CN101432227A

CN101432227A - Functionalized single walled carbon nanotubes

Info

Publication number: CN101432227A
Application number: CNA200580028660XA
Authority: CN
Inventors: R·霍奇; A·费希尔; H·滕嫩特
Original assignee: Hyperion Catalysis International Inc
Current assignee: Hyperion Catalysis International Inc
Priority date: 2004-06-23
Filing date: 2005-06-23
Publication date: 2009-05-13
Also published as: WO2006130150A9; AU2005332329A1; KR20070030282A; WO2006130150A3; JP2008513318A; EP1776125A4; CA2571575A1; EP1776125A2; WO2006130150A2

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.

Description

Functionalized single walled carbon nanotubes

Cross reference with related application

[0001] the application is the further part application of the U. S. application sequence number [proxy's summary 100647-3583] of submission on April 30th, 2004, its 100647-3583 is the U. S. application sequence number of submitting to present 6 days March in 1997 of having abandoned 08/812,856 follow-up, and 08/812, the 856th, the U. S. application sequence number 08/611 that submit to the 6 days March in 1996 of now having abandoned, 368 follow-up, 08/611,368 provisional application that required submit in 1996 9 about 25 days number 60/037,238 right of priority. the application also is the U. S. application sequence number of submitting on June 6th, 2,000 09/594,673 further part application, 09/594,673 is U. S. applications of submitting on December 8th, 1994 number 08/352,400, be U.S.6 now, a part of 203,814.All be incorporated herein in this every piece of content by reference with above-mentioned application.

Invention field

[0002] the present invention relates broadly to graphite nanotubes, it comprises piped soccerballene (be commonly referred to as " filter line organ pipe ") and protofibril, it replaces by chemistry or is functionalized by the absorption of functionalized position. more specifically, the present invention relates to Single Walled Carbon Nanotube, it evenly or is anisotropically replaced or has adsorbed some ring compound by chemical part, forms interconnection by these functionalized protofibril and chelation structure that form. the present invention also relates to guide the method for these compounds to the surface of Single Walled Carbon Nanotube.

Background of invention

[0003] the invention belongs to the graphite protofibril field of submicron, sometimes being called vapor-grown carbon fiber. carbon fibrils is helminthoid carbon deposits, its diameter is less than 0.1 μ, preferably less than 0.5 μ, even being more preferably less than 0.2 μ. they exist with various forms, by various carbonaceous gas catalytic decomposition make in the metallic surface.Since electron microscope has been arranged, just almost can observe these worm sample carbon deposits. reconnoitre preferably in early days and with reference to being by Baker and Harris, Chemistry and Physics of Carbon,Walker and Thrower ed., Vol.14,1978, p.83 find, be introduced into by reference at this. also be found in Rodriguez, N., J. Mater.Research.Vol.8, p.3233 (1993) are incorporated herein by reference.

[0004] in 1976, people such as Endo (referring to Obelin, A.and Endo, M., J.of Crystal Growth.Vol.32 (1976), pp.335-349 is by with reference to being incorporated herein) have explained the basic mechanism of this carbon fibrils growth.This process is begun by the metal catalytic particle, in the presence of the gas of hydrocarbon-containiproducts, becomes supersaturation in carbon. according to people such as Endo, extruded columned orderly graphite nuclei, immediately by outer field pyrolytic deposition graphite institute dressing.These protofibril diameters with pyrolytic outer coatings have surpassed 0.1 μ, more typically are 0.2 to 0.5 μ.

[0005] nineteen eighty-three, by the U.S.4 with reference to the Tennent that is incorporated herein, 663,230 are obtaining success aspect the cylindric orderly graphite nuclei of growth, and it is not polluted by RESEARCH OF PYROCARBON.Therefore the invention of Tennent provides diameter littler protofibril, and typically 35 to 700 (0.0035 to 0.070 μ), and obtained orderly, " the growth sample " graphite surface.Also grow the less protofibril carbon of perfect structure degree, but also do not contained the RESEARCH OF PYROCARBON skin.

[0006] functionalized in this application protofibril, filter line organ pipe and nanotube are to differentiate with the commercial available long carbon fiber as strengthening material. the protofibril limited with having required more greatly but inevitably long-width ratio is opposite, and the long-width ratio of long carbon fiber (L/D) is at least 10 ⁴, be generally 10 ⁶Or it is bigger.Long stapled diameter is also much bigger than protofibril, and is general〉1.0 μ, 5 to 7 μ typically.

[0007] long carbon fiber is by the organic precursor fiber, and normally the pyrolysis of artificial silk, polyacrylonitrile (PAN) and pitch is made.Therefore, can comprise heteroatoms in their structure." as prepared " and the graphite properties of long carbon fiber have nothing in common with each other, but they may be used to follow-up greying step.The degree of crystallinity of degree of graphitization, orientation and graphite plane different, if exist, then heteroatomic may exist and even the office of substrate diameter can make macrofiber become the more weak precursor of nanofiber chemistry to difference.

[0008] U.S.4 of Tennent, 663,230 carbon fibrils of describing, do not contain long hot carbon outer coatings and have parallel with the protofibril axle basically a plurality of graphite skins. like this, their feature just is to have the c-axle, this tangent line with the graphite buckled layer is vertical, and the axis of a cylinder with them is vertical basically.Their general diameters are no more than 0.1 μ, and length is at least 5 in the ratio of diameter.Estimate that they are substantially free of long hot carbon outer coatings, i.e. the pyrolytic deposit carbon that causes by the thermally splitting of the gas that is used to prepare they and charging.

[0009] by U.S.5 with reference to the people such as Tennent that are incorporated herein, 171,560 carbon fibrils of describing do not contain hot outer coatings and have parallel with the protofibril axle basically graphite linings, so that the distance of at least 2 protofibril diameters is extended in the described projection on described protofibril axle once.Typically, these protofibril are cylindric basically, the substantially invariable graphite nanotubes of diameter, comprising the basic and vertical cylindric graphite flake of cylinder axis of c-axle. they are substantially free of the carbon of pyrolytic deposition, these fibers just that diameter is mainly paid close attention to greater than 5. the present invention less than 0.1 μ and slenderness ratio.

[0010] can be for the more detailed description of carbon fibrils aggregation information referring to people such as Snyder, the U.S. patent application serial number 149 that on January 28th, 1988 submitted to, 573, with PCT application the US89/00322 (" carbon fibrils of submitting on January 28th, 1989 ") WO89/07163, submitted on September 28th, 1989 with people such as Moy, U.S. patent application serial number 413,837 and PCT application US90/05498 (" protofibril aggregation of submitting to September 27 nineteen ninety and preparation method thereof ") WO 91/05089, all these patents all have been assigned to the transferee identical with the present invention, they are incorporated herein by reference at this.

[0011] USSN 07/887 by submitting on May 22nd, 1992 with reference to the people such as Moy that are incorporated herein, 307 the protofibril of describing with the aggregation preparation, this aggregation has different macromorphology (determining by scanning electron microscopy (SEM)), and wherein their twine mutually to form at random and are similar to Bird's Nest (" BN ") fibriilar winding ball; Or this fiber prepares with following aggregation, this aggregation comprises from up to the bundle of the carbon fibrils of slight bending or kink, wherein carbon fibrils has substantially the same relative orientation, and have valley yarn (" CY ") phenomenon, for example the longitudinal axis of each fiber deposition individuality of kink (crooked or) extends with the direction identical with intrafascicular peripheral protofibril; Or this fiber is with the preparation of following aggregation, and this aggregation comprises that from up to the protofibril of slight bending or kink, it twines more loosely mutually and forms " open net " (" ON ") structure.In open web frame, the degree that fiber twines is greater than viewed degree in valley yarn aggregation (wherein individual fibers has substantially the same relative orientation), but less than the winding degree of Bird's Nest.CY and ON aggregation are easier to disperse than BN, and this makes them needing can be used for entire structure all to have the chelation structure of even character.

[0012] when graphite linings at the projection extended distance of protofibril layer on the protofibril axle during less than 2 protofibril diameters, in cross section, the shape of fish-bone will appear in the carbon plane of gnf.This is called the fish-bone protofibril with term.By the U.S.4 with reference to the Geus that is incorporated herein, 855,091 provide preparation to be substantially free of the fibriilar method of fish-bone of pyrolysis outer coatings. and these protofibril also can be used to implement the present invention.

[0013] with the form of the similar carbon nanotube of protofibril of above-mentioned catalytic growth can in the high temperature carbon arc, grow (Iijima, Nature 35456 1991).(Weaver, Science have generally been accepted now 2651994), the nanofiber of these arc-growths has the identical morphology of catalytic growth fiber with early Tennent.Arc growth carbon nanofiber also is useful in the present invention.

[0014] from 1993 " Single-shell carbon nanotube of 1-nm diameter ", SIijima and T Ichihashi, Nature, vol.363, p.603 (1993) and " Cobalt-catalysed growth of carbon nanotube with single-atomic-layer walls ", D SBethune, C H Kiang, M S DeVries, G Gorman, R Savoy and R BeyersNature, vol.363, p.605 since (1993) report, disclose Single Walled Carbon Nanotube and preparation method thereof, by reference these two pieces of articles be incorporated herein at this.

[0015] at the U.S.6 of Moy, also disclosed Single Walled Carbon Nanotube in 221,330, at this by with reference to being introduced into this paper.Moy discloses the method for preparing hollow Single Walled Carbon Nanotube, comprise that at first forming the gas phase mixture unstripped gas comes one or more gaseous carbon compounds of catalytic decomposition, wherein unstripped gas comprises one or more gaseous carbon compounds and the metallic compound of gas phase, wherein every kind of carbon compound comprises 1 to 6 carbon atom and only comprises H, O, N, S or Cl are as heteroatoms, choose wantonly and mix with hydrogen, the metallic compound of gas phase be under reaction conditions to described decomposition reaction unstable compounds, the metallic catalyzer of the metallic compound formation of gas phase is brought into play under reaction conditions and is decomposed katalysis; Under the decomposition reaction condition, carry out described decomposition reaction then, so just made described nanotube. the present invention relates to a kind of gas-phase reaction, wherein the metallic compound of gas phase is incorporated in the reaction mixture that also contains the gaseous state carbon source.This carbon source has the C of heteroatoms H, O, N, S or Cl typically ₁To C ₆Compound, optional and hydrogen mixture.Carbon monoxide or carbon monoxide and hydrogen are preferred carbon raw materials.It is believed that reaction zone temperature brings up to 1300 ℃ from about 400 ℃, pressure can make the metallic compound decomposition of gas phase become metallic catalyzer between about 0 to about 100p.s.i.g..Can resolve into the intermediate materials that atoms metal or part are decomposed. metallic catalyzer 1) also (2) catalysis SWNT formation of catalysis CO decomposition.Therefore, the present invention also relates to form SWNT. by the catalytic decomposition of carbon compound

[0016] U.S.6, aerosol technology has been used in 221,330 invention in some embodiments, and wherein the aerosol with metallic catalyzer is incorporated in the reaction mixture.The advantage of the aerosol processing of preparation SWNT is, can prepare the granules of catalyst of even size and ratio, and this method just can be used for effectively and successive commerce or industrial production like this.Previous arc-over and the laser deposition of discussing can not amplify economically, to be used for commerce or industrial production.Useful in the present invention metallic compound comprises that metal carbonyl, metal acetyl benzylacetone hydrochlorate or other can be used as the material that steam is introduced the metal catalyst that is decomposed to form non-carrier under decomposition condition.The effective metal of catalysis comprises Fe, Co, Mn, Ni and Mo.Molybdenum carbonyl compound and iron carbonyl compound are preferred metallic compounds, and wherein said compound can form the vapor phase catalyzer under reaction conditions.These solid-state metal hydroxy compounds can be delivered to pretreating zone, in this position they are gasified, and therefore become the vapor phase precursor of catalyzer.It is found that have two kinds of methods to may be used on the catalyzer of non-carrier, form SWNT.

[0017] first method is the direct injection volatile catalyst.In U.S. patent application serial numbers 08/459,534, described this direct injection, be incorporated herein by reference.It is found that, use hexacarbonylmolybdenum [Mo (CO) ₆] and cobalt octacarbonyl [CO ₂(CO) ₈] know from experience the formation that causes SWNT before the direct injection volatile catalyst.These two kinds of materials at room temperature all are solids, and environment depresses can distillation-molybdenum compound be heat-staple at least 150 degree but press or be similar at environment, and cobalt compound decomposes also and distils.(”Organic Syntheses via MetalCarbonyls，”Vol.1，1.Wender and P.Pino，eds.，IntersciencePublishers，New York，1968，p.40)。

[0018] second method uses vaporizer to introduce metallic compound (accompanying drawing 12).In embodiment preferred of the present invention, vaporizer 10 as shown in Figure 12 comprises the bottom and has about 1 " the thermopair conduit 20 that seals, and form second compartment.This chamber has two 1/4 " hole 26, this hole is open and is exposed in the reactant gases.Catalyzer is placed this compartment, gasify with vaporizer stove 32 under the temperature of wanting of what is the need in office then.With first thermopair, 22 these stoves of control.Metallic compound, preferred carbonyl compound gasifies in its temperature below decomposition point, and reactant gases CO or CO/H sub.2 sweep reaction zone 34 with precursor, control reaction zone 34 respectively by the reaction zone stove 38 and second thermopair 42.Although the applicant does not wish to be confined to a certain specific theory of operation, but it is believed that under temperature of reaction, metallic compound partly is decomposed into intermediate materials or is decomposed into atoms metal fully. and these intermediate materials and/or atoms metal are merged into bigger aggregated particles, becoming actual catalyzer. this particle growth is to suitable size then, decomposition that just can catalysis CO also can promote the SWNT growth.In the device of accompanying drawing 11, the form of collection catalyst particle and resulting carbon on quartzy tampon 36. particulate growth rate depends on the concentration of the metallic intermediate materials of gas phase.Determine this concentration by the vapour pressure in the vaporizer (with the temperature that therefore obtains).If excessive concentration, particle growth are too fast, will grow structure beyond the SWNT (for example MWNT, decolorizing carbon, onions or the like). with U.S.6,221,330 full content comprises that described embodiment is by with reference to being incorporated herein.

[0019] by U.S.5 with reference to the people such as Bethune that are incorporated herein, 424,054, described a kind of method for preparing Single Walled Carbon Nanotube, comprise carbon steam is contacted with cobalt catalyst.The electric-arc heating of this carbon steam-type by solid carbon obtains, and solid carbon can be decolorizing carbon, graphite, activated carbon or decolorizing carbon or its mixture. the additive method of heating carbon also has been discussed, for example LASER HEATING, electron beam heating and RF induction heating.

[0020] Smalley (Guo that is incorporated herein by reference, T., Nikoleev, P., Thess, A., Colbert, D.T., and Smally, R.E., Chem.Phys.Lett.243:1-12 (1995)) a kind of method for preparing Single Walled Carbon Nanotube has been described, wherein graphite post and transition metal gasify by high-temperature laser simultaneously.

[0021] Smalley (Thess, A., Lee, the R. that is incorporated herein by reference, Nikolaev, P., Dai, H., Petit, P., Robert, J., Xu, C, Lee, Y.H., Kim, S.G., Rinzler, A.G., Colbert, D.T., Scuseria, G.E., Tonarek, D., Fischer, J.E., and Smalley, R.E., Science, 273:483-487 (1996)) a kind of method for preparing Single Walled Carbon Nanotube has also been described, the graphite post that wherein comprises a small amount of transition metal in baking oven 1200 ℃ of following laser gasifications.The yield of the preparation single-walled nanotube of being reported surpasses 70%.

[0022] carrier metal catalyst that is used to produce SWNT also is known.By with reference to the Smalley (Dai., H., the Rinzler that are incorporated herein, A.G., Nikolaev, P., Thess, A., Colbert, D.T., and Smalley, R.E., Chem.Phys.Lett.260:471-475 (1996)) described support C o, Ni and Mo catalyzer and can be used for the growth of many walls nanotube and single-walled nanotube, and proposed the mechanism that their produce from CO.

[0023] the U.S. patent application serial number 351 by submitting to March 15 in 1989 with reference to the people such as McCarthy that are incorporated herein, 967 have described the method on a kind of carbonoxide protofibril surface, be included in that (for example time, temperature and pressure) contacts protofibril under the reaction conditions with oxygenant, oxygenant comprises sulfuric acid (H ₂SO ₄) and Potcrate (KClO ₃), with the fibriilar surface of abundant oxidation.Protofibril according to people's such as McCarthy method oxidation is non-homogeneous oxidation, that is to say that carbon atom is replaced by carboxyl, aldehyde, ketone, phenol and other carbonyls.

[0024] by nitric acid treatment also oxidation protofibril anisotropically.International Application PCT/US94/10168 has disclosed the fibriilar generation of oxidation of the mixture that comprises functional group.Hoogenvaad, M.S., Deng people's (" Metal Catalysts supported on a NovelCarbon Support ", Presented at Sixth International Conference onScientific Basis for the Preparation of Heterogeneous Catalysts, Brussels, Belgium, in September, 1994) also found, with the precious metal of nitric acid and protofibril-carrier at first the preparation on oxidation protofibril surface be favourable. be the standard step of preparation carbon-carrier noble metal catalyzer with sour pre-treatment, wherein, this catalyzer is common carbon source, and it cleans the surface of not wishing material as much as possible and makes it functionalized.

[0025] in disclosed work, McCarthy and Bening (PolymerPreprints ACS Div.ofPolymer Chem. 30(1) 420 (1990)) prepared the fibriilar derivative of oxidation, contain various oxide groups with proof list bread. for example color is vivid or show some other signal strong and that differentiate and distinguish easily according to their analysis availability, selects the compound phenylhydrazone that they prepare, assorted aromatic ester, thallium salt or the like.These compounds are unsegregated, and are different with the described derivative of not implementing meaning.

[0026],,, then can develop and many differences and important purposes if with the protofibril functionalisation of surfaces although the aggregation of carbon fibrils and carbon fibrils has had a lot of purposes as described in the above-mentioned patent and patent application that relates to.Protofibril that functionalized permission even or heterogeneous is functionalized and various substrate interaction and form unique compositions with peculiar property material, and can produce fibrillar structure according to the key between the protofibril functionalisation of surfaces site.

Goal of the invention

[0027] therefore, primary and foremost purpose of the present invention provides the functionalized single walled carbon nanotubes of diameter less than 5 nanometers, promptly surperficial even or non-homogeneous modification with functionalized chemical part bonded Single Walled Carbon Nanotube.

[0028] of the present inventionly further provides the Single Walled Carbon Nanotube of diameter, by with oxygenant or the reaction of other chemical mediators and with functionalisation of surfaces less than 5 nanometers with relevant purpose.

[0029] of the present inventionly further provide the Single Walled Carbon Nanotube of diameter less than 5 nanometers with relevant purpose, its surface is modified equably by chemical reaction or by the physical adsorption that chemically reactive material is arranged itself.

[0030] further purpose of the present invention provides the Single Walled Carbon Nanotube of diameter less than 5 nanometers, and its surface is for example by oxidative modification, then by further modifying with functional group reactions.

[0031] of the present inventionly further provide the Single Walled Carbon Nanotube of diameter less than 5 nanometers with relevant purpose, a series of modified with functional group is used on its surface, so that Single Walled Carbon Nanotube can be carried out chemical reaction or physical bond with the chemical group of various substrates.

[0032] of the present inventionly further provides the chelation structure of diameter, the wherein combination by large-scale chemical connexon mutually of the functional group on the Single Walled Carbon Nanotube less than the Single Walled Carbon Nanotube of 5 nanometers with relevant purpose.

[0033] of the present inventionly further provides the method on chemically modified protofibril surface and physical adsorption diameter method, so that the functionalized moiety with the protofibril surface bonding all is provided in both of these case less than the material on the Single Walled Carbon Nanotube surface of 5 nanometers with relevant purpose.

[0034] further purpose of the present invention provides based on the novel composition of diameter less than the material of the functionalized single walled carbon nanotubes of 5 nanometers.

The accompanying drawing summary

[0035] accompanying drawing 1 is the diagrammatic representation that the protofibril bonded BSA that modifies with Plain fiber, carboxyl protofibril and PEG-analyzes.

[0036] accompanying drawing 2 is diagrammatic representations of analyzing by the protofibril bonded beta-lactoglobulin of two kinds of diverse ways and carboxyl protofibril and PEG-modification.

[0037] accompanying drawing 3 is diagrammatic representations of bovine serum albumin (BSA) elution profile on tertiary amine protofibril post.

[0038] accompanying drawing 4 is diagrammatic representations of BSA elution profile on quaternary amine protofibril post.

[0039] accompanying drawing 5 is that preparation is based on the fibriilar reaction sequence of the dendrimeric of Methionin.

[0040] accompanying drawing 6 is diagrammatic representations of cyclic voltammogram, proves to use iron-phthalocyanine to modify protofibril in flow cell.

[0041] accompanying drawing 7 is by adding N _ε-(tertbutyloxycarbonyl)-L-Methionin prepares difunctional fibriilar reaction sequence.

[0042] accompanying drawing 8 is the diagrammatic representations with the result of protofibril fixed esterase synthetic butyric acid ethyl ester.

[0043] accompanying drawing 9 is the diagrammatic representations that separate the result of alkaline phosphatase (AP) with the protofibril that the AP inhibitor is modified from the mixture of AP and beta-galactosidase enzymes (β G).

[0044] accompanying drawing 10 is the diagrammatic representations that separate the result of β G with the protofibril that β G modifies from the mixture of AP and β G.

What [0045] accompanying drawing 11 showed is the reactor that can produce Single Walled Carbon Nanotube.

What [0046] accompanying drawing 12 showed is the evaporator assemblies of accompanying drawing 11 described reactors.

Detailed Description Of The Invention

[0047] the present invention relates to composition, it broadly has following general formula:

[C _nH _L]R _m

Wherein n is integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each R is identical, and is selected from SO₃H、COOH、NH ₂, OH, R ' CHOH, CHO, CN, COCl, halide, COSH, SH, COOR ', SR ', SiR '₃、 Si(-OR’-) _yR’ _3-y、Si(-O-SiR’ ₂-)OR’、R”、Li、AlR’ ₂、Hg-X、TlZ ₂And Mg-X,

Y is equal to or less than 3 integer,

R ' is hydrogen, alkyl, aryl, cycloalkyl or aralkyl, cyclophane base or poly-(alkyl ether),

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl, fluoro aralkyl or cyclophane base,

X is halide, and

Z is carboxylate radical or trifluoroacetic acid root.

[0048] carbon atom C _nIt is the surface carbon of the substantially invariable columned graphite nanotubes basically of diameter.This nanotube comprise slenderness ratio greater than 5 and diameter less than 0.5 μ, preferably less than those of 0.1 μ. this nanotube also can be columned basically graphite nanotubes, it is substantially free of the carbon of pyrolytic deposition, be characterised in that more preferably this nanotube is the distance of at least 2 times of protofibril diameters of the stone mill layer projection on the protofibril axle extending and/or has those of cylindric stone mill sheet, its c-axle is vertical with its cylinder axis basically. in a preferred embodiment, and carbon atom C _nBe the surface carbon of the substantially invariable or diameter of diameter less than the columned basically single wall graphite nanotubes of 5 nanometers.More preferably, carbon atom C _nBe the surface carbon of diameter less than the single wall graphite nanotubes of 5 nanometers.These compositions are that wherein each R is identical uniformly.

[0049] also can prepare the nanotube of non-homogeneous replacement. this comprises the composition of following general formula

[C _nH _L]R _m

Wherein n, L, m, R and nanotube itself as above-mentioned definition, condition is all oxygen-frees of each R, if perhaps each R is the oxygen containing group of bag, does not then have COOH.

[0050] the functionalized nanotube with following general formula is also included within the scope of the present invention

[C _nH _L]R _m

Wherein n, L, m, R and R ' have and above-mentioned same implication, and carbon atom is a slenderness ratio greater than the fibriilar surface carbon atom of 5 fish-bone or the diameter carbon atom less than the Single Walled Carbon Nanotube of 5 nanometers.They can be even or non-homogeneous replacements, and preferably, nanotube does not contain pyrolysis outer coatings and diameter less than 0.5 μ.

[0051] the present invention also comprises the functionalized nanotube with following general formula

[C _nH _L][R’-R] _m

Wherein n, L, m, R and R ' have and above-mentioned same implication.Carbon atom C _nIt is the surface carbon of the substantially invariable columned graphite nanotubes basically of diameter.The slenderness ratio of this nanotube greater than 5 and diameter less than 0.5 μ, preferably less than 0.1 μ.This nanotube can be the nanotube that is substantially free of pyrolytic deposition carbon.In addition, this nanotube be at least 2 times of protofibril diameters of the stone mill layer projection on the protofibril axle extending distance those and/or have cylindric stone mill sheet, its c-axle basically with its cylinder axis vertical those.In this embodiment preferred, carbon atom C _nBe the surface carbon of the substantially invariable or diameter of diameter less than the columned basically single wall graphite nanotubes of 5 nanometers.More preferably, carbon atom C _nBe the surface carbon of diameter less than the single wall graphite nanotubes of 5 nanometers.

[0052] in the nanotube of all even non-homogeneous replacements, surface atom C _nReact.As in graphite, is basal plane carbon at most of carbon atoms of the fibriilar upper layer of stone mill.Basal plane carbon is relative inertness for chemical attack.In damaged site, for example the stone mill face fail to extend to fully fibriilar around, the edge carbon atom that so just has carbon atom and stone mill face is (referring to Urry, Elementary Equilibrium Chemistry of Carbon, Wiley, the discussion of 1989 pairs of edges of New York and basal plane carbon) similar.

[0053] in damaged site, edge or basal plane carbon that nanotube hangs down more inner layer may expose.The term surface carbon comprises the outermost basal plane of nanotube and all carbon at edge, and the basal plane and/or the edge carbon of the lower level that exposes in outermost damaged site.Responding property of edge carbon must comprise some heteroatomss or group to satisfy the valence of carbon.

The nanotube of [0054] can be further advantageously functionalized above-mentioned replacement.These compositions comprise the combination of following general formula

[C _nH _L]A _m

Wherein carbon atom is the surface carbon of above-mentioned nanotube, and n, L and m are as mentioned above, A be selected from OY, NHY,

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, Nucleotide, oligonucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-N (R ') ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' N ⁺(R ') ₃X ^-, R ' SiR ' ₃, R ' Si (OR ') _yR ' _3-y, R ' Si (O-SiR ' ₂) OR ', R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ',

And w is greater than 1 and less than 200 integer.Carbon atom C _nBe the surface carbon of the substantially invariable columned graphite nanotubes basically of diameter. this nanotube comprise slenderness ratio greater than 5 and diameter less than 0.5 μ, preferably less than those of 0.1 μ.This nanotube also can be columned basically graphite nanotubes, and it is substantially free of the carbon of pyrolytic deposition.More preferably it is characterized in that the distance of at least 2 times of protofibril diameters of the stone mill layer projection on the protofibril axle extending and/or they comprise the c-axle basically with the vertical cylindric stone mill sheet of its cylinder axis.Preferably, this nanotube does not contain pyrolysis outer coatings and diameter less than 0.5 μ.In addition, this nanotube is diameter substantially constant or the diameter columned basically Single Walled Carbon Nanotube less than 5 nanometers.More preferably, this nanotube is the Single Walled Carbon Nanotube of diameter less than 5 nanometers.

[0055] structure of functionalized nanotube

[C _nH _L][R’-R] _m

Also can the functionalized composition that has following general formula with preparation

[C _nH _L][R’-A] _m

Wherein n, L, m, R ' and A as above define. carbon atom C _nBe the surface carbon of the substantially invariable columned graphite nanotubes basically of diameter. this nanotube comprise slenderness ratio greater than 5 and diameter less than 0.5 μ, preferably less than those of 0.1 μ. this nanotube also can be columned basically graphite nanotubes, and it is substantially free of the carbon of pyrolytic deposition. more preferably it is characterized in that the projection of stone mill layer on the protofibril axle extend the distance of at least 2 times of protofibril diameters and/or have the c-axle basically with the vertical cylindric stone mill sheet of its cylinder axis.Preferably, this nanotube does not contain pyrolysis outer coatings and diameter less than 0.5 μ. and in addition, this nanotube is diameter substantially constant or the diameter columned basically Single Walled Carbon Nanotube less than 5 nanometers.Most preferably, this carbon atom C _nBe the surface carbon of diameter less than the Single Walled Carbon Nanotube of 5 nanometers.

[0056] composition of the present invention also comprises the nanotube that some ring compound is adsorbed on it.This comprises the composition of the material of following general formula

[C _nH _L][X-R _a] _m

Wherein n is an integer, L is the numeral less than 0.1n, and m is the numeral less than 0.5n, and a is 0 or less than 10 numeral, X ' is the aromatic base of multinuclear aromatic base, many heteronuclears or the aromatic base part or the planar tetrathio oxalic acid metal-salt of the many heteronuclears of metal, and R is as mentioned above. carbon atom C _nBe the surface carbon of the substantially invariable columned graphite nanotubes basically of diameter. this nanotube comprise slenderness ratio greater than 5 and diameter less than 0.5 μ, preferably less than those of 0.1 μ.This nanotube also can be columned basically graphite nanotubes, it is substantially free of the carbon of pyrolytic deposition, more preferably it is characterized in that the distance of at least 2 times of protofibril diameters of the stone mill layer projection on the protofibril axle extending and/or have the c-axle basically with the vertical cylindric stone mill sheet of its cylinder axis.Preferably, this nanotube does not contain pyrolysis outer coatings and diameter less than 0.5 μ. and in addition, this nanotube is diameter substantially constant or the diameter columned basically Single Walled Carbon Nanotube less than 5 nanometers. most preferably, this carbon atom C _nBe the surface carbon of diameter less than the Single Walled Carbon Nanotube of 5 nanometers.

[0057] preferred ring compound is Cotton and Wilkinson, the 76th page of described plane macrocylc compound of AdvancedOrganic.The ring compound of preferred absorption is a porphyrin, phthalocyanine, or the trithio oxalate of Ni, Cu, Pd, Pt or Ag.

[0058] composition of the present invention also comprises the Single Walled Carbon Nanotube of diameter less than 5 nanometers, if wherein on the wall of Single Walled Carbon Nanotube, adsorbed some plane aromatics. adsorbed molecule is that itself is functionalized, so just can functionalized effectively sidewall and do not change their physical structure.Can further heat then this functionalized single walled carbon nanotubes or with absorption the molecular moiety thermolysis to avoid desorption.Can use for example crosslinked or oligomerization (to reduce solvability) of other mechanism of fixing adsorbed molecule.The plane aromatics of preferred absorption comprises porphyrin, phthalocyanine, or the trithio oxalate of Ni, Cu, Pd, Pt or Ag.

[0059] can functionalized adsorbed ring compound. these compositions comprise the compound of following general formula

[C _nH _L][X-A _a] _m

Wherein m, n, L, a, X and A such as above-mentioned definition, carbon are the surface carbon of aforesaid columned basically graphite or Single Walled Carbon Nanotube.

[0060] functionalized as mentioned above carbon fibrils can be incorporated in the matrix.Preferably, this matrix is organic polymer (for example, for example epoxy, dimaleimide, polymeric amide or vibrin of thermosetting resin; Thermoplastic resin; The reaction injection molded resin; Or elastomerics for example natural rubber, styrene butadiene rubbers or suitable-1); Inorganic polymer (for example polymeric inorganic oxide for example glass), metal (for example lead or copper), or stupalith (for example, silicate cement).Mix fibriilar matrix and can form globule.Alternately, functionalized protofibril can be incorporated into the outside surface of functionalized globule.

[0061] do not interrelate with particular theory, functionalized protofibril preferably is distributed in the polymer system, because the character on the surface of modifying more with polymer-compatible, perhaps, become end group on the polymkeric substance because functional group's (particularly hydroxyl or amido) of modifying directly is attached to.Like this, polymer system for example polycarbonate, polyurethane(s), polyester or polyamide/imide directly combines with protofibril, makes protofibril have the viscosity of improvement and is easier to disperse

[0062] the present invention also relates to functional group is incorporated into the lip-deep method of carbon fibrils, comprise carbon fibrils is contacted for some time with strong oxidizer, with the described fibriilar surface of oxidation fully, further described protofibril is contacted with the reagent that is fit to functional group is added to oxidized surface.In embodiment preferred of the present invention, this oxygenant comprises alkaline metal chlorate's strong acid solution.In another embodiment of the invention, the alkaline metal chlorate is sodium chlorate or Potcrate.In preferred embodiments, employed strong acid is sulfuric acid. fully the time of oxidation is about 0.5 hour to about 24 hours.

[0063] in a further preferred embodiment, has general formula [C _nH _L] [CH (R ') OH] _m(14 pages), the wherein formation of n, L, R ' and m composition as defined above comprises the CH with R ' ₂The surface carbon of OH and nanotube radical initiator for example benzoyl peroxide in the presence of react.

[0064] the present invention also relates to protein is connected to method on the nanotube that NHS modifies, be included between NHS ester and the proteinic amino and form covalent linkage.

[0065] the present invention also relates to prepare the method for the reticulation of carbon fibrils or Single Walled Carbon Nanotube, comprise carbon fibrils or Single Walled Carbon Nanotube are contacted the surface of for some time with abundant carbonoxide protofibril or Single Walled Carbon Nanotube with oxygenant, carbon fibrils that the surface is already oxidised or Single Walled Carbon Nanotube contact with the reagent that is fit to functional group is added to carbon fibrils or Single Walled Carbon Nanotube surface, further the protofibril with functionalisation of surfaces contacts effectively with linking agent, with the reticulation of preparation carbon fibrils or Single Walled Carbon Nanotube. and preferred cross-linking agents is a polyvalent alcohol, polyamine or polycarboxylic acid.

[0066] functionalized protofibril and Single Walled Carbon Nanotube also are useful in the reticulation of fibriilar hard reticulation of preparation or Single Walled Carbon Nanotube.The three-dimensional netted thing of the acid-functionalized protofibril or the good distribution of Single Walled Carbon Nanotube can be for example be cross-linked to form hard reticulation and stabilization by acidic group (between the protofibril) and polyvalent alcohol or polyamine.

[0067] the present invention also comprises three-dimensional netted thing, it is formed by connecting by functionalized protofibril or Single Walled Carbon Nanotube of the present invention. and these chelation structure comprise at least two functionalized protofibril or Single Walled Carbon Nanotube, and it connects by one or more connexons that comprise direct key or chemical part. and these reticulations comprise the very how empty medium of homogeneous phase equal aperture. and they can be used as sorbent material, support of the catalyst and separating medium.

[0068] although the gap between protofibril or the single armed carbon nanotube is irregular in size and vpg connection, can think that they are holes, be characterised in that this method of use makes the porous medium characterization.In these reticulations, the size in hole can be controlled by the concentration and the chain length of fibriilar concentration and dispersive level and linking agent. and this writes material and can be used as structural support of the catalyst, can adjust to get rid of or to comprise a certain size molecule.Except the Industrial Catalysis of routine, they can be applied to biological catalyst as macropore carrier especially.

[0069] hard reticulation also can be used as the main chain of intending the ecosystem and is used for molecular recognition.These systems are at U.S.5,110,833 and International Patent Publication No. W093/19844 in be described.Suitably select linking agent and complexing agent can be used for the stabilization of specific molecular framework.

The method of functionalized nanotube

[0070] can directly prepare evenly functionalized protofibril of the present invention to deoxidation protofibril surface and metallization by sulfurization, electrophilic addition.When using the arc grown nanofibers, they may be functionalized before extensive purifying.People such as Ebbesen (Nature 367 519 (1994)) have provided the method for this purifying.

[0071] preferably, with handle carbon fibrils before the functionalized agent contacts earlier.These processing can comprise protofibril is distributed in the solvent.In some instances, can filter carbon fibrils then, and dry before further contacting.

1. sulfurization

[0072] at March, J.P., Advanced Organic Chemistry, 3rd Ed.Wiley, New York 1985; House, H., Modern Synthetic Reactions, 2ndEd., Benjamin/Cumrnings, Menlo Park is described background technology among the CA 1972.

[0073] with oleum (oleum) but sulfonation activatory C-H (comprising aromatic C-H) key, oleum is to comprise up to 20% SO ₃Concentrated sulfuric acid solution.This ordinary method is to use oleum through liquid phase at 80 ℃; But also can in inertia, aprotic solvent, use SO ₃Or in gas phase, use SO ₃Coming sulfonation activatory c h bond. this reaction is

-C-H+SO ₃---->-C-SO ₃H

Above-mentioned reaction has caused the generation of sulfone, according to following reaction:

2-C-H+SO3----->-C-SO ₂-C-+H ₂O

Embodiment 1

Activate c h bond with sulfuric acid

[0074] is reflected in the gas phase neutralization solution and carries out, can not make the result produce significant difference.The vapor phase reaction is to carry out in the horizontal quartz tube reactor of Lindberg stove heating.Have being equipped with of gas feed/outlet pipe and comprise 20%SO ₃The multinecked flask of the vitriol oil as SO ₃The source.

[0075] sample of weighing of protofibril in the porcelain dish (BN or CC) is placed have inlet mouth 1 " pipe; Outlet links to each other with vitriol oil bubble trap.With argon gas whole reactor is washed away 20 minutes removing all gas, with sample be heated to 300 ℃ 1 hour to remove residual moisture.After the drying, under argon gas, adjust the temperature to temperature of reaction.

[0076] when required temperature change is stablized, with SO ₃The pipe that comes from reactor links to each other, with argon gas stream with SO ₃Steam is brought in the quartz tube reactor. in required time, reacts down temperature required, then at cooling reactor under the mobile argon gas. then at 90 ℃ and 5 " and dry protofibril in the Hg vacuum, the increase of acquisition dry weight.By with 0.100N NaOH reaction with 0.100N HCl back titration, be that terminal point is determined sulfuric acid (SO with pH6.0 ₃H) amount.

[0077] in multinecked flask, comprising 20%SO with gas feed/outlet pipe and magnetic stirring apparatus ₃The vitriol oil in carry out liquid phase reaction. will be at dense H ₂SO ₄(50) the protofibril slurry in places flask.Before adding reactor, oleum solution (20cc) is preheating to-60 ℃.After the reaction, the wintercherry body is poured in the trash ice, at once with the dilution of 11DI water.Filter this solid, wash until elutriant pH value no change as possible with DI water.Then at 100 ℃ and 5 " dry protofibril in the Hg vacuum.Because filtering transfer indfficiency can't obtain accurate weight increased value.The results are shown in table 1.

[0078]

Table 1

The reaction general introduction

SAMPLE FIBRIL DRY Wt SO ₃H CONC

Embodiment RUN# REACT Wt.g TYPE T ℃ TIME GAIN Me/g

1A 118-60A Vap 0.20 CY 110 15m 9.3％ 0.50

1B 118-61A Vap 0.20 BN 100 30m 8.5％ 0.31

1C 118-61B Vap 0.20 BN 65 15m 4.2％ 0.45

1D 118-56A Liq 1.2 CY 50 10m 0.33

1E 118-56B Liq 1.0 CY 25 20m 0.40

[0079] react in vapor phase or liquid phase, sulfuric acid amount does not have significant difference.Has temperature effective.Higher temperature of reaction (vapor phase) has obtained the sulfone of more amount.In 118-61B, the increase of 4.2%wt consistent with the vitriolic amount (being 0.51meq/g in theory).Operation 60A and 61A wt increase too high and can't only calculate by sulfuric acid amount.Therefore, supposition has also made appreciable sulfone.

2. join the protofibril surface of oxide-free

[0080] at Urry, G., Elementary Equilibrium Chemistry ofCarbon, Wiley is described background technology among the New York 1989.

[0081] character of fibriilar surface carbon and graphite is similar, that is, they are all in comprising six rib sheets of basal plane and edge carbon.When basal plane carbon was relative inertness for chemical attack, edge carbon relatively also must comprise some heteroatomss or group to satisfy the valence of carbon. and protofibril also has the surface damage site, and it is edge carbon basically and comprises heteroatoms or group.

[0082] the modal heteroatoms that is attached on the fibriilar surface carbon is a hydrogen, and it is a gaseous fraction main in the preparation process; Oxygen is owing to its hyperergy with owing to its trace is very difficult to avoid; And H ₂O, it always exists owing to the reason of catalyzer. in a vacuum～1000 ℃ of pyrolysis will be with the mechanism of the unknown but known stoichiometry in complex reaction with surperficial deoxidation.Product is that ratio is CO and the CO of 2:1 ₂Resulting protofibril surface comprises with C ₁-C ₄The atomic group of arranging, itself and activatory chain hydrocarbon have very big reactivity.This surface in a vacuum or rare gas element to exist down be stable, but still keep hyperergy, in being exposed to reactant gas.Therefore, can the pyrolysis protofibril under vacuum or inert atmosphere～1000 ℃, also can cool off under the same conditions, and obtain stable functional group with suitable molecular reaction at low temperatures.Typical example is:

Then:

X＝-OH、-Cl、-NH ₂、-H

-----------protofibril-R ' (COOH) for the RFS+ maleic anhydride ₂

RFS+ cyanogen--------------〉protofibril-CN

RFS+CH ₂=CH-CH ₂X--------------〉protofibril-R ' CH ₂X X=-NH ₂,-OH ,-halogen

RFS+H ₂O--------------〉protofibril=O (quinoidal)

RFS+CH ₂=CHCHO--------------〉protofibril-R ' CHO (aldehyde)

RFS+CH ₂=CH-CN--------------〉protofibril-R ' CN

Wherein R ' is alkyl (alkyl, cycloalkyl or the like)

Embodiment 2

The functionalized protofibril of prepared in reaction by vinylformic acid and oxide-free protofibril surface

[0083] the 1g BN protofibril in the porcelain dish is placed the level 1 that has thermopair and be arranged in the Lindberg tube furnace " silica tube. this end has gas feed/outlet.Argon cleaning with dry deoxidation should be managed 10 minutes, furnace temperature was elevated to 300 ℃ then, kept 30 minutes.Under the successive argon gas stream, temperature is elevated to 1000 ℃ then, kept 16 hours with 100 ℃ increments.After this section period finishes, with pipe cool to room temperature (RT) under argon gas.Then with argon gas shunting by pure vinylformic acid and have the multinecked flask of gas feed/outlet 50 ℃ comprise.Vinylformic acid/argon gas steam flow was kept 6 hours under RT.After finishing during this period of time, remove residual unreacted vinylformic acid, at first use argon cleaning, then 5 " in the vacuum 1000 ℃ of following vacuum-dryings.By with excessive 0.100N NaOH reaction and determine the amount of carboxylic acid to the terminal point of pH7.5 with 0.100N HCl back titration.

Embodiment 3

[0084] repeating this method, except 10 with the similar mode of aforesaid method ^-4Carry out pyrolysis and cooling in the vacuum of holder.As the above-mentioned method purified vinylformic acid steam of argon-dilution.

Embodiment 4

The functionalized protofibril of prepared in reaction by toxilic acid and oxide-free protofibril surface

[0085] repeat this method as embodiment 2, except reagent under RT is purified maleic anhydride (MAN), it is by being fed to the MAN bath of argon gas by fusing in the reactor at 80 ℃.

Embodiment 5

The functionalized protofibril of prepared in reaction by acrylate chloride and oxide-free protofibril surface

[0086] repeat this method as embodiment 2, except reagent under RT is purified acrylate chloride, it is by being fed to argon gas in the reactor by purified acrylate chloride MAN at 25 ℃.By with excessive 0.100N NaOH reaction and determine the amount of acrylate chloride with 0.100N HCl back titration.

[0087] protofibril pyrolysis in a vacuum makes protofibril surface deoxidation.In the TGA device, vacuum or in purified Ar stream in 1000 ℃ pyrolysis make fibriilar 3 the sample average wt of BN lose 3%.Gas chromatographic analysis only detects ratio and is respectively～CO and the CO of 2:1 ₂The reactivity on resulting surface is very big, the activatory chain hydrocarbon is vinylformic acid, acrylate chloride, acrylamide, propenal, maleic anhydride, allylamine, vinyl carbinol or allyl halide even the purified product of reaction generation at room temperature for example, and it only comprises and the functionalized bonded material of activatory chain hydrocarbon.Therefore, the surface that comprises carboxylic acid by vinylformic acid or maleic anhydride reaction is an available; By the surface that comprises chloride of acid with the acrylate chloride reaction is available; Only obtain aldehyde from propenal; Only obtain hydroxyl from vinyl carbinol; Only obtain amine from allylamine, only obtain halogenide from allyl halide.

3. metallization

[0088] at March, Advanced Organic Chemistry, 3rd ed., p ⁵⁴⁵In provided background technology.

[0089] aromatic c h bond can produce carbon-to-metal bond (C-M) with various organometallic reagent metallization.M is Li, Be, Mg, Al or Tl normally; But also can use other metals. this simple reaction is by direct substitution hydrogen in the activatory aromatics:

1. protofibril-H+R-Li--------〉protofibril-Li+RH

[0090] this reaction may need other highly basic, for example the diamines of uncle's fourth oxygen potassium or chelating.Aprotic solvent is essential (paraffin, benzene).

2. protofibril-H+AlR3--------〉protofibril-AlR ₂+ RH

3. protofibril-H+Tl (TFA) ₃--------〉protofibril-Tl (TFA) ₂+ HTFA

TFA=trifluoroacetate HTFA=trifluoroacetic acid

[0091] the metallization derivative is the main fibriilar example of monofunctional.But they can further react, and obtain the protofibril of other main monofunctional.Some reactions can be badly in need of carrying out in identical device, and do not need separation of intermediates.

4. protofibril-M+O ₂--------〉protofibril-OH+MO M=Li, Al, H+

Protofibril-M+S---------〉protofibril-SH+M ⁺

Protofibril-M+X ₂--------〉protofibril-X+MX X=halogen

Protofibril-T1 (TFA) ₂+ aq.KCN----------〉protofibril-CN+TlTFA+KTFA

Protofibril-CN+H ₂----------〉protofibril-CH ₂-NH ₂

Embodiment 6

The preparation of protofibril-Li

[0092] 1g CC protofibril is placed porcelain dish, and be inserted into wrap in the Lindberg tube furnace 1 " in the quartz tube reactor. the end of pipe has gas feed/outlet.At successive H ₂Flow down, with protofibril be heated to 700 ℃ 2 hours, so that all oxide on surface are transformed into c h bond.Then at H ₂Flow down cooling reactor to room temperature.

[0093] uses exsiccant deoxidation heptane (and LiAlH then ₄) the hydrogenant protofibril is transferred in many necks of the 1L round-bottomed flask that is equipped with the pure argon gas cleaning system to remove all gas and to keep inert atmosphere, this round-bottomed flask also has condenser, magnetic stirring apparatus and rubber septum, can add liquid by syringe by rubber septum.Under the argon gas atmosphere, add 2% n-heptane solution that comprises the 5mmol butyllithium by syringe, stirred this slurry 4 hours under the gentle reflux.After this finishes time, separate protofibril by gravity filtration in the atmospheric glove box of argon, on strainer, wash several with the heptane of dry deoxidation.Protofibril is transferred in the 50cc r.b. flask that tap is housed, and 10 ^-4Dry down under the holder vacuum at 50 ℃.The concentration of lithium is determined in DI water reaction by fibriilar sample and excessive 0.100N HCl and with the terminal point of 0.100N NaOH back titration to pH5.0.

Embodiment 7

Protofibril-T1 (TFA) ₂Preparation

[0094] as embodiment 5 with the hydrogenation of 1g CC protofibril, be loaded in the multinecked flask that contains HTFA, wherein by repeatedly wash degasification with dry argon gas to HTFA.In flask, add 2% 5mmol Tl (TFA) by rubber septum ₃HTFA solution, under gentle reflux, stirred slurry 6 hours.After the reaction, collect protofibril also as embodiment 1 drying.

Embodiment 8

The preparation of protofibril-OH

(only comprising the functionalized oxidized derivatives of OH)

[0095] heptane that is used in the dry deoxidation in the atmospheric bag glove of argon is transferred to the lithiumation protofibril of 0.5g such as embodiment 6 preparations in the single neck flask of the 50cc with tap and magnetic stirring bar.Flask is left bag glove, on magnetic stirring apparatus, stir.Open the tap ventilation then, slurry was stirred 24 hours.After this time finishes, the filtering separation protofibril, and use the MeOH solution washing, under 50 ℃ 5 " dry in the vacuum. determine the concentration of OH base; comprise by with the standardized solution of the diox (0.252M) of diacetyl oxide 80 ℃ down reaction change the OH base into acetic ester, do the acid anhydrides that has reacted that can discharge 1 normal acetate or 1 mole like this.The determining of the total acid content of free acetic acid and unreacted diacetyl oxide comprises, with the terminal point of 0.100N NaOH titration to pH7.5.

Embodiment 9

Protofibril-NH ₂Preparation

[0096] as the protofibril of embodiment 7 preparation 1g trifluoroacetic acidizations.This protofibril of pulp in the Zai diox.And be added in 0.5g triphenyl phosphorus in the diox.Under 50 ℃,, added the ammoniacal liquor gases 30 minutes down at 50 ℃ then with this slurry stirred for several minute.This protofibril of filtering separation then, in the Zai diox, in DI water, wash then, 80 ℃ down and 5 " dry in the vacuum.The determining of amine concentration comprises and excessive acetic acid anhydride reactant and with 0.100N NaOH back titration free acetic acid and unreacted acid anhydrides.

4. deutero-polynuclear aromatic family, many heteronuclears aromatic series and plane macrocylc compound

[0097] physical adsorption of fibriilar stone mill surface permission aromatics.This absorption is to pass through Van der Waals force.These power are important between the basal plane carbon on many ring heteronuclear aromatics and stone mill surface.Being that emulative surface adsorption or adsorbate have under the higher deliquescent condition, desorption may take place.

[0098] for example, have been found that and to come functionalized protofibril by the absorption of phthalocyanine derivates.The protofibril of these phthalocyanine derivates can be used as solid carrier and is used for protein immobilization then.Only just can introduce different chemical groups to the protofibril surface by the different derivatives of selecting phthalocyanine.

[0099] compare with proteocrasic method in the prior art, the application of the protofibril of phthalocyanine derivates in protein is fixing is very useful.Particularly, they are more simpler than covalent modification.In addition, the protofibril of phthalocyanine derivates has higher surperficial flesh, and all is stable in most of solvents under the temperature of a wider range and pH.

Embodiment 10

Porphyrin and phthalocyanine are to fibriilar absorption

[00100] the fibriilar preferred compound of physical adsorption is deutero-porphyrin or phthalocyanine, and they are materials of known strong adsorption stone mill and carbon black.Several compounds all is an available, for example tetracarboxylic acid porphyrin, phthalocyanine cobalt (II) or phthalocyanine two cobalts.Both can be derivatized to the form of carboxylic acid the back.

Phthalocyanineization two lithiums

[00101] usually, from phthalocyanine (Pc) base, 2 Li have been replaced by most metal (particularly multivalence complexing) ⁺Ion.Therefore, use that to replace 2 Li+ ions with not labile part bonded metal ion are a kind of methods of putting stable functional group on the protofibril surface.Almost transition metal complex all will be from Pc base replaces Li ⁺Ion and form stable, not labile inner complex.This point is with suitable part and melts combine then.

Phthalocyanine cobalt (II)

[00102] cobalt (II) complex compound is particularly suitable .Co to this ⁺⁺Ion can be by two Li ⁺Replace and form highly stable inner complex.Co then ⁺⁺Ion can with for example nicotinic acid coordination of part, nicotinic acid comprises the pyridine ring with side carboxyl, and known preferential and pyridyl combination.In the presence of excessive nicotinic acid, Co (II) Pc can change into Co (III) Pc by electrochemistry oxygen, with the not labile complex compound of pyridine moiety formation of nicotinic acid.Therefore, free carboxy acid's base of nicotinic acid part is firmly bonded to fibriilar surface.

[00103] other suitable part is aminopyridine or Aminocardol (side group NH ₂), dredge yl pyridines (SH) or other multi-functional part that comprises amino or pyridine moiety and required function is arranged at the other end at a terminal.

[00104] carrying capacity of porphyrin or phthalocyanine can by when add fashionable solution decolouring determine.Discharge dark solution (for the tetracarboxylic acid porphyrin in MeOH is rediance, is dark blue-green for phthalocyanine Co (II) or two lithiums in acetone or pyridine) and remove this molecule by absorption on fibriilar black surface.

[00105] estimate carrying capacity by this method, the footprinting of derivative is calculated by its proximate mensuration (～140 square angstroms).For fibriilar average surface area 250m ²/ g, maximum load is～0.3mmol/g.

[00106] analyzes the tetracarboxylic acid porphyrin by titration.By around with comparatively high temps under absorption is tested in the release of look in water system integrity.

[00107] mixes protofibril slurry (Wei Lin agitator) and stir during in load in when beginning.When no longer discharging color, some slurries of supersound process, but there is not effect.

[00108] after load, in identical solvent washing operation 169-11 ,-12 ,-14 and-19-1 (seeing Table II), to remove the pigment of obturation.In the washing elutriant, obtain the little light color of successive, so just be difficult to accurately determine saturation point.Operation 168-18 and-19-2 is used for the pigment of load calculation amount, and only washing very slightly after load.

[00109] tetracarboxylic acid porphyrin (from acetone) and Co phthalocyanine (from pyridine) load to and are used for recording and narrating further feature on the protofibril. (operation 169-18 and-19-2, respectively).

The analysis of tetracarboxylic acid porphyrin

[00110] adds excessive alkali (pH11-12) and cause this titration slurry pulverize redness at once.When this can not interact with titration, shown porphyrin desorption when higher pH.Determine the concentration of carboxylic acid as terminal point with pH7.5 by the excess NaOH back titration.Titration has provided the sour load of 1.10meq/g, is equivalent to the 0.275meq/g porphyrin.

The analysis of phthalocyanine two lithiums or cobalt

[00111] only estimate the concentration of these adsorbates from Decolorant Test, the indigo plant-green point that does not fade is exactly a saturation point after 30 minutes.

[00112] in the protofibril surface adsorption polynuclear aromatic family or many heteronuclears aromatics of a large amount of replacement.In order to adsorb, the quantity of aromatic nucleus should greater than 2/ring or side functional group.Therefore, comprise the anthracene, phenanthrene etc. of the replacement of 3 fused rings or comprise 4 or the multi-functional derivative of more fused rings can be used to replace porphyrin or phthalocyanine derivates. similarly, can use to comprise 4 or for example quinoline or polysubstituted hetero-aromatic ring of the aromatic heterocycle that replaces of polycyclic more.

[00113] table 2 has been summarized the result of 3 kinds of porphyrin/phthalocyanine derivative load tests.

TCAPorph=tetracarboxylic acid porphyrin (cal)=calculating

DiLiPhth=phthalocyanine two lithiums (T)=titration

CoPhth=phthalocyanine cobalt (H)

[00114] following

embodiment

11 and 12 has illustrated two kinds of different phthalocyanine derivates has been adsorbed onto method on the carbon nanotube.

Embodiment 11

By the functionalized protofibril of absorption phthalocyanine tetrasulfonic acid nickel (II)

[00115] with 2mg phthalocyanine tetrasulfonic acid nickel (II) (tetra-na salt) and the pure protofibril of 4.2mg at 1ml dH ₂Mix among the O.This mixture of sonication 50 minutes, and at room temperature rotation is spent the night.

[00116] with 3 x 1ml dH ₂O, 3 x 1ml MeOH and 3 x 1ml CH ₂Cl ₂Washing protofibril, and vacuum-drying.

[00117] on the protofibril of these phthalocyanine derivates, passes through fixedly thermolysin of absorption.The 0.5mg protofibril is suspended in 250 μ l dH ₂Among the O, sonication 20 minutes.Discharge supernatant liquor, protofibril is suspended in 250 μ l 0.05M Tris (pH=8.0) in, mixes with the 250 μ l 0.6mM thermophilic bacteria protein enzyme solution that same buffer is made.Mixture at room temperature rotated 2 hours, stored down at 4 ℃ and spent the night.Use 1ml 25mM Tris (pH=8) washing 3 times then.And be suspended in pH7.5 comprise 40mM Tris and 10mM CaCl ₂250 μ l damping fluids in.

[00118] determine the amount of thermolysin on the protofibril by measuring fibriilar enzymic activity. thermolysin can with substrate FAGLA (N-(3-[2-furyl] propionyl)-Gan ammonia-leucyl amine) reaction; generation causes the compound in the reduction of 345nm absorbancy, and optical extinction coefficient is-310M ^-1Cm ^-1The analysis buffer condition of this reaction is 40mM Tris, 10mMCaCl ₂With 1.75M NaCl, pH is 7.5.Be reflected in the 1ml test tube and carry out, with 5 μ lFAGLA mother liquors (at dH ₂25.5mM 30% DMF among the O) and 10 μ g thermolysin protofibril in the analysis buffer of 1ml, mix.Scanning detects 345nm place absorbancy and reduces more than 10 minutes.With optical extinction coefficient be-310M then ^-1Cm ^-1Calculate enzymic activity (μ M/ minute) from initial slope.The amount of active thermolysin is 0.61 μ mole in every g protofibril.

Embodiment 12

By adsorbing 1,4,8,11,15,18,22,25-eight butoxy-29H, the functionalized protofibril of 31H-phthalocyanine

[00119] get 3mg1,4,8,11,15,18,22,25-eight butoxy-29H, the pure protofibril of 31H-phthalocyanine and 5.3mg is at the CHCl of 1ml ₃The middle mixing.This mixture of sonication 50 minutes and rotation are at room temperature spent the night.

[00120] with the CH of 3 x 1ml ₂Cl ₂Wash this protofibril and vacuum-drying.

[00121] method according to embodiment 34 is fixed in thermolysin on the protofibril of phthalocyanine derivates by absorption.The amount of active thermolysin is 0.70 μ mole in every g protofibril.

Embodiment 13

With the protofibril of phthalocyanine derivates and the thermophilic bacteria protein enzymic synthesis aspartame precursor that is fixed thereon

[00122] protofibril of having fixed the phthalocyanine derivates of thermolysin can be used for precursor synthetic of catalysis artificial sweetner aspartame. and carry out this reaction, comprise that the protofibril that use 10 μ M have fixed thermolysin mixes with 80mM L-Z-Asp and 220mM L-PheOMe in ethyl acetate.Detect product Z-Asp-PheOMe to determine yield by HPLC.

5. the oxidation of chloric acid or nitric acid

[00123] pass through for example document of the Potcrate graphite oxide in the vitriol oil or nitric acid of strong oxidizer, comprise R.N.Smith, Quarterly Review 13,287 (1959); MJ.D.Low, Chem.Rev.60.267 (1960)). usually, attack the mixture that edge carbon can obtain (comprising deletion segment) carboxylic acid, phenol and other oxide groups.Its mechanism is complicated, comprises free radical reaction.

Embodiment 14

Prepare carboxylic acid functionalized protofibril with oxymuriate

[00124] the fibriilar sample of CC pulp in the vitriol oil comprises with shovel and mixing, and transfers to then in the reaction flask with gas feed/outlet and top agitator.Stirring and slowly under the argon gas stream, adding NaClO at run duration portioning under RT ₃. in the whole process of operation, produced chlorine steam, it is cleared out of entered in the reactor in the NaOH trap. aqueous solution.Behind the end of run, the protofibril slurry is poured in the trash ice vacuum filtration. then filter cake is transferred on the Soxhlet cylindrical filter paper, in the Soxhlet extractor, washed, every a few hours exchange fresh water with DI water.Continue washing can not change water after fibriilar sample is adding fresh DI water pH.Filtering separation protofibril then is 5 " vacuum and 100 ℃ of dried overnight.

[00125] determine the amount of carboxylic acid, comprise and say sample and excessive 0.100N NaOH reaction, and with the terminal point of 0.100N HCl back titration to pH7.5.The results are shown in following table.

Table III

Directly carry out the summary of oxidation

Embodiment 15

Prepare carboxylic acid functionalized protofibril with nitric acid

[00126] have in the reaction flask of trace fibriilar sample and the suitably nitric acid pulp of intensity of having weighed at the many necks of the round bottom with top agitator and water condenser. under constant speed stirs, attemperation, and react at specified time. when temperature surpasses 70 ℃, no matter the intensity of acid how, all discharged brown smog at once.After the reaction, slurry is poured in the trash ice, and dilute with DI.Filter this slurry, excessive acid is removed in washing in the Soxhlet extractor, replaces water in the container every several hours with fresh DI water, does not change the pH. of DI water 5 until the slurry sample " vacuum and 100 ℃ are the protofibril dried overnight.The protofibril of the part of having weighed and the 0.100N NaOH of standard react, and determine the content of carboxylic acid with 0.100N HCl back titration.Determine the content of surperficial oxygen by XPS.By mixing the dispersibility of determining 0.1wt% in water in 2 minutes in Wei Lin agitator high speed. the result is summarized in the table 4.

Table IV

Directly carry out the summary of oxidation

P=is poor; G=is good

6. fibriilar aminofunctional

[00127] amino can be introduced directly on the graphite protofibril, comprises according to following general formula, with nitric acid and vitriolization protofibril, obtains nitrated protofibril, then with reductive agent for example Sodium Hydrosulphite reduce this nitrated type and obtain the protofibril of aminofunctional:

Resulting fibril has numerous purposes, comprises fixing protein (for example enzyme and antibody) and affine and ion-exchange chromatography.

Embodiment 16

The protofibril for preparing aminofunctional with nitric acid

[00128] to the water 1.6ml of protofibril (70mg)) and acetate 0.8ml) add nitric acid (0.4ml) in the mode that drips in the cold suspension.At 0 ℃ reaction mixture was stirred 15 minutes, at room temperature restir is 1 hour.Slowly add the mixture of sulfuric acid (0.4ml) and hydrochloric acid (0.4ml), at room temperature stirred 1 hour.Stopped reaction is also centrifugal.Remove water layer, water (X5) washing protofibril.Handle residue with 10% sodium hydroxide (X3), water (X5) washing obtains nitrated protofibril.

[00129] in the suspension of nitrated fibriilar water (3ml) and ammoniacal liquor (2ml), dividing adding Sodium Hydrosulphite (200mg) three times to protofibril s. under 0 ℃.At room temperature stirred reaction mixture is 5 minutes, refluxes 1 hour down at 100 ℃. and stopped reaction is cooled to 0 ℃, regulate pH with acetate (pH4), after at room temperature placement is spent the night, filter this suspension, water (X10), methyl alcohol (X5) washing, vacuum-drying obtains amino protofibril.

[00130] in order to test the protofibril of aminofunctional, this protofibril is combined with horseradish peroxidase.To extensively dialyse with the amino protofibril of HRP bonded then.After dialysis, in next week, protofibril is washed 15 times.The protofibril that following enzyme analysis is modified:

[00131] result shows, is preserving above after the week, with protofibril-NH ₂Bonded HRP has shown good enzymic activity.

7. with the alcohol of radical initiator in conjunction with end

[00132] when it is used for severe rugged environment, the high stability of carbon nanotube makes them be difficult to activation and further modifies.Existing method comprises uses strong oxygenant and acid.Be surprisingly found out that now, with radical initiator for example benzoyl peroxide (BPO) can be attached to terminal alcohol on the carbon nanotube.Adding general formula on carbon nanotube is RCH ₂The alcohol of OH, wherein R is hydrogen, alkyl, aryl, cycloalkyl, aralkyl, aryl or poly-(alkyl oxide) and radical initiator, and is heated to about 90 ℃ from about 60 ℃.Preferred alcohol comprises methyl alcohol and ethanol.After past time that enough all radical initiators decompose, filter reaction mixture, washing carbon nanotube material is also dry, obtains the nanotube of the modification of general formula nanotube-CH (R) OH.This method also can be used to connect bifunctional alcohol.This just allows an end to connect carbon nanotube, and the other end is used for being connected indirectly with other materials on surface.

Embodiment 17

The nanotube for preparing carbinol-functionalization with benzoyl peroxide

[00133] is distributed among the MeOH with the carbon nanotube of probe ultrasonic disruption device 0.277g.Add the BPO of 0.126g under RT, temperature is elevated to 60 ℃, adds the BPO raw material of 0.129g, and mixture kept under 60 ℃ 30 minutes again.Filtering product on film washs several times with MeOH and EtOH, and is dry in 90 ℃ baking box.Yield is 0.285g.Esca analysis shows that oxygen level is 2.5% atom, and in contrast to this, the control sample that refluxes in the MeOH that does not contain BPO is 0.74%.

Embodiment 18

Modify the carbon nanotube that contains poly-(ethylene glycol) with benzoyl peroxide

[00134] poly-(ethylene glycol) avg.mol.wt.1000 (PEG-1000) of carbon nanotube, 0.5g BPO and the 10g of 0.1g is at room temperature mixed.Temperature is heated to 90 ℃ and makes the PEG fusing, mixture is remained on 90 ℃ of reactions to spend the night. filter whole mixtures then, excessive PEG is removed in washing, and is dry then. and can use resulting material, perhaps can further modify in conjunction with interested material by free terminal to PEG.

Embodiment 19

The application of the carbon nanotube that PEG modifies in reducing non-specific binding

[00135] be ubiquitous with the bigger carbon class material non-specific binding of surface-area. have been found that combine hydrophilic oligomers for example PEG to carbon nanotube, can reduce non-specific binding.In addition, have been found that at the surface bonding chain sample molecule of carbon nanotube PEG for example, its free terminal can comprise can with other substances of interest bonded functional groups, still kept the character that PEG (or other materials) layer reduces non-specific binding simultaneously.

The protofibril that PEG modifies reduces the non-specific binding of bovine serum albumin

[00136] female dispersion liquid of the 50mM dipotassium hydrogen phosphate damping fluid of the fibriilar pH7.0 of the protofibril of the protofibril of the unmodified of preparation 0.1mg/ml, oxymuriate oxidation and PEG modification comprises that every kind of material with 1.0mg is dispersed in the damping fluid of 10mis with sonication. in 9 polypropylene tube, put into 2 times of serial dilutions of every kind of 2mis.The same buffer solution of adding 100 μ l 0.2mg/ml bovine serum albumins in each pipe and three blank damping fluids.Also preparing nonprotein three damping fluid pipes. all pipes all mix on the vortex mixed thing, cultivate 30 minutes, and vortex was 30 seconds in wherein per 10 minutes.The all pipe of centrifugation is transferred to the supernatant liquor of 1ml five equilibrium in the new pipe, with little BCA analysis of protein method (Pierce) analyzing total protein content to separate protofibril.The proteinic level that keeps in supernatant liquor is the indirect measurement value with the amount of protofibril non-specific binding.For the protofibril that PEG modifies, all BSA are retained in the supernatant liquor, and they almost completely combine (accompanying drawing 1) with the protofibril of unmodified or oxymuriate oxidation simultaneously.

The protofibril of modifying with the PEG of benzoyl peroxide preparation with combine non-spy by NHS The comparison of anisogamy minimizing

[00137] use sonication at 50mM dipotassium hydrogen phosphate damping fluid, the protofibril of preparation 1.0mg/ml perchlorate oxidation among the pH7.0, protofibril of being modified by PEG with benzoyl peroxide and perchlorate oxidation, by the NHS ester in conjunction with fibriilar female dispersion liquid of being modified by PEG. in 7 polypropylene tube, put into 3 times of serial dilutions of every kind of 2mis.The same buffer solution of adding 100 μ l 0.2mg/ml beta-lactoglobulins (β LG) in each pipe and three blank damping fluids. also prepare nonprotein three damping fluid pipes.All pipes all mix on the vortex mixed thing, cultivate 60 minutes, and vortex was 30 seconds in wherein per 10 minutes.The all pipe of centrifugation is transferred to the supernatant liquor of 1ml five equilibrium in the new pipe, with little BCA analysis of protein method (Pierce) analyzing total protein content to separate protofibril.The proteinic level that keeps in supernatant liquor is the indirect measurement value (seeing accompanying drawing 2) with the amount of protofibril non-specific binding. for each pipe, through the protofibril of NHS ester approach with the PEG modification, β LG is retained in the supernatant liquor, shows not have non-specific binding.Only show the slight combination of β LG (about 10%) during the protofibril highest level of the protofibril of modifying with PEG through the BPO approach, when low-level, then do not have significant combination at 1.0mg/ml.On the contrary, when 1.0mg/ml and above-mentioned protofibril level, almost combine, be low to moderate still combination basically of 0.01mg/ml at protofibril with the protofibril of oxymuriate oxidation is complete.

8. the secondary derivative of functionalized nanotube

Carboxylic acid functionalized nanotube

[00138] can be only be unlimited basically by the quantity of the secondary derivative of carboxylic acid preparation.If alcohol or amine are easy to and acid combine and forms stable ester or acid amides. alcohol or amine are two of part-or difunctional polyfunctional molecules, will make other the functional side group that becomes so by O-or NH-connection.The exemplary embodiments of secondary reagent is:

General formula Side group Embodiment

HO-R, R=alkyl, aralkyl, aryl, R-methyl alcohol, phenol, three fluorocarbon based, the terminal polyester of OH-, silanol

Fluorinated alcohols, polyester, SiR ' 3

H ₂N-RR=R-amine same as described above, aniline, fluoroamine, silylation amine, the polymeric amide of amido end, protein

Cl-SiR ₃SiR ₃-chlorosilane

HO-R-OH, R=alkyl, HO-ethylene glycol, PEG, five erythritols, dihydroxyphenyl propane

Aralkyl, CH ₂O-

H ₂N-R-NH ₂, R=alkyl, H ₂N-quadrol aminophylline, polyvinylamine

Aralkyl

X-R-Y, R=alkyl etc.; Y-polyamine acid amides, mercaptoethanol

X=OH or NH ₂Y=SH, CN,

C=O, CHO, alkene, alkynes, aryl,

Heterocycle

[00139] this reaction can be carried out with any method, so that with alcohol or amine esterification or amination carboxylic acid.Wherein, use H.A.Staab, Anagew.Chem.Intemat.Edit., (1), and 351 (1962) method is used N, and N '-N,N'-carbonyldiimidazole (CDI) prepares ester or acid amides as acylating agent; Use G.W.Anderson, wait the people, J.Amer.Chem.Soc.86,1839 (1964) method uses N-hydroxy-succinamide (NHS) activating carboxy acid with amidation.

Embodiment 20

The preparation of functionalized fibriilar secondary derivative

N, N '-N,N'-carbonyldiimidazole

[00140] this method needs clarifying, exsiccant aprotic solvent (for example toluene Huo diox).The reagent of chemistry amount is enough.For ester, carboxylic acid cpd reacted 2 hours under RT with the CDI that is dissolved in the toluene of chemistry amount in toluene in rare gas element (argon gas).During this period, discharge CO ₂After 2 hours, add the sodium ethylate of pure and mild catalytic amount, continue 80 ℃ of reactions 4 hours.For usual alcohol, yield is quantitative.This reaction is:

1.R-COOH+Im-CO-Im----------R-CO-Im+HIm+CO ₂, the Im=imidazoles,

The HIm=imidazoles

2.

[00141] amidation of amine not catalysis under RT can take place.The first step of this method is identical.Discharging CO ₂After, at room temperature add the amine that chemistry is measured, reacted 1-2 hour.This reaction is quantitative.This reaction is:

3.R-CO-Im+R’NH ₂--------------->R-CO-NHR+Him

Silylation

[00142] trialkylsilanyl chlorine or trialkyl silanol and tart H are immediately according to following reaction formula reaction:

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

[00143] a spot of diazonium-1,1,1-bicyclooctane (DABCO) is as catalyzer.Appropriate solvent Shi diox and toluene.

Sulfonic acid-functionalized protofibril

[00144] aryl sulfonic acid for preparing as embodiment 1 can further react and obtain secondary derivative.Sulfonic acid can be by LiAlH ₄Or the combination of triphenylphosphine and iodine (p.1107) March, J.P. are reduced into mercaptan.They also can be transformed into sulphonate, i.e. protofibril～SO by reacting with dialkyl ether ₃H+R-O-R---------〉protofibril-SO ₂OR+ROH

N-hydroxy-succinamide

[00145] come the activating carboxy acid to finish with the primary amine amidation by N-hydroxyl succinyl-ester; Carbodiimide is used to hinder water and discharges with the urea that replaces. then under RT with the NHS ester by converting acid amides to primary amine reaction.This reaction is:

1.R-COOH+NHS+ carbodiimide-----------R-CONHS+ material urea

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

[00146] this method particularly can be used for protein and the graphite protofibril free NH through protein side chain ₂Covalent attachment. can comprise trypsinase, streptavidin and avidin by the proteinic example that this method is fixed on the protofibril.Streptavidin (or avidin) protofibril provides solid carrier for biotinylated substrate arbitrarily.

Embodiment 21

Protofibril and protein are through NHS ester covalent attachment

[00147] in order to prove that protein can be covalently bound on the protofibril through the NHS ester, is attached to streptavidin, avidin and trypsinase on the protofibril as follows.

[00148] the NHS-ester protofibril of usefulness 5mM sodium phosphate buffer (pH7.1) washing 0.5mg, abandoning supernatant.In protofibril, add 200 μ l streptavidin solution (in same buffer, 1.5mg), at room temperature rotated mixture 5.5 hours, wash protofibril successively with the following damping fluid of 1ml then: 5mM sodium phosphate (pH7.1), PBS (0.1mM sodium phosphate, 0.15mM NaCl, pH7.4), ORIGEN ^TMAnalysis buffer (IGEN, Inc., Gaithersburg, MD) and PBS.The streptavidin protofibril is stored in and is used for further application in the PBS damping fluid.

[00149] with the sonication 40 minutes in the 5mM sodium phosphate buffer (pH7.1) of 500 μ l of 2.25mg NHS-ester protofibril, abandoning supernatant.This protofibril is suspended in the 5mM sodium phosphate buffer (pH7.1) of 500 μ l, the avidin solution that adds 300 μ l, wherein avidin solution is the (Sigma that makes in comprising the identical damping fluid of 2mg avidin, A-9390). at room temperature with this mixture rotation 2 hours, under 4 ℃, store and spend the night, at room temperature rotated again 1 hour., wash 2 times protofibril washing 4 times with 5mM sodium phosphate buffer (pH7.1) with the PBS damping fluid. the avidin protofibril is suspended in the 200 μ lPBS damping fluids stores.

[00150] preparation of trypsinase protofibril, comprise 1.1mg NHS-ester protofibril (handling) and the 1.06mM trypsin solution mixing of 200 μ l of preparation in 5mM sodium phosphate buffer (pH7.1), at room temperature rotated 6.5 hours as the avidin protofibril.Then with the trypsinase protofibril with the 5mM sodium phosphate buffer (pH7.1) of 1ml washing 3 times, and be suspended in the same buffer of 400 μ l and store.

Embodiment 22

The mensuration of tryptic enzymic activity on the protofibril

[00151] trypsinase can with substrate L-BAPNA (N _α-benzoyl-L-arginine p-Nitroaniline) reaction discharges the light absorbing colored compound at 410nm.The analysis buffer of this reaction is 0.05M Tris, 0.02M CaCl ₂, pH8.2.This is reflected in the 1ml test tube and carries out, and comprises that L-BAPNA mother liquor with 5 μ l is (at H ₂The 37%DMSO of 50mM among the O) and the trypsinase protofibril of 10-25 μ g in the analysis buffer of 1ml, mix.After 10 minutes, detect absorbancy increases at the 410nm place. calculate enzymic activity (μ M/ minute) from the gradient of beginning then.

[00152] for covalently bound trypsinase protofibril, this activity is per 13 μ g protofibril 5.24 μ M/ minutes.This result also can be transformed into the tryptic amount of activation on protofibril, comprises the activity divided by the trypsin solution of concentration known, and it is per 1 μ M trypsinase 46 μ M/ minutes that the trypsin solution of concentration known is measured under the same analysis condition.Therefore, the tryptic amount of the fibriilar activation of every g is 8.3 μ moles (or 195mg).

Embodiment 23

Carbon nanotube with surperficial mercaptan

[00153] will be suspended in the 0.05M dipotassium hydrogen phosphate damping fluid of the 20mis pH8.0 that comprises 50mM EDTA by the amino-carbon nanotube (CN) with the 0.112g of quadrol aminophylline modification of embodiment 27 (hereinafter) preparation.This suspension is thought highly of sonication 5 minutes to disperse CN in Branson 450 Watt probe ultrasonications.Resulting suspension is the ten minutes heavy-gravity.Stir and feed argon gas foaming 30 minutes down. add the 2-imido grpup mercaptan .HCl of 50mg, mixture is continued to be stirred in continue reaction 70 minutes under the argon gas.On polycarbonate membrane filter, filter resulting material, wash 2X,,, all under argon gas covers, carry out with pure EtOH washing 2X with DI water washing 1X with damping fluid.The CN that mercaptan is modified places vacuum drier, and suction is spent the night

[00154] final weight=0.118g increases according to weight, and number turnover is 55%.

[00155] is suspended in the DI water of 10mis. with the 10mg sample of sonication, on 0.45 μ m nylon membrane, filters the material that forms the blanket sample the mercaptan nanotube.Earlier this material part is stored in the vacuum drier, uses esca analysis then, this analysis shows 0.46% sulphur and 1.69% nitrogen, proves successfully to be transformed into the CN that mercaptan is modified.

Embodiment 24

The carbon nanotube that mercaptan is modified with contain combining of gold surface

[00156] with 1 part of 30% H ₂O ₂Clean goldleaf (Alfa/Aesar) with the solution of 3 parts of vitriol oils, 2cm x 0.8cm, totally 10 minutes, and clean with DI water.The paper tinsel fragment link to each other with Au the end of a thread and its electrochemistry at 1M H ₂SO ₄In between 1.45V vs.Ag/AgCl, circulate at-0.35Vvs.Ag/AgCl with the speed of 50mv/ second, until cyclic voltammogram till not changing in about 10 minutes.Clean and drying with DI water then.Big fragment cuts into the bar of 4 0.5cm x 0.8cm.

[00157] the pure EtOH of 10mis that will be by the argon cleaning deoxidation places 2 vials respectively.In a bottle, CN (CN/SH) and 2 Au fragments that the mercaptan of the 16mg that suspended is modified, and in another bottle, make the mercaptan derivatize with the ethylene diamine-modified CN of 1 fragment of Au and 10mg.All operations are all carried out in being full of the bag glove of argon gas.Wherein seal this bottle at argon, placed the refrigerated ultra sonic bath 1 hour.The bottle of sealing was placed under RT 72 hours. remove the Au sample from bottle, wash 3X with EtOH, dry air also places the bottle of protectiveness.

[00158] by scanning electron microscopy (SEM) (SEM) check the Au foil sample be exposed among CN/ quadrol aminophylline and the CN/SH detect go up CN in the surface existence whether. 40, inspection under the 000X shows the distribution that has CN on the surface of CN/SH being exposed to, but does not observe CN on the Au foil sample that is exposed to CN/ quadrol aminophylline.

Embodiment 25

Prepare the maleimide protofibril by amino protofibril

[00159] according to the amino protofibril of embodiment 13 preparations.Then with amino protofibril (62.2mg) sodium phosphate buffer (5ml, 5mM, pH7.2) in sonication.In the protofibril suspension, add Sulfosuccinmidyl-4-(N-maleimide methyl) hexanaphthene-1-carboxylate salt (SMCC; 28.8mg, 0.66mmols; Pierce, Cat.No.22360). at room temperature the stirred reaction mixture fruit is also.Water and methanol wash protofibril, vacuum-drying product protofibril.The antibody that is fixed on the product has proved the fibriilar existence of maleimide. other maleimide (for example, sulfo group-SMCC, succinimidyl 4-[are to maleimide phenyl] butyric acid [SMPB], sulfo group-SMPB, m-maleoyl benzyl-N-hydroxy-succinamide ester [MBS], sulfo group-MBS etc.) protofibril with different connexons also can prepare by identical method.

[00160] resulting maleimide protofibril can be as the protein solid carrier of the Covalent Immobilization of antibody and enzyme for example, and the antibody Covalent Immobilization is on the activation protofibril of maleinamide.When using from amino protofibril that nitrated/reduction method (embodiment 13) obtains, the capacity of antibody is every g protofibril 1.84mg, and is every g protofibril 0.875mg when the amino protofibril that uses the carboxyl protofibril to prepare.

Embodiment 26

Prepare ester/alcohol derivatives from carboxylic acid-functionalized protofibril

[00161] as the carboxylic acid functionalized protofibril of embodiment 14 preparations. the content of carboxylic acid is 0.75meq/g.Protofibril is CDI reaction at room temperature in inert atmosphere of solvent with toluene with the chemistry amount, until CO ₂Stop to discharge.Then, the following macrogol (MW 600) with 10 times of molar excess at 80 ℃ of this slurry reacts, and adds small amount of N aOEt as catalyzer. and react after 2 hours, the filtering separation protofibril is used toluene wash, and 100 ℃ of dryings.

Embodiment 27

By carboxylic acid functionalized protofibril (177-041-1) preparation acid amides/sulfonamide derivatives

[00162] stirring down in having the 100ml RB flask of serum stopper protofibril (0.62meq/g) with the oxymuriate oxidation of 0.242g is suspended in 20ml and does not have in the water diox.The N-hydroxy-succinamide (0.299g) and the dissolving that add 20 times of molar excess. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDAC) that adds 20 times of molar excess then (0.510g) continues at room temperature to stir 2 hours.After finishing during this period of time, stop to stir, extract supernatant liquor,, on 0.45 micron polysulfone membrane, filter with no Shui diox and MeOH washing solid.Wash solid with MeOH again on filter membrane, vacuum-drying no longer reduces until observing weight. and increase by 6% according to the weight that is observed, the fibriilar yield of NHS-activatory oxidation is 100%.

[00163] 100 μ l quadrol aminophyllines (en) is joined 10ml 0.2M NaHCO ₃In the damping fluid.The acetate (HOAc) that adds equivalent is to keep pH about 8.The en and the 300 μ l HOAc that add 300 μ l after under brute force stirs, adding NHS-activatory oxidation protofibril (0.310g) and reacting 1 hour .10 minute again.On 0.45 micron polysulfone membrane, filter this solution, and use NaHCO ₃Damping fluid, 1%HCl, DI water and EtOH continuous washing.This solid of vacuum-drying spends the night. and by reacting with NaOH (177-046-1), HCl salt is converted back into free amine again and is used for further analyzing and reaction.

[00164] carries out ESCA to determine amination protofibril (GF/NH ₂) in the amount of N.The esca analysis of 177-046-1 shows and contains 0.90 at% N (177-059). in order further to estimate the amount of N in susceptible to and reactive amino, prepare a kind of derivative, comprise with penta fluoro benzene formaldehyde gas-phase reaction generating the corresponding Schiff alkali that effectively is connected with primary amine groups.Esca analysis still shown, as expected, contain 0.91 at%N and 1.68 at% F. this can to convert the N that exists as reactive primary amine on the amination protofibril to be 0.34 at% (each penta fluoro benzene formaldehyde molecule contains 5 F).Suppose the free-end complete reaction at each N, the level of N should be 0.45 at%.The level that is observed shows that N and the fibriilar reaction of NHS-activatory have very high yield, have proved the reactivity of available free amino.

[00165] by the level of ESCA data computation as the N of 0.34 at% of unhindered amina existence, this almost is fibriilar four corner by the free amido, and this just allows to be connected with other material.

[00166] the carboxyl protofibril also can be transformed into amino protofibril, comprise use list-protection 1 (with in six carbon connexons), rather than quadrol aminophylline (a kind of two carbon connexons).

Embodiment 28

Prepare sulfonamide derivatives by carboxylic acid functionalized protofibril

[00167] carboxyl on the modification protofibril; comprising amino a reaction on carboxyl and the compound (at least one amino is the radical protection that not have t-Boc for example or CBZ) with two or more amino. Zhi Bei protofibril is an amide derivatives like this; wherein amidocarbonylation is from fibriilar carboxyl, and the group of the involved one or more primary amine of amide nitrogen (for example alkyl) replaces.Amino can use effectively or further modify then.

[00168] carbon fibrils of getting 1g places exsiccant scintered glass strainer tube, and its outlet clogs tightly with rubber serum partition, adds anhydrous methylene chloride and covers.(758 μ L, 7mmol), the medication shovel is assisted and is mixed suspension to add N-methylmorpholine.(915 μ L, 7mmol), suspension regularly mixed 1 hour to add isobutyl chlorocarbonate then.By Parafilm cover as much as possible so that this mixture away from the water vapor in the atmosphere.

[00169] simultaneously, hydrochloric acid N-boc-l, 6-diamino base alkane (1.94g, 7.7mmol) layering between methylene dichloride (10mL) and 1M NaOH (10mL).Lower organic phase is dry on Anhydrous potassium carbonate, filters by comprising the disposable pasteur pipet of cotton plug, add N-methylmorpholine (758 μ L, 7mmol).

[00170] remove the serum deprivation partition from filter funnel, reagent is removed in vacuum filtration from protofibril, wash protofibril with anhydrous methylene chloride.The serum partition is put back to, in protofibril, added the diamino hexane of N-methylmorpholine and single protection.Periodically stirred the mixture 1 hour.Then, remove by filter reagent, once use methylene dichloride, methyl alcohol, water, methyl alcohol and washed with dichloromethane protofibril.

[00171] 50% mixture of adding three fluoric acids and methylene dichloride in protofibril periodically stirred the mixture 20 minutes.Remove by filter solvent, use methylene dichloride, methyl alcohol, water, 0.1M NaOH and water washing protofibril successively.

[00172] for the validity of method of proof, with amino fibriilar small sample with modified with amino specific reaction " activatory " horseradish peroxidase (HRP; 5mg, Pierce) reaction.Repetitive scrubbing protofibril in several days (suspendible, rotation and centrifugal in the Eppendorf Eppendorf tube) keeps cooling simultaneously.After about 2 weeks of washing, use H ₂O ₂/ ABTS is at glycine buffer, and solution shows green in enzyme analysis .10 minute has shown the existence of enzyme among the pH4.4.The protofibril of contrast (with the COOH protofibril of activatory HRP processing and in same time washing) shows if any catalytic activity is arranged, also almost do not have enzyme.

Embodiment 29

Prepare the silicomethane derivative by carboxylic acid functionalized protofibril

[00173] in inert atmosphere, will be as acid-functionalized protofibril pulp in diox of embodiment 14 preparations.Stirring down, add the chlorine triethyl silicane of chemistry amount, reacted 0.5 hour, drip the dioxane solution of several 5%DABCO then. system reacted 1 hour again, filtered then and collected protofibril, washed in diox.This protofibril is at 100 ℃ 5 " dried overnight in the vacuum.

[00174] Table V has been summarized the preparation of secondary derivative.The surperficial content of C, O, N, Si and F by analyzing the esca analysis product.

Table V

The general introduction of secondary derivative preparation

Embodiment 30

Prepare silane derivative by carboxylic acid-functionalized protofibril

[00175] in inert atmosphere, will be as acid-functionalized protofibril pulp in diox of embodiment 14 preparations.Stir down, add the chlorine triethyl silicane of chemistry amount, reacted 0.5 hour, drip several 5%DABCO De dioxane solutions then.System reacted 1 hour again, filtered then and collected protofibril, washed in the Zai diox.This protofibril is at 100 ℃ 5 " dried overnight in the vacuum.

[00176] Table VI has been summarized the preparation of secondary derivative.By analyzing the esca analysis product.This analysis revealed has mixed required side group.The surperficial content of C, O, N, Si and F by analyzing the esca analysis product.

Table VI

The general introduction of secondary derivative preparation

Embodiment 31

Prepare tertiary amine and quaternary ammonium derivative by carboxylic acid functionalized protofibril

[00177] tertiary amine and quaternary amine functional group can be through acid amides or ester bond carboxyl and the amine of tertiary amine and quaternary amine precursor or the surfaces that hydroxyl is attached to carbon nanotube through nanotube.These tertiary amines can be used as chromatography matrix with the first fiber of quaternary amine and are used for separating of biomolecules.Tertiary amine and quaternary amine protofibril can be assembled in the discous pad or be mixed for isolating purpose with the chromatography matrix (for example agarose) of routine.

The preparation of iodate triethyl thanomin precursor

[00178] in the 100ml round-bottomed flask, with 10g N, N-diethylethanolamine (85.3mmole) mixes with the 10ml anhydrous methanol.Drip the mixture of 20g iodoethane (127.95mmole) and 10ml anhydrous methanol then with valinche.With reaction mixture refluxed 30 minutes.When reaction mixture is cooled to room temperature, formed the white crystal product.To filter and collect white solid product, and wash with anhydrous methanol. product further vacuum-drying in moisture eliminator is spent the night.(10.3g, 37.7mmole) yield is 33% to the product that obtains.

The fibriilar preparation of graphite that quaternary amine is functionalized

[00179] in the disposable scintillation vial of vacuum drying 25ml Wheaton, with dry carboxyl protofibril of 100mg (the about 0.7mmole COOH of every g protofibril) and 2ml anhydrous dimethyl base acid amides, with mixture sonication 60 seconds. in reaction flask, add 2L dimethylformamide dimethylformamide, 39mg dimethyl-aminopyridine (0.316mmole) and 50 μ l DIC (0.316mmole).At room temperature reaction mixture was stirred 1 hour, add 88mg iodate triethyl thanomin (0.316mmole) then in this bottle, reaction is spent the night.Resulting protofibril is used 20ml washed with dichloromethane 3 times with 20ml dimethylformamide washing 3 times, uses 20ml methanol wash 3 times, uses deionized water wash at last 3 times.This product of vacuum-drying. to the result of nitrogen ultimate analysis show on the protofibril about 50% carboxyl with the primary amino reaction of quaternary amine part

Embodiment 32

The chromatography of bovine serum albumin (BSA) on the functionalized graphite protofibril of tertiary amine

[00180] (aqueous slurry Sweden) is at room temperature preserved and is spent the night for Pharmacia, Uppsala, to guarantee the complete hydration of solid carrier will to comprise carboxyl protofibril that 60mg 2-ethylamino ethamine modifies and the hyperfine resin of 180mg sephadex G-25.Slurry is encased in the 1cm x 3.5cm post.This post is with the flow velocity balance of 5mM sodium phosphate buffer (pH7.3) with 0.2ml/ minute. load BSA on post (0.6mg is in the 0.1ml deionized water).With 5mM sodium phosphate buffer (pH7.3) this post of flow velocity wash-out, collect the part of 0.6ml with 0.2ml/ minute.Detect elution profile with the visible detector of UV-, as shown in Figure 3.Because this detector shows there is not protein wash-out from post, by being added in the BSA of the 1M KCl elution of bound in the 5mM sodium phosphate (pH7.3).Proteinic existence can (Pierce, Rockford II) differentiate by little BCA analysis in each part.

Embodiment 33

The chromatography of bovine serum albumin (BSA) on the functionalized graphite protofibril of quaternary amine

[00181] (aqueous slurry Sweden) is at room temperature preserved and is spent the night for Pharmacia, Uppsala will to comprise carboxyl protofibril that 100mg 2-(2-triethyl amino ethoxy) ethanol modifies and the hyperfine resin of 300mg sephadex G-25.Resulting slurry is used for being encased in the post of 1cm diameter.This post is with the flow velocity balance of 5mM sodium phosphate buffer (pH7.3) with 0.1-0.6ml/ minute.Load BSA on post (2.70.6mg is in the 0.2ml deionized water).With 5mM sodium phosphate this post of flow velocity wash-out, collect the part of 0.6ml with 0.2ml/ minute.Detect elution profile (accompanying drawing 4) with the visible detector of UV-. because this detector shows that protein can be by 5mM sodium phosphate buffer wash-out, the change solvent becomes the 1M KCl in the 5mM sodium phosphate (pH7.3).Proteinic existence can (Pierce, Rockford II) differentiate by little BCA analysis in each part.

9. the enzyme of graphite carbon is functionalized

[00182] biological catalyst can be used for functional group is incorporated into graphite carbon, particularly the surface of carbon nanotube. and up to now, be to modify graphite carbon (referring to for example, the U.S. patent application serial numbers 08/352,400 that on December 8th, 1994 submitted to) by the pure chemistry method.These chemical processes have following shortcoming: (1) condition harshness (is used high temperature, very big acidity or toxic chemical substance), (2) lack specificity (for example, oxidation can be introduced COOH, COH and CHO yl). the solid graphite carbon of preparation (carbon fibrils for example; Hyperion, aqueous suspension Inc.) comprise one or more and can accept graphite carbon and carry out the enzyme that chemical reaction forms the graphite carbon of chemically modified as substrate.Aqueous suspension is remained on enzyme react acceptable terms (temperature, pH, salt concn or the like) following for some time, this time is enough to make enzyme catalysis to modify the surface of graphite carbon.During reaction, continue to mix suspension so that enzyme leads to the surface of graphite carbon.After reaction reached the gratifying degree acceptable response time, filtration washing was removed enzyme from carbon.

[00183] carbon nanotube. up to now, use two types enzyme: cytopigment p450 enzyme and peroxidase.In two kinds of situations, two types enzymes have all been carried out good research, they accept the substrate of aromatic series type, and have found their The optimum reaction conditions.Two types enzyme is incorporated into hydroxyl in their substrate, and hydroxyl can be incorporated in the graphite carbon. except enzyme, other biological catalyst is ribozyme and catalytic antibody for example, or the abiology mimicry of enzyme can be used for catalysis functionalized.

Embodiment 34

It is functionalized to carry out enzyme with rat liver microsomes

[00184] cytopigment p450 enzyme it is generally acknowledged as detoxicant in liver, bring into play function (F.Peter Guengerich, American Scientist, 81,440-447 and F.PeterGuengerich, J.Biol.Chem., 266,10019-10022).They are with for example poly-aromatics toxic chemical hydroxylation of exogenous compounds.Hydroxylation makes that these compounds become can be water-soluble, so that they can be discharged from health through urine.A lot of different cytopigment p450 enzymes are arranged in liver, and each all has different substrate specificities. owing to need the toxicide environmental toxin in extensive range, therefore it is believed that the specificity of these relative broad ranges is very important.Although individual cells pigment p450 is commercial available, whether do not accept any information of carbon nanotube as substrate about them.Because this uncertainty, we determine to begin to cultivate carbon nanotube with the rats'liver extract that comprises many different cytopigment p450.

[00185] in its tap water, give 2 rats (" tentative " rat) use phenylethyl barbituric acid (1g/L, pH7.0) 1 week, the expression that comes inducing cell pigment p450 enzyme.In addition two rats (" contrast " rat) give not contain the water of phenylethyl barbituric acid.Put to death rat then, by standard method (referring to, Methods in Enzymology for example, Vol.206) comprise the microsome of cytopigment p450 by their liver preparation.

[00186] plastosome mixes with carbon nanotube (protofibril), so that cytopigment p450 and graphite carbon reaction.In these trials, the protofibril (" of 5mg is pure " or non-functionalized and " COOH " or the protofibril of oxidation) comprising 0.1M Tris, 1.0mM NADPH, 0.01% NaN with microsome (test and contrast microsome) ₃, 10mM G-6-P salt, glucose-6-phosphate dehydrogenase (G6PD) (1 unit/mL), mix in the buffered soln of pH7.4.Comprise NADPH, add glucose-6-phosphate dehydrogenase (G6PD) with from NADP as the cosubstrate of cytopigment p450 and G-6-P salt ⁺Middle regeneration (if NADP ⁺Produce by cytopigment p450).In the Eppendorf Eppendorf tube, reactant was at room temperature rotated this mixture about 1.5 days. after cultivation, extensively wash protofibril at deionized water, 1M HCl, 1M NaOH, 0.05% Triton X-100,0.05%Tween, methyl alcohol and 1M NaCl.After the washing, little BCA of protein (Pierce) analyzes (Pierce) and shows that protofibril looks still have the protein (although not detecting protein in washing soln) that is connected with them.

[00187] in order to determine whether hydroxyl is incorporated into fibriilar surface, with protofibril and the reaction of N-FMOC-Isoleucine.The dry DMF solution reaction of protofibril of different batches (contrast and experiment) (every group of 1.5mg) and 333ml, DMF solution comprises the 4.45mg/mLFMOC-Isoleucine, 1.54mg/mL dimethyl aminopyridine (DMAP) and 2.6mg/mL 1,3-dicyclohexylcarbodiimide (DCC).Reaction is (by continuous rotation) two days later, with DMF, piperidines, methyl alcohol, water, DMF, methyl alcohol, methylene dichloride (every kind of 600ml) washing protofibril. this washing sequence is repeated 3 times.Protofibril is sent to GalbraithLaboratories (Knoxville, TN) be used for amino acid analysis in, being used to analyze the existence of Isoleucine. this result is having a double meaning, because visible many other amino acid show that the protein, peptide and the amino acid that are present in the rat liver microsomes extract can not be fully from the protofibril flush awaies except Isoleucine.Therefore, because the technical difficulty aspect washing and analysis can not determine whether cytopigment p450 makes protofibril functionalized.

Embodiment 35

With the functionalized protofibril of commercial available reconstitution cell pigment p450 enzyme

[00188] for fear of with use rat liver microsomes as the relevant impurity in cytopigment p450 source, buy individual cells pigment p450 enzyme (GENTEST, Woburn, MA).Because cytopigment p450 enzyme only has active to film, provide these enzymes to be used for MC preparation. with reaction method similar to the above, we have tested following cytopigment p450:CYPlAl (cat.# Mlllb), CYP1A2 (cat.# M103c), CYP2B6 (cat.# HOa), CYP3A4 (containing reductase enzyme, cat J 107r).In reaction soln, also can comprise MgCh (0.67mg/mL).In this test, with Soxhlet device washing protofibril.

The analysis of the hydroxyl of [00189] introducing, the protofibril and the colouring reagents 3 that comprise cytopigment p450 reaction, washing, 5-dinitrobenzoic acid (DNBA) reaction. carry out the combination of N-FMOC-Isoleucine as mentioned above. after reacting with DNBA, wash protofibril with DMF, with 6M HCl or 46 (covalent attachment) DNBA that unit/mL Pig Liver Esterase (Sigma) hydrolysis is residual.After the hydrolysis treatment, analyzing the analysis of the DNBA that discharges by the HPLC of the supernatant liquor around the protofibril. the HPLC of the DNBA of release analyzes and carries out in the WatersHPLC system, and this system equipment has Vydac C 18 anti-phase analytical columns (cat.#218TP54) and the gradient elution from the deionized water that comprises 0.1%TFA (solvent orange 2 A) to the acetonitrile that comprises 0.1%TFA (solvent B).

Embodiment 36

With the functionalized protofibril of peroxidase

[00190] the specific document description of peroxidase substrate is shown, carbon nanotube can be these enzymes substrate (people such as J.S.Dorick, Biochemistry(1986), 25,2946-2951; People such as D.R.Buhler, Arch.Biochem.Biophys. (1961) 92,424-437; H.S.Mason, Adyances in Enzymology, (1957) 19,79; People such as G.D.Nordblom, Arch Biochem.Biophys.(1976) 175,524-533).In order to determine peroxidase (catalase, the H type, Sigma) whether hydroxyl can be incorporated into fibriilar surface, protofibril (11mg) is mixed into comprises 50mM sodium acetate (1.25mL, pH5.0), in the solution of horseradish peroxidase (200nM), at preceding 3 hours each 5g dihydroxy fumaric acids (15mg altogether) that add of reaction.Carried out altogether 5 hours 4 ℃ of following reactions, have gas oxygen to bubble by spells.After the reaction, water, 1N NaOH, methyl alcohol and methylene dichloride (every kind of 200mL) washing protofibril.Carry out control reaction (100 ℃ are following 5 minutes) with heat-killed peroxidase.

[00191] in order to analyze the degree of the enzymatic protofibril hydroxylation of superoxide, the reaction in the presence of imidazoles in dry DMF with protofibril and tertiary butyl dimethylsilane chlorine (Aldrich).Behind the washing protofibril, protofibril is sent into Robertson Microlit Laboratories, (Madison NJ) carries out ultimate analysis to Inc, analyzes the silicon that is incorporated in the protofibril.The result who analyzes is having a double meaning for the existence of the silicon on protofibril surface.It is believed that, silicon in the glassware that uses in this test is present in the protofibril with the form of small shreds, for ultimate analysis. this causes the high level of silicon in test and control sample. and the conclusion of test is, peroxidase has been incorporated into hydroxyl in the protofibril, determines the existence of the hydroxyl of introducing arbitrarily but technical difficulty has hindered us.

By electrophilic addition to oxygen-free protofibril substrate or by functionalized the receiving of metallizing Mitron

[00192] by on oxygen-free protofibril surface, add primary product that the activatory electrophilic reagent obtains be side group-COOH ,-COCl ,-CN ,-CH ₂NH ₂,-CH ₂OH ,-CH ₂-halogen or HC=O. can be transformed into secondary derivative according to following reaction formula:

Protofibril-COOH------------〉see on

Protofibril-COCl (chloride of acid)+HO-R-Y------------〉F-COO-R-Y (Sec.4/5)

Protofibril-COCl+NH ₂-R-Y------------〉F-CONH-R-Y

Protofibril-CN+H ₂------------〉F-CH ₂-NH ₂

Protofibril-CH ₂NH ₂+ HOOC-R-Y------------〉F-CH ₂NHCO-R-Y

Protofibril-CH ₂NH ₂+ O=CR-R ' Y------------〉F-CH ₂N=CR-R '-Y

Protofibril-CH ₂OH+O (COR-Y) ₂------------〉F-CH ₂OCOR-Y

Protofibril-CH ₂OH+HOOC-R-Y------------〉F-CH ₂OCOR-Y

Protofibril-CH ₂-halogen+Y-------------〉F-CH ₂-Y+X-Y-=NCO-,-OR-

Protofibril-C=O+H ₂N-R-Y------------〉F-C=N-R-Y

11. the nanotube of dendrimer

[00193] can reduce the concentration of the functional group of nanotube surface, comprise that multi-functional dose with a series of each generation modified this nanotube, this multi-functional dose cause with the quantity of specificity functional group along with each for increase, thereby form the dendrimer spline structure.Resulting dendroid nanotube is useful especially as the solid carrier with the protein Covalent Immobilization, because they have increased the proteinic density that is fixed on nanotube surface.The present invention has proved that the high-density of specificity chemistry functionality can be delivered to the surface of the higher particulate carbon of surface-area, and this is difficult for the higher carbon of previous surface-area.

Embodiment 37

Preparation based on the dendrimer of Methionin

[00194] reaction sequence is as shown in Figure 5.

[00195] (5ml, 0.2M pH8.6) add N α, N ε-di-t-boc-L-Methionin N-hydroxy-succinamide ester (120mg, 0.27mmol) De diox (5ml) solution in the suspension to the sodium bicarbonate of amino protofibril (90mg).At room temperature stirred reaction mixture spends the night.Water, methyl alcohol and methylene dichloride extensively wash the Methionin protofibril and the vacuum-drying of tert.-butoxy protection.At room temperature use trifluoroacetic acid (5ml) in methylene dichloride (5ml), to handle the Methionin protofibril 2 hours of this tert.-butoxy protection then. extensively wash the amino Methionin protofibril of this product with methylene dichloride, first alcohol and water. prepare second and third generation Methionin protofibril with same procedure then.The amino acid analysis data show, every g protofibril comprises 0.6 μ mol Methionin in the Methionin protofibril of the first-generation, every g protofibril comprises 1.8 μ mol Methionins in the Methionin protofibril of the s-generation, and every g protofibril comprises 3.6 μ mol Methionins in the Methionin protofibril of the first-generation.

[00196] carboxyl dendrimer protofibril can pass through same procedure, uses aspartic acid or L-glutamic acid and carboxyl protofibril to prepare.

Embodiment 38

The preparation of the dendrimer of carboxylate salt-end

[00197] preparation of dendrimer with carboxylate salt-end of carbon nanotube (CN) nuclear comprises, amino butyl-nitrilotriacetic acid (NTA) continuously, combination in order, begin to prepare with the NHS of the carbon nanotube of oxymuriate oxidation.

The preparation of NTA

[00198] method according to Hochuli (E.Hochuli, H.Dobeli, and A.Schacher, J.Chromatography.411,177-184 (1987)) prepares NTA, by reference its content is incorporated herein.

The preparation of CN/NHS

[00199] method according to embodiment 20 prepares CN/NHS.

The preparation of CN/NTA

[00200] NTA-HCl of 0.4g is dissolved in the 0.2M NaHCO of 25mis ₃, among the pH8.1.Adding 1M NaOH adjusts back to pH up to 7.8.The CN/NHS that adds 0.5g, this mixture of sonication to be to disperse CN, stirs down resulting slurry and stayed in the reaction 30 minutes.On 0.45 μ m nylon membrane, filter this slurry, on strainer,, use DI water washing 2X with the carbonate buffer solution washing 2X of pH8.1.Twice resuspending of CN that sonication will be modified down filters and obtain solids cake compresses in the MeOH of 25mis, and be dry in vacuum drier at last.

The preparation of CN/NTA/NTA

[00201] at first CN/NTA is transformed into the NHS active ester..The CN/NTA that gets 0.396g 90 ℃ of dryings 30 minutes, places the 100ml RB bottle that the no Shui diox of 30mis is housed then, and uses flushed with argon gas in vacuum drier.Stir the N-hydroxy-succinamide that adds 0.4g down, continue then to stir add 0.67g after 1 hour EDC. during this period, CN assembles together.Discharge diox, with no Shui diox 2X washing solid of 20mis.When aggregation is broken up, with the anhydrous MeOH washing solid of 20mis.On 0.45 μ m nylon membrane, filter this solid, be suspended among the MeOH, filter and on strainer, wash with MeOH.

[00202] NTA that gets 0.2g joins in the flask of 50ml, and adds 10 1M NaOH and make its dissolving.The 20mis 0.2M NaHCO that adds pH8.1 ₃, adding whole CN/NTA/NHS then, with probe sonicator this solution of sonication lightly. and mixture was at room temperature stayed in the reaction 2.5 hours.Filter the CN of this modification on 0.45 μ m nylon membrane, wash 2X with carbonate buffer solution, resuspending filters and uses the DI water washing under the sonication in DI water.Then they are placed vacuum drier dry.

The preparation of CN/NTA/NTA/NTA

[00203], adds the NTA. of interpolation level according to above-mentioned method

The preparation of CN/NTA/NTA/NTA/NTA

[00204], adds the NTA of interpolation level according to above-mentioned method.

[00205] under the sonication each four generation NTA additive sample (about 10mg) is suspended in the DI water of 10mis, on 0.45 μ m nylon membrane, filters the material that forms the blanket sample.This material part is stored in the vacuum drier, by the relative quantity of esca analysis nitrogen (N) with indication NTA.The results are shown in the following table.

Material is by esca analysis %N

CN/NTA 0

CN/NTA/NTA 1.45

CN/NTA/NTA/NTA 1.87

CN/NTA/NTA/NTA/NTA 2.20

[00206] ESCA result confirms, each is continuously for the increasing amount of mixing.

Embodiment 39

The carbon nanotube dendrimer is as protein carrier

[00207] derives by protofibril and have dendrimer and can greatly improve the proteinic density that is fixed on the carbon nanotube.According to following method horseradish peroxidase (HRP) is fixed on the carbon nanotube.

[00208] at room temperature, with pure protofibril (0.49mg), amino protofibril (0.32mg), first-generation Methionin protofibril (0.82mg), s-generation Methionin protofibril and third generation Methionin protofibril and sodium bicarbonate conjugation damping fluid (600 μ l, 0.1M, comprise 0.9%NaCl) and sonication 15 minutes.At room temperature, sodium bicarbonate conjugation damping fluid (490ml, the enzyme stock solution of 5.6mg/ml) water culture of they and HRP is 19 hours.Be fixed with the protofibril of HRP with following damping fluid (1ml) washing: the 10mMNaHCO that comprises 0.9% NaCl ₃PH of buffer 9.5 (IX lavation buffer solution) washing 7 this, the 0.1% Triton X-100 washing in the IX lavation buffer solution 5 times, the 50% glycol washing in the IX lavation buffer solution 3 times.(50mM, the pH4.4) superoxol in (10 μ l, 10mM mother liquor) and 2,2-azino two (3-second class benzene thiazoline)-6-sulfonic acid di-ammonium salts (ABTS, 3 μ l, mM mother liquor) are analyzed the activity of HRP at the 414nm place to be used in the glycine analysis buffer.The results are shown in the following table:

Protofibril Nmol HRP/g protofibril

Pure protofibril 3.82

Protofibril-NH ₂8.58

Protofibril-NH-Lys 28.09

Protofibril-NH-Lys (Lys) ₂28.30

Protofibril-NH-Lys (Lys) ₄46.28

12. difunctional protofibril

[00209] functional group's (for example carboxyl and amino) that has been found that more than one types can be incorporated on the protofibril simultaneously, comprises functionalized carbon nanotube carboxyl nanotube for example, reacts with amino acid.These bifunctional protofibril can be used for fixing a plurality of molecules, particularly in chemistry amount and the mode closely to close on of 1:1.

Embodiment 40

Prepare difunctional protofibril by adding Methionin

N _α Synthesizing of-CBZ-L-Methionin benzyl ester

[00210] reaction sequence is as shown in Figure 7. get N _ε(2g 8.12mmol) is dissolved in methyl alcohol (40ml) and the water (40ml)-(tertbutyloxycarbonyl)-L-Methionin, regulates pH to 8 with triethylamine.In said mixture, add in the solution of diox (2.4g, 9.7mmol is in 20ml) of N-(carbobenzoxy-(Cbz)-oxygen) amber line imines, keep pH at 8-9 with triethylamine.Stirred reaction mixture spends the night.Rotary evaporation removes and desolvates then, obtains thick N _α-CBZ-N _ε-(tertbutyloxycarbonyl)-L-Methionin.With 0.2M lime carbonate (4ml) washing N _α-CBZ-N _ε-(tertbutyloxycarbonyl)-L-Methionin is removed water layer and is obtained white solid. and this solid suspension is in N, N-dimethylformamide (40ml) and bromotoluene (1.16ml).Stir under the reaction mixture room temperature and spend the night.With ethyl acetate and this reaction mixture of water treatment, dry organic layer on sal epsom.Removing desolvates obtains thick N _α-CBZ-N _ε-(tertbutyloxycarbonyl)-L-Methionin benzyl ester passes through silica gel purification with the ethyl acetate of 25% hexane as solvent.Under 0 ℃ to N _α-CBZ-N _ε(1g joins trifluoroacetic acid in methylene dichloride 2.2mmol) (10ml) solution to-(tertbutyloxycarbonyl)-L-Methionin benzyl ester.Reaction mixture stirred 10 minutes at 0 ℃, then restir 2.5 hours at room temperature.Remove and desolvate, obtain crude product.Obtain N by silica gel chromatography _α-CBZ-L-Methionin benzyl ester.

N _α -CBZ-L-Methionin benzyl ester is fibriilar synthetic

[00211] in methylene dichloride (18ml) suspension of carboxyl protofibril (300mg), adds N _αThe solution of-CBZ-L-Methionin benzyl ester (148mg, 0.32mmol is in 20ml methylene dichloride and 176 μ l triethylamines). add then HOBT (43.3mg, 0.32mmol) and EDC (61.3mg, 0.32mmol).Reaction mixture at room temperature stirs and spends the night, and obtains crude product. with methyl alcohol, methylene dichloride and the extensive washed product protofibril of water, and vacuum-drying then.

Difunctional protofibril Fib-Lys (COOH) NH ₂ Synthetic

[00212] to N _α(1N, 4ml), reaction mixture stirs and spends the night to add sodium hydroxide in the methyl alcohol (4ml) of-CBZ-L-Methionin benzyl ester protofibril (113mg).The extensive washed product N of water and methyl alcohol _α-CBZ-L-Methionin benzyl ester protofibril, this protofibril of vacuum-drying.To N _αAdding trimethyl silane iodine (1ml) in acetonitrile (4ml) suspension of-CBZ-L-Methionin benzyl ester protofibril (50mg). mixture stirred 3 hours down at 40 ℃.Water, methyl alcohol, 0.5N sodium hydroxide, acetonitrile and methylene dichloride be the final difunctional protofibril of washing extensively.Amino acid analysis shows that every g protofibril contains 0.3 μ mols Methionin.

[00213] use Serine, Threonine or tyrosine, by preparing hydroxyl and the difunctional protofibril of carboxyl (or amino) with the similar method of aforesaid method. can prepare mercaptanization and the difunctional protofibril of carboxyl (or amino) with halfcystine. can prepare carboxyl and amino difunctional protofibril with aspartic acid and L-glutamic acid.

The purposes of functionalized nanotube

[00214] owing to its higher porosity, chemistry and thermostability and higher surface area, functionalized graphite nanotubes can be as solid carrier in many biotechnology applications.Have been found that they adapt to harsh chemistry and thermal treatment, are adapted to chemical functionalization very much.

[00215] for example, enzyme can Covalent Immobilization on the nanotube of modifying, kept its biological activity simultaneously.In addition, nanotube also is adapted at being used as in the bio-molecular separation carrier of affinity chromatography.For example, in multistep is synthesized suddenly, on nanotube, prepared enzyme inhibitors, made that these fixed inhibitor can be approaching with macromole, the identification of reversible specific biological has taken place between the protofibril of protein and modification.

[00216] hydrophobicity of nanotube surface is not enough to by adsorbing fixing highdensity protein.Expand into three-dimensional for the hydrophobicity that increases nanotube surface with hydrophobic environment from the plane, the alkyl chain of different lengths is attached to nanotube surface. comprise trypsinase, alkaline phosphatase, lipase and avidin by the protein that is absorbed and fixed on the alkyl nanotube. the activity of proteinic enzymic activity of these fixed and resolvase is suitable, and this can prove by the catalytic effect to substrate hydrolysis in the aqueous solution.

[00217] in addition, the terminal phenyl of alkyl chain joins the alkyl nanotube and phenyl-alkyl nanotube of forming has also prepared to come out.This modification has been introduced and the interactional aryl structure of proteinic amino acid phenylalanine, tyrosine and tryptophane on by π-π.Alkaline phosphatase and lipase is adsorbed in C on phenyl-alkyl nanotube ₈Absorption on the-alkyl nanotube is suitable.

[00218] has been found that also functionalized protofibril can be as the solid carrier of protein synthesis.

1. the functionalized nanotube that is used for enzyme as solid carrier

Embodiment 41

Enzyme by absorption is fixed

The fibriilar preparation of alkyl

[00219] preparation alkyl protofibril, comprise will comprise about 0.007mmoles-the 10mg carboxyl protofibril of COOH (protofibril=0.007 mmoles of 10mg protofibril x 0.7 mmoles-COOH/mg), 1.5ml DMF (N with the alkylamine of 0.14mmoles, the N-dimethylformamide) solution reacts with the EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide) of 0.14 mmoles and the DMAP (4-dimethylaminopyridine) of 0.14 mmoles.Chemical reaction is as follows:

Protofibril-COOH+NH ₂(CH ₂) _nCH ₂R (R=H or OH)-------------

Protofibril-CONH (CH ₂) nCH ₂R

With the preparation of this method comprise the different lengths alkyl chain (n=5,7,9,17, when n=5, former county Party committee of alkyl that R=OH) several are different.Stir at ambient temperature after the reaction and spend the night, with 3 x 25ml CH ₂Cl ₂, 3 x 25ml MeOH and 3 x 25ml dH ₂The strict washing of O protofibril.Nitrogen content in this protofibril is carried out ultimate analysis show that the yield of reaction is 65-100%.

The absorption of enzyme

[00220] by absorption lipase, trypsinase, alkaline phosphatase and avidin are fixed on the alkyl protofibril of this embodiment.At room temperature mixed alkyl protofibril and enzyme 3-4 hour, use 5mM sodium phosphate (pH7.1) washing 2 to 4 times then.Alkaline phosphatase is fixed on C ₈-protofibril and C ₆On the OH-protofibril; Trypsinase is at C ₆, C ₈, and C ₁₈On-the protofibril, lipase is at C ₆OH-, C ₈-, C ₁₀-and C ₁₈On-the protofibril, avidin is at C ₁₈On-the protofibril.The results are shown in following table.

[00221] kinetic property and the resolvase of discovery fixed enzyme are suitable, the results are shown in figure below:

Enzyme	K _m(M)	K _cat(s ^-1)	K _cat/K _m(M ^-1s ^-1)
Enzyme	K _m(M)	K _cat(s ^-1)	K _cat/K _m(M ^-1s ^-1)	Lipase	40 x 10 ^-6	0.040	0.99 x 10 ³
Trypsinase	36 x 10 ^-6	0.048	1.34 x 10 ³	Lipase	40 x 10 ^-6	0.040	0.99 x 10 ³
Trypsinase	36 x 10 ^-6	0.048	1.34 x 10 ³	Alkaline phosphatase	1.2 x 10 ^-3	4.8	4.17 x 10 ³
Avidin	7.9 x 10 ^-3	19.1	2.43 x 10 ³	Alkaline phosphatase	1.2 x 10 ^-3	4.8	4.17 x 10 ³

Substrate: lipase 1,2-O-dilauryl-rac-glycerine-3-pentanedioic acid resorufin ester

Trypsinase N-benzoyl-L-arginine-p-Nitroaniline

Embodiment 42

Turn usefulness (synthesizing of ethyl butyrate) into by protofibril-lipase-catalyzed paper

[00222] method according to embodiment 41 is fixed on C with lipase ₈On-alkyl the protofibril.At first the Yong diox washs the prolipase fiber, and the mixture of Yong diox and hexane washing is then used hexane wash, at last so that protofibril is dispersed in the hexane.For synthetic butyric acid ethyl ester (a kind of foodstuff additive provide pineapple-banana flavor), in the hexane that contains 6.2 μ m protofibril-fixed lipase, ethanol (0.4M) and butyric acid (0.25M) are mixed.Stirred reaction mixture under the room temperature is 60% at 7 hours yields, and this can determine by the alcohol concn that uses confirmed method assaying reaction mixing species.Reaction and result are as shown in Figure 8.

Embodiment 43

Fixed base acid phosphatase on phenyl-alkyl protofibril

Phenyl-alkyl is fibriilar synthetic

[00223] protofibril is by two different prepared in reaction phenyl-alkyl protofibril.Reaction 1 with 20mg carboxyl protofibril (comprise about 0.014mmoles-COOH) and the 4-PHENTERMINE of 0.28mmoles, 0.28mmoles EDC and 0.28mmoles DMAP (4-dimethylaminopyridine) be mixing in the DMF of 1.5ml (N, N-dimethylformamide).The reaction 2 6-phenyl-1-hexanols with 20mg carboxyl protofibril and 0.28mmoles, 0.28mmolesDCC (1, the 3-dicyclohexylcarbodiimide) and 0.28 mmoles DMAP mix in the DMF of 1.5ml.Reaction is at room temperature carried out, and stirring is spent the night.Use 3 x 25ml CH then ₂Cl ₂, 3 x 25ml MeOH and 3 x 25ml dH ₂This protofibril of the strict flushing of O.

The preparation of fixed alkaline phosphatase

[phenyl of 00224l0.5mg-alkyl protofibril is suspended among the 0.05M Tris (pH8.6) of 400 μ l and sonication 20 minutes.(1.67mg/ml pH7.0), rotates mixture 2 hours under the room temperature in the 5mM sodium phosphate buffer alkaline phosphatase enzyme solution of adding 150 μ l in these protofibril then, stores down at 4 ℃ and spends the night.Use the 5mM sodium phosphate buffer (pH7.1) of 600 μ l to wash protofibril 2 times then, and be suspended in the same buffer of 200 μ l.

By measuring the quantitative specificity fixed of catalytic activity alkaline phosphatase

[00225] alkaline phosphatase enzyme-to-substrate p-nitrophenyl phosphate reaction discharges the absorb light at the 405nm place, optical extinction coefficient 18,200M ^-1Cm ^-1Colored compound.The analysis buffer condition of this reaction is 10mM Tris, 1mM MgCl ₂With 0.1mM ZnCl ₂, pH=8.4.Be reflected in the 1ml test tube and carry out, comprise the p-nitrophenyl phosphate solution of 5 μ l (0.5M at 33% DMSO in analysis buffer) and the alkaline phosphatase protofibril of 13 μ g are mixed in the analysis buffer of 1ml.Increase by the absorbancy that detects the 405nm place in the time scan more than 0 minute.Use optical extinction coefficient 18 then, 200M ^-1Cm ^-1Gradient is from the outset calculated enzymic activity (μ M/ minute).

[00226] for the alkaline phosphatase on the phenyl protofibril of being adsorbed on of reaction 1, activity is per 13 μ g protofibril 6.95 μ M/ minutes.For the alkaline phosphatase on the phenyl protofibril of being adsorbed on of reaction 2, activity is per 13 μ g protofibril 2.58 μ M/ minutes.These results change every g protofibril 0.63 μ moles (or 54mg) and 0.23 μ moles (or 20mg) active alkali acid phosphatase into, method of converting is the alkaline phosphatase enzyme solution divided by concentration known, and the alkaline phosphatase enzyme solution of concentration known was determined as per 1 μ M alkaline phosphatase 879.8 μ M/ minutes under the same analysis condition.

Embodiment 44

Fixed fat enzyme on the phenylalkyl protofibril

The fibriilar preparation that lipase is modified

[00227] phenyl-alkyl protofibril with 0.5mg is suspended in the 5niM sodium phosphate buffer (pH7.1) of 50 μ l sonication 20 minutes.(0.2mM pH7.1), with mixture rotation 5 hours, stores under 4 ℃ and spends the night under the room temperature in the 5mM sodium phosphate buffer lipase solution of adding 350 μ l in these protofibril.Use the 5mM sodium phosphate buffer (pH7.1) of 600 μ l to wash protofibril 3 times then, and be suspended in the same buffer of 200 μ l.

By measuring the quantitative specificity fixed of catalytic activity lipase

[00228] lipase can with substrate 1, the reaction of 2-o-dilauryl-rac-glycerine-3-pentanedioic acid-resorufin ester (Boehringer Mannheim, 1179943) is created in 572nm place absorb light, optical extinction coefficient 60,000M ^-1Cm ^-1Colored compound.The analysis buffer condition of this reaction is 0.1M KH ₂PO ₄, pH=6.8 is reflected in the 1ml test tube and carries out, and comprises the substrate mother liquor of 5 μ l (7.6mM at 50% diox in Thesit) and the alkaline phosphatase protofibril of 13 μ g are mixed in the analysis buffer of 1ml.Increase by the absorbancy that detects the 572nm place in the time scan more than 10 minutes.Use optical extinction coefficient 60 then, 000M ^-1Cm ^-1Gradient is from the outset calculated enzymic activity (μ M/ minute).

[00229] is adsorbed on lipase on the phenylalkyl protofibril in the reaction 1 for embodiment 43, activity is per 13 μ g protofibril 0.078 μ M/ minute. be adsorbed on the lipase on the phenylalkyl protofibril in the reaction 2 for embodiment 43, activity is per 13 μ g protofibril 0.054 μ M/ minute.These results change every g protofibril 4.7 μ moles (or 564mg) and 3.3 μ moles (or 396mg) active alkali acid phosphatases into, method of converting is the alkaline phosphatase enzyme solution divided by concentration known, and the alkaline phosphatase enzyme solution of concentration known was determined as per 1 μ M lipase 1.3 μ M/ minutes under the same analysis condition.

Embodiment 45

Fixing of horseradish peroxidase on the protofibril of aminoalkyl group-modification (HRP) The preparation of the protofibril (carboxyl protofibril) of carboxylic acid-functionalized

[00230] mix by medicine shovel, with 10.0g graphite protofibril sample at the dense H of 450mL ₂SO ₄Middle pulp is transferred in the reaction flask with outlet/inlet and top agitator then.Stirring and slowly in the argon gas stream, at room temperature in 24 hours time by a NaClO who part adds 8.68g ₃The chlorine steam that produces in the whole process of reaction is discharged in the NaOH trap aqueous solution from reaction.Behind end of run, be poured in the trash ice protofibril slurry and vacuum filtration.Then filter cake is transferred on the Soxhlet cylindrical filter paper, in the Soxhlet extractor, use deionized water wash, each renewed bright water in several hours. continue washing until when adding fresh deionized water, the pH. that fibriilar sample can not change water is filtered and recycled carboxylate salt protofibril then, at 100 ℃ and 5 " dried overnight in the vacuum.Obtain 10.0g.

The fibriilar preparation of HRP-fixed

[00231] method of taking embodiment 27 joins conjugation damping fluid (0.1M NaHCO by the amino protofibril (1.2mg) of 1 preparation ₃, 0.9% NaCl, pH9.5) in, this suspension of sonication 20 minutes.In the Eppendorf Eppendorf tube, this protofibril is washed 2 times then with the conjugation damping fluid, and be suspended in the 430 μ L conjugation damping fluids. get this suspension (0.14mg protofibril) and the 4.0mg activatory HRP (Pierce that is dissolved in the 50 μ L deionized waters of 50-μ L equal portions, Rockford, IL) mix, resulting suspension spends the night 4 ℃ of rotations.The combination with following solution in Eppendorf tube of HRP-conjugated protofibril is extensively washed: conjugation damping fluid, lavation buffer solution (20mM KH ₂PO ₄, 0.45%NaCl pH6.2), comprises the lavation buffer solution and the lavation buffer solution that comprises 50% ethylene glycol of 0.03-0.1% TritonX-100.In contrast, carry out identical operations, proved HRP and amino fibriilar bonded specificity covalent linkage really with activatory HRP and pure (underivatized) protofibril.

By measuring the quantitative specificity fixed of catalytic activity HRP

[00232] extensively the enzyme of most non-specific binding is removed in washing.By substrate upset H ₂O ₂With chromogenic substrate 2,2 '-azine-two (3-ethylbenzene thiazoline-6-sulfonic acid), di-ammonium salts (ABTS) come quantitative fixed activation HRP.With 100 μ M H ₂O ₂With 30 μ M ABTS as the enzymic activity of substrate at 414nm punishment light photometric detection HRP.In these preliminary study, the total amount of the enzyme that is connected with amino protofibril is 0.0230 μ mol HRP/g protofibril.As a comparison, contrast (pure protofibril) non-specific binding 0.0048 μ mol HRP/g protofibril.Two are subtracted each other, and the amount of the HRP of covalency (specificity combination) is 0.0182 μ mol/g protofibril.

Embodiment 46

Affinity chromatography is separated in alkaline phosphatase (AP) and the beta-galactosidase enzymes (β G) on the protofibril with fixed enzyme inhibitors

The fibriilar preparation of alkaline phosphatase enzyme inhibitors

[00233] according to people such as Brenna (1975), Biochem J., the method for 151:291-296 prepares the AP-inhibitor.The carboxylate salt protofibril is used for preparing NHS ester protofibril as described in above-mentioned embodiment 50. and get NHS ester protofibril (114mg) and be suspended in 4mL acetone and add 10 times of normal (according to every g protofibril 0.7meq NHS ester estimation) tyrasamines.Add exsiccant triethylamine (10 times of equivalents), at room temperature mixture was stirred 3 hours.In the scintered glass funnel, this tyrasamine base protofibril is used washing with acetone earlier under the vacuum, extensively wash with deionized water then.

[00234] getting 4-(to aminophenyl nitrogen)-phenylarsonic acid (66mg) is suspended among the 1NHCl of 4mL.Suspension be cooled to 4 ℃ and with the 0.5M NaNO of 0.36mL ₂Slowly mix.After 15 minutes, in tyrasamine base protofibril, add arsenic acid/NaNO ₂Mixture is suspended in the 0.1M NaCO of 10mL with it ₃(pH10.0) in.Reaction mixture (pH ≈ 10) spends the night 4 ℃ of stirrings. use 0.1M Na then ₂CO ₃(pH10.0), 8M guanidine HCl, 25mMNaOH and water continuous washing handle this protofibril, becomes clarification until effluent liquid.(Knoxville TN) carries out atomic absorption analysis to the arsenic in the protofibril of AP-inhibitor by GalbraithLaboratories.By atomic absorption analysis, the protofibril of finding to comprise the AP-inhibitor of the side chain with 1 arsenic atom has 0.4% total arsenic content.This shows that about 10% the initial COOH base of having estimated has been transformed into the AP-inhibitor in multistep is synthetic suddenly.According to fibriilar surface-area, this means every

Surface-area has 1 inhibitor molecules (enzyme binding site).

The fibriilar preparation of beta-galactosidase enzymes-inhibitor

[00235] according to Ullman, (1984) Gene, the method preparation of 29:27-31 is to amino-phenyl-β-D-thiogalactoside (TPEG) deutero-protofibril.In carboxylate salt protofibril, add the pH value to 4.0 that 2.24mg TPEG. regulates suspension with 0.1M HCl, adding 15mg EDAC at the 8mg of 0.2mL deionized water.Mixture stirred 3 hours under pH4.0 and room temperature.Centrifugation comes stopped reaction fast in the Eppendorf Eppendorf tube, removes liquid.By resuspending and centrifugal repeatedly in deionized water with beta-galactosidase enzymes-inhibitor protofibril washing 5 times.

Affine separation

[00236] alkaline phosphatase (AP) (from intestinal bacteria, the III type; Ullman, (1984) Gene, 29:27-31) and beta-galactosidase enzymes (BG) (from intestinal bacteria; Calbiochem, La Jolla, CA) in the Eppendorf Eppendorf tube with AP-inhibitor protofibril or β G-inhibitor protofibril batch fermentation and separate.For carrying out affine separation, get the 1.0mL load buffer (20mMTris, 10mM MgCl, the 1.6M NaCl that comprise AP (general about 10 units) and BG (general about 280 units), 10mM halfcystine, solution pH7.4) join among the AP-or BG-inhibitor protofibril of 0.8-1.0mg.The slight resulting suspension of vortex at room temperature rotated 2 hours then.After the enzyme combination, by simple centrifugal this protofibril that precipitates in desk centrifuge, recovery comprises the not liquid phase of desmoenzyme, preserves to be used for the enzyme analysis.By adding damping fluid, slight vortex, rotation 15 minutes, simple centrifugal and reclaim solvent with pasteur pipet and come to wash (7 x 1.0mL) repeatedly with load buffer. after washing 7 times, with suitable elution buffer to β G-inhibitor protofibril (100mM borax, the 10mM halfcystine, the 10mM halfcystine, pH10.0) or AP-inhibitor protofibril (40mM NaHPO ₄, 10mM Tris, 1.0mM MgCl ₂, 0.1mM ZnCl ₂, pH8.4) repeat identical operations.

[00237] analyzes AP and β G activity in all parts (not desmoenzyme, washes and eluate).By determining alkaline phosphatase activity (Δ ε=18,000M with the hydrolysis of Spectrophotometric Assays 500 μ M p-nitrophenyl phosphates (PNPP) at the 410nm place ^-1Cm ^-1).At the 10mM of pH8.4 Tris, 1.0mM MgCl ₂And 0.1mM ZnCl ₂In carry out the mensuration of alkaline phosphatase activities. the ability by Spectrophotometric Assays enzymic hydrolysis 2-nitro-gala-β-D-pyranoside (ONPG) is analyzed beta-galactosidase enzymes.5.0mM the initial rate of beta-galactosidase enzymes-catalytic hydrolysis of ONPG is in 10mM Tris at 405nm, 10mM MgCl ₂, 1.6M NaCl, the 10mM halfcystine carries out (Δ ε=3500M among the pH7.4 ^-1Cm ^-1) middle mensuration.

[00238], adds the mixture of AP and β G for AP-inhibitor and β G-inhibitor protofibril.In order to promote the mensuration of specificity binding capacity, the concentration of the enzyme of adding is very big excessive with respect to the fixed inhibitor concentration.For AP-inhibitor protofibril, combine 0.550 μ mol AP/g protofibril (on the contrary, non-specific binding is a 0.020Amol β G/g protofibril). for β G-inhibitor protofibril, determine that its capacity is 0.093 μ mol β G/g protofibril (on the contrary, non-specific binding is 0.012 μ mol AP/g protofibril).The results are shown in accompanying drawing 9 and 10 of affinity chromatography test.AP-inhibitor bibril strand does not combine with β G, but combine with AP, when in damping fluid, adding the emulative inhibitor phosphate salt of 40mM, its specificity wash-out (accompanying drawing 9). the AP combination that β G deutero-protofibril discord is relatively large, but combine with β G, when pH raises binding ability with weakening enzyme-inhibitor, its specificity wash-out.These results show inhibitor successfully with the protofibril covalent attachment, fixed inhibitor and macromole are approaching, in conjunction with being useful, and when the specificity wash-out, enzyme has still kept activity to inhibitor for specific enzymes.In accompanying drawing 10, continue from β G-inhibitor protofibril, to have leached β G.This may be the result of natural more weak enzyme-inhibitor avidity, rather than fibriilar shortcoming, because do not observe identical phenomenon in the AP-of accompanying drawing 9 inhibitor protofibril.

2. functionalized carbon nanotubes is as the solid carrier of antibody

[00239] have been found that antibody can be fixed on the functionalized nanotube, these antibody nanotubes have unique advantage for many application, and this is because they have higher every weight surface-area, electroconductibility and chemistry and physical stability.For example the antibody nanotube can be as the affinity reagent of molecular separation.The antibody nanometer is rolled the application that also can be used for analyzing, and comprises diagnostic immunoassay, for example based on the immunoassay of ECL.

[00240] antibody can be fixed by covalent attachment or non-covalent absorption.Covalent Immobilization can be finished by diverse ways: comprise that the reduction activation of antibody carbohydrate group, carboxylate salt protofibril (see embodiment 27, NHS ester activation above), react with protofibril and (to see embodiment 23 and 25, above) with reduction or the amine-modified antibody of maleimide and mercaptanization or maleimide amination.

[00241] antibody and the nanotube bonded best approach will depend on their application.For isolating application, preferable methods can be non-covalent attachment, because the capacity of protein bound is the highest for this method.For the method that comprises ECL, wherein fibriilar electroconductibility is important, preferably (the alkyl annexation is the weak conductor of electricity to covalent approach, can expect to make the protofibril insulation). the best method that reductive amination is a covalent attachment antibody to the protofibril, because by this method, antibody is correctly directed so that their binding site points to outer (away from protofibril).

3. on functionalized nanotube, add NAD ⁺

[00242] has been found that for example NAD of cofactor ⁺Can add and as the solid carrier in the proteinic biologic specificity affinity chromatography, wherein protein combines with enzyme co-factor.For example, NAD ⁺Protofibril is as the solid carrier of purifying desaturase. uses fibriilar main specific be that their relatively large accessible surfaces amass.The affinity matrix of high surface area needs, because they have higher latent capacity.Protofibril can loose dispersion or is fixed in the post or in the material.

Embodiment 47

At NAD ⁺The affinity chromatography of desaturase is separated on the protofibril

NAD ⁺ Fibriilar preparation

[00243] according to embodiment 14 and 15 oxidation protofibril, to introduce carboxyl.(3ml, 0.2M add N in suspension pH8.6) to the sodium hydrogen carbonate solution of protofibril (31mg) ⁶-[ammonia hexyl] carboxamide methyl)-lithium salt solution (25mg is from Sigma, in the 5ml sodium hydrogen carbonate solution) of nadide.Reaction mixture at room temperature stirs and spends the night.Product protofibril water, N, N-dimethylformamide and methyl alcohol extensively wash.The ultimate analysis data show that protofibril comprises, and analyze the NAD molecule of every g protofibril 147mmols by the NAD molecular phosphorus of the every g product of nitrogen analysis 130mmols. other have with amino the NAD of the connexon that is end ⁺Analogue can be used to prepare NAD ⁺Protofibril.

Affine separation

[00244] under 40 ℃, with NAD ⁺Sodium phosphate (1ml, 0.1M, t pH7.1) the solution sonication of fixed protofibril (0.26mg) and pure protofibril (0.37mg) and 0.1% polyoxyethylene glycol (PEG, MW 1000) 30 minutes was cultivated 30 minutes at 40 ℃ then.Centrifugation protofibril suspension is removed supernatant liquor.Under 4 ℃, the mixture of 0.1%PEG (1000) sodium phosphate buffer (250 μ l, the ratio of LDH solution and 0.1% PEG damping fluid is 1:1) of protofibril and L-serum lactic dehydrogenase (LDH) was cultivated 90 minutes.Then with mixture balance 30 minutes at room temperature.After protofibril and LDH cultivation, with sodium phosphate buffer (5 X, 1000 μ l) the washing protofibril of 0.1% PEG (1000), each washing was carried out 15 minutes under rotation.With the eluant solution LDH. of 0.1% PEG (1000) sodium phosphate buffer (5mM3X1000 μ l) of 5mM NADH during each eluate washing, protofibril rotation 15 minutes.The active mensuration of LDH comprises in elutriant, is determined at the change of reduction pyruvate salt period detecting 340nm place absorbancy.This analysis mixed solution comprises sodium phosphate buffer (980 μ l), pyruvate salt (3.3 μ l, 100mM mother liquor) and each wash-out part (16.7 μ l) of 0.1% PEG (1000). and reactive magnesium is as follows:

[00245] result shows, NAD ⁺The capacity of LDH on the fixed protofibril is every g protofibril 484nmols, and the capacity of the LDH on pure protofibril (contrast) is every g protofibril 3.68nmols.The non-specific binding of LDH is 5.6%.

Functionalized nanotube is as the solid carrier of protein synthesis

Embodiment 48

Functionalized protofibril is as the application of solid carrier in peptide is synthetic

[00246] amino protofibril (400mg) in methylene dichloride (20ml) and 4-(methylol)-phenylium suspension (255mg, 1.4mmol) mixture in add 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC, 268mg, 1.40mmol) and I-hydroxybenzotriazole hydrate (HOBT, 189mg, 1.4mmol). reaction mixture at room temperature stirs under nitrogen and spends the night. with methylene dichloride, the extensive washed product protofibril of first alcohol and water. and vacuum-drying obtains protofibril then.To fibriilar N, N-dimethylformamide (DMF, 2ml) and add in the suspension of methylene dichloride (8ml) N-(9-fluorenyl methoxycarbonyl)-O-butyl-L-Serine (215mg, 0.56mmol), 1,3-dicyclohexylcarbodiimide (DCC, 115mg, 0.56mmol) and 4 dimethyl aminopyridines (DMAP, 3.4mg, 0.028mmol). reaction mixture at room temperature stirs and spends the night, DMF solution (5 X40ml soaked 1 minute at every turn) with 20% pyridine is handled the product protofibril.Use DMF, water, sodium hydroxide (1N) and the extensive washed product protofibril of methylene dichloride then. vacuum-drying product Fib-handle-Ser (O+)-COOH (ninhydrin test is positive). for synthesizing of dipeptides, use identical method to add arginine .Fib-handle-Ser (O+)-Arg (N ^ε-2,2,5,7,8-pentamethyl-chrornan-6-alkylsulfonyl) amino acid analysis data show that it comprises 6.5 μ mol/g protofibril and 7.6 μ mol arginine/g protofibril.The peptide that can prepare any other by same procedure.

5. biotinylated protofibril and biotinylated alkyl protofibril

[00247] having been found that and can come functionalized fibriilar surface by biotinylation or alkylation and biotinylation. the protofibril that comprises these modifiers then can be in conjunction with streptavidin conjugated material arbitrarily for example streptavidin pearl and streptavidin enzyme.

[00248] owing to its higher surface area, protofibril provides very big benefit as solid carrier.The pearl that can produce strong magnetic is exceedingly useful in compartment analysis.Described biotinylated protofibril has the characteristics of protofibril and pearl concurrently. and biotinylated alkyl protofibril is the expansion of same concept, but has also shown the fibriilar adsorption property of alkyl.

[00249] streptavidin-and the protofibril of vitamin H-dressing can be used for diagnostics, can be used as trapping agent and be used to analyze for example electrochemiluminescence analysis.

[00250] new feature of the present invention is the combination of two kinds of solid carriers on a kind of protofibril, to make bifunctional protofibril. in addition, disclosed method has increased the surface-area of pearl and has amplified fibriilar magnetization.

Embodiment 49

Biotinylated fibriilar preparation

[00251] prepares biotinylated protofibril, comprise that NHS ester long-chain vitamin H with the amino protofibril of the 2.4mg of preparation as described in embodiment 16 and 19 and 9mg is at the damping fluid 0.2M of pH8.15 NaHCO ₃The middle mixing.At room temperature rotated mixture 4 hours, with identical damping fluid washing 2 times.

Embodiment 50

The fibriilar preparation of biotinylated alkyl

[00252] prepares biotinylated alkyl protofibril by two-step reaction.At first, the difunctional protofibril (comprising amino and carboxyl) of 4.25mg and the NHS ester long-chain vitamin H of 25mg are mixed.Washing protofibril and vacuum-drying.

The carrying out of [00253] second reaction comprises the biotinylated difunctional protofibril of 4mg and the EDC of 11mg (1-ethyl-3,3-dimethylamino-propyl) carbodiimide), the NH of the DMAP of 7.5mg (4-dimethylaminopyridine) and 10 μ l ₂(CH ₂) ₇CH ₃0.5mlDMF solution mix.This mixture at room temperature stirs and spends the night.Use CH at last ₂Cl ₂, MeOH and dH ₂The biotinylated alkyl protofibril that the O washing is final.

Embodiment 51

Biotinylated protofibril in analysis as solid carrier

[00254] biotinylated protofibril can be used for analyzing, and this analysis comprises streptavidin-vitamin H or the interactional mode of avidin-vitamin H of needing.Biotinylated protofibril can for example further be used the streptavidin derivatize.Can form and the strong non-covalent interaction in combination of streptavidin with the covalently bound vitamin H of protofibril (seeing embodiment 50).Because streptavidin is four poly-hoof protein with 4 identical combination sites; therefore almost necessarily have the combinable idle binding site of other biotinylation agent with biotinylated protofibril bonded streptavidin. therefore, biotinylated protofibril will convert the protofibril of streptavidin-dressing to.

[00255] there is a lot of analytical tests to carry out with these protofibril-vitamin Hs-streptavidin (FBS) carrier. for example; can catch biotinylated anti-analyte antibody (in this antibodies before or after the analyte) on the FBS carrier. finished the analysis of using biotinylated anti-analyte antibody well. these analyses comprise competitive analysis, and the analyte of wherein interested analyte and mark combines with anti-analyte antibody competitively.The labelled analyte of the analyte of free (not combination) and free (not combination) can wash out from protofibril fixed antibody.Washing step depend on by common practice in comprise centrifugal, filter or by being attracted on the magnetite physical sepn from solid phase.

[00256] except competitive analysis, the interlayer immunoassay also can be carried out on the PBS carrier.The interlayer immunoassay is known in diagnostics. this analysis comprises two kinds of antibody bonded analyte simultaneously; By for example with biotin labeling solid surface catch first " mainly " antibody; do not caught by solid carrier but with report disjunction mark note " secondary " antibody. the protofibril that this sandwich assay can be used as the solid capturing carrier carries out; wherein as described in above-mentioned paragraph, catch former county Party committee. therefore; in this analysis; protofibril and vitamin H covalent attachment; it combines with streptavidin; its successively with biotinylated main antibodies; it combines (if existence), the secondary antibodies combination of all the other marks with analyte.

[00257] similarly, the dna probe analysis can be carried out with the FBS carrier.Biotinylated singly-bound DNA can combine with the FBS carrier, and competitive hybridization can occur between additional singly-bound analyte dna molecular and the additional labeled oligonucleotide.

[00258] the biotinylated protofibril of other types, biotinylated alkylation protofibril can be used for immunoassay and DNA analysis. as described in embodiment 51; modify difunctional protofibril, can comprise vitamin H and one type functional group and functional group's covalent attachment of alkyl chain and other types.Resulting alkylation, biotinylated protofibril can be used for combining with the specificity of streptavidin or avidin (through vitamin H). also can be used for absorption of proteins (through alkyl chain).

[00259] the alkyl protofibril can be used for and the combining of other solid carriers, for example the magnetic bead of streptavidin-dressing.Fibriilar advantage on these pearls of One advantage of is that they have much higher surface-area (per unit weight).Therefore, if protofibril can combine with the outside surface of magnetic bead, this will remarkably improve surface-area, with the kind binding capacity that improves pearl.Can infer; alkylation, biotinylated protofibril can combine with the pearl of c streptavidin-dressing; streptavidin (the pearl)-vitamin H (protofibril) that produces high-affinity interacts; therefore the pearl of protofibril dressing has high surface-area. because the alkyl protofibril can be by absorption and protein bound, the pearl of protofibril dressing can further comprise that with the protein that adsorbs streptavidin and antibody come derivatize.As mentioned above, the protofibril of streptavidin or antibody-coating can be used for immunoassay and DNA analysis.Therefore the pearl of protofibril dressing can be by remarkably increasing the character that surface-area improves pearl, so that have only pearl seldom to have identical result in given analysis.

6.3-dimension structure

[00260] compare with unoxidized protofibril, the protofibril of oxidation is easier to be dispersed in the aqueous medium.Having neutralizes fears greatly (hole〉2nm) stable, many empty three-dimensional structures are very useful as catalyzer or chromosorb.Because protofibril can be dispersed in the personalized matrix, the sample of the good distribution by crosslinked and stabilization makes people remove to construct such carrier.Functionalized protofibril is an ideal for this application because they be dispersed in aqueous medium or the polarizable medium, functionality provides cross-linking set. in addition, functionality provides the point in supported catalyst or chromatogram site.End-result is hard three-dimensional structure, and it has the total surface area that influenced by functionalized site, supports promoting agent on functionalized position.

[00261] typically used of these carriers in catalysis comprise they as highly porous carrier by soaking into the metallic carrier of installing, the for example application in the noble metal hydrogenation catalyst. in addition, by hitching carrier, the ability that hitches the catalyzer of molecule through the functionality combination with the very high porosity of structure allows us to carry out the homogeneous phase reflection in a different manner. and the molecular catalyst that hitches waves in the successive liquid phase basically, similar with homogeneous reactor, wherein it can make and have benefit in the application of the selection carried out and speed in equal qualitative responses.But the solid carrier that hitches makes and to be easy to separate and activation recovering, in many cases, is unusual expensive catalysts.

[00262] these stable, hard structures also allow to carry out up to now the very reaction of difficulty, for example asymmetric synthesis or by suitable corresponding body catalyst or selective substrate and carrier-bound affinity chromatography.Derivatize by metal-Pc or metalloporphyrin complexing also allows to be used for to reclaim and metal ion bonded part, and by secondary derivatize and any molecule of part bonded.For example, when functionalized fibriilar three-dimensional structure is electrode or electrode a part of, the functionalized absorption that causes Co (II) Pc becomes Co (III) will produce stable Co (HI)-pyridyl complex compound Co (II) electrochemical oxidation in the presence of nicotinic acid, and it contains carboxylic acid as side group.To allow optionally to separate with additive method in conjunction with suitable antigen, antibody, catalysis antigen or other site-specific trapping agents and be very difficult to isolating molecule (affinity chromatography).In washing after electrode removes inaccessible material, Co (III) complex compound that comprises target molecule can electrochemical reduction and reclaim unsettled Co (II) complex compound.Can reclaim by the mass action replacement of unsettled Co (II) part at the part that comprises on the Co of target molecule (II) then, therefore, the separation and the recovery of molecule have been influenced, wherein the unusual difficulty and expensive of the words finished with additive method (for example chiral drug) of this molecule

[00263] before, it is believed that the Kong Taixiao in functionalized what is said or talked about protofibril material, and enough flowing can not be arranged, and therefore, can not be used to flow through electrode. use particulate carbon or other materials (for example reticulated vitreous carbon (RVC)) relevant problem also to be arranged as electrode substance based on carbon.For example, the porous electrode substance can not form in the original place, fill too closely and formation hole or passage, this makes and when changing solvent and flow condition the planar unstable can take place, and can not form extremely thin electrode. in flow cell, use functionalized carbon fibrils to solve these problems as electrode.

[00264] carbon fibrils that is used as electrode in flow cell can be modified by the surface treatment of electroactive agents.This protofibril also can be modified with non-electroactive substance, the effect that this non-electroactive substance performance catalysis or electrocatalysis function or performance suppress undesirable reaction or suppress adsorbent from flow.

[00265] these to flow through electrode be useful in isolation technique, for example electrochromatography, electrochemistry are regulated affinity chromatography, electrosynthesis or electrochemistry and are regulated ion-exchange chromatography. they also can be used for separating and/or analyzing the diagnostic device that is trapped in the material on the carbon fibrils material.

[00266] the combination material comprises functionalized carbon fibrils, also can use other fibers or particle.These fibers or particle can join in the suspension to change final porosity or the electric conductivity of carbon fibrils material.

Embodiment 52

The functionalized protofibril of FePC in flow cell as the application of electrode

[00267] iron-phthalocyanine-(Aldrich 41 for two-pyridine (FePc-2Py) by absorption, 016-0) modify the graphite protofibril. get the protofibril of 0.403g and the FePc-2Py of 0.130g and join among the pure EtOH of 150mis, with 450 Watt Branson probe sonicator sonications 5 minutes.Resulting slurry filters in the pipe of 47mm millipore filtration vacuum filter on 0.45 μ m MSI NF, and the water flushing is spent the night 35 ℃ of vacuum-dryings. and final weight is 0.528g, shows basically and has all adsorbed.Remaining FeP-2Py is calculated in the spectrophotometric analysis that filtrate is carried out.

[00268] getting the protofibril that 5mg FePc-2Py modifies is dispersed under sonication in the DI water of 10mis.Dispersion liquid placed on the knitting sieve of 200 purpose stainless steels (SS) with 25mm membrane filtration pipe and at room temperature dry.Downcut the protofibril material of the SS sieve support of 0.5 inch of diameter with arc punch tool.

[00269] the electrochemistry flow cell is to use the film filter paper clipping of 13mm, plastics, Swinney type to become, and comprises the top that the plate-like gold system sieve (400 orders, Ladd Industries) of diameter 13mm is placed membrane carrier, uses platinum line and sieve to take place electrically to contact, and uses

Heat-shrinkable tube insulation, this heat-shrinkable tube charging are carried out outside the connection by the wall of filter holder with the working electrode as three electrode potentiostat loops. externally fix gold system sieve in the edge with minimum epoxy.A goldleaf is changed into ring-type, and bottom being placed on, the downstream part of filter holder also links to each other with insulating Pt the end of a thread, counter electrode as three electrode potentiostat loops connects. and the ring-type of the diameter 0.5mm of electrochemical oxidation silver line connects as reference electrode with a top that places filter holder of insulating in 1M HCl.

[00270] the CN dish of the FePc-2Py of 0.5 inch of diameter being modified places flow cell, flow cell and EG﹠amp then; The suitable joint of G PAR 273 potentiostats links to each other.Flow cell links to each other with the filtering Sage syringe pump of 0.1M KCl in the 0.1M dipotassium hydrogen phosphate damping fluid that is used in pH7.0. under do not flow (static) and flow (0.4mls/ minute), write down cyclic voltammogram (CVs) (seeing accompanying drawing 6) with the electric potential scanning speed of 20mv/sec.Cvs flow and do not flow equal substantially down, show two kinds enduringly, reversible oxidation and reduction wave be consistent with the FePc-2Py of surface-limited.The persistence of reduction peak shows under fluid flow conditions, and FePc-2Py combines with carbon fibrils is strong.Use fibriilar function that FePC modifies and to flow through electrode substance identical.

[00271] other examples of three-dimensional structure are mixtures of protofibril-pottery.

Embodiment 53`

Synthetic (185-02-01) of aluminum oxide-protofibril mixture

[00272] with and U/S pulverizer with protofibril (185-01-02) high dispersing of the nitric acid oxidation of 1g in 100cc DI water. heating protofibril slurry to 90 ℃ also slowly adds the 0.04mol three fourth oxygen aluminium that are dissolved in the 20cc propyl alcohol. continue to reflux 4 hours, remove condenser then to get rid of alcohol.After 30 minutes, condenser is put back to, slurry spends the night 100 ℃ of backflows.Acquisition has the black colloidal sol of uniform outer appearance.Colloidal sol is cooled to room temperature, after 1 week, forms black gel with smooth-flat-surface.In air, this gel was heated 12 hours at 300 ℃.

[00273] checks aluminum oxide-protofibril mixture by SEM.The fiber photo of crack surface shows protofibril homodisperse in gel.

Embodiment 54

The preparation of silica-protofibril mixture (173-85-03)

[00274] with supersound process with protofibril protofibril (173-83-03) high dispersing of the nitric acid oxidation of 2g in 200cc ethanol.At room temperature in this slurry, slowly add the 0.1mol tetraethoxy that is dissolved in the 50cc ethanol, add the dense HCl. of 3cc then with mixture heating up to 85 ℃ and under this temperature, keep, be decreased to 100cc. until volume and cool off this mixture, place, form the black solid gel until it.In air, heat these gels at 300 ℃.

[00275] checks silica-protofibril mixture by SEM.The fiber photo of crack surface shows protofibril homodisperse in gel.

[00276] with other ceramic-like material, for example zirconium, titanium, rare earth oxide and trioxide can prepare similarly, also can prepare trioxide.

7. on polymeric beads, mix graphite nanotubes

[00277] polymeric beads particularly comprises Fe ₃O ₄The magnetic polymer pearl of nuclear for example by Dynal and other people preparation those, has a lot of application in diagnostics.But, compare with the nanotube effective surface area, these pearls have less surface-area. and functionalized protofibril can be incorporated into the surface of pearl, and this just makes that polymkeric substance/protofibril mixture can be as the solid carrier in gifts for a ceremony or the analytical applications (for example electrochemiluminescence analysis, the immobilization of enzyme).

Embodiment 55

Functionalized protofibril combines with functionalized pearl

[00278] use the 0.1M sodium phosphate buffer, (Norway) washing is three times for Dynal, Oslo with magnetic tolylsulfonyl--activatory Dynabeads M-450 (30mg/ml) pearl of 7.5mg for pH7.5.The 0.1M sodium phosphate buffer that adds 0.9ml then in this pearl, pH8.4 adds the amine protofibril of 0.1ml then.Mixture was at room temperature rotated 16-24 hour.

[00279] when examining under a microscope, the protofibril group that has pearl on the protofibril surface is very tangible

[00280] it will be apparent to those skilled in the art that disclosed any method can be come repetition less than the routine test of the Single Walled Carbon Nanotube of 5 nanometers with diameter in previous embodiment 1-55, thereby obtain required functionalized result.

Embodiment 56

Confirm the absorption of Yamamoto Methylene Blue ZF

[00281] went out all metallic impurity in 72 hours with 6M HCl treatment S WNT (University of Kentucky).Carbon nanotube with acid elution is dispersed in the DI water and filtration with sonication.Repeat to wash 4 times to neutral pH.

[00282] the SWNT resuspending disperses with sonication in DI water.The 2X serial dilution is placed the 8PP centrifuge tube.Equivalent adds Yamamoto Methylene Blue ZF,, pipe placed on the turner stirred 4 hours.Then pipe was rotated on whizzer 4 minutes, obtain being deposited on the SWNT of pipe bottom.The pipe that contains high density SWNT has clarifying filtrate, and two pipe colors with minimum SWNT concentration are similarly, with the dissolving of control MB.6 pipes with intermediate concentration SWNT have shown the reduction along with concentration, the blue enhancing.

The absorption of confirmation FePC

SWNT (University of Kentucky) is handled 72 hours to remove any metallic impurity with 6M HCl.Adopt ultrasonic nanotube to be dispersed in the DI water, and filter acid elution.Should operate repetition washs to neutral pH for 4 times.

Under supersound process, 7mg Fe phthalocyanine-two pyridines (FePc-2Py) are dissolved with 40ml EtOH.Add 22mg SWNT, and continued supersound process 5 minutes.Allow the cooling of this solution, and supersound process repeatedly.Hot solution is filled on 0.45 micron pvdf membrane with drying, then with fresh EtOH washing.Be filtered to dried. dry weight=27mg. is adsorbed onto 5mg FePc-2Py on the SWNT.

The filter membrane that will have the SWNT/FePC of connection places 40ml DI water, and supersound process is to lift SWNT from filter membrane.Filter membrane is taken out from suspension, and add 20mg Whatman #42 filter paper.The effect that (40% duty cycle) ultrasonic 2 * 5 ' come the SWNT tackiness agent with pulp WH42 paper under superpower.On filtration of material to the 0.45 micron PVDF filter that suspends, and with DI water washing 3 times.Be filtered to dried.On hot plate @ in 150 ℃ of dryings 1 hour.With exsiccant SWNT/WH42 pad as complete pad-36mm diameter, about 25 micron thickness-separate from film.The sheet resistivity of measuring is 1500ohm/ square metre (square).The final weight of pad is 45mg; Weight=the 20mg of WH42 component; Weight=22mg of SWNT; The weight of estimating that is adsorbed onto the lip-deep FePc-2Py of SWNT is 5mg.

Be adsorbed onto the electrochemistry of the FePc-2Py on the SWNT

By print, the section of pad is used for forming electrode with current collector between 400 * 400 order SS screen clothes.An edge of SS screen cloth is connected on the Cu silk that inserts in the glass volumetric pipette.With the Cu insulation of epoxide with any exposure.With electrode 100 ℃ of heating 1 hour with epoxide slaking 5 minutes.

By cyclic voltammetry SS screen cloth counter and Ag/AgCl reference electrode are measured this electrode.Boronising hydrochlorate buffer saline such as ionogen is.Observed two peaks, one is approaching-.-4, and it is covered by the H2 air-flow.Second peak concentrates on pact+0.1V vs Ag/Ag/Cl.For concentrating on+peak of 0.1V vs Ag/AgCl, peak current is consistent along with scan rate is linear with the electroactive substance of surface-limited.The cyclic voltammogram trace is repeatably, and this shows that FePc-2PY is limited on the electrode very securely, rather than is lost in the ionogen.

[00288] as described above shown in book and the embodiment, the present invention relates to formation of the functionalized widely carbon nanotube of kind and uses thereof.

[00289] employed term and expression are the term uses of book as an illustration, rather than limit, nor plan to use exceed any as term or the expression in addition of the shown and described feature same range as of its part, should recognize that various possible changes all within the scope of the invention.

Claims

2. the composition of a following general formula material:

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each R is identical, and is selected from SO ₃H, COOH, NH ₂, OH, R ' CHOH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X,

Y is equal to or less than 3 integer,

R ' is hydrogen, alkyl, aryl, cycloalkyl or aralkyl, cyclophane base or poly-(alkyl oxide),

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide, and

Z is carboxylate radical or trifluoroacetic acid root.
3. the composition of a following general formula material:

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each R can be identical or different, and be selected from SO ₃H, COOH, NH ₂, OH, R ' CHOH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X,

Y is equal to or less than 3 integer,

R ' is selected from hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cyclophane base or gathers (alkyl oxide),

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide, and

Z is carboxylate radical or trifluoroacetic acid root,

And condition is if each R is the oxygen containing group of bag further, does not then have COOH.
4. the composition of a following general formula material:

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each R ' is alkyl, aryl, cycloalkyl, aralkyl, cyclophane base or poly-(alkyl oxide),

A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, Nucleotide, oligonucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-N (R ') ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' N ⁺(R ') ₃X ^-, R ' SiR ' ₃, R ' Si (OR ') _yR ' _3-y, R ' Si (O-SiR ' ₂) OR ', R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

Y is equal to or less than 3 integer,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide,

Z is carboxylate radical or trifluoroacetic acid root, and

W is greater than 1 and less than 200 integer.
5. the composition of claim 3, wherein

A is

Or

R ' is H, and

Y is selected from following amino acid: Methionin, Serine, Threonine, tyrosine, aspartic acid and L-glutamic acid.
6. the composition of a following general formula material:

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n, and a is the integer less than 10,

Each A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, Nucleotide, oligonucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-N (R ') ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' N ⁺(R ') ₃X ^-, R ' SiR ' ₃, R ' Si (OR ') _yR ' _3-y, R ' Si (O-SiR ' ₂) OR ', R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

Y is equal to or less than 3 integer,

R ' is alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide,

X ' is polynuclear aromatic, many heteronuclears aromatics or many heteronuclears of metal aromatics part or planar tetrathio oxalic acid metal-salt,

Z is carboxylate radical or trifluoroacetic acid root, and

W is greater than 1 and less than 200 integer.
7. method for compositions that produces following general formula material, wherein

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

R ' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cyclophane base or poly-(alkyl oxide),

Described method comprises the following steps: to have general formula being enough to form

The condition of functionalized nanotube under, with surface carbon with have general formula R ' CH ₂The compound of OH reacts in the presence of radical initiator.
8. the method for claim 6, wherein said radical initiator is a benzoyl peroxide.
9. method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, oligonucleotide, Nucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-N (R ') ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' SiR ' ₃, R ' N ⁺(R ') ₃X ^-, R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

R ' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide,

Z is carboxylate radical or trifluoroacetic acid root, and

W is greater than 1 and less than 200 integer,

Described method comprises the following steps:

(a) with surface carbon and at least a suitable reagent react, reaction conditions is enough to formation and has general formula

The Single Walled Carbon Nanotube of replacement, wherein each R is identical and be selected from SO ₃H, COOH, NH ₂, OH, CH (R ') OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X, and y is equal to or less than 3 integer; With

(b) with the Single Walled Carbon Nanotube that replaces

With at least a suitable reagent react, reaction conditions is enough to form general formula

Functionalized single walled carbon nanotubes.
10. method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, oligonucleotide, Nucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-N (R ') ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' SiR ' ₃, R ' N ⁺(R ') ₃X ^-, R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

R ' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide,

Z is carboxylate radical or trifluoroacetic acid root, and

W is greater than 1 and less than 200 integer,

Described method comprises the following steps:

(a) with surface carbon and at least a suitable reagent react, reaction conditions is enough to formation and has general formula

The Single Walled Carbon Nanotube of replacement, wherein each R is selected from SO ₃H, COOH, NH ₂, OH, CH (R ') OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X, and y is equal to or less than 3 integer; With

(b) with the Single Walled Carbon Nanotube that replaces

With at least a suitable reagent react, reaction conditions is enough to form general formula

Functionalized single walled carbon nanotubes.
11. a method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, oligonucleotide, Nucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-N (R ') ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' SiR ' ₃, R ' N ⁺(R ') ₃X ^-, R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

R ' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide,

Z is carboxylate radical or trifluoroacetic acid root, and

W is greater than 1 and less than 200 integer,

Described method comprises the following steps: the Single Walled Carbon Nanotube that will replace

With at least a suitable reagent react, reaction conditions is enough to form general formula

Functionalized single walled carbon nanotubes, wherein each R is identical, and is selected from SO ₃H, COOH, NH ₂, OH, CH (R ') OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X, and y is equal to or less than 3 integer.
12. a method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, oligonucleotide, Nucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-N (R ') ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' SiR ' ₃, R ' N ⁺(R ') ₃X ^-, R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

R ' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide,

Z is carboxylate radical or trifluoroacetic acid root, and

W is greater than 1 and less than 200 integer,

Described method comprises the following steps: the Single Walled Carbon Nanotube that will replace

With at least a suitable reagent react, reaction conditions is enough to form general formula

Functionalized single walled carbon nanotubes, wherein each R is identical, and is selected from SO ₃H, COOH, NH ₂, OH, CH (R ') OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X, and y is equal to or less than 3 integer.
13. a method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

R ' is alkyl, aryl, cycloalkyl, aralkyl, cyclophane base or poly-(alkyl oxide),

X is a halogenide,

Each A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, oligonucleotide, Nucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-NH ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' SiR ' ₃, R ' R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl, and

Z is carboxylate radical or trifluoroacetic acid root,

Described method comprises the following steps: to have formula

The Single Walled Carbon Nanotube and at least a suitable reagent react of replacement, reaction conditions is enough to form and has general formula

Functionalized single walled carbon nanotubes, wherein each R is identical, and is selected from SO ₃H, COOH, NH ₂, OH, CH (R ') OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X, and y is equal to or less than 3 integer.
14. a method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

Wherein n is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n, and a is 0 or less than 10 integer,

Each R is selected from SO ₃H, COOH, NH ₂, OH, CH (R ') OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X,

Y is equal to or less than 3 integer,

R ' is alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

X is a halogenide,

X ' is polynuclear aromatic, many heteronuclears aromatics or many heteronuclears of metal aromatics part or planar tetrathio oxalic acid metal-salt,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl, and

Z is carboxylate radical or trifluoroacetic acid root,

Said method comprising the steps of: have general formula being enough to form

The condition of functionalized single walled carbon nanotubes under, at least a suitable macrocylc compound is adsorbed onto on the surface of Single Walled Carbon Nanotube.
15. a method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n, and a is the integer less than 10,

Each A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, oligonucleotide, Nucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-NH ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' SiR ' ₃, R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

R ' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide,

X ' is polynuclear aromatic, many heteronuclears aromatics or many heteronuclears of metal aromatics part or planar tetrathio oxalic acid metal-salt,

Z is carboxylate radical or trifluoroacetic acid root, and

W is greater than 1 and less than 200 integer,

Described method comprises the following steps:

(a) at least a suitable macrocylc compound is adsorbed onto on the surface of Single Walled Carbon Nanotube, adsorption conditions is enough to formation and has general formula

The Single Walled Carbon Nanotube of replacement, wherein each R is selected from SO ₃H, COOH, NH ₂, OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X, and y is equal to or less than 3 integer; With

(b) with the nanotube that replaces

With at least a suitable reagent react, reaction conditions is enough to formation and has general formula

Functionalized single walled carbon nanotubes.
16. a method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n, and a is the integer less than 10,

Each A is selected from

Or C=Y,

Y be protein, peptide, amino acid, enzyme, antibody, oligonucleotide, Nucleotide, antigen or enzyme substrates, enzyme inhibitors or enzyme substrates transition state analog suitable functional group or be selected from R '-OH, R '-NH ₂, R ' SH, R ' CHO, R ' CN, R ' X, R ' SiR ' ₃, R '-R ", R '-N-CO, (C ₂H ₄O) _wH, (C ₃H ₆O) _wH, (C ₂H ₄O) _w-R ', (C ₃H ₆O) _w-R ', R ' and

R ' is alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide,

X ' is polynuclear aromatic, many heteronuclears aromatics or many heteronuclears of metal aromatics part or planar tetrathio oxalic acid metal-salt,

Z is carboxylate radical or trifluoroacetic acid root, and

W is greater than 1 and less than 200 integer,

Described method comprises the following steps: the Single Walled Carbon Nanotube that will replace

With at least a suitable reagent react, reaction conditions is enough to formation and has general formula

Functionalized single walled carbon nanotubes, wherein each R is selected from SO ₃H, COOH, NH ₂, OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X, and y is equal to or less than 3 integer.
17. a method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

Wherein n is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

R ' is alkyl, aryl, cycloalkyl or aralkyl,

Described method comprises the following steps:

With surface carbon and at least a suitable reagent react, reaction conditions is enough to formation and has general formula

Functionalized single walled carbon nanotubes;

With functionalized single walled carbon nanotubes and the compound reaction with 2 or a plurality of amino, reaction conditions is enough to form and has general formula

Functionalized single walled carbon nanotubes.
18. method for compositions that forms following general formula material

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Each R is identical, and is selected from SO ₃H, COOH, NH ₂, OH, CH (R ') OH, CHO, CN, COCl, halogenide, COSH, SH, COOR ', SR ', SiR ' ₃, Si (OR '-) _yR ' _3-y, Si (O-SiR ' ₂-) OR ', R ", Li, AlR ' ₂, Hg-X, TlZ ₂And Mg-X,

Y is equal to or less than 3 integer,

R ' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or cyclophane base,

R " be fluoro-alkyl, fluorinated aryl, fluoro cycloalkyl or fluoro aralkyl,

X is a halogenide, and

Z is carboxylate radical or trifluoroacetic acid root,

Described method comprises the following steps: that with surface carbon and at least a enzyme reaction this endonuclease capable is accepted Single Walled Carbon Nanotube as substrate and carried out carrying out chemical reaction under the chemical reaction acceptable terms at least a enzyme, forms general formula [C in aqueous suspension _nH _L] R _mThe composition of material.
19. the method for claim 17, wherein R _mBe-OH and enzyme are cytopigment p450 enzyme or peroxidase.
20. a method for compositions that forms following general formula material,

Carbon atom C wherein _nBe the surface carbon of diameter less than the columned Single Walled Carbon Nanotube basically of 5 nanometers,

N is an integer, and L is the numeral less than 0.1n, and m is the numeral less than 0.5n,

Described method comprises the following steps:

With surface carbon and nitric acid or sulfuric acid reaction to form nitrated Single Walled Carbon Nanotube; With

Reduce this Single Walled Carbon Nanotube to form
21. one kind evenly replaces the method for diameter less than the surface of the Single Walled Carbon Nanotube of 5 nanometers with functional group, described method comprises the Single Walled Carbon Nanotube of described diameter less than 5 nanometers is contacted with the reagent of the functional group that can evenly replace described Single Walled Carbon Nanotube surface of significant quantity.
22. the method for claim 20, wherein reagent is phthalocyanine.
23. the method for claim 20, wherein reagent is phthalocyanine tetrasulfonic acid nickel (II) (tetra-na salt) or 1,4,8,11,15,18,22,25-eight butoxy-29H, 31H-phthalocyanine.
24. the diameter that makes by the following method is less than the Single Walled Carbon Nanotube of the finishing of 5 nanometers: described Single Walled Carbon Nanotube is contacted with the reagent of significant quantity, to replace the functional group on described Single Walled Carbon Nanotube surface.
25. the Single Walled Carbon Nanotube of the finishing of claim 23, wherein reagent is phthalocyanine.
26. the Single Walled Carbon Nanotube of the finishing of claim 23, wherein reagent is phthalocyanine tetrasulfonic acid nickel (II) (tetra-na salt) or 1,4,8,11,15,18,22,25-eight butoxy-29H, 31H-phthalocyanine.
27. a method that connects protein and nanotube, described method comprises the following steps:

Be enough under the condition that forms covalent linkage between NHS ester and the proteinic amido, the diameter that carries the NHS ester group functionalized single walled carbon nanotubes less than 5 nanometers is being contacted with protein.
28. an electrode, described electrode comprise the functionalized single walled carbon nanotubes of diameter less than 5 nanometers.
29. the electrode of claim 27, wherein this electrode is the porous flow electrify electrode.
30. the described electrode of claim 27, wherein functionalized single walled carbon nanotubes is the nanotube that phthalocyanine replaces.
31. porous material, described material comprises a plurality of functionalized single walled carbon nanotubes reticulations, wherein said functionalized single walled carbon nanotubes reticulation comprises at least two functionalized protofibril that are connected with functional group by at least one connection portion, and wherein said connection portion is bifunctional or multi-functional.
32. a method of separating interested solute from sample, described method comprises the following steps:

Be enough to form under the condition of functionalized single walled carbon nanotubes, with at least a suitable reagent physics or chemically modified diameter Single Walled Carbon Nanotube less than 5 nanometers;

The fixing material that interested solute can be attached on the functionalized single walled carbon nanotubes; With

Under the condition that material on being enough to make interested solute and be fixed on functionalized single walled carbon nanotubes combines, the Single Walled Carbon Nanotube that replaces is exposed in the composition that contains interested solute.
33. the method for claim 31, wherein interested solute is a protein.
34. the method for claim 31, described method also comprises the step of recovering this functionalized single walled carbon nanotubes.
35. the method for claim 31, wherein functionalized single walled carbon nanotubes is the form of porosity.
36. the method for claim 31, wherein functionalized single walled carbon nanotubes is the form of packed column.
37. the method for claim 31, wherein this combination is a reversible.
38. the method for claim 31, wherein this combination is an ionic interaction.
39. the method for claim 31, wherein this combination is a hydrophobic interaction.
40. the method for claim 31, wherein this combination is by specific molecular recognition.
41. a polymeric beads, described polymeric beads comprise the basically spheric pearl of diameter less than 25 μ, it links to each other with the functionalized single walled carbon nanotubes of a plurality of diameters less than 5 nanometers.
42. the polymeric beads of claim 40, wherein this pearl is a magnetic.
43. the method for a catalyzed reaction, wherein at least a reactant is transformed at least a product, and described method comprises the following steps:

Be enough to form under the condition of functionalized single walled carbon nanotubes, with at least a suitable reagent physics or chemically modified diameter surface carbon less than the Single Walled Carbon Nanotube of 5 nanometers;

The biological catalyst of the reaction on the fixing energy catalysis functionalized single walled carbon nanotubes; With

Being enough to that reagent is transformed under the condition of product, functionalized single walled carbon nanotubes is contacted with reagent.
44. the method for claim 42, described method also are included in the step of recovering functionalized Single Walled Carbon Nanotube after the complete reaction.
45. the method for claim 42, wherein functionalized single walled carbon nanotubes is the form of porosity.
46. the method for claim 42, wherein functionalized single walled carbon nanotubes is the form of packed column.
47. the method for synthetic peptide, described method comprises the following steps: the end amino acid of peptide is attached on the Single Walled Carbon Nanotube of diameter less than 5 nanometers by the reversible connexon.
48. the method for claim 46, wherein connexon is 4-(methylol) phenylium.