as all the carbon of the base plane and the edges present in the outermost layer of the nanotubes. The marginal carbon is reactive and must contain some heteroatom or group to satisfy the carbon valence.

The substituted nanotubes described above may preferably be attached with additional functional groups. Such compounds include those compounds represented by the formula:

[C _n H _L ] -A _m

-CR'₂-OY, N=Y,

또는 C=Y 중에서 선택되는 것이며, 여기서 Y는 단백질, 펩타이드, 아미노산, 효소, 항체, 뉴클레오타이드, 올리고뉴클레오타이드, 항원 또는 효소 기질, 효소 억제제 또는 효소 기질의 전이 상태 유사체의 적당한 작용기이거나 또는 R'-OH, R'-NR'₂, R'SH, R'CHO, R'CN, R'X, R'N⁺(R')₃X^-, R'SiR'₃, R'Si-(OR')_y-R'_{3 -y}, R'Si-(O-SiR'₂)-OR', R'-R", R'-N-CO, (C₂H₄O)_w-H, -(C₃H₆O)_w-H, -(C₂H₄O)_w-R', (C₃H₆O)_w-R', R' 및

중에서 선택되는 것이며, 여기서 w는 1보다 크고 200보다 작은 정수이다. 탄소원자 C_n은 직경이 실질적으로 일정한, 실질적으로 원기둥형의 흑연 나노튜브의 표면 탄소이다. 이러한 나노튜브에는 길이 대 직경 비가 5보다 크고, 직경이 0.1μ 미만, 바람직하게는 0.05μ 미만인 것이 포함된다. 또한, 이 나노튜브는 열분해 침착된 탄소가 실질적으로 없는, 실질적으로 원기둥형의 흑연 나노튜브일 수 있다. 더욱 바람직하게는, 원섬유 축 위에 적어도 2개의 원섬유 직경 거리 만큼 전개되어 있는 흑연 층의 돌출부를 보유한 것 및/또는 c 축이 원기둥 축에 실질적으로 수직인 원기둥형 흑연 시트로 구성된 것을 특징으로 하는 것이다. 특히, 나노튜브는 열적 오버코트가 없고, 직경이 0.5μ 미만인 것이 바람직하다. 또한, 나노튜브는 직경이 실질적으로 일정하거나 직경이 5나노미터 미만인 실질적으로 원기둥형의 단일벽 탄소 나노튜브일 수 있다. 가장 바람직하게는 탄소 원자 C_n은 직경이 5 나노미터 미만인 단일벽 탄소 나노튜브의 표면 원자인 것이다.Wherein carbon is the surface carbon of the nanotubes described above, n, L and m are as described above, and A is ON, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, wherein Y is a suitable functional group of a protein, peptide, amino acid, enzyme, antibody, nucleotide, oligonucleotide, antigen or enzyme substrate, enzyme inhibitor or transition state analog of an enzyme substrate, or _{, R'-NR '2, R'SH} , R'CHO, R'CN, R'X, R'N + (R') 3 X -, R'SiR '3, R'Si- (OR') _y -R ' _{3 -y} , R'Si- (O-SiR' ₂ ) -OR ', R'-R ", R'-N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w -H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Wherein w is an integer greater than 1 and less than 200. The carbon atom C _n is the surface carbon of the substantially cylindrical graphite nanotubes having a substantially constant diameter. Such nanotubes include those having a length to diameter ratio of greater than 5 and a diameter of less than 0.1 μ, preferably less than 0.05 μ. The nanotubes can also be substantially cylindrical graphite nanotubes, substantially free of pyrolytically deposited carbon. More preferably, it has a projection of the graphite layer which extends at least two fibrous diameter distances on the fibrous axis and / or the c-axis consists of a cylindrical graphite sheet substantially perpendicular to the cylindrical axis. will be. In particular, the nanotubes are free of thermal overcoat and preferably have a diameter of less than 0.5 microns. In addition, the nanotubes may be substantially cylindrical single-walled carbon nanotubes having a substantially constant diameter or less than 5 nanometers in diameter. Most preferably the carbon atom C _n is the surface atom of single wall carbon nanotubes of less than 5 nanometers in diameter.

In addition, functional nanotubes represented by the formula [C _n H _L ]-[R'-R] _{m can} be attached to a functional group to produce a compound represented by the formula [C _n H _L ]-[R'-A] _m . Where n, L, m, R 'and A are as described above. The carbon atom C _n is the surface carbon of the substantially cylindrical graphite nanotubes having a substantially constant diameter. Such nanotubes include those having a length to diameter ratio of greater than 5 and a diameter of less than 0.5 microns, preferably less than 0.1 microns. The nanotubes can also be substantially cylindrical graphite nanotubes, substantially free of pyrolytically deposited carbon. More preferably, it has a projection of the graphite layer which extends over at least two fibrous diameter distances on the fibrous axis and / or has a cylindrical graphite sheet substantially perpendicular to the cylindrical axis. will be. In particular, the nanotubes are free of thermal overcoat and preferably have a diameter of less than 0.5 microns. In addition, the nanotubes may be substantially cylindrical single-walled carbon nanotubes having a substantially constant diameter or less than 5 nanometers in diameter. Most preferably the carbon atom C _n is the surface atom of single wall carbon nanotubes of less than 5 nanometers in diameter.

In addition, the composite of the present invention includes a nanotube to which a specific cyclic compound is adsorbed. Examples include composites of the material represented by the formula [C _n H _L ]-[XR _a ] _m (wherein n is an integer, L is a number less than 0.1n, and m is less than 0.5n). Is a number less than 0 or 10, X is a multinuclear aromatic moiety, a polyheteronuclear aromatic moiety or a metallopolyheteronuclear aromatic moiety, or a planar metal tetrathiooxalate, and R is as described above Same thing). The carbon atom C _n is the surface carbon of the substantially cylindrical graphite nanotubes having a substantially constant diameter. Such nanotubes include those having a length to diameter ratio of greater than 5 and a diameter of less than 0.5 microns, preferably less than 0.1 microns. In addition, the nanotubes may be substantially cylindrical graphite nanotubes, substantially free of pyrolytically deposited carbon, and more preferably, at least two of the fibrous diameter distances that extend over the fibrous axis. And a cylindrical graphite sheet having a projection and / or a c axis substantially perpendicular to the cylindrical axis. In particular, the nanotubes are free of thermal overcoat and preferably have a diameter of less than 0.5 microns. In addition, the nanotubes may be substantially cylindrical single-walled carbon nanotubes having a substantially constant diameter or less than 5 nanometers in diameter. Most preferably the carbon atom C _n is the surface atom of single wall carbon nanotubes of less than 5 nanometers in diameter.

Preferred cyclic compounds are planar macrocycles as described on page 76 of Cotton and Wilkinson, Advanced Organic Chemistry . More preferred cyclic compounds for adsorption are porphyrins and phthalocyanines or tetrathiooxalates of Ni, Cu, Pd, Pt or Ag.

In addition, the composites of the present invention include single-walled carbon nanotubes less than 5 nanometers in diameter, in which certain planar aromatic compounds are adsorbed on the walls of single-walled carbon nanotubes. If functional groups are attached to the adsorbed molecules themselves, the sidewalls are effectively functionalized (combined with "functional group attachment") without constituting the physical structure. Such functionally attached single-walled carbon nanotubes can then be subjected to partial pyrolysis so that further heated or adsorbed molecules do not desorb. Other mechanisms such as crosslinking or oligomerization (for the purpose of reducing solubility) may also be used to fix the adsorbed molecules. Preferred planar aromatic compounds for adsorption include porphyrins and phthalocyanines or tetrathiooxalates of Ni, Cu, Pd, Pt or Ag.

The adsorbed cyclic compound may be attached to a functional group. Such compounds include compounds represented by the formula [C _n H _L ]-[XA _a ] _m where m, n, L, a, X and A are as described above and carbon is as described above. Substantially surface carbon of cylindrical graphite or single-walled carbon nanotubes].

As described above, the functional fiber attached carbon fibrils may be incorporated into a matrix. The substrate may be an organic polymer (e.g., a thermosetting resin such as epoxy, bismaleimide, polyamide or polyester resin; thermoplastic resin; reaction injection molding resin; or an elastomer such as natural rubber, styrene-butadiene rubber or cis-1,4 Polybutadiene); Preferred are inorganic polymers (eg polymer inorganic oxides such as glass), metals (eg lead or copper), or ceramic materials (eg portland cement). From this substrate, beads incorporating fibrils can be formed. Alternatively, the functionally attached fibrils may be attached to the outer surface of the functionally attached beads.

Without wishing to be bound by a particular theory, the functionalized fibrillars are polymer systems because the modified surface properties are more compatible with the polymer, or because the modified functional groups (especially hydroxy or amine groups) bind directly to the polymer as terminal groups. Is better dispersed in. In this way, polymer systems such as polycarbonates, polyurethanes, polyesters or polyamides / imides bind directly to the fibrils, allowing the fibrils to more easily disperse with improved adhesion.

The present invention also provides the steps of contacting the carbon fibrils with a strong oxidant for a period of time sufficient to oxidize the surfaces of the fibrils, and contacting the fibrils with reactants suitable for adding functional groups to the oxidized surface again. It provides a method for introducing a functional group to the surface of the carbon fibrils through. According to a preferred embodiment of the invention, the oxidant comprises a solution of alkali metal chlorate dissolved in a strong acid. According to another aspect of the invention, the alkali metal chlorate is sodium or potassium chlorate. In a preferred embodiment, the strong acid used is sulfuric acid. Sufficient time periods for oxidation range from about 0.5 hours to about 24 hours.

According to another preferred embodiment, the composite represented by the formula [C _n H _L ]-[CH (R ') OH] _m (wherein n, L, R' and m are as described above) is a free radical initiator. For example, by reacting the surface carbon of the nanotubes with R′CH ₂ OH in the presence of benzoyl peroxide.

The present invention also provides a method of binding a protein to a nanotube modified with NHS ester by forming a covalent bond between the NHS ester and the amino group of the protein.

The present invention also provides a method of contacting a carbon fibrous or single-walled carbon nanotube for a time period sufficient to oxidize the surface of the carbon fibrous or single-walled carbon nanotube, such that the surface oxidized carbon fibrous or single Contacting the wall carbon nanotubes with a reactant suitable for adding functional groups to the surface of the carbon fibres or single-walled carbon nanotubes, and thus attaching the fibrous functional groups attached to the surface to these carbon fibres or single-walled carbon nanotubes Provided is a method for producing a network of carbon fibrils or single-walled carbon nanotubes, further comprising contacting with a crosslinking agent effective to produce a network of tubes.

In addition, functional group-attached fibrous or single-walled carbon nanotubes are also useful for preparing rigid networks of fibrous or single-walled carbon nanotubes. Three-dimensional networks having good dispersion of acid-functional fibrils or single-walled carbon nanotubes can be stabilized, for example, by crosslinking acid groups (in fibrils) with polyols or polyamines to form a rigid network. .

In addition, the present invention provides a three-dimensional network structure formed by combining the functional group-attached fibrils or single-walled carbon nanotubes described above. Such composites include those in which at least two functionally attached fibrils or single-walled carbon nanotubes are bound by one or more linkers that bind directly or through chemical moieties. These networks contain porous media that are very uniform in pore size and are the same. Such networks are useful as adsorbents, catalyst supports and separation media.

Interstices between such fibrillar or single-walled carbon nanotubes are irregular in size and shape, but they can also be regarded as pores and can be produced by the methods used to prepare porous media. The size of the thermode present in this network structure can be controlled through the concentration and dispersion of the fibrils and the concentration and chain length of the crosslinking agent. Such materials can act as structured catalyst supports and can be processed to include or exclude molecules of a particular size. These materials can be used for special purposes as large pore supports for biocatalysts, in addition to conventional industrial catalysis.

Rigid networks can also act as the backbone of a biomimetic system for molecular recognition. Such systems are described in US Pat. No. 5,110,833 and International Publication No. WO 93/19844. Proper selection of crosslinkers and complexing agents can stabilize the backbone of certain molecules.

Method of attaching functional group of nanotube

The uniformly functionally attached fibrils of the present invention can be prepared directly through electrophilic addition or metallization reactions to sulfonated, deoxygenated fibrous surfaces. If arc grown nanofibers are used, sufficient purification should be performed prior to attachment of the functional groups. Everson (Ebbesen et al., Nature 367 519 (1994)) presents this purification procedure.

The carbon fibril is preferably treated before contacting with the functionalizing agent. Such treatment may include dispersing the fibrils in a solvent. In some cases, it may be filtered and dried before the carbon fibrils are contacted.

Sulfonation

Background art is described in March, J. P., Advanced Organic Chemistry, 3rd Ed. Wiley, New York 1985; House, H., Modern Synthetic Reactions, 2nd Ed., Benjamin / Cummings, Menlo Park, CA 1972.

Activated CH (including aromatic CH) bonds can be sulfonated using fumed sulfuric acid (oleum), a concentrated sulfuric acid solution containing up to 20% SO ₃ . Prior art has been conducted through the fluid in a T-80 ℃ using fuming sulfuric acid, the activated CH bonds may hwahal sulfone using SO ₃ on the use of SO ₃ gas, or in an inert aprotic solvent. The reaction is as follows:

-CH + SO ₃ ----> -C-SO ₃ H

The excess reaction forms sulfones according to the following scheme:

2 -CH + SO ₃ ----> -C-SO ₂ -C- + H ₂ O

Example  One

Activation of C-H Bond with Sulfuric Acid

This reaction can be carried out in the gas phase and in the solution without any significant difference in results. The gas phase reaction was carried out in a horizontal quartz tube reactor heated by a Lindberg furnace. As a source of SO _3, a multi-necked flask equipped with a gas inlet / outlet was used containing 20% SO ₃ solution in concentrated sulfuric acid.

An amount of fibril (BN or CC) sample in a homemade boat-type vessel was placed in a 1 "tube equipped with a gas inlet, and the outlet was connected to a concentrated sulfuric acid foam trap. Argon was flowed through the reactor for 20 minutes to allow air All were removed and the sample was heated for 1 hour at 300 ° C. to remove residual moisture After drying, the temperature was adjusted to the reaction temperature under argon.

When stable to the desired temperature, the SO ₃ source was connected to the reactor tube, and argon was used to transport the SO ₃ vapor into the quartz tube reactor. The reaction was carried out at the desired temperature for the desired time, after which the reactor was cooled under flowing argon. The fibrils were then dried under vacuum at 5 ° C. at 90 ° C. to determine the increase in anhydrous weight. The sulfonic acid (-SO ₃ H) content was reacted with 0.100 N NaOH and then 0.100 N HCl to the end point of pH 6.0. It was measured by reverse titration.

The liquid phase reaction was carried out in concentrated sulfuric acid containing 20% SO ₃ in a multi-necked 100 cc flask equipped with a thermometer / thermostat and a magnetic stirrer. The fibril slurry in concentrated sulfuric acid 50 was added to the flask. The fuming sulfuric acid solution (20 cc) was preheated to -60 ° C before addition to the reactor. After the reaction, the acid slurry was poured onto ice cubes and immediately diluted with 1 L deionized water. The solid was filtered off and washed thoroughly with deionized water until there was no change in pH of the wash effluent. The fibrils were dried under vacuum at 5 "Hg at 100 ° C. Accurate weight gain could not be obtained due to migration losses in filtration. The results are summarized in Table I.

Table I

Reaction Theorem

There was no significant difference in the sulfonic acid content obtained by reaction in the gas phase or liquid phase. In the temperature effect, the high temperature reaction (gas phase) provided a greater amount of sulfone. The 4.2% weight gain at 118-61B was consistent with the sulfonic acid content (theoretical value 0.51 meq / g). In experiments 60A and 61A the weight gain was too high than that obtained solely by the sulfonic acid content. Therefore, it was assumed that a significant amount of sulfone was further prepared.

2. Addition to Oxidized Fibrous Surface

Background art is described in Urry, G., Elementary Equilibrium Chemistry of Carbon, Wiley, New York, 1989.

The surface carbon of the fibrils is similar to graphite and is arranged in a hexagonal sheet containing basic planar carbon and edge carbon. The base planar carbon is relatively inert to chemical attack, while the marginal carbon must be reactive and contain some heteroatoms or groups to meet the carbon valence. In addition, fibrils are basically edge carbons and have surface defect sites containing heteroatoms or groups.

The most common heteroatoms attached to the surface carbon of the fibrils are hydrogen, the main gas component during manufacture; Oxygen due to high reactivity and traces that are very difficult to remove; And H ₂ O, which is always present due to the catalyst. Pyrolysis at about 1000 ° C. under vacuum removes oxygen from the surface in a complex reaction in which the reaction mechanism is unknown but stoichiometry is known. The product is CO and CO ₂ in a 2: 1 ratio. The fibrillar surface obtained contains radicals of the C ₁ -C ₄ configuration which are highly reactive with the activated olefins. This surface is stable in the presence of vacuum or inert gas, but retains high reactivity until exposure to reactive gas. That is, the fibrils can be pyrolyzed at about 1000 ° C. in a vacuum or inert atmosphere, cooled under the same conditions, and reacted with appropriate molecules at low temperatures to provide stable functional groups. Typical reaction examples are as follows:

1000 ℃

Fibrillar-O ------------> Reactive Fibrillar Surface (RFS) + 2CO + CO ₂ , then

1000 ℃

RFS + CH ₂ = CHCOX ------------>Fibre-R'COX (X = -OH, -Cl, -NH ₂ , -H)

RFS + Maleic Anhydride -----------> Fibrillar-R '(COOH) ₂

RFS + Cyanogen ----------> Fibrillar-CN

RFS + CH ₂ = CH-CH ₂ X ----------->Fibrils-R'CH ₂ X (X = -NH ₂ , -OH, -halogen)

RFS + H ₂ O ------------> Fibres = O (quinone)

RFS + CH ₂ = CHCHO --------->Fibrillar-R'CHO (aldehyde type)

RFS + CH ₂ = CH-CN ---------->Fibre-R'CN

Wherein R 'is a hydrocarbon radical (alkyl, cycloalkyl, etc.).

Example 2

Preparation of Fibrils with Functional Group by Reaction of Oxygen-Removed Fibrillar Surface with Acrylic Acid

One gram of BN fibres in a boat-made vessel was placed in a horizontal 1 "quartz tube equipped with a thermocouple and placed in a Lindbergh tube furnace. The end was equipped with a gas inlet / outlet. These tubes were anhydrous deoxygenated argon. After 10 minutes of rinsing, the temperature of the furnace was raised to 300 ° C. and maintained for 30 minutes, then under continuous argon, the temperature was raised to 1000 ° C. at 100 ° C. and maintained for 16 hours. The tube was cooled under argon to room temperature (RT) The argon was then flowed into a multi-necked flask containing pure purified acrylic acid equipped with a gas inlet / outlet and at 50 ° C. The acrylic acid / argon gas stream was at RT. It was continued for 6 hours. The remaining unreacted acrylic acid was then removed, first washed with argon and then vacuum dried under 100 ° C., <5 ”vacuum. The carboxylic acid content was measured by reacting with excess 0.100N NaOH and back titrating with 0.100N HCl to the end point of pH 7.5.

Example 3

Preparation of Fibrils with Functional Group by Reaction of Oxygen-Removed Fibrillar Surface with Acrylic Acid

The experiment was repeated in a similar manner to the above experiment except that pyrolysis and cooling were carried out under 10 ^-4 Torr vacuum. Purified acrylic acid vapor was diluted with argon as in the previous experiment.

Example 4

Preparation of functional fiber with functional group by reaction of oxide-removed fiber surface with maleic acid

The experiment was repeated as in Example 2 except that the purified maleic anhydride which was passed through the melt purified maleic anhydride (MAN) bath at 80 ° C. and then fed to the reactor was a reactant of RT.

Example  5

Preparation of Fibrils with Functional Groups by Reaction of Oxidized Fibrous Surface with Acryloyl Chloride

This experiment was repeated as in Example 2 except that the reaction at RT was purified acryloyl chloride, where the reaction was passed through argon over pure acryloyl chloride at 25 ° C. and fed to the reactor. . Acid chloride content was measured by reacting with excess 0.100N NaOH followed by back titration with 0.100N HCl.

Pyrolysis of fibrils under vacuum removes oxygen from the fibrillar surface. In the TGA apparatus, the average weight loss of the three BN fibril samples was 3% as a result of pyrolysis at 1000 ° C. under vacuum or purified Ar. Gas chromatographic analysis detected only CO and CO _{2 in a} ratio of about 2: 1, respectively. The surface obtained is so reactive that activated olefins, such as acrylic acid, acryloyl chloride, acrylamide, acrolein, maleic anhydride, allyl amine, allyl alcohol or allyl halide, react even at room temperature to bind only activated olefins. It will form a clean product containing deception. That is, the surface containing only carboxylic acid can be obtained by reaction with acrylic acid or maleic anhydride, the surface containing only acid chloride can be obtained by reaction with acryloyl chloride, and the surface containing only aldehyde is By reaction with acrolein, the surface containing only hydroxy groups is reacted with allyl alcohol, the surface containing only amines is reacted with allyl amine, and the surface containing only halides is reacted with allyl halide. Can be obtained.

3. Metallization

Background techniques are described in March, Advanced Organic Chemistry , 3rd ed., P 545.

Aromatic C-H bonds can be metalized by various organometallic reagents to form carbon-metal bonds (C-M). M is generally Li, Be, Mg, Al or Tl, although other metals may be used. The simplest reaction is by direct substitution of hydrogen in the activated aromatic:

1.Fiber-H + R-Li ----------> Fibrillar-Li + RH

This reaction may further require strong bases such as t-butoxylated or chelated diamines. An aprotic solvent is necessary (paraffins, benzene).

2. Fibrillar-H + AlR ₃ ----------> Fibrillar-AlR ₂ + RH

3. Fibrils-H + Tl (TFA) ₃ ----------> Fibrils-Tl (TFA) ₂ + HTFA

TFA = trifluoroacetate, HTFA = trifluoroacetic acid

Metallized derivatives are examples of primary single functionally attached fibrils. However, additional reactions may provide other primary single functionally attached fibrils. Some reactions can be carried out continuously in the same device without intermediate separation.

4. Fibrils-M + O ₂ ---------> Fibrils-OH + MO (M = Li, Al)

H ⁺

Fibrils-M + S ---------> Fibrils-SH + M ⁺

Fibrils-M + X ₂ ----------> Fibrils-X + MX (X = halogen)

                          catalyst

Fibrils _{-M + CH 3 ONH 2 · HCl} --------> fibrils -NH ₂ + ₃ MOCH

ether

catalyst

Fibrils-Tl (TFA) ₂ + NaOH ---------> Fibrils-OH

catalyst

Fibrils-Tl (TFA) ₂ + NH ₃ OH ---------> Fibrils-NH ₂ + HTFA

Fibrillar-Tl (TFA) ₂ + KCN aqueous solution --------> Fibrillar-CN + TlTFA + KTFA

Fibrils-CN + H ₂ ----------> Fibrils-CH ₂ -NH ₂

Example 6

Preparation of Fibrillar-Li

Put CC fibrils 1g to porcelain boat-shaped vessel, were charged to a 1 "quartz tube reactor wrapped in Lindberg tube furnace. Ends of the tubes was fitted with a gas inlet / outlet. In a continuous H ₂ flow, the fibrils 700 Any surface oxygenate was converted to CH bonds by heating to 2 ° C. The reactor was then cooled to room temperature in H ₂ stream.

Hydrogenated fibres, together with anhydrous deoxygenated heptane (with LiAlH ₄ ), are rubber diaphragms that can be added to liquids with a purified argon cleaning system, condenser, magnetic stirrer and syringe to remove all air and maintain an inert atmosphere. Transfer to a fitted 1 liter multi-ball round bottom flask. A 2% solution containing 5 mmol butyllithium in heptane under argon atmosphere was added by syringe and the slurry was stirred for 4 hours under gentle reflux. The fibrils were then separated by gravity filtration in an argon atmospheric glove box and washed with anhydrous deoxygenated heptane on the filter. The fibrils were transferred to a 50 cc rb flask equipped with stop plugs and dried under vacuum at 10 ° C., 10 ⁻⁴ torr. The lithium concentration was measured by reacting a fibril sample with 0.100 N HCl in excess deionized water and then back titrating with 0.100 N NaOH to the end point of pH 5.0.

Example 7

Fibrillar-Tl (TFA) ₂ Manufacture

1 g of CC fibril was hydrogenated as in Example 5 and loaded into a multi-necked flask with HTFA degassed by repeated washing with anhydrous argon. To this flask, a 5% solution of 5 mmol Tl (TFA) ₃ dissolved in HTFA was added through a rubber septum, and the slurry was stirred for 6 hours under gentle reflux. After the reaction, the fibrils were collected and dried as in Example 1.

Example 8

Preparation of Fibrillar-OH (Oxygenated Derivatives Containing Only OH Functional Groups)

0.5 g of the lithiated fibril prepared in Example 6 was placed in a 50 cc one-necked flask equipped with a stopper and a magnetic stirrer rod with anhydrous deoxygenated heptane in an argon atmospheric glove bag. This flask was removed from the glove bag and stirred on a magnetic stirrer. The stopper was opened and exposed to the atmosphere and the slurry was stirred for 24 hours. The fibrils were then separated by filtration, washed with aqueous MeOH solution and dried under vacuum at 5 ° C. at 50 ° C. The concentration of OH groups converted OH groups to acetate esters and one equivalent of acetic acid per mole of anhydride reacted during this process. Measurement was made by reacting acetic anhydride standard solution in dioxane (0.252 M) to release it at 80 ° C. Total acid content, free acetic acid and unreacted acetic anhydride were measured by titration to the end point of pH 7.5 with 0.100 N NaOH.

Example 9

Fibrillar-NH ₂ Manufacture

1 g of thalliumated fibrils were prepared as in Example 7. Fibrils were slurried in dioxane and then 0.5 g triphenyl phosphine dissolved in dioxane was added. This slurry was stirred at 50 ° C. for several minutes and then ammonia gas was added at 50 ° C. for 30 minutes. The fibrils were then filtered off, washed with dioxane and then deionized water and dried under vacuum at 5 ° C. at 80 ° C. The amine concentration was reacted with excess acetic anhydride, followed by free acetic acid with 0.100 N NaOH. And unreacted anhydride were measured by back titration.

4. Derivatized Multinuclear Aromatic Compounds, Heteropolynuclear Aromatic Compounds, and Planar Macrocyclic Compounds

The graphite surface of the fibrils can physically adsorb aromatic compounds. The attraction is van der Waals forces. This force is significant between the polycyclic heteronuclear aromatic compound and the underlying planar carbon of the graphite surface. Desorption can occur when competitive surface adsorption is possible or when the adsorbate is high in solubility.

For example, it has been found that fibrils can be attached to functional groups by adsorption of phthalocyanine derivatives. Such phthalocyanine derivative fibrils can be used as a solid support for protein fixation. Other phthalocyanine derivatives may also be selected to simply introduce other chemical groups onto the fibrous surface.

The use of phthalocyanine derivative fibrils for protein fixation has significant advantages over conventional protein fixation methods. Specifically, it is a simpler method than covalent modifications. In addition, phthalocyanine derivative fibrils have a wide surface area and are stable in almost all kinds of solvents over a wide range of temperatures and pH ranges.

Example 10

Adsorption of Porphyrin and Phthalocyanine on Fibrillar

Preferred compounds for physical adsorption on fibrils are derivatized porphyrins or phthalocyanines known to adsorb strongly on graphite or carbon black. Some of the compounds available include tetracarboxylic acid porphyrin, cobalt (II) phthalocyanine or dilithium phthalocyanine. The last two can be derivatized in carboxylic acid form.

Dilithium Phthalocyanine

In general, two Li ⁺ ions are substituted from phthalocyanine (Pc) groups by most metal (particularly multivalent) complexes. Thus, substitution of Li ⁺ ions with metal ions bound to an unstable ligand is a method of introducing stable functional groups on the surface of fibril. Almost all transition metal complexes replace Li ⁺ from Pc to form a stable, insecure chelate. This site is then the site that couples the metal to the appropriate ligand.

Cobalt (II) Phthalocyanine

Cobalt (II) complexes are particularly suitable. Co ⁺⁺ Ions are substituted in place of two Li ⁺ ions to form very stable chelates. Co ⁺⁺ ions may then coordinate to ligands such as nicotinic acid which contain a pyridine ring with carboxylic acid side groups and are known to preferentially bind to pyridine groups. In the presence of excess nicotinic acid Co (II) Pc is electrochemically oxidized to Co (III) Pc to form an instability complex with the pyridine moiety of nicotinic acid. That is, the free carboxylic acid group of the nicotinic acid ligand is firmly attached to the fibril surface.

Other suitable ligands are aminopyridine or ethylenediamine (NH ₂ side groups), mercaptopyridine (SH) or other multifunctional ligands containing amino or pyridyl moieties at one end and any desired functional groups at the other end. .

The loading of porphyrin or phthalocyanine can be measured through decolorization of the solution when the amount of addition is increased. The dark color of the solution (tetracarboxylic acid porphyrin in MeOH is dark pink, Co (II) or dilithium phthalocyanine in dark blue green in acetone or pyridine) discolors when the molecules are adsorbed and removed on the black surface of the fibrils.

The loading was measured by this method and the residual amount of the derivative was calculated from this approximate measurement (about 140 mm ² ). Fibres with an average surface area of 250 m 2 / g will have a maximum loading of about 0.3 mmol / g.

Tetracarboxylic porphyrin was analyzed by titration. Adsorption retention was examined by decolorization in an aqueous system at room temperature and elevated temperature.

The fibril slurry was first mixed (Waring blender) and stirred during loading. Some of the slurry was sonicated when the color no longer bleached, but had no effect.

After loading, experiments 169-11, -12, -14 and -19-1 (see Table II) were washed with the same solvent to remove the contained pigments. Faint hues continued to appear in the wash eluate, making it difficult to accurately determine the saturation point. In Experiments 168-18 and -19-2, the calculated amount of pigment was used for loading, and very little was washed after loading.

Tetracarboxylic porphyrin (from acetone) and Co phthalocyanine (from pyridine) were loaded onto fibril for further analysis (Experiments 169-18 and -19-2, respectively).

Tetracarboxylic acid porphyrin assay

The addition of excess base (pH 11-12) immediately discolored the slurry under titration. This did not interfere with titration but showed desorption of porphyrin at high pH. The carboxylic acid concentration was measured by back titration with excess NaOH to the end point of pH 7.5. As a result of the titration, a load of 1.10 meq per gram of acid corresponding to 0.275 meq per gram of porphyrin was obtained.

Cobalt or Dilithium Phthalocyanine Assay

The concentration of the adsorbate was evaluated only through decolorization experiments. The point where the bluish hue disappeared after 30 minutes was regarded as the saturation point.

Many substituted multinuclear aromatic or heteropolynuclear aromatic compounds were adsorbed onto the fibrillar surface. For attachment, the number of aromatic rings should be more than two per ring / side chain functional group. That is, substituted anthracene, phenanthrene, etc., containing three fused rings, or multifunctional derivatives containing four or more fused rings can be used in place of porphyrin or phthalocyanine derivatives. Likewise, substituted aromatic heterocycles such as quinoline or multiple substituted heteroaromatics containing four or more rings can also be used.

Table II summarizes the loading test results of the three porphyrin / phthalocyanine derivatives.

Table II

Adsorption experiment

TCAPorph = tetracarboxylic porphyrin

DiLiPhth = Dilithium Phthalocyanine

CoPhth = cobalt (II) phthalocyanine

(cal) = calculated value

(T) = proper value

Examples 11 and 12 below illustrate the process of adsorbing two different phthalocyanine derivatives on carbon nanotubes.

Example 11

Fibrillar with functional groups by adsorption of nickel (II) phthalocyanine tetrasulfonic acid

In 1 ml of dH ₂ O, 4.2 mg of basic fibril and 2 mg of nickel (II) phthalocyanine-tetrasulfonic acid (tetrasodium salt) were mixed. This mixture was sonicated for 50 minutes and spun overnight at room temperature.

Fibrils were washed three times with 1 ml of dH ₂ O, three times with 1 ml of MeOH, and three times with 1 ml of CH ₂ Cl ₂ and vacuum dried.

Thermolysin was adsorbed and immobilized on the phthalocyanine derivative fibrils. 0.5 mg of fibrils were suspended in 250 μl of dH ₂ O and sonicated for 20 minutes. The supernatant was discarded and the fibrils were again suspended in 250 μl of 0.05 M Tris (pH 8.0) and mixed with 250 μl of 0.6 mM thermolysine solution prepared with the same buffer. The mixture was spun at room temperature for 2 hours and stored at 4 ° C. overnight. The fibrils were then washed three times with 1 ml of 25 mM Tris (pH = 8) and suspended in 250 μl of buffer (pH 7.5) containing 40 mM Tris and 10 mM CaCl ₂ .

The amount of thermolysine on the fibrils was confirmed by measuring the enzyme activity of the fibrils. Thermolysine reacts with the substrate FAGLA (N-3 [2-furyl] acryloyl) -gly ^- leuamide) to produce a compound with an absorption coefficient of -310M ^-1 cm ^-1 , which reduces the absorbance at 345 nm. Assay buffer conditions for this reaction were 40 mM Tris, 10 mM CaCl ₂ and 1.75 M NaCl, pH 7.5. The reaction was carried out in 1 ml cuvette by mixing 5 μl of FAGLA stock solution (30% DMF in dH ₂ O, 25.5 mM) and 10 μg of thermolysine fibril in 1 ml of assay buffer and mixing. Absorbance reduction at 345 nm was monitored via timed scan for 10 minutes. Enzyme activity (μM / min) was calculated from the initial slope using the extinction coefficient -310M ^-1 cm ^-1 . The amount of active thermolysine per gram of fibril was 0.61 μΜ.

Example  12

Fibrillars with functional groups attached by adsorption of 1,4,8,11,15,18,22,25-octabutoxy-29H, 31H-phthalocyanine

3 mg of 1,4,8,11,15,22,25-octabutoxy-29H, 31H-phthalocyanine and 5.3 mg of the basic fibrils were mixed in 1 ml of CHCl ₃ . This mixture was sonicated for 50 minutes and spun overnight at room temperature.

Fibrils were washed three times with 1 ml of CH ₂ Cl ₂ and vacuum dried.

Thermolysine was adsorbed and immobilized on the phthalocyanine derivative fibril according to the method of Example 34. The amount of active thermolysine per gram of fibril was 0.70 μΜ.

Example 13

Synthesis of Aspartame Precursor Using Raw Fiber of Phthalocyanine Derivative Fixed with Thermolysine

Phthalocyanine derivative fibrils immobilized with thermolysine can be used to catalyze the synthesis of aspartame precursors, artificial sweeteners. This reaction was carried out by mixing 80 mM L-Z-Asp and 220 mM L-PheOMe in ethyl acetate with 10 μM of thermolysin fixed fibril. The product Z-Asp-PheOMe measured the yield by monitoring the HPLC.

5. Chlorate or Nitrate Oxidation

Literature on the oxidation of graphite with strong oxidants such as potassium chlorate in concentrated sulfuric acid or nitric acid is described in RNSmith, Quarterly Review 13 , 287 (1959); MJDLow, Chem . Rev. 60 , 267 (1960). In general, the marginal carbon (at the defect site) is attacked to provide a mixture of carboxylic acids, phenols and other oxygenated groups. This reaction mechanism is complicated with radical reactions.

Example 14

Preparation of Fibrils with a Carboxylic Acid Functional Group Using Chlorates

CC fibril samples were mixed with a spatula, slurried in concentrated sulfuric acid, and then transferred to a reactor flask equipped with a gas inlet / outlet and an upper stirrer. Under stirring and slow argon, the NaClO ₃ charge was added in portions at RT during the experiment. Chlorine vapor was generated during the entire experiment and removed from the reactor with a NaOH aqueous solution trap. At the end of the experiment, the fibril slurry was poured onto ice cubes and vacuum filtered. The filter cake was transferred to Soxhlet Timber, washed with deionized water in a Soxhlet extractor and replaced with fresh deionized water every few hours. Washing continued until the fibril sample did not change the pH of the water when fresh deionized water was added. The fibrils were then filtered off and dried overnight at 100 ° C., 5 ”vacuum.

The carboxylic acid content was measured by reacting an excess of 0.100N NaOH with the sample and back titrating with 0.100N HCl to the end point of pH 7.5. The results are summarized in the table below.

TABLE III

Direct Oxidation Experiment Results

Example 15

Preparation of Fibrils Attached with Carboxylic Acid Functional Group Using Nitric Acid

In a round bottom multi-hole concave reactor flask equipped with an upper stirrer and a water condenser, nitric acid of appropriate strength was added thereto, and a constant weight of fibril sample was added to slurry. Under constant stirring, the temperature was adjusted and the reaction proceeded for a certain time. Immediately after the temperature exceeded 70 ° C., brown smoke was released regardless of the strength of the acid. After the reaction, the slurry was poured into ice cubes and diluted with deionized water. The slurry was filtered and excess acid was washed off in a Soxhlet extractor and exchanged with fresh deionized water every few hours until no pH change of deionized water due to the slurried sample was observed. The fibrils were dried overnight at 100 ° C., 5 ”vacuum. Some of the weighed fibrils were reacted with standard 0.100 N NaOH and back titrated with 0.100 N HCl to determine the carboxylic acid content. Surface oxygen content was determined by XPS. The water dispersibility was tested by mixing 0.1 wt% concentration in a wear ring blender at high speed for 2 minutes.

Table IV

Direct Oxidation Experiment Results

6. Amino Functionalization of Fibrils

The amino group is treated with nitric acid and sulfuric acid according to the following scheme to obtain nitrated fibril, and then the nitrated form is reduced with a reducing agent such as sodium dithionite to form an amino functional group attached. By obtaining the fibers, they can be introduced directly onto the graphite fibres:

The resulting fibrils are of great utility, such as can be used for protein (eg, enzymes and antibodies) immobilization, and affinity and ion exchange chromatography.

Example 16

Preparation of Amino Functional Group Attached Fibrils Using Nitric Acid

Fibril (70 mg) was added dropwise to nitric acid (0.4 ml) in a cooling suspension (0 ° C.) suspended in water (1.6 ml) and acetic acid (0.8 ml). The reaction mixture was stirred at 0 ° C. for 15 minutes and at room temperature for an additional 1 hour. A mixture of sulfuric acid (0.4 ml) and hydrochloric acid (0.4 ml) was added slowly and stirred at room temperature for 1 hour. The reaction was stopped and centrifuged. The aqueous layer was removed and the fibrils were washed with water (X5). The residue was treated with 10% sodium hydroxide (X3) and washed with water (X5) to give nitrated fibrils.

To the suspension in which the nitrated fibril was suspended in water (3 ml) and ammonium hydroxide (2 ml), sodium ionic acid (200 mg) was added in 3 portions at 0 ° C. The reaction mixture was stirred at room temperature for 5 minutes and refluxed at 100 ° C. for 1 hour. The reaction was stopped, cooled to 0 ° C., and the pH was adjusted with acetic acid (pH 4). After standing at room temperature overnight, the suspension was filtered, washed with water (X10), methanol (X5) and dried in vacuo to afford amino fibrils.

To examine the amino functional group attached fibrils, horseradish peroxidase was coupled to the fibrils. The HRP coupled amino fibrils were then sufficiently dialyzed. After dialysis, the fibrils were washed 15 times until the following week. Enzyme modified fibrils were analyzed as follows:

                       HRP

H ₂ O ₂ + ABTS (transparent) --------------> 2H ₂ O + product (green)

As a result, HRP coupled with Fib-NH ₂ showed good enzymatic activity maintained for 1 week.

7. Attachment of terminal alcohols with free radical initiators

The high stability of carbon nanotubes allows them to be used in harsh environments but is difficult to activate for further modification. Conventional methods have involved the use of harsh oxidants and acids. However, it has now been surprisingly found that free radical initiators such as benzoyl peroxide (BPO) can be used to attach terminal alcohols to carbon nanotubes. The carbon nanotubes are added with a free radical initiator to an alcohol represented by the formula RCH ₂ OH, wherein R is hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cycloaryl or poly (alkylether), and is about 60 Heated to ° C to about 90 ° C. Preferred alcohols include ethanol and methanol. After sufficient time has elapsed for all free radical initiators to decompose, the reaction mixture is filtered, the carbon nanotube material is washed and dried to yield modified nanotubes represented by the formula nanotube-CH (R) OH. This method can also be used for the coupling of difunctional alcohols. This method allows one end to be bonded to the carbon nanotube and the other end to be used to indirectly bond the other material to the surface.

Example 17

Preparation of Alcohol-functional Nanotubes Attached with Benzoyl Peroxide

0.277 g of carbon nanotubes were dispersed in MeOH using a probe sonicator. 0.126 g of BPO was added at RT and this temperature was increased to 60 ° C. followed by an additional 0.128 g of BPO. After 45 minutes at 60 ° C., 0.129 g of the final BPO charge was added and the mixture was held at 60 ° C. for an additional 30 minutes. The product was filtered over the membrane, washed several times with MeOH and EtOH and dried in a 90 ° C. oven. The yield was 0.285 g. ESCA analysis showed that the oxygen content was 2.5 atomic percent compared to 0.74 percent of the control sample refluxed in MeOH without BPO.

Example 18

Modification of Carbon Nanotubes to Poly (ethylene Glycol) Using Benzoyl Peroxide

0.1 g of carbon nanotubes, 0.5 g of BPO and 10 g of poly (ethylene glycol) average molecular weight 1000 (PEG-1000) were mixed together at room temperature. The mixture was heated to 90 ° C. to dissolve PEG, and the mixture was reacted at 90 ° C. overnight. The total mixture was then filtered and washed to remove excess PEG and dried. The material obtained can be used as such or further modified by attaching the material to the free end of the PEG.

Example 19

Use of PEG-Modified Carbon Nanotubes to Reduce Nonspecific Binding

Nonspecific binding to a large surface area carbon material occurs everywhere. It has been found that attachment of hydrophilic oligomers such as PEG to carbon nanotubes can reduce nonspecific binding. In addition, by attaching one end of a chained molecule, such as PEG, to the surface of the nanotube, the free end may contain a functional group that can be used for attachment of the other material, while reducing PEG (or other substance), which reduces nonspecific binding It has been found that it can retain the properties of the layer.

PEG modified With fiber Bovine serum  Reduced nonspecific binding of albumin

Stock dispersions in which unmodified fibrils, chlorate oxidized fibrils and PEG-modified fibrils were dispersed in 50 mM potassium phosphate at pH 7.0 at a concentration of 0.1 mg / ml were prepared by dispersing 1.0 mg each by sonication in 10 ml of buffer. did. Two serial dilutions each were injected into 9 polypropylene tubes, 2 ml each. 100 μl of a solution of bovine serum albumin (BSA) dissolved in 0.2 mg / ml concentration in the same buffer was also added to each tube and three buffer blank tubes. Three buffer tubes except protein were also prepared. All tubes were mixed with a vortex mixer and incubated for 30 minutes, vortexing every 10 minutes for 30 seconds. The tubes were all centrifuged to separate the fibrils, and 1 ml of the supernatant was placed in a new tube to analyze the total protein content using the Micro BCA protein assay (Pierce). Protein concentration remaining in the supernatant is an indirect measure of the amount of nonspecific binding to fibril. BSA remained in the supernatant of PEG-modified fibrils, while almost completely bound to unmodified fibrillar or chlorate oxidized fibrils (see FIG. 1).

Comparison of nonspecific binding reduction by PEG modified fibrils prepared with benzoyl peroxide and NHS ester coupling

Stock dispersions of chlorate oxidized fibrils, PEG-modified fibrils with benzoyl peroxide, and chlorate oxidized fibrils modified with PEG by NHS ester coupling were subjected to sonication in 50 mM potassium phosphate buffer (pH 7.0). To 1.0 mg / ml. Three-fold serial dilutions of each dispersion were injected into 7 polypropylene tubes of 2 ml each. 100 μl of a solution of β-lactoglobulin (βLG) dissolved in 0.2 mg / ml in the same buffer was also added to each tube and three buffer blank tubes. Three buffer tubes except protein were also prepared. All tubes were mixed with a vortex mixer and incubated for 60 minutes, vortexing every 10 minutes for 30 seconds. The tubes were all centrifuged to separate the fibrils, and 1 ml of the supernatant was placed in a new tube to analyze the total protein content using the Micro BCA protein assay (Pierce). Protein concentration remaining in the supernatant is an indirect measure of the amount of nonspecific binding to fibril (see FIG. 2). ΒLG remained in the supernatant of PEG-modified fibrils via the NHS ester pathway in each tube, so no nonspecific binding was seen. PEG-modified fibrils via the BPO pathway showed slight (about 10%) βLG binding only at the highest fibrillar concentration of 1.0 mg / ml, with no significant binding at lower concentrations. In contrast, almost complete binding was observed for chlorate oxidized fibres with a fiber concentration of 1 mg / ml or higher, and a significant decrease in binding for 0.01 mg / ml of the fiber.

8. Secondary Derivatives of Functionally Attached Nanotubes

Nanotubes with carboxylic acid functional groups

The number of secondary derivatives that can be produced solely from carboxylic acids is essentially infinite. Alcohols or amines readily bind to acids to form stable esters or amides. If the alcohol or amine is part of a bifunctional polyvalent molecule, the bond through O- or NH- leaves other functional groups as side groups. Typical examples of secondary reagents are as follows:

General formula Instrument Yes HO-R, R = alkyl, aralkyl, aryl, fluoroethanol, polymer, SiR ' ₃ R- Methanol, phenol, trifluorocarbon, OH-terminated polyester, silanol H ₂ NR, R = same as above R- Amines, anilines, fluorinated amines, silylamines, amine terminated polyamides, proteins Cl-SiR ₃ SiR _3- Chlorosilanes HO-R-OH, R = alkyl, aralkyl, CH ₂ O- HO- Ethylene Glycol, PEG, Pentaerythritol, Bisphenol A H ₂ NR-NH ₂ , R = alkyl, aralkyl H ₂ N- Ethylenediamine, Polyethyleneamine XRY, R = alkyl and the like; X = OH or NH ₂ ; Y = SH, CN, C = O, CHO, Alkene, Alkyne, Aromatic, Heterocycle Y- Polyamine amide, mercaptoethanol

The reaction can be carried out using any method developed for esterifying or aminating carboxylic acids with alcohols or amines. Among these, HAStaab, Anagew. Chem. Internat. Edit., (1), 351 (1962) and amide using N, N'-carbonyl diimidazole (CDI) as acylating agents of esters or amides; Activation of carboxylic acids with N-hydroxysuccinimide (NHS) for oxidation [GW] Anderson, et al., J. Amer. Chem. Soc. 86, 1839 (1964).

Example 20

Preparation of Secondary Derivatives of Fibrils with Functional Groups

N, N'-carbonyl diimidazole

This experiment requires a clean, anhydrous aprotic solvent (eg toluene or dioxane). The stoichiometric content is sufficient. For the esters, the carboxylic acid compounds were reacted in toluene under inert atmosphere (argon) with the stoichiometric content of CDI dissolved in toluene for 2 hours at room temperature. During this reaction, CO ₂ was generated. After 2 hours, alcohol was added together with the catalytic amount of sodium ethoxide and the reaction continued at 80 ° C. for 4 hours. The yield was quantitative for normal alcohol. The scheme is as follows:

1.R-COOH + Im-CO-Im ----------> R-CO-Im + HIm + CO ₂ ,

Im = imidazolid, HIm = imidazole

                     NaOEt

2.R-CO-Im + R'OH -----------> R-CO-OR '+ HIm

Amidation of amines is carried out in a noncatalytic reaction at RT. The first step of the experiment is the same. After CO ₂ generation, stoichiometric content of amine was added at RT and reacted for 1-2 hours. The reaction was quantitative. The scheme is as follows:

3.R-CO-Im + R'NH ₂ -----------> R-CO-NHR + HIm

Silylation

Trialkylsilylchloride or trialkylsilanol reacts immediately with acid H according to the following scheme:

R-COOH + Cl-SiR ' ₃ -------->R-CO-SiR' ₃ + HCl

Small amounts of diaza-1,1,1-bicyclooctane (DABCO) were used as catalyst. Suitable solvents are dioxane and toloene.

Fibrillars with sulfonic acid functional groups

Aryl sulfonic acid as prepared in Example 1 may be further reacted to produce secondary derivatives. Sulfonic acid can be reduced to mercaptan by LiAlH ₄ or a mixture of triphenyl phosphine and iodine (March, JP, p. 1107). In addition, sulfonic acids can be converted to sulfonate esters by reaction with dialkyl ethers:

Fibres--SO ₃ H + ROR ------> Fibres--SO ₂ OR + ROH

N-hydroxysuccinimide

Activation of the carboxylic acid for amidation to the primary amine occurs via the N-hydroxysuccinyl esters; Carbodiimide was used to remove the water released with the substituted urea. The NHS ester is then converted to an amide by reaction with the primary amine at room temperature. The scheme is as follows:

1.R-COOH + NHS + Carbodiimide ---------> R-CONHS + Substituted Urea

2.R-CONHS + R'NH ₂ ---------> R-CO-NHR '

This method is particularly useful for covalent attachment of proteins to graphite fibrils via free NH ₂ on the protein side chain. Examples of proteins that can be immobilized on fibrils by this method are trypsin, streptavidin and avidin. Streptavidin (or avidin) fibrils serve as solid carriers for all biotinylated materials.

Example 21

Covalent attachment to fibrils of proteins through NHS esters

Streptavidin, avidin and trypsin were attached to the fibrils as follows to demonstrate whether the protein can be covalently bound to the fibrils via the NHS ester.

0.5 mg of NHS-ester fibril was washed with 5 mM sodium phosphate buffer (pH 7.1) and the supernatant was removed. 200 µl of streptavidin solution (1.5 mg in the same buffer) was added to the fibrils, and the mixture was stirred at room temperature for 5.5 hours. Fibrils were then washed sequentially with 1 ml of the following buffer: sodium phosphate (pH 7.1), PBS (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.4), ORIGEN ™ Assay Buffer (IGEN, Inc., Gaithersburg, MD ) And PBS. Streptavidin fibrils were stored in PBS buffer for later use.

2.25 mg of NHS-ester fibril was sonicated in 500 μl of 5 mM sodium phosphate buffer (pH 7.1) for 40 minutes and the supernatant was removed. The fibrils were suspended in 500 µl of 5 mM sodium phosphate buffer (pH 7.1), and 300 µl of an avidin solution prepared by adding 2 mg avidin (Sigma, A-9390) to the same buffer. The mixture was spun for 2 hours at room temperature, stored overnight at 4 ° C. and then for an additional 1 hour at room temperature. Fibrils were washed four times with 1 ml of 5 mM sodium phosphate buffer (pH 7.1) and twice with PBS buffer. Avidin fibrils were suspended in 200 μl of PBS buffer for storage.

Trypsin fibrils were mixed with 200 μl of 1.06 mM trypsin solution prepared in 5 mM sodium phosphate buffer (pH 7.1) and 1.1 mg of NHS-ester fibrils (treated as in avidin fibrils) for 6.5 hours at room temperature. It was made by rotating. Trypsin fibrils were then washed three times with 1 ml of 5 mM sodium phosphate buffer (pH 7.1) and suspended in 400 μl of the same buffer.

Example  22

Determination of the enzyme activity of trypsin present on fibrils

Trypsin can react with the substrate L-BAPNA (N _α -benzoyl-L-arginine p-nitroanilide) to release colored compounds that absorb light at 410 nm. As an analysis buffer of this reaction, 0.05M Tris, 0.02M CaCl ₂ and pH 8.2 were used. This reaction was performed by mixing 5 μl of L-BAPNA stock solution (50 mM in 37% DMSO aqueous solution) and 10-25 μg trypsin fibrils in 1 ml assay buffer in 1 ml cuvette. Absorbance increase at 410 nm was monitored for 10 minutes. The enzyme activity (μM / min) was then calculated from the initial slope.

For covalently bound trypsin fibrils, the enzyme activity was 5.24 μM / min per 13 μg fibrils. This result can be converted into the amount of active trypsin present in fibrils by dividing the activity of a known concentration of trypsin solution measured at 46 μM / min per 1 μM trypsin under the same assay conditions. Therefore, the amount of active trypsin per gram of fibril was 8.3 μM (or 195 mg).

Example 23

Carbon Nanotubes with Surface Thiols

0.112 g of amino carbon nanotubes (CN) prepared by modification with ethylenediamine as described in Example 27 (below) were suspended in 20 ml of 0.05 M potassium phosphate buffer (pH 8.0) containing 50 mM EDTA. This suspension was sonicated for 5 minutes with a Branson 450 Watt probe sonicator for CN dispersion. The suspension obtained was quite thick. Argon was bubbled through this suspension under injection for 30 minutes under stirring. 50 mg of 2-iminothiolane-HCl was added, and the mixture was stirred under argon continuously for 70 minutes. The material obtained was filtered using a polycarbonate membrane filter, washed twice with 2X with buffer, 1X with deionized water and anhydrous EtOH, all under an argon blanket. Thiol modified CN was placed in a vacuum desiccator and degassed overnight.

Final weight = 0.18 g, conversion 55% based on weight increase.

A 10 mg sample of thiolated nanotubes was suspended by ultrasonication in 10 ml of deionized water and filtered using a 0.45 μm nylon membrane to form a felt-based mat. This piece of mat was stored in a vacuum desiccator and analyzed by ESCA, showing 0.46% sulfur and 1.69% nitrogen, demonstrating the successful conversion to thiol modified CN.

Example 24

Attachment of Thiol Modified Carbon Nanotubes to Gold Surfaces

Gold foil (Alfa / Aesar), ₂ cm × 0.8 cm was washed with a solution of 30 parts H ₂ O ₂ 1 part and 3 parts concentrated sulfuric acid for 10 minutes and then washed with deionized water. The foil piece was connected to the Au wire lead to give -0.35 V vs. 50 mV / sec for about 10 minutes until the cyclic voltammetry did not change. Ag / AgCl and 1.45 V vs. An electrochemical cycle occurred between Ag / AgCl in 1M H ₂ SO ₄ . Then it was washed with deionized water and then dried. The large fragment was cut into four 0.5 cm x 0.8 cm pieces.

10 ml of deoxygenated anhydrous EtOH by argon rinse for 30 minutes was injected into two glass vials, respectively. One bay was suspended with 16 mg of thiol-modified CN (CN / SH) and two Au fragments, while the other was suspended with one Au fragment and 10 mg of ethylene diamine modified CN used to prepare thiol derivatives. All manipulations were performed in an Ar filled glove bag. The bayer was sealed under argon and left for 1 hour in a cooled ultrasonic bath. The sealed vial was left at room temperature for 72 hours. The Au sample was taken from the bayer, washed 3x with EtOH, air dried and placed in a protective bayer.

Au foil samples exposed to CN / ethylenediamine and CN / SH were irradiated with a scanning electron microscope (SEM) to detect the presence of CN on the surface. When irradiated at 40,000 ×, the distribution of CN was observed on the surface exposed to CN / SH, but no CN was observed in the Au foil sample exposed to CN / ethylenediamine.

Example 25

Preparation of Maleimide Fiber from Amino Fiber

Amino fibrils were prepared according to Example 13. This amino fibril (62.2 mg) was sonicated in sodium phosphate buffer (5 ml, 5 mM, pH 7.2). Sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC; 28.8 mg, 0.66 mM; Pierce, Cat. No. 22360) was added to this fibril suspension. The reaction mixture was stirred at rt overnight. Fibrils were washed with water and methanol, and fibrils were vacuum dried. Antibody immobilization on this product meant the presence of maleimide fibrils. In the same manner, other linkers (eg sulfo-SMCC, succinimidyl 4- [p-maleimidophenyl] butyrate [SMPB], sulfo-SMPB, m-maleimidobenzyl-N-hydroxysuccinimide esters [ MBS], sulfo-MBS, etc.) The other maleimide which has fibrils was also manufactured.

The obtained maleimide fibril can be used as a solid support for covalent fixation of proteins such as antibodies and enzymes. Antibodies were covalently immobilized on maleimide activated fibrils. The loading amount of the antibody was 1.84 mg per gram of fibril when the amino fibrils obtained by the nitration / reduction method (Example 13) were used, and 0.875 per gram of fibril when the amino fibrils derivatized from the carboxy fibrils were used. mg.

Example 26

Preparation of Esters / Alcohol Derivatives from Fibrils with a Carboxylic Acid Functional Group

Fibrils with carboxylic acid functional groups were prepared as in Example 14. The carboxylic acid content was 0.75 meq / g. Fibrils were reacted at room temperature with toluene as stoichiometric content of CDI and solvent until CO ₂ evolution ceased under inert atmosphere. The slurry was then reacted at 80 ° C. with a 10-fold excess mole of polyethylene glycol (MW600) and a small amount of NaOEt as catalyst. After 2 hours of reaction, the fibrils were separated by filtration, washed with toluene and dried at 100 ° C.

Example  27

Preparation of Amide / Amine Derivatives from Fibrils with a Carboxylic Acid Functional Group (177-041-1)

0.242 g of chlorate oxidized fibril (0.62 meq / g) was suspended with stirring in 20 ml anhydrous dioxane in a 100 ml RB flask equipped with a serum stopper. A 20-fold excess mole of N-hydroxysuccinimide (0.299 g) was added to dissolve. Then 20-fold excess molar 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) (0.510 g) was added and stirred at RT for 2 h. After 2 hours, stirring was stopped, the supernatant was aspirated off, the solid was washed with anhydrous dioxane and MeOH, and filtered using a 0.45 micron polysulfone membrane. The solid was washed with additional MeOH on the filtration membrane and vacuum dried until no weight loss was observed anymore. The yield of NHS activated fibrils was 100% based on the observed 6% weight gain.

100 μl of ethylenediamine (en) was added to 10 ml of 0.2 M NaHCO ₃ buffer. The same amount of acetic acid (HOAc) was added to maintain the pH at about 8. NHS activated fibril oxidized fiber (0.310 g) was added under vigorous stirring and reacted for 1 hour. In addition, 300 μl en and 300 μl HOAc were added for an additional 10 minutes. The solution was filtered over 0.45 micron polysulfone membrane and washed sequentially with NaHCO ₃ buffer, 1% HCl, deionized water and EtOH. The solid was vacuum dried overnight. The HCl salt was converted back to the free amine (177-046-1) by reaction with NaOH for subsequent analysis and reaction.

ESCA was performed to quantify the amount of N present on the aminated fibrils (GF / NH ₂ ). ESCA analysis of 177-046-1 showed 0.90 at% N (177-059). To further analyze whether any of these N amounts are accessible and reactive amine groups, derivatives were prepared by gas phase reaction with pentafluorobenzaldehyde to form Schiff base bonds having available primary amine groups. As a result of ESCA analysis, the content of N was 0.91 at% as expected and F 1.68 at%. This result is interpreted to be 0.34 at% of N present as reactive primary amine present on the aminated fibrils (5F per pentafluorobenzaldehyde molecule). A concentration of 0.45 at% N can be considered to be obtained upon complete reaction with the free end of each N. Thus, the measured concentration suggests a very high yield by the reaction of NHS activated fibril with N, confirming the reactivity of the available free amine groups.

At concentrations of 0.34 at% N present as free amines calculated from ESCA data, almost all of the fibrils will be covered with free amine groups that can couple with other materials.

Carboxy fibrils were also converted to amino fibrils by 1,6-diaminohexane (6 carbon linkers) in which one group was protected rather than ethylenediamine (2 carbon linkers).

Example 28

Preparation of Amine Derivatives from Fibrils Attached with Carboxylic Acid Functional Groups

The carboxyl group on the fibril can be modified by the reaction of a carboxy group with one amino group of a compound having at least two amino groups, at least one of which is deprotected with a group such as t-Boc or CBZ. The fibril thus formed is an amide derivative from which an amide carbonyl is derived from a fibrous carboxy group and in which the amide nitrogen is substituted with a group containing at least one primary amine (eg an alkyl group). Such amino groups can be used in use or further modification.

The outlet was placed in a dry sintered glass filter tunnel completely covered with a rubber serum diaphragm and 1 g of carbon fibril was added to cover anhydrous dichloromethane. N-methylmorphine (758 μl, 7 mmol) was added and the suspension was mixed using a spatula. Then isobutyl chloroformate (915 μl, 7 mmol) was added and the suspension was periodically mixed for 1 hour. Parafilm was applied as practically helpful to protect this mixture from atmospheric moisture.

On the other hand, N-boc-1,6-diaminohexane hydrochloride (1.94 g, 7.7 mmol) was fractionated with dichloromethane (10 ml) and 1 M NaOH (10 ml). The lower organic layer was dehydrated over anhydrous potassium carbonate, filtered through an applied disposable parser pipette, and N-methylmorpholine (758 μl, 7 mmol) was added.

The serum septum was removed from the filter tunnel, vacuum filtered to remove reagents from the fibrils, and the fibrils were washed with anhydrous dichloromethane. The serum diaphragm was reinserted and a mixture of N-methylmorpholine and one group protected diaminohexane was added to the fibrils. The mixture was stirred periodically for 1 hour. The reagent was then filtered off and the fibrils were washed sequentially with dichloromethane, methanol, water, methanol and dichloromethane.

Then, a 50% mixture of trifluoric acid and dichloromethane was added to the fibrils and the mixture was stirred periodically for 20 minutes. The solvent was filtered off and the fibrils were washed sequentially with dichloromethane, methanol, water, 0.1 M NaOH and water.

To demonstrate the efficacy of this method, a small amount of amino fibril sample was reacted with "activated" horseradish peroxidase (HRP; 5 mg, Pierce) modified to specifically react with amino groups. The fibrils were washed repeatedly for several days while maintaining the low temperature (suspended, stirred and centrifuged in an Eppendorf tube). After washing for about 2 weeks, the enzyme was analyzed by H ₂ O ₂ / ABTS in glycine buffer (pH 4.4). Within 10 minutes the solution appeared green, indicating the presence of the enzyme. Control fibrils (COOH fibrils treated with activated HRP and washed for the same period of time) showed little catalytic activity.

Example 29

Preparation of Silyl Derivatives from Fibrils Attached with Carboxylic Acid Functional Groups

Acid functional group-attached fibrils prepared as in Example 14 were slurried in dioxane under an inert atmosphere. Under stirring, a stoichiometric content of chlorotriethyl silane was added and allowed to react for 0.5 hours, followed by several drops of DABCO 5% solution in dioxane. The system was allowed to react for an additional hour, then the fibrils were collected by filtration and washed with dioxane. The fibrils were dried overnight at 100 ° C., 5 ”vacuum.

Table V summarizes the preparations of secondary derivatives. The C, O, N, Si and F surface contents of the product were analyzed by ESCA.

Table V

Secondary derivative preparation summary

Example 30

Preparation of Silyl Derivatives from Fibrils Attached with Carboxylic Acid Functional Groups

The acid functional grouped fibrils prepared as in Example 14 were slurried in dioxane under an inert atmosphere. While stirring, a stoichiometric content of chlorotriethyl silane was added and reacted for 0.5 hour, followed by several drops of DABCO 5% solution in dioxane. The system was allowed to react for an additional hour, then the fibrils were collected by filtration and washed with dioxane. The fibrils were dried overnight at 100 ° C., 5 ”vacuum.

Table VI summarizes the preparations of the secondary derivatives. The product was analyzed by ESCA. As a result of this analysis, it was confirmed that necessary instruments were mixed. The C, O, N, Si and F surface contents of the product were analyzed by ESCA.

Table VI

Secondary derivative preparation summary

Example 31

Preparation of tertiary and quaternary amine derivatives from fibrils with carboxylic acid functional groups

Tertiary and quaternary amine functionalities may be attached to the surface of the carbon nanotubes by amide or ester linkages through either the carboxyl groups on the nanotubes and the amine and hydroxy groups of the tertiary or quaternary amine precursors. Such tertiary or quaternary amine fibrils are useful as chromatographic substrates for the separation of biomolecules. Tertiary or quaternary amine fibres can be processed into discoid mats or mixed with conventional separation chromatography media (eg agarose).

Preparation of Iodide Triethylethanolamine Precursor

In a 100 ml round bottom flask, 10 g of N, N-diethylethanolamine (85.3 mmol) was mixed with 10 ml of anhydrous methanol. Then, a mixture of 20 g of ethyl iodide (127.95 mmol) and 10 ml of anhydrous methanol was added dropwise using a pipette. The reaction mixture was refluxed for 30 minutes. When the reaction mixture was cooled to room temperature, a white crystalline product was formed. This white solid product was collected by filtration and washed with anhydrous methanol. The product was dried overnight in a vacuum desiccator. The product was obtained in a yield of 33% (10.3 g, 37.7 mmol).

Preparation of Graphite Fibrils with Quaternary Amine Functional Group

In a vacuum dried 25 ml Wheaton disposable scintillation vial, 100 mg of anhydrous carboxy fibrils (approximately 0.7 mmol COOH per gram of fibrils) were mixed with 2 ml of anhydrous dimethylformamide and the mixture was sonicated for 60 seconds. At least 2 ml of dimethylformamide, 39 mg dimethyl-aminopyridine (0.316 mmol) and 50 μl diisopropylcarbodiimide (0.316 mmol) were added to the reaction bay. After the reaction mixture was stirred at room temperature for 1 hour, 88 mg triethylethanolamine (0.316 mmol) was added to the vial, followed by overnight reaction. The fibrils obtained 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 deionized water. The product was vacuum dried. As a result of nitrogen element analysis, it was found that about 50% of the carboxy groups present in the fibrils reacted with the primary amino groups in the quaternary amine moiety.

Example 32

Chromatography of Bovine Serum Albumin (BSA) on Graphite Fibrils Attached with Tertiary Amine Functional Groups

An aqueous slurry containing 60 mg of 2-diethylamino ethylamine modified carboxy fibrils and 180 mg of Sephadex G-25 ultrafine resin (Pharmacia, Uppsala, Sweden) was left at room temperature overnight to fully hydrate the solid support. The slurry was packed into a 1 cm x 3.5 cm column. The column was equilibrated with 5 mM sodium phosphate buffer (pH 7.3) at a flow rate of 0.2 ml / min. BSA (0.6 mg in 0.1 ml deionized water) was loaded onto the column. The column was eluted with 5 mM sodium phosphate at a flow rate of 0.2 ml / min, and the fractions were collected by 0.6 ml. The results of monitoring the elution profile with a UV-visible detector are shown in FIG. 3. When no more protein was eluted from the column through the detector, the bound BSA was immediately eluted by addition of 1M KCl in 5 mM sodium phosphate, pH 7.3. The presence of protein in each fraction was confirmed by micro BCA analysis (Pierce, Rockford, Il).

Example 33

Chromatography of Bovine Serum Albumin (BSA) on Graphite Fibrils Attached with Quaternary Amine Functional Groups

An aqueous slurry containing 100 mg 2- (2-triethylamino ethoxy) ethanol modified carboxy fibrils and 300 mg Sephadex G-25 ultrafine resin was left at room temperature overnight. The resulting slurry was packed into a 1 cm diameter column. The column was equilibrated with 5 mM sodium phosphate buffer (pH 7.3) at a flow rate of 0.1 to 0.6 ml / min. BSA (2.7 mg in 0.2 ml deionized water) was loaded onto the column. The column was eluted with 5 mM sodium phosphate at a flow rate of 0.2 ml / min and fractions were collected at 0.6 ml. Elution profiles were monitored with a UV-visible detector (FIG. 4). When the detector was observed to no longer elute the protein with 5 mM sodium phosphate buffer, the solvent was immediately exchanged with 1 M KCl in 5 mM sodium phosphate, pH 7.3. The presence of protein in each fraction was confirmed by micro BCA analysis (Pierce, Rockford, Il).

9. Enzymatic Functionalization of Graphite Carbon

Biocatalysts can be used to introduce functional groups on the surface of graphite carbon, especially carbon nanotubes. To date, graphite carbon has been modified by pure chemical methods (eg, US patent application Ser. No. 08 / 352,400, 1994.12.8). These chemical methods have disadvantages such as (1) harsh conditions (maximum temperature, maximum acidic or toxic chemical use) and (2) lack of specificity (eg, oxidation can introduce COOH, COH and CHO groups). Aqueous suspensions of solid graphite carbon (e.g. carbon fibrils; Hyperion, Inc.) are prepared by adding one or more enzymes capable of accepting graphite carbon as a substrate and carrying out a chemical reaction to yield chemically modified graphite carbon. . The aqueous suspension is maintained for a time sufficient for the enzyme to catalytically modify the surface of the graphite carbon under conditions where the enzyme can carry out the reaction (temperature, pH, salt concentration, etc.). During the reaction, the suspension is continuously mixed so that the enzyme can access the surface of the graphite carbon. After a reaction time sufficient to allow the reaction to proceed to a satisfactory degree, the enzyme is filtered off to remove from the carbon.

To date, two enzymes have been used: cytochrome p450 enzymes and peroxidase enzymes. Both of these enzymes have been thoroughly studied in their type, and both have been found to accommodate aromatic substrates, and their optimum reaction conditions have also been found. Both types of enzymes can introduce hydroxy groups into the substrate, thereby introducing hydroxy groups into the graphite carbon. In addition to enzymes, other biocatalysts such as ribozymes and catalytic antibodies, or non-biological mimetics of enzymes, can also be designed to catalytically functionalize carbon nanotubes.

Example  34

Enzymatic Functionalization Using Rat Liver Microsomes

Cytochrome p450 enzymes are generally thought to act in the liver as detoxifying agents (F. Peter Guengerich, American Scientist, 81, 440-447 and F. Peter Guengerich, J. Biol. Chem., 266, 10019-10022 ). This enzyme hydroxylates heterogeneous compounds such as polyaromatic toxic compounds. Hydroxation renders the compound water soluble so that it can be removed from the body through urine. There are many types of cytochrome p450 enzymes in the liver, each with different substrate specificities. This wide specificity is considered to be an important feature because of the wide variety of environmental toxins that require detoxification. Each cytochrome p450 is commercially available, but it is not known which cytochrome p450 can accept carbon nanotubes as a substrate. Because of this uncertainty, we decided for the first time to incubate rat liver extract and carbon nanotubes containing many different cytochrome p450s.

Two rats (“experimental” rats) were administered pentobarbital (1 g / L, pH 7.0) in drinking water for one week to induce expression of the cytochrome p450 enzyme. The other two rats ("control" rats) received water without pentobarbital. Rats were then slaughtered and cytochrome p450 containing microsomes were prepared from the liver according to standard methods (see, for example, Methods in Enzymology, Vol. 206).

These microsomes were mixed with carbon nanotubes (fibers) to cause cytochrome p450 to react with graphite carbon. In this experiment, 5 mg of fibrils ("basic" or non-functional fibrils and "COOH" or oxidized fibrils) were mixed with microsomes (experimental and control microsomes) and 0.1M Tris, 1.0 mM NADPH, 0.01% NaN. ₃ , 10 mM glucose-6-phosphate and glucose-6-phosphate dehydrogenase (1 unit / ml) were mixed in a buffer solution (pH 7.4). NADPH was added as co-substrate of cytochrome p450 and glucose-6-phosphate, and glucose-6-phosphate dehydrogenase was added to regenerate NADPH from NADP ⁺ (if NADP ⁺ was generated by cytochrome p450). This mixture was placed in a microcentrifuge tube and spun at room temperature for about 1.5 days. After incubation, the fibrils were washed sufficiently with deionized water, 1M HCl, 1M NaOH, 0.05% Triton X-100, 0.05% Tween, methanol and 1M NaCl. After washing, microBCA analysis of the protein (Pierce) observed that the fibrils still retain the protein bound to them (no protein was detected in the wash solution).

To confirm the introduction of hydroxy groups on the fiber surface, the fiber was reacted with N-FMOC-isoleucine. Multiple batches of fibrils (control and experimental) (1.5 mg each) were used for 4.45 mg / ml FMOC-isoleucine, 1.54 mg / ml dimethylaminopyridine (DMAP) and 2.6 mg / ml 1,3-dicyclohexylcarbodiimide It was reacted with 333 [mu] l of anhydrous DMF solution containing (DCC). After reaction (rotating continuously) for 2 days, the fibrils were washed with DMF, piperidine, methanol, water, DMF, methanol, methylene chloride (600 μl each). This washing sequence was repeated three times. The fibrils were sent to Galbraith Laboratories (Knoxville, TN) for amino acid analysis of isoleucine present in the fibrils. The results were not clear because many other amino acids were observed in addition to isoleucine, probably because proteins, peptides and amino acids present in rat liver microsome extracts were not completely washed from fibril. Therefore, due to technical difficulties in washing and analysis, it was not possible to confirm whether cytochrome p450 was functionalized on fibrils.

Example 35

Fibrillar functionalization using commercially available recombinant cytochrome p450 enzymes

In order to eliminate impurities, a problem associated with using rat liver microsomes as a source of cytochrome p450, each cytochrome p450 enzyme was purchased (GENTEST, Woburn, MA). The cytochrome p450 enzyme is supplied as a microsome preparation because the enzyme binds to and acts only on the membrane. Cytochrome p450 was tested using a reaction procedure similar to that described above: CYP1A1 (cat. # M111b), CYP1A2 (cat. # M103c), CYP2B6 (cat. # 110a), CYP3A4 (with reductase, cat. # 107r). MgCl ₂ (0.67 mg / ml) was also added to the reaction solution. In this experiment, the fibrils were washed using a Soxhlet apparatus.

Analysis of the introduced hydroxyl group was carried out by reacting cytochrome p450 and washed fibril with 3,5-dinitrobenzoic acid (DNBA), a color developing reagent. Coupling was carried out as previously described for N-FMOC-isoleucine. After reaction with DNBA, the fibrils were washed with DMF and residual (covalently attached) DNBA was hydrolyzed using 6M HCl or 46 units / ml swine liver esterase (Sigma). Analysis of dissociated DNBA was performed by HPLC analysis of the fibrous periphery supernatant after hydrolysis treatment. HPLC analysis of dissociated DNBA showed a linear gradient of 0.1% TFA containing deionized water (solvent A) to 0.1% TFA containing acetonitrile (solvent B) in a Waters HPLC system equipped with a Vydac C18 reverse phase analysis column (cat. # 218TP54). It carried out using.

Example 36

Functional Vaporization of Fibrils Using Peroxidase

The literature description of peroxidase substrate specificity suggested that carbon nanotubes could be substrates of this enzyme (JS Dorick et al., Biochemistry (1986), 25, 2946-2951; DRBuhler et al., Arch ... Biochem Biophys (1961) 92, 424-437; HSMason, Advances in Enzymology, (1957) 19, 79;... GDNordblom et al, Arch Biochem Biophys (1976) 175, 524-533). To determine whether peroxidase (hydrogen peroxidase, type II, Sigma) can introduce hydroxyl groups to the surface of the fibrils, fibrillar (11 mg) was added to 50 mM sodium acetate (1.25 ml, pH 5.0) In a solution containing wasabi peroxidase (200 nM), dihydroxyfumaric acid (15 mg) was added 5 mg at a time for the first 3 hours of reaction. This reaction was carried out intermittently at 4 ° C. for a total of 5 hours under foaming of gaseous oxygen. After the reaction, the fibrils were washed with water, 1N NaOH, methanol and methylene chloride (200 ml each). Control reactions were performed using heat inactivated (100 ° C., 5 min) peroxidase.

To analyze the degree of hydroxylation of the fibrils catalyzed by peroxidase, the fibrils were reacted with t-butyldimethylsilyl chloride (Aldrich) in anhydrous DMF in the presence of imidazole. After the fibrils were washed, they were sent to Robertson Microlit Laboratories, Inc. (Madison, NJ) for elemental analysis of the silicon incorporated into the fibres. As a result of the analysis, it was unclear whether silicon was present on the surface of the fibril. The reason for this is that the silicon derived from the glass used in this experiment is thought to exist in small pieces in the fibres for elemental analysis. This resulted in high silicon concentration in both the experimental and control samples. As a result of this experiment, peroxidase can introduce hydroxy groups into the fibrils, but due to technical difficulties it was impossible to measure the presence of any hydroxy groups introduced.

10. Anaerobic Fiber  For the surface Electrophile  Addition or On metallization  Nanotubes attached by functional groups

Primary products obtainable by the addition of activated electrophiles to the surface of anoxic fibrils are the side groups —COOH, —COCl, —CN, —CH ₂ NH ₂ , —CH ₂ OH, —CH ₂ —halogen or HC═O. To hold. These groups can be converted into secondary derivatives as follows:

Fibrils-COOH --------> As described above.

Fibrillar-COCl (Acid Chloride) + HO-R-Y --------> F-COO-R-Y (Sec. 4/5)

Fibril-COCl + NH ₂ -RY --------> F-CONH-RY

Fibrillar-CN + H ₂ --------> F-CH ₂ -NH ₂

Fibre-CH ₂ NH ₂ + HOOC-RY --------> F-CH ₂ NHCO-RY

Fibril-CH ₂ NH ₂ + O = CR-R'Y --------> F-CH ₂ N = CR-R'-Y

Fibre-CH ₂ OH + O (COR-Y) ₂ --------> F-CH ₂ OCOR-Y

Fibre-CH ₂ OH + HOOC-RY --------> F-CH ₂ OCOR-Y

Fibrils -CH ₂ - halogen ^{+ Y - --------> F-CH} 2 -Y + X - Y ^- = NCO ^-, -OR ^-

Fibre-C = O + H ₂ NRY --------> FC = NRY

11.Dendrimer Type Nanotubes

The concentration of functional groups present on the surface of the nanotubes can be increased by transforming the nanotubes into sequentially produced multivalent reagents, and as a result, the number of specific functional groups increases in each step to form a dendrimer structure. The resulting dendrimer-type nanotubes are particularly useful as a solid support for covalently immobilizing proteins because these nanotubes can increase the density of the proteins immobilized on the nanotube surface. The present invention demonstrates that the high density of certain chemical functional groups, which has been difficult for conventional high surface area carbon, can be imparted to the surface of the high surface area particulate carbon.

Example  37

Preparation of Lysine Dendrimer

The reaction sequence is shown in FIG. 5.

Nα, Nε-di-t-boc-L-lysine N-hydroxysuccinimide ester (120 mg, 0.27 mmol) in a suspension in which amino fibril (90 mg) was suspended in sodium bicarbonate (5 ml, 0.2 M, pH 8.6). ) Was added to a solution in which dioxane (5 ml) was dissolved. The reaction mixture was stirred at rt overnight. The tert-butoxycarbonyl protected lysine fibril was thoroughly washed with water, methanol and methylene chloride and dried in vacuo. The tert-butoxycarbonyl protected lysine fibrils were then treated with trifluoroacetic acid (5 ml) in methylene chloride (5 ml) at room temperature for 2 hours. The product amino lysine fibril was thoroughly washed with methylene chloride, methanol and water and dried in vacuo. In the same procedure, secondary and tertiary lysine fibrils were also prepared. As a result of amino acid analysis, primary lysine fibrils contain 0.6 μmol of lysine per gram of fibrils, secondary lysine fibrils contain 1.8 μmol lysine per gram of fibrils, and tertiary lysine fibrils per 1 gram of fibrils It was found to contain 3.6 μmol of lysine.

Carboxy dendrimer fibrils can also be prepared using carboxy fibrils and aspartic acid or glutamic acid according to the same method.

Example 38

Preparation of carboxylate terminated dendrimers

Carboxylate terminated dendrimers with carbon nanotube (CN) cores are produced by continuous sequential coupling of aminobutylnitrilotriacetic acid (NTA), starting with NHS esters of chlorate oxidized carbon nanotubes.

Manufacture of NTA

NTA was prepared according to the method of the cited literature reference herein (E.Hochuli, H.Dobeli and A.Schacher, J.Chromatography. 411, 177-184 ( 1987)).

Manufacture of CN / NHS

CN / NHS was prepared according to the method of Example 20.

Manufacture of CN / NTA

0.4 g of NTA.HCl was dissolved in 25 ml of 0.2 M NaHCO ₃ (pH 8.1). The pH was again adjusted to 7.8 or lower by adding 1M NaOH. 0.5 g CN / NHS was added, the mixture was sonicated to disperse the CN, and the resulting slurry was reacted for 30 minutes with stirring. This slurry was filtered over a 0.45 μm nylon membrane and washed 2 × with pH 8.1 carbonate buffer and 2 × with deionized water on a filter. The modified CN was resuspended in 25 ml of MeOH again by sonication, filtered to give a solid cake, and finally dried in a vacuum desiccator.

Manufacture of CN / NTA / NTA

CN / NTA was first converted to NHS active esters. 0.396 g of CN / NTA was dried in a 90 ° C. oven for 30 minutes, then placed in a 100 ml RB flask containing 30 ml of anhydrous dioxane and washed with argon. 0.4 g of N-hydroxysuccinimide was added with stirring followed by 0.67 g of EDC with continued stirring for an additional hour. At this time, CN tended to aggregate together. Dioxane was decanted and the solid was washed 2 × with 20 ml of anhydrous dioxane. The solid was then washed with 20 ml of anhydrous MeOH, during which the aggregates were milled. The solid was filtered over 0.45 μm nylon membrane, resuspended in MeOH, filtered and washed with MeOH on the filter.

0.2 g of NTA was added to a 50 ml flask and dissolved in 10 drops of 1 M NaOH. After adding 20 ml of 0.2 M NaHCO ₃ (pH 8.1), both CN / NTA / NHS were added and the solution was sonicated lightly with a probe sonicator. The mixture was reacted at room temperature for 2.5 hours. The modified CN was filtered over a 0.45 μm nylon membrane, washed 2 × with carbonate buffer, resuspended in deionized water with sonication, filtered and washed with deionized water. It was then placed in a vacuum desiccator and dried.

Manufacture of CN / NTA / NTA / NTA

Addition of NTA was added according to the procedure described previously.

Manufacture of CN / NTA / NTA / NTA / NTA

Addition of NTA was added according to the procedure described previously.

Samples of each step (approximately 10 mg) in four steps of NTA addition were suspended in 10 ml of deionized water and sonicated, and then filtered over a 0.45 μm nylon membrane to form a felt-based mat. This piece of mat was stored in a vacuum desiccator and analyzed by ESCA for nitrogen (N) to obtain the relative content of NTA. The results were as follows.

N% by material ESCA

CN / NTA 0

CN / NTA / NTA 1.45

CN / NTA / NTA / NTA 1.87

CN / NTA / NTA / NTA / NTA 2.20

The ESCA results demonstrated that the increase content was fed in with each sequential step.

Example 39

Carbon Nanotube Dendrimers as Protein Supports

The density of proteins immobilized on carbon nanotubes can be greatly increased by using derivatized fibrils to produce dendrimers. Horseradish peroxidase (HRP) was immobilized on the dendrimer nanotubes according to the following method:

Sodium bicarbonate conjugated buffer (600 μl, 0.1 M, 0.9) for basic fibrillar (0.49 mg), amino fibrillar (0.32 mg), first-stage lysine fibrous (0.82 mg), second-stage lysine fibrous and three-stage lysine Sonicated at room temperature for 15 minutes). It was then incubated with HRP solution (490 ml, 5.6 mg / ml enzyme stock solution) in bicarbonate conjugation buffer for 19 hours at room temperature. The HRP immobilized fibres were thus washed with the following buffer (1 ml): 10 mM NaHCO ₃ buffer containing 0.9% NaCl, pH 9.5 (1 × wash buffer) 7 times, 0.1% Triton X-100 in 1 × wash buffer 5 times, 3 times 50% ethylene glycol in IX wash buffer. The activity of HRP was determined using 2,2-azinobis (3-ethylbenzothiazoline) -6-sulfonic acid diammonium salt (ABTS, 3 μl, mM stock solution) and hydrogen peroxide solution in glycine assay buffer (50 mM, pH 4.4). (10 μl, 10 mM stock solution) was used to analyze at 414 nm. The results are shown in the following table:

Fibrillar HRP nmol / Fibrillar g

Primary Fibrous Fiber 3.82

Fibrillar-NH ₂ 8.58

Fibrils-NH-Lys 28.09

Fibril-NH-Lys (Lys) ₂ 28.30

Fibril-NH-Lys (Lys) ₄ 46.28

12. Bifunctional Fibrillar

It has been found that by reacting functionally attached nanotubes, such as carboxy nanotubes with amino acids, one or more kinds of functional groups (eg, carboxyl and amino groups) can be introduced simultaneously on fibril. Such bifunctional fibrils can be used to immobilize a number of molecules, particularly at 1: 1 stoichiometry and similar ratios.

Example 40

Preparation of bifunctional fibril by adding lysine

N _α Synthesis of -CBZ-L-lysine Benzyl Ester

The reaction sequence is shown in FIG. 7. N _ε - (tert--hydroxy-carbonyl) -L- lysine (2g, 8.12mmol) was dissolved in a methanol (40ml) and water (40ml) and adjusted to pH 8 with triethylamine. To this mixture was added a solution of N- (benzyloxycarbonyl-oxy) succinimide in dioxane (2.4 g in 20 ml, 9.7 mmol) and the pH was maintained at 8-9 with triethylamine. The reaction mixture was stirred overnight. The solvent was removed by rotary evaporation the crude N _α -CBZ-N _ε - to give the (tert- butoxycarbonyl) -L- lysine. _{N α -CBZ-N ε - (} tert- butoxycarbonyl) -L- lysine was treated with 0.2M calcium carbonate (4ml), and the aqueous layer was removed to give a white solid. This solid was resuspended in N, N-dimethylformamide (40 ml) and benzyl bromide (1.16 ml). The reaction mixture was stirred at rt overnight. The reaction mixture was finished with ethyl acetate and water, and the organic layer was dried over magnesium sulfate. The solvent was removed and the crude N _α -CBZ-N _ε - to give (tert- butoxycarbonyl) -L- lysine benzyl ester, followed by purification by silica gel chromatography using as solvent a 25% hexane in ethyl acetate. Was added (tert- butoxycarbonyl) -L- lysine benzyl ester trifluoroacetic acid to (1g, 2.2mmol) at 0 ℃ - methylene chloride (10ml) of N _α N _ε -CBZ-. The reaction mixture was stirred at 0 ° C. for 10 minutes and then at room temperature for an additional 2.5 hours. Solvent was removed and crude product was obtained. Silicagel chromatography gave pure N _α -CBZ-L-lysine benzyl ester.

N _α Synthesis of -CBZ-L-lysine-benzyl ester fibrils

To a suspension in which carboxy fibrils (300 mg) were suspended in methylene chloride (18 ml) was added a solution of N _α -CBZ-L-lysine benzyl ester (148 mg, 0.32 mmol in 20 ml methylene chloride and 176 μl triethylamine). Then HOBT (43.3 mg, 0.32 mmol) and EDC (61.3 mg, 0.32 mmol) were added. The reaction mixture was stirred at rt overnight to afford crude product. This product fibrils were washed sufficiently with methanol, methylene chloride and water and dried in vacuo.

Bifunctional Fiber, Fiber-Lys (COOH) NH ₂ Synthesis of

To the N _α -CBZ-L-lysine benzyl ester fibril (113 mg) in methanol (4 ml) was added sodium hydroxide (1N, 4 ml) and the reaction mixture was stirred overnight. The product N _alpha -CBZ-L-lysine fibrils were sufficiently washed with water and methanol, and the fibrils were vacuum dried. Trimethyl silyl iodide (1 ml) was added to a suspension in which N _α -CBZ-L-lysine fibril (50 mg) was suspended in acetonitrile (4 ml). The mixture was stirred at 40 ° C. for 3 hours. The final bifunctional fibril was thoroughly washed with water, methanol, 0.5N sodium hydroxide, acetonitrile and methylene chloride. Amino acid analysis showed that lysine was 0.3 μmol per gram of fibril.

Hydroxy and carboxy (or amino) bifunctional fibrils can also be prepared using serine, threonine or tyrosine according to methods analogous to those described above. Thiolated, carboxy (or amino) difunctional fibrils can be prepared using cysteine. Carboxy and amino difunctional fibrils can be prepared using aspartic acid or glutamic acid.

Use of functionally attached nanotubes

Functionally attached graphite nanotubes are useful as solid supports in many biotech applications due to their high porosity, chemical and thermal stability, and large surface area. These nanotubes have been found to be able to withstand harsh chemical and thermal treatments and to be very easy to chemically functionalize.

For example, enzymes can be covalently immobilized on modified nanotubes while maintaining their biological activity. In addition, nanotubes are also suitable for use as a support for affinity chromatography in biomolecule separation. For example, enzyme inhibitors have been prepared on nanotubes by multistep synthesis such that immobilized inhibitors have access to macromolecules, resulting in specific reversible biological recognition between modified fibrils and proteins.

The hydrophobicity of the surface of the nanotubes is not sufficient to fix the protein at high density by adsorption. Thus, in order to increase the hydrophobicity of the nanotube surface and extend the hydrophobic environment from two to three dimensions, alkyl chains of various lengths were coupled to the nanotube surface. Proteins immobilized by adsorption on such alkyl nanotubes include trypsin, alkaline phosphatase, lipase and avidin. This enzymatic activity of the immobilized protein will be similar to the enzymatic activity of the free enzyme, provided that the catalytic efficacy of hydrolysis of each substrate in aqueous solution is demonstrated.

Phenyl-alkyl nanotubes were also prepared as alkyl nanotubes in which phenyl groups were added at the ends of the alkyl chains. This modification introduces an aromatic structure that interacts via π-π interactions with the amino acids phenylalanine, tyrosine and tryptophan of the protein. Adsorption of alkaline phosphatase and lipase on phenyl-alkyl nanotubes was comparable to that on C ₈ alkyl nanotubes.

Functionally attached fibrils have also been found to be useful as solid supports for protein synthesis.

1. Functionally attached nanotubes as solid supports of enzymes

Example 41

Enzyme Immobilization by Adsorption

Preparation of Alkyl Fibres

Alkyl fibrils are 10 mg of carboxy fibrils containing about 0.007 mmol (10 mg fibrils x 0.7 mmol -COOH / mg fibrils = 0.007 mmol) of -COOH groups, and EDC (1-ethyl-3- (3-dimethylamino). Propyl) carbodiimide) and 0.14 mmol of DMAP (4-dimethylaminopyridine) with 0.14 mmol of alkylamine in 1.5 ml DMF (N, N-dimethylformamide). The chemical scheme is as follows:

Fibrils-COOH + NH ₂ (CH ₂ ) _n CH ₂ R (R = H or OH) -------> Fibrils-CONH (CH ₂ ) _n CH ₂ R

This procedure produced several alkyl fibrils of different alkyl chain lengths (n = 5, 7, 9, 17; R = OH for n = 5). After the reaction was stirred at room temperature, the fibrils were washed thoroughly with 3 x 25 ml CH ₂ Cl ₂ , 3 x 25 ml MeOH and 3 x 25 ml dH ₂ O. Elemental analysis of the nitrogen content of the fibrils showed that the yield of the reaction was 65-100%.

Enzyme adsorption

Enzyme 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 3-4 hours and then washed 2-4 times with 5 mM sodium phosphate, pH 7.1. Alkaline phosphatase on C ₈ -fibers and C ₆ OH-fibers; Trypsin is on C _6- , C _8- , C ₁₀ -and C ₁₈ -fibrils; Lipase on C ₆ OH-, C _8- , C ₁₀ -and C ₁₈ -fibers; Avidin was immobilized on C ₈ -fibrils. The results are summarized in the following table:

enzyme μmol / g fibril mg / g fibril Lipase 6.8 816 Trypsin 1.7 40 Alkaline phosphatase 0.66 56 Avidin Not measured

The kinetic properties of the immobilized enzymes were found to be similar to those of the free enzymes as shown in the following table:

enzyme K _m (M) k _cat (s ^-1 ) k _cat / K _m (M ^-1 s ^-1 ) Lipase 40 x 10 ^-6 0.040 0.99 x 10 ³ Lipase-fibrils 36 x 10 ^-6 0.048 1.34 x 10 ³ Trypsin 1.2 x 10 ^-3 4.8 4.17 x 10 ³ Trypsin-Fibrillar 7.9 x 10 ^-3 19.1 2.43 x 10 ³

temperament:

Lipase 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorphin ester

Trypsin N-benzoyl-L-arginine-p-nitroanilide

Example  42

Esterification catalyzed by fibril-lipase (synthesis of ethyl butyrate)

Lipase was immobilized on C ₈ -alkyl fibrils following the procedure of Example 41. These lipase fibrils were first washed with dioxane, then with a mixture of dioxane and heptane, and finally with heptane to disperse the fibrils in heptane. To synthesize ethyl butyrate (a food additive providing pineapple-banana flavor), ethanol (0.4M) and butyric acid (0.25M) were mixed with 6.2 μm fibrillar fixed lipase in heptane. The reaction mixture was stirred at room temperature. The yield was 60% after 7 hours (yield was determined by measuring the ethanol concentration in the reaction mixture using conventional methods). The reaction and the results are as shown in FIG.

Example 43

Immobilization of Alkali Phosphatase on Phenyl-Alkyl Fibrils

Preparation of Phenyl-Alkyl Fibrils

Phenyl-alkyl fibrils were prepared according to two other reactions. In Reaction 1, 20 mg of carboxy fibril (containing about 0.014 mmol of -COOH group) was added to 0.28 mmol 4-phenylbutylamine, 0.28 mmol EDC, and 0.28 mmol DMAP in 1.5 ml DMF (N, N-dimethylformamide). Dimethylaminopyridine). In Reaction 2, 20 mg of carboxy fibrils were mixed with 0.28 mmol 6-phenyl-1-hexanol, 0.28 mmol DCC (1,3-dicyclohexylcarbodiimide) and 0.28 mmol DMAP in 1.5 ml of DMF. The reaction was stirred at room temperature overnight. The fibrils were then thoroughly washed with 3 x 25 ml CH ₂ Cl ₂ , 3 x 25 ml MeOH and 3 x 25 ml dH ₂ O.

Preparation of Alkali Phosphatase Fixed Fibril

0.5 mg of phenyl-alkyl fibrils were suspended in 400 μl of 0.05 M Tris pH 8.6 and sonicated for 20 minutes. 150 μl of alkaline phosphatase solution (1.67 mg / ml in 5 mM sodium phosphate buffer pH 7.0) was added to this fibril and the mixture was spun at room temperature for 2 hours and stored at 4 ° C. overnight. The fibrils were then washed twice with 600 [mu] l of 5 mM sodium phosphate buffer (pH7.1) and suspended in 200 [mu] l of the same buffer.

Quantification of Specificly Fixed Alkali Phosphatase by Measurement of Catalytic Activity

Alkali phosphatase emits a colored compound having an extinction coefficient of 18,200 M ⁻¹ cm ⁻¹ , which reacts with the substrate p-nitrophenyl phosphate to absorb light at 405 nm. Assay buffer conditions for this reaction were 10 mM Tris, 1 mM CaCl ₂ and 0.1 mM ZnCl ₂ , pH 8.4. This reaction was carried out in a 1 ml cuvette by mixing 5 μl of nitrophenyl phosphate stock solution (0.5 M in 33% DMSO in assay buffer) with 13 μg of alkaline phosphatase fibrils in 1 ml assay buffer. Absorbance increase at 405 nm was monitored via timed scan for 10 minutes. Enzyme activity (μM / min) was calculated from the initial slope using the extinction coefficient 18200M ^-1 cm ^-1 .

The alkaline phosphatase adsorbed to the phenyl fibril from reaction 1 had an activity of 6.95 µM / min of 13 µg raw island lactose. Alkali phosphatase adsorbed to phenyl fibril from reaction 2 had an activity of 2.58 μM / min per 13 μg fibrils. These results were divided by the activity of known alkaline phosphatase solutions, measured at 879.8 μM / min per 1 μM alkaline phosphatase under the same analytical conditions. μmol (or 20 mg).

Example 44

Immobilization of Lipase on Phenyl Alkyl Fibrils

Preparation of Lipase Fixed Fibril

0.5 mg of phenyl-alkyl fibrils were suspended in 50 μl of 5 mM sodium phosphate buffer, pH 7.1 and sonicated for 20 minutes. To the fibrils were added 350 μl of lipase solution (0.2 mM in 5 mM sodium phosphate buffer, pH 7.1) and the mixture was spun at room temperature for 5 hours and then stored at 4 ° C. overnight. The fibrils were then washed three times with 600 μl 5 mM sodium phosphate buffer (pH 7.1) and suspended in 200 μl of this buffer.

Specifically fixed Lipase  Quantitative analysis by measuring catalytic activity

Lipase reacts with substrate 1,2-o-dilauryl-rac-glycero-3-glutaric acid-resorufine ester (Boehringer Mannheim, 1179943) to absorb light at 572 nm with an absorption coefficient of 60,000 M ^-1 cm ^It produces colored compounds that are ^-1 . Analytical buffer conditions of this reaction were 0.1M KH ₂ PO ₄ , pH 6.8. This reaction was performed by mixing 5 μl of substrate stock solution (7.6 mM in 50% dioxane in Thesit) and 13 μg of alkaline phosphatase fibrils in 1 ml assay buffer in 1 ml cuvette. Absorbance increase at 572 nm was monitored via timed scan for 10 minutes. Enzyme activity (μM / min) was calculated from the initial slope using an extinction coefficient of 60,000 M ⁻¹ cm ⁻¹ .

The activity of the lipase adsorbed on the phenylalkyl fibrils from reaction 1 of Example 43 was 0.078 μM / min per 13 μg fibrils. The activity of the lipase adsorbed on the phenylalkyl fibrils from reaction 2 of Example 43 was 0.054 μM / min per 13 μg fibrils. These results were obtained by dividing the activity of known lipase solutions with known concentrations, measured at 1.3 μM / min per 1 μM lipase under the same assay conditions, with active lipase concentrations of 4.7 μmol (or 564 mg) and 3.3 μmol, Or 396 mg).

Example 45

Immobilization of Horseradish Peroxidase (HRP) on Amino Alkyl Modified Fibrils

Preparation of Fibrils with a Carboxylic Acid Functional Group (Carboxy fibrils)

A sample of 10.0 g of graphite fibril was mixed with a spatula in 450 ml of concentrated sulfuric acid and slurried, and then injected into a reactor flask equipped with an inlet / outlet and an upper stirrer. Under stirring and slow argon, a charge of 8.68 g of NaClO ₃ was added in portions over 24 hours at room temperature. Chlorine vapors generated during the entire experiment were removed from the reactor with an aqueous NaOH trap. At the end of the experiment, the fibril slurry was poured onto ice cubes and vacuum filtered. The filter cake was then transferred to Soxhlet Tim, washed with deionized water in a Soxhlet extractor and replaced with fresh water every few hours. Washing was continued until fresh deionized water was added until the fibril sample did not change the pH of the water. The carboxylated fibrils were collected by filtration and dried overnight under vacuum at 5 ° C. at 100 ° C. The yield was 10.0 g.

Preparation of HRP Fixed Fibril

Amino fibrils (1.2 mg) prepared from 1,6-diaminohexane were added to the conjugate buffer (0.1M NaHCO ₃ , 0.9% NaCl, pH 9.5) using the method of Example 27 and the suspension was added for 20 minutes. Ultrasonicated. The fibrils were then washed twice with conjugation buffer in an Eppendorf tube and suspended in 430 μL conjugation buffer. 50 μl portions of the suspension were mixed with 4.0 mg active HRP (Pierce, Rockford, IL) dissolved in 50 μl deionized water and the resulting suspension was spun at 4 ° C. overnight. HRP conjugated fibrils were thoroughly washed in an Eppendorf centrifuge tube with a combination of the following solutions; Conjugation buffer, wash buffer (20 mM KH ₂ PO ₄ , 0.45% NaCl, pH 6.2), wash buffer containing 0.03 to 0.1% Triton X-100 and wash buffer containing 50% ethylene glycol. As a control, the same manipulation with active HRP was performed with the base (non-derivatized) fibrils, indicating that the attachment of HRP to amino fibrils was actually specific covalent bonds.

Quantification of Specific Immobilized HRP by Measurement of Catalytic Activity

Thorough washing removed most of the nonspecifically bound enzymes. Fixed active HRP was quantitated via substrate conversion using H ₂ O ₂ and a chromogenic substrate 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), disoammonium salt (ABTS). The catalytic activity of HRP was monitored spectroscopically at 414 nm using 100 μM H ₂ O ₂ and 30 μM ABTS as substrates. In this preliminary study, the total content of enzyme bound to amino fibrils was 0.0230 μmol HRP per gram of fibrils. For comparison, the control (basic fibrils) had nonspecific binding of 0.0048 μmol HRP per gram of fibrils. Except for this, the content of covalently (specifically) attached HRP was 0.0182 μmol / g (fibrils).

Example 46

Affinity Chromatographic Separation of Alkaline Phosphatase (AP) and β-Galactosidase (βG) on Fibrillars with Immobilized Enzyme Inhibitor

alkali Phosphatase  Inhibitor Fibril  Produce

Preparation of AP inhibitor modified fibrils was based on the method of Brenna et al. (1975), Biochem J. , 151: 291-296. Carboxylated fibrils were used to prepare NHS ester fibrils as described in Example 50 below. NHS ester fibril (114 mg) was suspended in 4 ml acetone and 10 equivalents of tyramine (based on the calculation of 0.7 meq NHS ester per gram of fibril) was added. Anhydrous triethylamine (10 equiv) was added and the mixture was stirred at rt for 3 h. The tiraminil fibrils were first washed with acetone under vacuum in a sintered glass funnel and then thoroughly with deionized water.

4- (p-aminophenylazo) -phenylarsonic acid (66 mg) was suspended in 4 ml of 1N HCl. The suspension was cooled to 4 ° C. and slowly mixed with 0.36 ml of 0.5 M NaNO ₂ . After 15 minutes, an arsonic acid / NaNO ₂ mixture was added to the tiraminil fibrils suspended in 10 ml of 0.1 M NaCO ₃ (pH 10.0). The reaction mixture (pH about 10) was stirred at 4 ° C. overnight. The fibrils were then washed sequentially with 0.1 M Na ₂ CO ₃ (pH 10.0), 8 M guanidine HCl, 25 mM NaOH and water until the eluate became clear. Atomic absorption analysis of arsenic present in AP-inhibitor fibrils was performed at Galbrain Laboratories (Knoxville, TN). AP inhibitor fibrils with side chains containing one atom of arsenic were found to have an optional arsenic content of 0.4% in atomic absorption analysis. This indicates that about 10% of the calculated initial COOH groups were converted to AP inhibitors following this multistep synthesis. Based on the fibrous surface area, it means that there is one inhibitor molecule (enzyme binding site) per 500 m ² surface area.

Preparation of β-galactosidase inhibitor fibrils

p-amino-phenyl-β-D-thiogalactoside (TPEG) derivatized fibrils were prepared based on the method of Ullman, (1984) Gene , 29: 27-31. 2.24 mg of TPEG was added to 8 mg of carboxylated fibril in 0.2 ml of deionized water. The pH of this suspension was adjusted to 4.0 with 0.1 M HCl and 15 mg of EDAC was added. The mixture was stirred at pH 4.0 at room temperature for 3 hours. The reaction was stopped by high speed centrifugation in an Eppendorf tube and removal of liquid. The β-galactosidase inhibitor fibrils were washed five times by resuspending and centrifuging in deionized water.

Affinity separation

A mixture of E. coli-derived alkaline phosphatase (AP) (type III; Sigma Chemical Co., St. Louis, MO) and β-galactosidase (βG) (derived E. coli; Calbiochem., La Jolla, CA) Batch separation was carried out on AP-inhibitor fibrils or βG inhibitor fibrils in a microcentrifuge tube. For affinity separation, 1.0 ml of a loading buffer (20 mM Tris, 10 mM MgCl, 1.6 M NaCl, 10 mM cysteine, pH 7.4) solution containing both AP (typically about 10 units) and βG (typically about 280 units) was added. 0.8-1.0 mg of AP-inhibitor fibrils or βG inhibitor fibrils were added. The resulting suspension was gently vortexed and then spun for 2 hours at room temperature. After enzymatic binding, the fibrils were instantaneously centrifuged in a tabletop centrifuge to precipitate the fibrils, and the liquid phase containing the unbound enzyme was recovered and stored for enzymatic analysis. Washing with loading buffer (7 × 1.0 ml) was performed through repeated buffer addition, gentle vortexing, 15 minute rotation, flashing centrifugation and solvent removal with a parser pipette. After 7 washes, elution buffer appropriate for βG inhibitor fibrils (100 mM sodium borate, 10 mM cysteine, pH 10.0) or elution buffer appropriate for AP inhibitor fibrils (40 mM NaHPO ₄ , 10 mM Tris, 1.0 mM MgCl ₂ , 0.1 mM ZnCl ₂ , pH 8.4), and the same operation was repeated (5 x 1.0 mL).

All fractions (unbound enzymes, washes and eluates) were analyzed for AP and βG activity. Alkali phosphatase activity was determined by spectroscopically monitoring the hydrolysis rate of 500 μM p-nitro-phenylphosphate (PNPP) at 410 nm (Δε = 18,000 M ⁻¹ cm ⁻¹ ). Alkali phosphatase activity measurements were performed on 10 mM Tris, 1.0 mM MaCl ₂ and 0.1 mM ZnCl ₂ (pH 8.4). β-galactosidase was determined by spectroscopically monitoring the activity of an enzyme that hydrolyzes 2-nitro-galacto-β-D-pyranoside (ONPG). Initial rates of β-galactosidase catalyzed hydrolysis of 5.0 mM ONPG were measured at 405 nm (Δε = 3500 M ⁻¹ cm ⁻¹ ) at 10 mM Tris, 10 mM MgCl ₂ , 1.6M NaCl, 10 mM cysteine, pH 7.4.

For AP inhibitor fibrils and βG inhibitor fibrils, a mixture of AP and βG was added. To facilitate the measurement of specific binding capacity, the concentration of added enzyme was added in significant excess than the fixed inhibitor concentration. In the AP inhibitor fibrils 0.550 μmol AP / g fibrils were bound (as opposed to the nonspecific binding of 0.020 μmol βG / g fibrils). In the βG inhibitor fibrils, the binding capacity was determined to be 0.093 μmol βG / g fibrils (as opposed to the nonspecific binding of 0.012 μmol AP / g fibrils). The results of the affinity chromatography experiments are presented in FIGS. 9 and 10. AP inhibitor fibrils did not bind βG recognizably, but they bound AP, which was specifically eluted when 40 mM phosphate, a competitive inhibitor, was added to the buffer (FIG. 9). The fibrils derivatized with βG did not bind significant amounts of AP but bound βG, which was specifically eluted when the pH was raised to weaken enzyme-inhibitor binding (FIG. 10). These results indicate that the inhibitor has been successfully covalently attached to the fibrillar, the immobilized inhibitor can access large molecules, the inhibitor is available for specific enzyme binding, and the enzyme remains active when specifically eluted. Shows. As observed in FIG. 10, βG appears to be continuously leached from the βG inhibitor fibrils. This phenomenon was not observed in the case of AP inhibitor fibrils (Fig. 9), it is thought that the result is due to the weak innate enzyme-inhibitor affinity rather than the disadvantage of fibrils.

2. Nanotubes with Functional Groups as Solid Supports for Antibodies

Antibodies can be immobilized on functionally attached nanotubes, and these antibody nanotubes have been found to exhibit unique advantages in many applications due to their high surface area, electrical conductivity and chemical and physical stability on a weight basis. For example, antibody nanotubes can be used as affinity reagents for molecular separation. Antibody nanotubes are also useful for analytical applications, including diagnostic immunoassays such as ECL immunoassays.

Antibodies can be immobilized via covalent or non-covalent adsorption. Covalent immobilization can be achieved by a variety of methods, including: reductive amination of antibody carbohydrate groups, NHS ester activation of carboxylated fibrils (see Example 27 above) and reduced or maleimido modified antibodies; Reaction of thiolated or maleimidized fibril (see Examples 23 and 25 above).

The best way to attach antibodies to nanotubes depends on the use in which the complex is used. For separation, the preferred method is non-covalent adsorption because it appears to have the highest protein binding capacity in this method. Covalent methods may be preferred in methods involving ECL where the electrical conductivity of the fibrils may be important (the alkyl adduct is a weak electrical conductor, which is expected to insulate the fibrils). Reductive amination may be the best method for covalent attachment of an antibody to fibrillar, because using this method the antibody is oriented correctly such that the binding site of the antibody is directed outwards (opposite to the fibrillar).

3. NAD on functionally attached nanotubes ⁺ Addition of

It has been found that cofactors such as NAD ⁺ can be added to solid supports for biospecific affinity chromatography of proteins that bind enzyme cofactors and used as solid supports. For example, NAD ⁺ fibrils were used as a solid support for purification of dehydrogenases. The main advantage of the use of fibres is the large accessible surface area. Affinity substrates with large surface areas are preferred because of their high potential. The fibrils may be loosely dispersed or fixed in the column or mat.

Example 47

NAD ⁺  Affinity Chromatography Separation of Dehydrogenase from Fibrils

NAD ⁺ Preparation of fibril

Fibrils were oxidized according to Examples 14 and 15 to introduce carboxy groups. In a suspension suspended in fibrillar (31 mg) in sodium bicarbonate solution (3 ml, 0.2 M, pH 8.6) N ^6- [aminohexyl] carbamoylmethyl) -nicotinamide adenine dinucleotide lithium salt solution (in 5 ml sodium bicarbonate solution) 25 mg of Sigma) was added. The reaction mixture was stirred at rt overnight. The product fibrils were washed sufficiently with water, N, N-dimethylformamide and methanol. Elemental analysis data showed that the product fibril contains 130 mmol of NAD molecules per gram of fibril in nitrogen analysis, and 147 mmol of NAD molecules per gram of fibril in phosphorous analysis. Other NAD ⁺ analogs with linkers terminated with amino groups can also be used to make NAD ⁺ fibrils.

Affinity separation

NAD ⁺ immobilized fibrils (0.26 mg) and basic fibrils (0.37 mg) were sonicated at 40 ° C. for 30 minutes with 0.1% polyethylene glycol (PEG, MW1000) in sodium phosphate (1 ml, 0.1 M, pH7.1). After treatment, the cells were incubated at 40 ° C. for 30 minutes. This fibril suspension was centrifuged and the supernatant was removed. The fibrils were incubated for 90 minutes at 4 ° C. with a mixture of L-lactate dehydrogenase (LDH) in 0.1% PEG (1000) sodium phosphate buffer (250 μl, ratio of LDH solution to 0.1% PEG buffer is 1: 1). did. This mixture was then equilibrated for 30 minutes at room temperature. After incubation of the LDH and the fibrils, the fibrils were washed with 0.1% PEG (1000) (5 × 1000 μl) in sodium phosphate buffer and spun for 15 minutes per wash. LDH was eluted with a 5 mM NADH solution (5 mM 3 × 1000 μl) in 0.1% PEG (1000) sodium phosphate buffer. During each elution the fibrils were spun for 15 minutes. LDH activity in the eluate was analyzed by measuring the change in absorbance at 340 nm during pyruvate reduction. The assay mixture contained 0.1% PEG (1000) (980 μl), pyruvate (3.3 μl, 100 mM stock solution) and each elution fraction (16.7 μl) in sodium phosphate buffer. The enzymatic reaction is as follows:

LDH

Pyruvate + NADH -------------> Lactate Dehydrogenase + NAD ⁺

The analysis showed that the capacity of LDH on NAD ⁺ immobilized fibrils was 484 nmol per gram of fibrils and LDH on base fibrils (control) was 3.68 nmol per gram of fibrils. Nonspecific binding of LDH was 5.6%.

4. Nanotubes with functional groups attached as solid supports for protein synthesis

Example 48

Use of functional group-attached fibrils as solid support for peptide synthesis

To methylene chloride (20 ml) was added 1-ethyl-3- (3-dimethylaminopropyl) carbodii to a mixture of amino fibril (400 mg) and 4- (hydroxymethyl) phenoxyacetic acid suspension (255 mg, 1.4 mmol). Mid (EDC, 268 mg, 1.40 mmol) and 1-hydroxybenzotriazole hydrate (HOBT, 189 mg, 1.4 mmol) were added. The reaction mixture was stirred overnight at room temperature under argon gas. The product fibril was sufficiently washed with methylene chloride, methanol and water and then dried in vacuo to give a fibril. The fibrils were suspended in N, N-dimethylformamide (DMF, 2 ml) and methylene chloride (8 ml) in a suspension of N- (9-fluorenylmethoxycarbonyl) -O-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) were added. The reaction mixture was stirred overnight at room temperature and the product fibrils were treated with 20% piperidine in DMF (5 × 40 ml, immersed for 1 min for each treatment). The product fibril was sufficiently washed with DMF, water, sodium hydroxide (1N), methanol and methylene chloride. The product Fib-Handle-Ser (O +)-COOH (the ninhydrin test was positive) was vacuum dried. For the synthesis of dipeptides, arginine was added by repeating the same procedure. Amino acid analysis data of Fib-Handle-Ser (O +)-Arg (N ^ε -2,2,5,7,8-pentamethylchroman-6-sulfonyl) shows that 6.5 μmol of serine and 7.6 of arginine per gram of fibril It showed that it contained μmol. Any other peptide can be prepared by the same method.

5. Biotinylated Fiber  And Biotinylated Alkyl Fiber

It has been found that fibrillar surfaces can be functionally attached by biotinylation or alkylation and biotinylation. Fibrils containing this modification can then bind to any streptavidin conjugated material such as streptavidin beads and streptavidin enzymes.

Fibrils provide a very useful advantage as a solid carrier because of their large surface area. Beads that can be produced with strong magnetism are very useful for separation analysis. The biotinylated fibrils described herein combine the advantages of fibrils and the advantages of beads. Biotinylated alkyl fibrils are extensions of the same concept as alkyl fibrils but exhibit additional protein adsorption.

Streptavidin and biotin coated fibrils can be used in diagnostics and as capture agents in assays such as electrochemiluminescence assays.

A novel feature of the present invention is a bifunctional fibrils made by combining two solid carriers on one fibrils. Moreover, the published method increases the surface area of the beads and increases the fibrous magnetism.

Example 49

Preparation of Biotinylated Fibrils

Biotinylated fibrils were prepared by mixing 2.4 mg amino fibrils prepared as described in Example 16 with 9 mg NHS ester long chain biotin in 0.2M NaHCO ₃ buffer at pH 8.15. This mixture was spun for 4 hours at room temperature and washed twice with the same buffer.

Example 50

Preparation of Biotinylated Alkyl Fibrils

Biotinylated alkyl fibrils were prepared in a two step reaction. First, 4.25 mg of bifunctional fibrils (containing both amino and carboxy) and 25 mg of NHS ester long chain biotin were mixed. The fibrils were washed and dried in vacuo.

The second reaction consisted of 11 mg of EDC (1-ethyl-3,3-dimethylaminopropyl) carbodiimide), 10 mg of NH ₂ (CH ₂ ) ₇ CH ₃ in 7.5 mg of DMAP (4-dimethylaminopyridine) and 0.5 ml of DMF. It was mixed with and carried out. The mixture was stirred at rt overnight. The final biotinylated alkyl fibrils were washed with CH ₂ Cl ₂ , MeOH and dH ₂ O.

Example 51

Biotinylated fibrils as solid supports in the assay

Biotinylated fibrils can be used in assays involving formats requiring streptavidin-biotin or avidin-biotin interactions. Biotinylated fibrils can be further derivatized with, for example, streptavidin. Biotin covalently bound to fibrils (see Example 50) can form strong non-covalent interactions with streptavidin. Since streptavidin is a tetrameric protein with four equivalent binding sites, it is almost certain that streptavidin bound to biotinylated fibrils will have an empty binding site to which additional biotinylated reagents can bind. . That is, the biotinylated fibrils may be converted to streptavidin coated fibrils.

There are a number of assays that can be performed with such fibril-biotin-streptavidin (FBS) supports. For example, biotinylated anti-analyte antibodies can be captured on the FBS support (before or after the antibody binds to the analyte). Assays with biotinylated anti-analyte antibodies are very well established. Such assays include competitive assays in which the analyte of interest competes with a labeled analyte in binding to an anti-analyte antibody. Free (unbound) analytes and free (unbound) labeled analytes can be washed from antibody immobilized fibrils. The washing step depends on the fibrous fibers which are physically separated from the solution phase by general experiments involving centrifugation, filtration or by attraction to the magnets.

In addition to competitive assays, sandwich immunoassays can be performed on FBS supports. Sandwich immunoassays are known in the diagnostic arts. This assay consists of two antibodies, eg a "primary antibody" labeled with biotin and captured on a solid surface, and a "secondary" antibody labeled with a reporter group but not captured on a solid surface. It involves analytes that are bound at the same time. Such sandwich assays can be performed using fibrils as a solid capture support, where fibrils are captured as described in the preceding paragraph. According to this assay, the fibrils will be covalently bound to biotin, which is bound to streptavidin, which in turn binds to the biotinylated primary antibody, which is then analyzed for the analyte (which is present). Case) and this analyte will bind to a labeled secondary antibody.

Similarly, DNA probe assays can also be performed using FBS supports. Biotinylated single stranded DNA can be bound to the FBS support and competitive hybridization can occur between complementary single stranded analyte DNA molecules and complementary labeled oligonucleotides.

Another type of biotinylated fibrils, biotinylated alkylated fibrils can also be used in immunoassays and DNA probe assays. As described in Example 51, bifunctional fibrils can be modified by covalently attaching biotin to one type of functional group and an alkyl chain to another type of functional group. The alkylated, biotinylated fibrils obtained can be used for the adsorption (via the alkyl chain) of proteins as well as for specific binding (via biotin) with streptavidin or avidin.

Alkyl fibrils can be used with other solid supports, such as streptavidin coated magnetic beads. One advantage of these fibers on the beads is that they have a larger surface area (based on unit weight). In other words, if the fibrils can be attached to the outer surface of the magnetic beads, this can lead to a sharp increase in the surface area and thus an increase in the binding capacity of the beads. Thus, alkylated, biotinylated fibrils can be mixed with streptavidin coated beads to produce high affinity streptavidin (bead) -biotin (fibrin) interactions, thus fibrils having a very high surface area It is expected to produce coated beads. Since alkyl fibrils can bind to proteins by adsorption, fibrilla coated beads can be further derivatized by adsorption proteins, including streptavidin and antibodies. As mentioned above, streptavidin or antibody coated fibrils can be used in immunoassays and DNA probe assays. That is, fibrillar coated beads can improve the properties of the beads by dramatically increasing the surface area such that the number of beads required to provide the same result in a given assay is at least smaller.

6. 3D structure

Oxidized fibrils are more readily dispersed in an aqueous medium than unoxidized fibrils. Stable porous three-dimensional structures with mesopores and macropores (pore> 2 nm) are very useful as catalysts or chromatographic supports. Since the fibrils can be dispersed according to individual criteria, well dispersed samples stabilized by crosslinking may be used in the construction of the support. Functionally attached fibrils are ideal for this use because they are readily dispersed in aqueous or polar media and the functional groups provide crosslinking points. The functional groups also provide a point of support for the catalyst or chromatography site. The end result is a rigid three-dimensional structure with a total surface area accessible by the functional site that supports the active agent.

Typical uses of such supports in catalysis include their use as highly porous supports for metal catalysts loaded by impregnation, such as precious metal hydrogenation catalysts. Moreover, the ability to bind and fix the molecular catalyst to the support via functional groups along with the very high porosity of the structure makes it possible to carry out a uniform reaction in a heterogeneous manner. The bound molecular catalyst is essentially suspended in a continuous liquid phase, similar to a homogeneous reactor, in that it can take advantage of the selectivity and rate of ancillary to homogeneous reactions. However, binding to a solid support can facilitate the separation and recovery of the active ingredient and in most cases very expensive catalysts.

This stable rigid structure also allows for the attachment of suitable enantiomeric catalysts or selective substrates to the support to carry out very difficult reactions such as asymmetric synthesis or affinity chromatography. In addition, derivatization via metal-Pc or metal-porphyrin complexes allows the recovery of ligands bound to metal ions, and further any molecules bound to ligands via secondary derivatives. For example, when the three-dimensional structure of the fibrils to which the functional groups are attached is the electrode or part of the electrode and the functionalization is by adsorption of Co (II) Pc, the Co (III) of Co (II) in the presence of nicotinic acid The electrochemical oxidation of will produce a labile carboxylic acid and an unstable Co (III) -pyridyl complex. Attachment of suitable antigens, antibodies, catalytic antibodies or other site specific trapping agents will allow selective separation (affinity chromatography) of molecules that are otherwise very difficult to achieve. After washing the electrode to remove occluded material, the Co (III) complex containing the target molecule can be electrochemically reduced to recover the labile Co (II) complex. Ligands on Co (II) containing the target molecule can then be recovered by mass functional substitution of the labile Co (II) ligands, thereby making molecules very difficult or expensive to perform in other ways (eg, chiral Drug) can be separated and recovered.

It has conventionally been thought that the pores in a functional fibrous carbon fibrous mat are too small to deliver significant flow, and thus are not useful for flow through the electrode. In addition, there have been related problems when using particulate carbon or other carbon-based material (eg, reticulated glassy carbon (RVC)) as an electrode material. For example, porous electrode materials may not be formed in situ, are too densely packed and form voids or channels, and may exhibit very thin electrodes that are dimensional instability during changes in solvent and flow conditions. There was no. The use of functional fibrous carbon fibrils as electrodes in flow cells solved this problem.

The functionally attached carbon fibrils used as electrodes of the flow cell can be modified by surface treatment with an electroactive agent. In addition, these fibrils can also be transformed into non-electroactive materials used to function as catalysts or electrocatalysts or to inhibit the adsorption or unwanted reaction of materials from the stream.

This flow through the electrodes is useful for separation techniques such as electrochromatography, electrochemically modified affinity chromatography, electrosynthetic or electrochemically modified ion exchange chromatography. It can also be used in diagnostic devices for performing separation and / or analysis of materials captured on carbon fibrous mats.

Composite mats composed of functionally attached carbon fibrils and other fibers or particulates may also be used. Such fibers or particulates can be added to the suspension to alter the final porosity or conductivity of the carbon fibrous mat.

Example 52

Use of fibrils with iron phthalocyanine functional groups as electrodes in flow batteries

Graphite fibrils were modified by adsorbing iron (III) phthalocyanine-bis-pyridine (FePc-2Py) (Aldrich, 41,016-0). 0.403 g of fibrils and 0.130 g of FePc-2Py were added to 150 ml of anhydrous ethanol and sonicated for 5 minutes with a 450 Watt Branson probe sonicator. The resulting slurry was filtered over a 0.45 μm MSI nylon filter in a 47 mm Millipore membrane vacuum filter manifold, washed with water and then dried in a vacuum oven at 35 ° C. overnight. The final weight was 0.528 g, which showed significant adsorption. The remaining FeP-2Py was calculated by spectroscopic analysis of the filtrate.

5 mg of FePc-2Py modified fibril was dispersed by sonication in 10 ml of deionized water. This dispersion was deposited on a piece of 200 mesh stainless steel (SS) woven screen secured to a 25 mm membrane filter manifold and dried at room temperature. Fibrous mats supported on 0.5 inch diameter SS screen discs were cut using an arch punch.

The electrochemical flow cell placed a 13 mm diameter disc gold mesh (400 mesh, Ladd Industries) on the membrane support and as a working electrode in a three-electrode constant-potential circuit, Teflon® heat-shrink perfusion flowed through the wall of the filter holder for external connection. Electrical contact was made to the screen with a platinum wire, insulated with, and made into a 13 mm plastic Swinney type membrane filter holder. This gold mesh was held in place with the least amount of epoxy around the outer edges. A strip of gold foil was looped and placed in the bottom downstream section of the filter holder and connected to an insulated Pt lead lead for connection as a counter electrode of a three-electrode constant potential circuit. A 0.5 mm diameter silver wire ring electrochemically oxidized in 1 M HCl was placed on top of the filter holder with insulated lead to connect as a control electrode.

A 0.5 inch diameter disk-shaped FePc-2Py modified CN was installed in the flow cell and then connected to the appropriate lead of the EG & G PAR 273 constant potential. This flow cell was connected to a Sage syringe pump filled with 0.1 M KCl in 0.1 M potassium phosphate buffer at pH 7.0. The cyclic voltammogram (CV) was recorded under no flow (stop) and flow (0.4 ml / min) at a potential scan rate of 20 mv / sec (see FIG. 6). The CV was nearly identical in flow and under no flow, showing two sustained reversible oxidation and reduction waves consistent with the surface-defined FePc-2Py. The continuation of the redox peak under fluid flow conditions demonstrates that FePc-2Py is strongly bound to the carbon fibrils, and that the use of iron phthalocyanine modified fibrils function well for flow through the electrode material.

Another example of a three-dimensional structure is a fibrillar-ceramic composite.

Example 53

Preparation of Alumina-Fiber Fiber Composite (185-02-01)

1 g of nitric acid oxidized fibril (185-01-02) was highly dispersed in 100c deionized water using a U / S grinder. This fibril slurry was heated to 90 ° C., and a 0.04 mol aluminum tributoxide solution dissolved in 20 cc propanol was slowly added. After refluxing was continued for 4 hours, the condenser was removed for alcohol discharge. After 30 minutes, the condenser was again loaded and the slurry was refluxed at 100 ° C. overnight. As a result, a black sol having a 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 to 300 ° C. in air for 12 hours.

The alumina-fiber composite was examined by SEM. Micrographs of the cracked surface showed uniform distribution of fibrils in the gel.

Example 54

Preparation of Silica-Fiber Fiber Composite (173-85-03)

2 g of nitrate oxidized fibrils (173-83-03) were highly dispersed on 200c ethanol using ultrasound. To this slurry, a solution in which 0.1 mol tetraethoxysilane was dissolved in 50 cc ethanol was slowly added at room temperature, followed by 3 cc concentrated hydrochloric acid. The mixture was heated to 85 ° C. and kept at this temperature until the volume was reduced to 100 cc. The mixture was cooled down and left until a black solid gel formed. This gel was heated in air at 300 ° C.

Silica-fiber composites were examined by SEM. Micrographs of the cracked surface showed uniform distribution of fibrils in the gel.

Similar preparations can be made with other ceramics such as zirconia, titania, rare earth oxides as well as ternary oxides.

7. Polymer Bead  Merging of Graphite Nanotubes on Top

Polymer beads, such as those prepared by Dynal et al., In particular magnetic polymer beads containing Fe ₃ O ₄ cores, are used in a variety of applications in diagnostics. However, these beads have less surface area available than nanotubes. The functional groups attached fibrils can be incorporated onto the surface of these beads, and such polymer / fibrils composites can be used as solid supports for separation or analysis (eg, for electrochemiluminescence analysis, for enzyme immobilization).

Example 55

Attachment of functional groups attached fibrils to functional group attached beads

7.5 mg of magnetic tosyl activated Dynabeads M-450 (30 mg / ml) beads (Dynal, Oslo, Norway) was washed three times with 0.1 M sodium phosphate buffer pH 7.5. Next, 0.9 ml of 0.1 M sodium phosphate buffer (pH 8.4) was added to the beads, and 0.1 ml of amine fibril was added. This mixture was spun at room temperature for 16-24 hours.

Upon microscopic examination, a fibrous mass with beads on the fibrous surface was clearly visible.

Those skilled in the art will appreciate that the desired functionalization results can be obtained by repeating any of the methods disclosed in Examples 1-55 above with single-walled carbon nanotubes of less than 5 nanometers in diameter in accordance with conventional experiments. .

Example  56

Methylene blue absorption proof

The University of Kentucky (SWNT) was treated with 6M HCl for 72 hours to remove any metal impurities. The acid washed nanotubes were dispersed in deionized water by sonication and filtered. This treatment was repeated four times and washed to neutral pH.

This SWNT was resuspended in deionized water and dispersed by sonication. 2 × serial dilutions were made in 8 PP centrifuge tubes. An amount of methylene blue solution was added and the centrifuge tube was placed on a rotator and shaken for 4 hours. The centrifuge tube was centrifuged for 4 minutes to obtain pelleted SWNTs at the bottom of the tube. The tube with the highest concentration of SWNTs was clear in filtrate, and the two tubes with the lowest concentration of SWNTs were similar in color to the control MB solution. Six tubes with medium SWNT concentrations increased blue with decreasing concentrations.

Proof of Iron Phthalocyanine Absorption

The University of Kentucky (SWNT) was treated with 6M HCl for 72 hours to remove any metal impurities. The acid washed nanotubes were dispersed in deionized water by sonication and filtered. This treatment was repeated four times and washed to neutral pH.

7 mg of iron phthalocyanine-bispyridine (FePc-2Py) was sonicated in 40 ml ethanol. 22 mg of SWNTs were added and sonication was continued for 5 minutes. The solution was cooled and sonication was repeated. The hot solution was filtered dry over 0.45 micron PVDF membrane and washed with fresh EtOH. Filtered to dryness. Dry weight = 27 mg. 5 mg of FePc-2Py was adsorbed onto SWNTs.

SWNT / FePC attached filter membrane was added to 40 ml of deionized water and sonicated to dissociate SWNTs from the membrane. The membrane was removed from the suspension and 20 mg of Whatman # 42 filter paper was added. Sonication was performed at 2 × 5 ′ under high power (40% workload cycle) to pulp the WH42 paper to act as a binder of SWNTs. The suspended material was filtered over a 0.45 micron PVDF filter and washed 3 × with deionized water. Filtered to dryness. Dry for 1 hour on a 150 ° C. hotplate. The dried SWNT / WH42 mat was separated from the membrane as a pure mat (36 mm diameter; about 25 microns thick). Sheet resistivity was measured at 1500 ohms / area. The final weight of the mat was 45 mg; Weight of WH42 component = 20 mg; SWNT wt = 22 mg; FePc-2Py wt was estimated to be 5 mg adsorbed on the surface of SWNTs.

Electrochemistry of FePc-2Py Adsorbed on SWNTs

The electrode was fabricated by pressing a mat zone between 400x400 mesh SS mesh for use as a current collector. One edge of the SS mesh was attached to a Cu wire inserted in a glass pipette. Any exposed Cu was insulated with epoxy. This electrode was heated at 100 ° C. for 1 hour to cure 5 minute epoxy.

The electrodes were examined with cyclic voltammetry for the SS mesh counter electrode and Ag / AgCl reference electrode. The electrolyte used isotonic borate buffered saline. Two peaks were observed, one near 0.04, but unclear due to the H2 emission current. The second peak was centered around about +0.1 V vs Ag / AgCl. The peak current of the peak centered at +0.1 V vs Ag / AgCl was linear with scan rate, which coincided with the surface-limited electroactive agent. This cyclic voltammetry was also repeated, demonstrating that FePc-2PY is strongly bound to the electrode and not lost to the electrolyte.

As illustrated by the foregoing detailed description and examples, the present invention is applicable to the manufacture of various functionally attached nanotubes and their use.

The terms and expressions used are for the purpose of description and not of limitation, and the use of such terms or expressions is not intended to exclude any equivalent of the described features set forth as part of the present invention and, therefore, It is to be understood that various variations are possible within the scope.

Claims (47)

Composite of material represented by the formula: [C _n H _L ] -R _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, R are the same each, SO ₃ H, COOH, NH ₂ , OH, R'CHOH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR') _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR' ₂ , Hg-X, TlZ ₂ and Mg-X, y is an integer of 3 or less, R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cycloaryl or poly (alkylether), R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, Z is a carboxylate or trifluoroacetate. Composite of material represented by the formula: [C _n H _L ] -R _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, R is the same or different, respectively, SO ₃ H, COOH, NH ₂ , OH, R'CHOH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR' ) _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR' ₂ , Hg-X, TlZ ₂ and Mg-X, y is an integer of 3 or less, R 'is selected from hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cycloaryl or poly (alkylether), R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, Z is a carboxylate or trifluoroacetate, In addition, if each R is oxygen-containing, it should not be COOH. Composite of material represented by the formula: [C _n H _L ]-[R'-A] _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, Each R 'is alkyl, aryl, cycloalkyl, aralkyl, cycloaryl or poly (alkylether), A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, nucleotide, oligonucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-N (R ') ₂ , R'SH, R'CHO, R'CN, R'X , R'N + (R ') 3 X -, R'SiR' 3, R'Si- (OR ') y -R' 3-y , R'Si- (O-SiR ' ₂ ) -OR', R'-R ", R'-N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w- H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Is selected from y is an integer of 3 or less, R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, Z is a carboxylate or trifluoroacetate, w is an integer greater than 1 and less than 200. The compound of claim 3 wherein A is

or

ego, R 'is H And Y is an amino acid selected from the group consisting of lysine, serine, threonine, tyrosine, aspartic acid and glutamic acid. Composite of material represented by the formula: [C _n H _L ]-[X'-A _a ] _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, a is an integer less than 10, Each A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, nucleotide, oligonucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-N (R ') ₂ , R'SH, R'CHO, R'CN, R'X , R'N + (R ') 3 X -, R'SiR' 3, R'Si- (OR ') y -R' 3-y , R'Si- (O-SiR ' ₂ ) -OR', R'-R ", R'-N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w- H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Is selected from y is an integer of 3 or less, R 'is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, X 'is a polynuclear aromatic, heteropolynuclear aromatic or metal heteropolynuclear aromatic moiety or a planar metal tetrathiooxalate, Z is a carboxylate or trifluoroacetate, w is an integer greater than 1 and less than 200. Reacting the surface carbon of the carbon nanotubes in the presence of a compound represented by the formula R′CH ₂ OH with a free radical initiator under conditions sufficient to form a functionally attached nanotube represented by the formula To prepare a composite of a material represented by the formula: [C _n H _L ]-[CH (R ') OH] _m In this formula, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube having a diameter less than 5 nanometers, n is an integer, L is a number less than 0.1 n, and m is a number less than 0.5 n. And R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cycloaryl or poly (alkylether). 7. The process of claim 6 wherein the free radical initiator is benzoyl peroxide. (a) The surface carbon of the carbon nanotubes is represented by the formula [C _n H _L ] -R _m (wherein each R is the same, SO ₃ H, COOH, NH ₂ , OH, CH (R ′) OH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR') _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", At least one suitable condition under conditions sufficient to form a substituted single-walled carbon nanotube represented by Li, AlR ' ₂ , Hg-X, TlZ ₂ and Mg-X, y being an integer of 3 or less). Reacting with a reagent; and (b) reacting such substituted single-walled carbon nanotubes [C _n H _L ] -R _m with at least one suitable reagent under conditions sufficient to form a functionally attached single-walled carbon nanotubes represented by the formula: Including the steps, Method for preparing a composite of the material represented by the formula: [C _n H _L ] -A _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, Each A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, oligonucleotide, nucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-N (R ') ₂ , R'SH, R'CHO, R'CN, R'X , R'SiR '3, R'N + (R') 3 X -, R'-R ", R'N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w -H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Is selected from R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, Z is a carboxylate or trifluoroacetate, w is an integer greater than 1 and less than 200. (a) The surface carbon of the carbon nanotubes is represented by the formula [C _n H _L ] -R _m (wherein each R is SO ₃ H, COOH, NH ₂ , OH, CH (R ') OH, CHO, CN, COCl , Halide, COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR') _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR Reacted with at least one suitable reagent under conditions sufficient to form a substituted single-walled carbon nanotube represented by ' ₂ , Hg-X, TlZ ₂ and Mg-X, y being an integer of 3 or less). Step; and (b) reacting such substituted single-walled carbon nanotubes [C _n H _L ] -R _m with at least one suitable reagent under conditions sufficient to form a functionally attached single-walled carbon nanotubes represented by the formula: Including the steps, Method for preparing a composite of the material represented by the formula: [C _n H _L ] -A _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, Each A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, oligonucleotide, nucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-N (R ') ₂ , R'SH, R'CHO, R'CN, R'X , R'SiR '3, R'N + (R') 3 X -, R'-R ", R'N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w -H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Is selected from R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, Z is a carboxylate or trifluoroacetate, w is an integer greater than 1 and less than 200. Formula [C _n H _L ] -R _m , wherein each R is the same, SO ₃ H, COOH, NH ₂ , OH, CH (R ′) OH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR') _y -R ' _{3 -y} , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR' ₂ , Hg-X, A substituted single-walled carbon nanotube represented by TlZ ₂ and Mg-X, y is an integer of 3 or less), under conditions sufficient to form a functionally attached single-walled carbon nanotube represented by the following formula: Reacting with one suitable reagent, Method for preparing a composite of the material represented by the formula: [C _n H _L ] -A _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, Each A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, oligonucleotide, nucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-N (R ' _{) 2, R'SH, R'CHO, R'CN} , R'X, R'SiR '3, R'N + (R') 3 X -, R'-R ", R'N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w -H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R ' And

Is selected from R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, Z is a carboxylate or trifluoroacetate, w is an integer greater than 1 and less than 200. Formula [C _n H _L ] -R _m , wherein each R is the same, SO ₃ H, COOH, NH ₂ , OH, CH (R ′) OH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR') _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR' ₂ , Hg-X, Substituted single-walled carbon nanotubes represented by TlZ ₂ and Mg-X, y being an integer of 3 or less), under conditions sufficient to form a functionally attached single-walled carbon nanotube represented by the following formula: Reacting with at least one suitable reagent, Method for preparing a composite of the material represented by the formula: [C _n H _L ] -A _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, Each A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, oligonucleotide, nucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-N (R ') ₂ , R'SH, R'CHO, R'CN, R'X , R'SiR '3, R'N + (R') 3 X -, R'-R ", R'N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w -H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Is selected from R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, R "is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, Z is a carboxylate or trifluoroacetate, w is an integer greater than 1 and less than 200. Formula [C _n H _L ]-[R'-R] _m (wherein each R is the same, SO ₃ H, COOH, NH ₂ , OH, CH (R ') OH, CHO, CN, COCl, halide , COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR') _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR' ₂ To form a functionally-attached single-walled carbon nanotube represented by the following formula: a substituted single-walled carbon nanotube represented by Hg-X, TlZ ₂ and Mg-X, and y is an integer of 3 or less). Reacting with at least one suitable reagent under conditions sufficient for Method for preparing a composite of the material represented by the formula: [C _n H _L ]-[R'-A] _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, R 'is alkyl, aryl, cycloalkyl, aralkyl, cycloaryl or poly (alkylether), X is a halide, Each A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, oligonucleotide, nucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-NH ₂ , R'SH , R'CHO, R'CN, R'X, R'SiR ' ₃ , R'R ", R'-N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w -H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Is selected from R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, Z is a carboxylate or trifluoroacetate. Adsorbing at least one suitable macrocyclic compound on the surface of the single-walled carbon nanotubes under conditions sufficient to form a functionally attached single-walled carbon nanotubes represented by the formula: Method for preparing a composite of the material represented by the formula: [C _n H _L ]-[X'-R _a ] _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, a is an integer less than 0 or 10, Each R is SO ₃ H, COOH, NH ₂ , OH, CH (R ') OH, CHO, CN, COCl, halide, COSH, SH, COOR', SR ', SiR' ₃ , Si- (OR ') _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR' ₂ , Hg-X, TlZ ₂ and Mg-X, y is an integer of 3 or less, R 'is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, X is a halide, X 'is a multinuclear aromatic, heteronuclear aromatic or metal heteropolynuclear aromatic moiety or a planar metal tetrathiooxalate, R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, Z is a carboxylate or trifluoroacetate. (a) Formula [C _n H _L ]-[X'-R _a ] _m (wherein each R is SO ₃ H, COOH, NH ₂ , OH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR', SiR ' ₃ , Si- (OR') _y -R ' _3-y , Si- (O-SiR' ₂ ) -OR ', R ", Li, AlR' ₂ , Hg-X, TlZ At least one suitable macrocyclic compound on the surface of the single-walled carbon nanotubes under conditions sufficient to form substituted single-walled carbon nanotubes, selected from ₂ and Mg-X and y is an integer of 3 or less). Adsorbing; (b) reacting the substituted nanotubes [C _n H _L ]-[X'-R _a ] _m with at least one suitable reagent under conditions sufficient to form a functionally attached single-walled carbon nanotube represented by the formula Including the step of, Method for preparing a composite of the material represented by the formula: [C _n H _L ]-[X'-A _a ] _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, a is an integer less than 10, Each A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, oligonucleotide, nucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-NH ₂ , R 'SH, R'CHO, R'CN, R'X, R'SiR' ₃ , R'R ", R'-N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w -H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Is selected from R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, X 'is a multinuclear aromatic, heteronuclear aromatic or metal heteropolynuclear aromatic moiety or a planar metal tetrathiooxalate, Z is carboxylate or trifluoroacetate, w is an integer greater than 1 and less than 200. Formula [C _n H _L ]-[X'-R _a ] _m (wherein each R is SO ₃ H, COOH, NH ₂ , OH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR ', SiR' ₃ , Si- (OR ') _y -R' _3-y , Si- (O-SiR ' ₂ ) -OR', R ", Li, AlR ' ₂ , Hg-X, TlZ ₂ and Mg At least one suitable reagent under conditions sufficient to form a substituted single-walled carbon nanotube, wherein the substituted single-walled carbon nanotubes are selected from -X and y is an integer of 3 or less) Reacting with, Method for preparing a composite of the material represented by the formula: [C _n H _L ]-[X'-A _a ] _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, a is an integer less than 10, Each A is OY, NHY,

-CR ' ₂ -OY, N = Y,

Or C = Y, Y is a suitable functional group of a transition state analog of a protein, peptide, amino acid, enzyme, antibody, oligonucleotide, nucleotide, antigen or enzyme substrate, enzyme inhibitor or enzyme substrate or R'-OH, R'-NH ₂ , R'SH , R'CHO, R'CN, R'X, R'SiR ' ₃ , R'R ", R'-N-CO, (C ₂ H ₄ O) _w -H,-(C ₃ H ₆ O) _w -H,-(C ₂ H ₄ O) _w -R ', (C ₃ H ₆ O) _w -R', R 'and

Is selected from R 'is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, R ″ is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, X 'is a multinuclear aromatic, heteronuclear aromatic or metal heteropolynuclear aromatic moiety or a planar metal tetrathiooxalate, Z is carboxylate or trifluoroacetate, w is an integer greater than 1 and less than 200. Reacting the surface carbon of the carbon nanotubes with at least one suitable reagent under conditions sufficient to form a functionally attached single-walled carbon nanotube represented by the formula [C _n H _L ] — (COOH) _m ; And Reacting the functional group-attached single-walled carbon nanotubes with a compound having two or more amino groups under conditions sufficient to form a functional-grouped single-walled carbon nanotube represented by the following formula: Method for preparing a composite of the material represented by the formula:

In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, R 'is alkyl, aryl, cycloalkyl or cycloaryl. Reacting surface carbon with at least one enzyme capable of accepting single-walled carbon nanotubes as a substrate and performing a chemical reaction in an aqueous suspension under the conditions under which at least one enzyme can carry out a chemical reaction, represented by the following formula A method for preparing a composite of a material represented by the following formula, comprising the step of producing a composite of the material: [C _n H _L ] -R _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, m is a number less than 0.5n, R are the same each, SO ₃ H, COOH, NH ₂ , OH, CH (R ') OH, CHO, CN, COCl, halide, COSH, SH, COOR', SR ', SiR' ₃ , Si- (OR ') _y -R' _3-y , Si- (O-SiR ' ₂ ) -OR', R ", Li, AlR ' ₂ , Hg-X, TlZ ₂ and Mg-X, y is an integer of 3 or less, R 'is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cycloaryl, R "is fluoroalkyl, fluoroaryl, fluorocycloalkyl or fluoroaralkyl, X is a halide, Z is a carboxylate or trifluoroacetate. 18. The method of claim 17, wherein R _m is -OH and the enzyme is a cytochrome p450 enzyme or peroxidase. Reacting the surface carbon of the carbon nanotubes with nitric acid and sulfuric acid to form nitrated single wall carbon nanotubes; And Reducing the nitrated single-walled carbon nanotubes as described above to obtain a compound represented by the following formula Including, a method of producing a composite of the material represented by the following formula: [C _n H _L ]-(NH ₂ ) _m In this equation, the carbon atom C _n is the surface carbon of a substantially cylindrical single-walled carbon nanotube, less than 5 nanometers in diameter, n is an integer, L is a number less than 0.1n, and m is a number less than 0.5n. Single-walled carbon nanotubes of less than 5 nanometers in diameter, including contacting single-walled carbon nanotubes less than 5 nanometers in diameter with an effective amount of reactants capable of uniformly displacing functional groups on the surface of the single-walled carbon nanotubes. Method of uniformly replacing the surface of the tube with a functional group. The method of claim 20, wherein the reactant is phthalocyanine. 21. The method of claim 20 wherein the reactant is nickel (II) phthalocyanine tetrasulfonic acid (tetrasodium salt) or 1,4,8,11,15,18,22,25-octabutoxy-29H, 31H-phthalocyanine. How to be. Surface modified less than 5 nanometers in diameter prepared by a method comprising contacting a single-walled carbon nanotube less than 5 nanometers in diameter with an effective amount of reactants capable of substituting functional groups on the surface of the single-walled carbon nanotubes. Single wall carbon nanotubes. 24. The surface modified single wall carbon nanotubes of claim 23, wherein the reactant is phthalocyanine. 24. The method of claim 23 wherein the reactant is nickel (II) phthalocyanine tetrasulfonic acid (tetrasodium salt) or 1,4,8,11,15,18,22,25-octabutoxy-29H, 31H-phthalocyanine. Surface modified single-walled carbon nanotubes. Contacting the protein with a single-wall carbon nanotube having a NHS ester group and less than 5 nanometers in diameter, under conditions sufficient to form a covalent bond between the NHS ester and the amine group of the protein. How to bind to. An electrode containing functionally attached single-walled carbon nanotubes less than 5 nanometers in diameter. 28. The electrode of claim 27, wherein the electrode is a porous flow through electrode. The electrode of claim 27, wherein the functionally attached single-walled carbon nanotubes are phthalocyanine substituted nanotubes. A plurality of functional group attachments wherein the functionally attached single-walled carbon nanotube network contains at least two functional fibrils bonded by at least one linker moiety to the functional group, wherein the linker addition is bifunctional or multifunctional. Porous material containing a single-walled carbon nanotube network. Physically or chemically modifying the surface carbon of single-walled carbon nanotubes less than 5 nanometers in diameter with at least one suitable reagent under conditions sufficient to form a functionally attached single-walled carbon nanotubes; Immobilizing a material to which the solute of interest is bound on the single-walled carbon nanotube to which the functional group is attached; And Exposing the substituted single-walled carbon nanotubes to the fraction containing the solute under conditions sufficient to bind the solute to the material immobilized on the functionally attached single-walled carbon nanotube. Method of separating the solute of interest. 32. The method of claim 31 wherein the solute of interest is a protein. 32. The method of claim 31, further comprising recovering the functionally attached single wall carbon nanotubes. 32. The method of claim 31, wherein the functionally attached single wall carbon nanotubes are in the form of a porous mat. 32. The method of claim 31, wherein the functionally attached single-walled carbon nanotubes are in the form of packed columns. The method of claim 31, wherein the binding is reversible. 32. The method of claim 31, wherein the bond is an ionic interaction. 32. The method of claim 31, wherein the bond is a hydrophobic interaction. 32. The method of claim 31, wherein the binding is via specific molecular recognition. A polymer bead containing substantially spherical beads of less than 25 microns in diameter, wherein a plurality of functionally attached single-walled carbon nanotubes of less than 5 nanometers in diameter are joined. 41. The polymer bead of claim 40 wherein the bead is magnetic. Physically or chemically modifying the surface carbon of single-walled carbon nanotubes less than 5 nanometers in diameter with at least one suitable reagent under conditions sufficient to form a functionally attached single-walled carbon nanotubes; Immobilizing a biocatalyst capable of catalyzing the reaction on the functionally attached single-walled carbon nanotube; And Contacting the functionally attached single-walled carbon nanotube with the reactant under conditions sufficient to convert the reactant into a product. A method of catalyzing a reaction to convert at least one reactant into at least one product, including. 43. The method of claim 42, further comprising recovering the functionally attached single-walled carbon nanotubes after the reaction is complete. 43. The method of claim 42, wherein the functionally attached single wall carbon nanotubes are in the form of a porous mat. 43. The method of claim 42, wherein the functionally attached single-walled carbon nanotubes are in the form of packed columns. Attaching the terminal amino acid of the peptide to a single-walled carbon nanotube less than 5 nanometers in diameter via a reversible linker. 47. The method of claim 46, wherein the linker is 4- (hydroxymethyl) phenoxyacetic acid.

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