English:
[0005] In 1983, Tennent, U.S. Patent No. 4,663,230, hereby incorporated by reference, succeeded in growing cylindrical ordered graphite cores, uncontaminated with pyrolytic carbon. Thus, the Tennent invention provided access to smaller diameter fibrils, typically 35 to 700 A (0.0035 to 0.070μ) and to an ordered, "as grown" graphitic surface. Fibrillar carbons of less perfect structure, but also without a pyrolytic carbon outer layer have also been grown.
[0019] U.S. Pat. No. 5,424,054 to Bethune et al., hereby incorporated by reference, describes a process for producing single-walled carbon nanotubes by contacting carbon vapor with cobalt catalyst. The carbon vapor is produced by electric arc heating of solid carbon, which can be amorphous carbon, graphite, activated or decolorizing carbon or mixtures thereof. Other techniques of carbon heating are discussed, for instance laser heating, electron beam heating and RF induction heating
[0027] It is therefore a primary object of this invention to provide functionalized single walled carbon nanotubes having diameters less than 5 nanometers, i.e., single walled carbon
[0028] It is a further and related object of this invention to provide single walled carbon nanotubes having diameters less than 5 nanometers whose surfaces are functionalized by reaction with oxidizing or other chemical media.
[0030] It is a further object to provide single walled carbon nanotubes having diameters less than 5 nanometers whose surfaces have been modified e.g., by oxidation which are then further modified by reaction with functional groups.
[0039] Fig. 5 is the reaction sequence for the preparation lysine-based dendrimeric fibrils.
[0041] Fig. 7 is the reaction sequence for the preparation of bifunctional fibrils by the addition of N ε -(tert-butoxycarbonyl)-L-lysine.
[0043] Fig. 9 is a graphical representation of the results of separation of alkaline phosphatase (AP) from a mixture of AP and β -galactosidase (βG) using AP inhibitor- modified fibrils.
[0044] Fig. 10 is a graphical representation of the results of separation of βG from a mixture of AP and βG using βG-modified fibrils.
[0062] The invention is also in methods of introducing functional groups onto the surface of carbon fibrils by contacting carbon fibrils with a strong oxidizing agent for a period of time sufficient to oxidize the surface of said fibrils and further contacting said fibrils with a reactant suitable for adding a functional group to the oxidized surface. In a preferred embodiment of the invention, the oxidizing agent is comprised of a solution of an alkali metal chlorate in a strong acid. In other embodiments of the invention the alkali metal chlorate is sodium chlorate or potassium chlorate. In preferred embodiments the strong acid used is sulfuric acid. Periods of time sufficient for oxidation are from about 0.5 hours to about 24 hours.
[0070] The uniformly functionalized fibrils of the invention can be directly prepared by sulfonation, electrophilic addition to deoxygenated fibril surfaces or metallation. When arc grown nanofibers are used, they may require extensive purification prior to functionalization. Ebbesen et al. (Nature 367 519 (1994)) give a procedure for such purification.
[0071] Preferably, the carbon fibrils are processed prior to contacting them with the functionalizing agent. Such processing may include dispersing the fibrils in a solvent. In some instances the carbon fibrils may then be filtered and dried prior to further contact.
[0073] Activated C-H (including aromatic C-H) bonds can be sulfonated using fuming sulfuric acid (oleum), which is a solution of cone, sulfuric acid containing up to 20% SO3. The conventional method is via liquid phase at T-80°C using oleum; however, activated C-H bonds can also be sulfonated using SO 3 in inert, aprotic solvents, or SO 3 in the vapor phase. The reaction is:
[0076] When the desired temperature was stabilized, the SO3 source was connected to the reactor tube and an argon stream was used to carry SO 3 vapors into the quartz tube reactor. Reaction was carried out for the desired time at the desired temperature, after which the reactor was cooled under flowing argon. The fibrils were then dried at 90°C at 5" Hg vacuum to obtain the dry weight gain. Sulfonic acid (-SO 3 H) content was determined by reaction with 0.10ON NaOH and back-titration with 0.10ON HCl using pH 6.0 as the end point.
[0085] The procedure is repeated as in Ex. 2, except that the reactant at RT is purified maleic anhydride (MAN) which is fed to the reactor by passing argon gas through a molten MAN bath at 80 0 C.
[0089] Aromatic C-H bonds can be metallated with a variety of organometallic reagents to produce carbon-metal bonds (C-M). M is usually Li, Be, Mg, Al, or Tl; however, other metals can also be used. The simplest reaction is by direct displacement of hydrogen in activated aromatics:
[0092] One gram of CC fibrils is placed in a porcelain boat and inserted into a 1" quartz tube reactor which is enclosed in a Lindberg tube furnace. The ends of the tube are fitted with gas inlet/outlets. Under continuous flow of H 2 , the fibrils are heated to 700 0 C for 2 hours to convert any surface oxygenates to C-H bonds. The reactor is then cooled to RT under flowing H 2 .
[00114] The following Examples 11 and 12 illustrate methods for the adsorption of two different phthalocyanine derivatives on carbon nanotubes.
[00126] A weighed sample of fibrils was slurried with nitric acid of the appropriate strength in a round bottom multi-neck indented reactor flask equipped with an overhead stirrer and a water condenser. With constant stirring, the temperature was adjusted and the reaction carried out for the specified time. Brown fumes were liberated shortly after the temperature exceeded 70 0 C, regardless of acid strength. After the reaction, the slurry was poured onto cracked ice and diluted with DI water. The slurry was filtered and excess acid removed by washing in a Soxhlet extractor, replacing the reservoir with fresh DI water every several hours, until a slurried sample gave no change in Ph from DI water. The fibrils were dried at 100 0 C at 5" vacuum overnight. A weighed portion of fibrils was reacted with standard 0.100 N NaOH and the carboxylic acid content determined by back-titration with 0.100 N HCl. Surface oxygen content was determined by XPS. Dispersibility in water was tested at 0.1 wt% by mixing in a Waring Blender at high for 2 min. Results are summarized in Table 4.
[00133] 0.277 grams of carbon nanotubes were dispersed in MeOH using a probe sonicator. 0.126 grams of BPO were added at RT and the temperature was increased to 60 0 C and an additional 0.128 grams of BPO were added. After an additional 45 minutes at 60 0 C, a final BPO charge of 0.129 grams was added and the mixture was kept at 6O 0 C for an additional 30 minutes. The product was filtered onto a membrane and washed several times with MeOH and EtOH and dried in an oven at 90 0 C. The yield was 0.285 grams. ESCA analysis showed an oxygen content of 2.5 atomic percent compared with 0.74% for a control
[00134] 0.1 grams of carbon nanotubes, 0.5 grams BPO and 10 grams poly(ethyleneglycol), avg. mol. wt. 1000 (PEG-1000) were mixed together at room temperature. The mixture was heated to 90 0 C to melt the PEG and the mixture was left to react at 90 0 C overnight. The entire mixture was then filtered and washed to remove the excess PEG and was then dried. The resultant material can be used either as is, or it can be further modified by attaching materials of interest to the free end of the PEG.
[00137] Stock dispersions of chlorate oxidized fibrils, Fibrils modified with PEG using benzoyl peroxide and chlorate oxidized fibrils modified with PEG by NHS ester coupling were prepared at 1.0 mg/ml in 50 mM potassium phosphate buffer, pH 7.0 with sonication. 2 mis of 3-fold serial dilutions of each were placed in each of 7 polypropylene tubes. lOOμl of a 0.2 mg/ml solution of β-lactoglobυlin (βLG) in the same buffer was added to each tube and to 3 buffer blanks. Three buffer tubes without protein were also prepared. All tubes were mixed on a vortex mixer and allowed to incubate for 60 minutes with 30 seconds of vortexing every 10 minutes. All tubes were centrifuged to separate the fibrils and 1 ml aliquots of the supernatant were transferred to new tubes and analyzed for total protein content using a Micro BCA protein assay (Pierce). The level of protein remaining in the supernatant was an indirect measure of the amount that had been non-specifically bound to the protein (see Fig. T). For each of the tubes the βLG remained in the supernatant for the fibrils modified with PEG via the NHS ester route signifying no non-specific binding. The fibrils modified with PEG via the BPO route exhibited only slight (approx. 10%) binding of the βLG at the highest fibril level of 1.0 mg/ml and no significant binding at lower levels. In contrast, there was nearly complete binding to the chlorate oxidized fibrils at fibril levels of 0.1 mg/ml and above and substantial binding down to 0.01 mg/ml of these fibrils.
[00139] The reactions can be carried out using any of the methods developed for esterifying or aminating carboxylic acids with alcohols or amines. Of these, the methods of H.A. Staab, Anagew. Chem. Intemat. Edit., (1), 351 (1962) using N,N'-carbonyl diimidazole (CDI) as the acylating agent for esters or amides, and of G. W. Anderson, et al., J. Amer. Chem. Soc. 86, 1839 (1964), using N-hydroxysuccinimide (NHS) to activate carboxylic acids for amidation were used.
[00142] Trialkylsilylchlorides or trialkylsilanols react immediately with acidic H according to:
[00151] Trypsin can react with substrate L-BAPNA (N α -benzoyl-L-arginine p- nitroanilide) and release a colored compound that absorbs light at 410 nm. The assay buffer for this reaction was 0.05 M Tris, 0.02 M CaCl 2 , pH 8.2. The reaction was performed in 1 ml cuvette by mixing 5 μl of L-BAPNA stock solution (50 mM in 37% DMSO in H 2 O) and 10-25 μg of trypsin fibrils in a 1 ml of assay buffer. The absorbance increase at 410 nm was monitored over 10 minutes. The enzyme activity (μM/min) was then calculated from the initial slope.
[00155] A 10 mg sample of thiolated nanotubes was suspended in 10 mis. of DI water with sonication and filtered onto 0.45μm nylon membrane to form a felt-like mat. The mat section was stored in a vacuum desiccator prior to analysis by ESCA which showed 0.46% sulfur and 1.69% nitrogen, confirming successful conversion to thiol-modified CN.
[00164] ESCA was carried out to quantify the amount of N present on the aminated fibrils (GFZNH 2 ). ESCA analysis of 177-046-1 showed 0.90 at% N (177-059). To further assess how much of this N is present as both accessible and reactive amine groups, a derivative was made by the gas phase reaction with pentafluorobenzaldehyde to produce the corresponding Schiff Base linkages with available primary amine groups. ESCA analysis still showed the 0.91 at% N, as expected, and 1.68 at%F. This translates into a 0.34 at% of N present as reactive primary amine on the aminated fibrils (5 F per pentafluorobenzaldehyde molecule). A level of 0.45 at% N would be expected assuming complete reaction with the free ends of each N. The observed level indicates a very high yield from the reaction of N with NHS-activated fibril and confirms the reactivity of the available free amine groups.
[00172] To demonstrate the efficacy of the method, a small sample of amino fibrils were reacted with "activated" horseradish peroxidase (HRP; 5 mg, Pierce) which was modified to specifically react with amino groups. The fibrils were washed repeatedly for several days (by suspension, rotation, and centrifugation in an Eppendorf tube) while kept cold. After approximately two weeks of washing, the enzyme was assayed with H 2 O 2 /ABTS in glycine buffer, pH 4.4. A green color appeared in the solution within 10 minutes indicating the presence of enzyme. Control fibrils (COOH fibrils treated with activated HRP and washed for the same period of time) showed little if any catalytic activity.
[00176] Table VI summarizes the secondary derivative preparations. Products are analyzed by ESCA. The analysis confirms the incorporation of the desired pendant groups. The products are analyzed by ESCA for C, O, N, Si and F surface contents.
[00180] An aqueous slurry containing 60 mg 2-diethylamino ethylamine modified carboxyl fibrils and 180 mg Sephadex G-25 superfine resin (Pharmacia, Uppsala, Sweden) was allowed to stand overnight at room temperature to ensure full hydration of the solid support. The slurry was packed into a 1 cm x 3.5 cm column. The column was equilibrated with 5 mM sodium phosphate buffer (pH 7.3) at a flow rate of 0.2 ml/min. BSA (0.6 mg in 0.1 ml de-ionized water) was loaded on the column. The column was eluted with 5 mM sodium phosphate at a flow rate of 0.2 ml/min and 0.6 ml fractions were collected. The elution profile was monitored using a UV- visible detector, and is shown in Fig 3. Once the detector indicated that no more protein was eluting from the column, bound BSA was eluted by adding 1 M KCl in 5 mM sodium phosphate (pH 7.3). The presence of the protein in each fraction was identified by micro BCA assay (Pierce, Rockford, II).
[00194] The reaction sequence is shown in Fig. 5.
[00199] CN/NHS were prepared according to the method of Example 20.
[00205] Samples (approx. 10 mg) of each of the four generation of NTA addition were suspended in 10 mis of DI water with sonication and filtered onto 0.45 μm nylon membranes to form felt-like mats. The mat sections were stored in a vacuum desiccator and analyzed by ESCA for nitrogen (N) to indicate relative amounts of NTA. The results are shown in the table below.
[00227] 0.5 mg of phenyl-alkyl fibrils were suspended in 50 μl of 5 niM sodium phosphate buffer (pH 7.1) and sonicated for 20 minutes. To these fibrils 350 μl of lipase solution (0.2 mM in 5 mM sodium phosphate buffer, pH 7.1) were added and the mixture was rotated at room temperature for 5 hours and stored at 4°C overnight. The fibrils were then washed with 600 μl of 5 mM sodium phosphate buffer (pH 7.1) three times and suspended in 200 μl of the same buffer.
[00246] To a mixture of amino fibrils (400 mg) and a 4-(hydroxymethyl)- phenoxyacetic acid suspension (255 mg, 1.4 mmol) in methylene chloride (20 ml) were added l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 268 mg, 1.40 mmol) and 1- hydroxybenzotriazole hydrate (HOBT, 189 mg, 1.4mmol). The reaction mixture was stirred overnight at room temperature under argon gas. The product fibrils were extensively washed with methylene chloride, methanol and water, then dried under vacuum to get fibrils. To the suspension of fibrils in N,N-dimethylformamide (DMF, 2 ml) and methylene chloride (8 ml) were added N-(9-fluorenylmethoxycarbonyl)-O-butyl-L-serine (215 mg, 0.56 mmol), 1,3- dicyclohexylcarbodiimide (DCC, 115 mg, 0.56 mmol) and 4 dimethylaminopyridine (DMAP, 3.4 mg, 0.028 mmol). The reaction mixture was stirred overnight at room temperature and the product fibrils were treated with 20% piperidine in DMF (5 X 40 ml, each time soaked 1 min.). The product fibrils were then extensively washed with DMF, water, sodium hydroxide (IN), methanol and methylene chloride. The product Fib-Handle- Ser(O+)-COOH (ninhydrin test was positive) was dried under vacuum. For synthesis of dipeptide, the same procedure was used to add arginine. The amino acid analysis data of Fib- Handle-Ser(O+)- Arg(N ε -2,2,5,7,8-pentamethylchrornan-6-sulfonyi) shows that it contains 6.5 μmol serine per gram fibrils and 7.6 μmol arginine per gram fibrils. Any other peptide can be made by the same method.
[00276] Similar preparations with other ceramics, such as zirconia, titania, rare earth oxides as well as ternary oxides can be prepared.
[00280] One skilled in the art will appreciate that any of the process disclosed in the previous Examples 1-55 may be repeated within routine experimentation with single walled carbon nanotubes having diameters less than 5 nanometers to obtain the desired functionalization result.
[00289] The terms and expressions which have been employed are used as terms of description and not of limitations, and there is no intention in the use of such terms or expressions of excluding any equivalents of the features shown and described as portions thereof, its being recognized that various modifications are possible within the scope of the invention.

中文:
[0005]1983年,通过参考引入本文的Tennent的U.S.4,663,230在生长的圆柱状有序的石墨核心方面取得了成功,其没有被热解碳所污染。因此Tennent的发明提供了直径更小的原纤维,典型地是35到700(0.0035到0.070μ),并得到了有序的,”生长样的”石墨表面。也生长出了结构完美程度较小的原纤维碳,但也不含热解碳外层。
[0019]通过参考引入本文的Bethune等人的U.S.5,424,054、描述了一种制备单壁碳纳米管的方法,包括将碳蒸汽与钴催化剂接触。该碳蒸汽式通过固体碳的电弧加热得到的,固体碳可以是无定形碳、石墨、活性碳或脱色碳或其混合物.也讨论了加热碳的其他方法,例如激光加热、电子束加热和RF感应加热.
[0027]因此,本发明的首要目的是提供直径小于5纳米的官能化的单壁碳纳米管,即表面均匀或非均匀修饰以与官能化的化学部分结合的单壁碳纳米管.
[0028]本发明的进一步和相关的目的是提供直径小于5纳米的单壁碳纳米管,通过与氧化剂或其他化学介质反应而将表面官能化。
[0030]本发明的进一步目的是提供直径小于5纳米的单壁碳纳米管,其表面已经例如通过氧化修饰,然后通过与官能团反应而进一步修饰。
[0039]附图5是制备基于赖氨酸的dendrimeric原纤维的反应顺序。
[0041]附图7是通过加入Nε-(叔丁氧羰基)-L-赖氨酸制备双功能原纤维的反应顺序。
[0043]附图9是用AP抑制剂修饰的原纤维从AP和β-半乳糖苷酶(βG)的混合物中分离碱性磷酸酶(AP)的结果的图形表示。
[0044]附图10是用βG修饰的原纤维从AP和βG的混合物中分离βG的结果的图形表示。
[0062]本发明也涉及将官能团引入到碳原纤维表面上的方法,包括将碳原纤维与强氧化剂接触一段时间,以充分地氧化所述原纤维的表面,进一步将所述原纤维与适合将官能团加到氧化表面的试剂接触。在本发明一个优选的实施方案中,该氧化剂包括碱金属氯酸盐的强酸溶液。在本发明的另一个实施方案中,碱金属氯酸盐是氯酸钠或氯酸钾。在优选的实施方案中,所使用的强酸是硫酸.充分氧化的时间是约0.5小时到约24小时。
[0070]可以通过磺化作用、亲电子加成到去氧原纤维表面和金属化来直接制备本发明的均匀官能化的原纤维。当使用弧生长纳米纤维时,它们可能需要在官能化前广泛纯化。Ebbesen等人(Nature 367 519(1994))给出了该纯化的方法。
[0071]优选地,在与官能化剂接触前先处理碳原纤维。这些处理可以包括将原纤维分散到溶剂中。在一些例子中,然后可以过滤碳原纤维,并在进一步接触前干燥。
[0073]用发烟硫酸(oleum)可磺化活化的C-H(包括芳香族的C-H)键,发烟硫酸是包含高达20% SO3的浓硫酸溶液。该常规方法是经液相在80°C使用发烟硫酸;但是也可以在惰性、非质子溶剂中使用SO3或在气相中使用SO3来磺化活化的C-H键.该反应是
[0076]当所需的温度变稳定时,将SO3源于反应器的管相连,用氩气流将SO3蒸汽带入到石英管反应器中.在所需温度下在所需时间里进行反应,然后在流动的氩气下冷却反应器.然后在90°C和5”Hg真空中干燥原纤维,获得干重的增加。通过与0.100N NaOH反应和用0.100N HCl反滴定,以pH6.0为终点来确定硫酸(-SO3H)的量。
[0085]如实施例2重复该方法,除了在RT下试剂是纯净的马来酸酐(MAN),其通过在80°C将氩气通过熔化的MAN浴进料到反应器中。
[0089]芳香族的C-H键可以用各种的有机金属试剂金属化而产生碳-金属键(C-M)。M通常是Li、Be、Mg、Al、或Tl;但是也可以使用其他金属.该简单反应是通过在活化的芳香族化合物中直接取代氢:
[0092]将1g CC原纤维置于瓷皿中,并插入到包入在Lindberg管形炉中的1”石英管反应器中.管的末端具有气体进口/出口。在连续的H2流下,将原纤维加热到700°C 2小时,以将所有的表面氧化物转变成C-H键。然后在H2流下冷却反应器至室温。
[00114]下列的实施例11和12说明了将两种不同的酞菁衍生物吸附到碳纳米管上的方法.
[00126]在具有顶搅拌器和水冷凝器的圆底多颈有痕的反应烧瓶中将原纤维的已称重样品与适当强度的硝酸浆化.在恒速搅拌下,调节温度,并在特定时间进行反应.在温度超过70°C时,不论酸的强度如何,都立刻释放出了棕色的烟雾。反应后,将浆体倾倒入碎冰中,并用DI稀释。过滤该浆体,在Soxhlet提取器中洗涤除去过量的酸,每隔几小时用新鲜的DI水取代容器中的水,直至浆体样品不改变DI水的pH.在5”真空和100°C将原纤维干燥过夜。已称重部分的原纤维与标准的0.100N NaOH反应,用0.100N HCl反滴定来确定羧酸的含量。通过XPS确定表面氧的含量。通过在韦林搅拌器中高速混合2分钟来确定在水中0.1wt%的可分散性.结果概述于表4中。
[00133]用探针超声波破碎器将0.277g的碳纳米管分散到MeOH中。在RT下加入0.126g的BPO,温度升高到60°C,加入0.129g的BPO原料,混合物在60°C下再保持30分钟。在膜上过滤产品,用MeOH和EtOH洗涤几次,在90°C的烤箱中干燥。收率是0.285g。ESCA分析表明,氧含量是2.5%原子,与此相比,在不含BPO的MeOH中回流的对照样品是0.74%。
[00134]将0.1g的碳纳米管、0.5g BPO和10g聚(乙二醇)avg.mol.wt.1000(PEG-1000)在室温下混合在一起。温度加热到90°C使PEG熔化,将混合物保持在90°C反应过夜.然后过滤全部混合物,洗涤除去过量的PEG,然后干燥.可以使用所得到的物质,或者可以通过向PEG的自由末端结合感兴趣的物质而进一步修饰。
[00137]用声处理在50mM磷酸氢二钾缓冲液,pH7.0中制备1.0mg/ml过氯酸盐氧化的原纤维,用过氧化苯甲酰被PEG修饰的原纤维和过氯酸盐氧化的,通过NHS酯结合被PEG修饰的原纤维的母分散液.在7个聚丙烯管中放入2mis的每种的3倍连续稀释液。向每个管和三个空白缓冲液中加入100μl 0.2mg/ml β-乳球蛋白(βLG)的相同缓冲液溶液.也制备不含蛋白质的三个缓冲液管。所有管都在涡旋混合物上混合,培养60分钟,其中每10分钟涡旋30秒。离心分离所有的管以分离原纤维,将1ml等分的上清液转移到新管中,用微BCA蛋白分析法(Pierce)分析总蛋白含量。在上清液中保留的蛋白质的水平是与原纤维非特异性结合的量的间接测定值(见附图2).对于每个管,经NHS酯途径用PEG修饰的原纤维,βLG都保留在上清液中,表明不存在非特异性结合。经BPO途径用PEG修饰的原纤维在1.0mg/ml的原纤维最高水平时仅显示βLG轻微的结合(约10%),在低水平时则没有显著的结合。相反地,在1.0mg/ml和上述的原纤维水平时几乎与氯酸盐氧化的原纤维完全结合,在原纤维低至0.01mg/ml仍基本上结合。
[00139]该反应可以用任何方法来进行,以便用醇或胺酯化或胺化羧酸。其中,使用H.A.Staab,Anagew.Chem.Intemat.Edit.,(1),351(1962)的方法,用N,N’-羰二咪唑(CDI)作为酰化剂制备酯或酰胺;使用G.W.Anderson,等人,J.Amer.Chem.Soc.86,1839(1964)的方法,用N-羟基琥珀酰亚胺(NHS)活化羧酸以酰胺化。
[00142]三烷基硅烷基氯或三烷基硅醇与酸性的H立即根据如下反应式反应:
[00151]胰蛋白酶可以与底物L-BAPNA(Nα-苯甲酰基-L-精氨酸对硝基苯胺)反应,释放出在410nm吸收光的有色化合物。该反应的分析缓冲液是0.05M Tris,0.02M CaCl2,pH8.2。该反应在1ml试管中进行,包括将5μl的L-BAPNA母液(在H2O中50mM的37%DMSO)和10-25μg的胰蛋白酶原纤维在1ml的分析缓冲液中混合。在10分钟后,在410nm处检测到吸光度增加.然后从开始的斜度计算酶活性(μM/分钟)。
[00155]用声处理将硫醇化纳米管的10mg样品悬浮于10mis.的DI水中,在0.45μm尼龙膜上过滤形成毯样的物质。先将该物质部分贮存在真空干燥器中,然后用ESCA分析,该分析显示有0.46%硫和1.69%氮,证明成功地转变成了硫醇修饰的CN。
[00164]进行ESCA以确定胺化原纤维(GF/NH2)中N的存在量。177-046-1的ESCA分析表明含有0.90 at% N(177-059).为了进一步评价在易受影响和反应性胺基中N的量,制备一种衍生物,包括与五氟苯甲醛气相反应生成相应的与伯胺基有效连接的Schiff碱。ESCA分析仍然表明了,如所期望,含0.91 at%N和1.68 at% F.这可以转换成在胺化原纤维上作为反应性伯胺存在的N是0.34 at%(每个五氟苯甲醛分子含5个F)。假设在每个N的游离末端完全反应的话,N的水平应当是0.45 at%。所观测到的水平表明,N与NHS-活化的原纤维的反应具有非常高的收率,证明了可用的游离胺基的反应性.
[00172]为了证明方法的有效性,用氨基原纤维的小样品与被修饰与氨基特异性反应的”活化的”辣根过氧化物酶(HRP;5mg,Pierce)反应。在几天内反复洗涤原纤维(混悬、旋转、和在Eppendorf微量离心管中离心),同时保持冷却。在洗涤约2周后,用H2O2/ABTS在甘氨酸缓冲液,pH4.4中分析酶.10分钟内溶液显现绿色,表明了酶的存在。对照的原纤维(用活化的HRP处理的COOH原纤维并在同样的时间洗涤)显示,如果有任何的催化活性,也几乎没有酶。
[00176]表VI概述了次级衍生物的制备。通过分析ESCA分析产物。该分析表明掺入了所需的侧基。通过分析ESCA分析产物的C、O、N、Si和F的表面含量。
[00180]将包含60mg 2-乙基氨基乙胺修饰的羧基原纤维和180mg葡聚糖凝胶G-25超精细树脂(Pharmacia,Uppsala,Sweden)的含水浆体在室温下保存过夜,以确保固体载体的完全水合。将浆体装入到1cm x 3.5cm柱中。该柱用5mM磷酸钠缓冲液(pH7.3)以0.2ml/分钟的流速平衡.在柱上负载BSA(0.6mg,在0.1ml去离子水中)。用5mM磷酸钠缓冲液(pH7.3)以0.2ml/分钟的流速洗脱该柱,收集0.6ml的部分。用UV-可见探测器检测洗脱图,如附图3所示。由于该探测器表明没有蛋白质从柱中洗脱,通过加入在5mM磷酸钠(pH7.3)中的1M KCl洗脱结合的BSA。在每个部分中蛋白质的存在可以通过微BCA分析(Pierce,Rockford,II)来鉴别。
[00194]反应顺序如附图5所示.
[00199]根据实施例20的方法制备CN/NHS。
[00205]声处理下将每个四代NTA添加物样品(约10mg)悬浮于10mis的DI水中,在0.45μm尼龙膜上过滤形成毯样的物质。该物质部分贮存于真空干燥器中,通过ESCA分析氮(N)以指示NTA的相对量。结果显示于下表中。
[00227]将0.5mg的苯基-烷基原纤维悬浮于50μl的5niM磷酸钠缓冲液(pH7.1)中,声处理20分钟。向这些原纤维中加入350μl的脂肪酶溶液(0.2mM在5mM磷酸钠缓冲液中,pH7.1),室温下将混合物旋转5小时,在4°C下贮存过夜。然后用600μl的5mM磷酸钠缓冲液(pH7.1)洗涤原纤维3次,并悬浮于200μl的相同缓冲液中。
[00246]向二氯甲烷(20ml)中的氨基原纤维(400mg)和4-(羟甲基)-苯氧基乙酸混悬液(255mg,1.4mmol)的混合物中加入1-乙基-3-(3-二甲氨基丙基)碳二亚胺(EDC,268mg,1.40mmol)和1-羟基苯并三唑水合物(HOBT,189mg,1.4mmol).反应混合物在氮气下在室温下搅拌过夜.用二氯甲烷、甲醇和水广泛洗涤产物原纤维.然后真空干燥得到原纤维。向原纤维的N,N-二甲基酰胺(DMF,2ml)和二氯甲烷(8ml)的混悬液中加入N-(9-fluorenyl甲氧羰基)-O-丁基-L-丝氨酸(215mg,0.56mmol)、1,3-二环己基碳二亚胺(DCC,115mg,0.56mmol)和4二甲基氨基吡啶(DMAP,3.4mg,0.028mmol).反应混合物在室温下搅拌过夜,用20%吡啶的DMF溶液(5 X40ml,每次浸湿1分钟)处理产物原纤维。然后用DMF、水、氢氧化钠(1N)和二氯甲烷广泛洗涤产物原纤维.真空干燥产物Fib-柄-Ser(O+)-COOH(水合茚三酮试验呈阳性).对于二肽的合成,使用相同的方法加入精氨酸.Fib-柄-Ser(O+)-Arg(Nε-2,2,5,7,8-五甲基chrornan-6-磺酰基)的氨基酸分析数据表明,它包含6.5μmol/g原纤维和7.6μmol精氨酸/g原纤维。通过相同方法可以制备任何其他的肽。
[00276]用其他的陶瓷类物质,例如锆、钛、稀土元素氧化物和三氧化物可以类似地制备,也可以制备三氧化物.
[00280]本领域技术人员将会明白,在前述实施例1-55中公开的任何方法都可以用直径小于5纳米的单壁碳纳米管的例行试验来重复,从而获得所需的官能化结果.
[00289]所使用的术语和表达是作为说明书的术语使用,而不是进行限制,而且也不打算使用超出任何作为其一部分所显示和所描述的特征相同范围以外的术语或表达,应当意识到,各种可能的改变都在本发明的范围内。