Software Technology for “Length-based seperation of carbon nanotubes

Nanotechnology – Zahra Borzooeian, Mohammad E. Taslim, Nanolc 12 LLC

Abstract for “Length-based seperation of carbon nanotubes

“Disclosed is a method for separating carbon nanotubes based on a specific parameter, such length. These methods include labelling carbon nanotubes with a biological moiety and then SDS-PAGE and staining to separate and/or characterize the length. Some embodiments use egg-white enzyme, which is conjugated covalently to single-walled carbon-nanotube surfaces using the carbodiimide technique. Next, SDS-PAGE is used and silver staining is used to visualize the single-walled nanotubes. This allows for high resolution characterisation of single-walled length. This simple, fast, precise, and inexpensive separation technique eliminates the need to centrifuge, perform additional chemical analyses, or use expensive spectroscopic techniques like Raman spectroscopy for carbon nanotube band visualization. These methods are useful in quality control in the production of carbon nanotubes with specific lengths.

Background for “Length-based seperation of carbon nanotubes

There are many ways to synthesize carbon nanotubes (CNTs). They come in different sizes, lengths, and shapes. When nanotubes are used in typical applications (e.g. conductive and high strength composites), probes and interconnects [1], sensors[2], energy storage devices and energy conversion devices], hydrogen storage [3-5], and nanotube transistors[6, 7], etc.], the structural parameters can have a significant impact on the properties (e.g. reactivity) carbon nanotubes.

“Any new method that aims to separate and purify nanotubes in an scalable, reproducible and simple way should measure certain morphological parameters (e.g. diameter and length) of CNTs. The length of nanotubes has been shown to correlate positively with electrical and thermal activities. This means that longer multi-wall carbon nanotubes, (MWCNTs), typically have higher thermal conductivities and electrical conductivities. Nanotube length can also impact transistor performance [10], electromagnetic interference shielding and the mechanical properties of CNT-based epoxy compounds [11].

“The length of nanotubes can have a significant impact on the biological response, human health, and the environment. It is becoming more common to recognize that the characterization of nanotubes is an important step towards assessing the potential toxic effects of nanomaterials within biological systems. Cheng [13] demonstrated that the length of carbon nanotubes plays a significant role in in-vivo toxicology of functionalized CNTs in Zebra Fish embryos in his study. A study of single-walled carbon tubes (SWCNTs), cytotoxicity, showed that cells’ cytotoxic response to functionalized CNTs is dependent upon their degree of functionalization [14]. To manage and decrease the toxicity of CNTs, it is crucial to measure length and control the measurement of length to maximize the green chemistry potential.

Accordingly, the length-based separation and measurement of CNTs has received particular attention. The measurement of basic parameters such as the diameter and length CNTs remains a challenge for nanotechnology researchers as well as commercial scale CNT production. There are several methods for size selection and purification. These include ultrafiltration (e.g., toluene and solvent) treatment of nanotubes, floculation in aqueous surfactants (17), oxidation and acid wash coupled with centrifugation [18, 19], chromatographic purification (22, 23), field-flow fractionation [24], size-exclusion analysis on nanotubes suspended in sodium sulfate [25, 26].

“Electrophoretic methods also allow nanomaterials purification [27-29] as well as characterization [30 and 31]. Separation based on parameters like size, shape and length [27, 31,] is possible. Electrophoresis techniques have been shown to be effective in separating CNTS by length-, diameter-, or curvature-based methods. These include AC electrophoresis with isopropyl alcohol [29], capillary electrodephoresis for SDS-coated SWCNTs (27], and agarose electrophoresis [32] to fractionate SWCNT/nucleic acids complexes. Capillary electrophoresis was also used to determine the complexity of DNA-suspended WCNTs by streptavidin/biotin bind [33]. Some efforts have been made to separate CNTs by length using a gel permeation column and an inhomogeneous magnet field.

Nanotubes can be made in many lengths, diameters, or structures. Because some properties of nanotubes are dependent upon their structure, much of the research has been focused on measuring these parameters accurately. Nanotube science can benefit from length-based separation. Accurate measurement of the length of nanotubes is crucial for understanding how they grow and cut in CNTs synthesis [39]. Franklin and his collaborators provided the first experimental evidence on the effects of contact length on nanotube transistors. They created sets of devices with different lengths of nanotubes [10]. The length-based separation (or length-based) of CNTs is a crucial step in enabling their use in biologically relevant settings, such as drug delivery.

“There have been over 200 papers published in the literature on the subject of the separation of SWNTs. They are classified according to their conductivity, diameter and handedness. Several chromatographic methods have been reported for length-based separation of CNT’s by Duesberg [25, 44, 45] using size-exclusion chromatography (SEC), Rinzler [46] using high performance liquid chromatography (HPLC) and Fotios Papadimitrakopolous’s group[47] using gel permeation chromatography. It is not possible to predict when a carbon nanotube will leave the chromatography column. This is due to the stochastic nature particle-pore interactions. The ability to extract carbon nanotubes with specific lengths from chromatographic separation processes is not yet known.

“Besides centrifugation[48], the length-based separations of carbon nanotubes based on their electrical properties is more often performed using capillary electrodephoresis (CE) or agarose gel electrophoresis[31?32]. There are no electrophoretic methods that can accurately and quickly separate CNTs along their length.

“The above methods, which are not precise and scaleable, were performed using the UV/vis, AFM and Raman Spectroscopy. These techniques can be complicated and costly. There are no pre-electrophoretic methods that can be used to quickly, cost-effectively, and precisely separate CNTs from their lengths. Therefore, there is a need for CNT characterization and length-based separation that is precise, quick, simple, and economical.

“Another aspect of the disclosure is methods for determining average lengths of labelled Carbon Nanotubes. These include: labelling a plurality carbon tubes with a biological moiety to produce labelled nanotubes; electrophoresis of the labelled nanotubes with an electrophoresis agent to create a gel; staining the electrophoresis Gel containing the labelled nanotubes with visualizing agents to provide stained, clearly labelled nanotubes; and measuring the intensity of the distance traveled in the gel.

The present disclosure is based on methods of separating carbon Nanotubes. This includes labelling a plurality carbon-nanotubes with a biological moiety to produce labelled nanotubes; using gel electrophoresis to treat the labelled nanotubes with an agent that can be used to visualize the target parameter to remove stained carbon nanotubes and then isolating them from the gel. The method may also include measuring the average length of stained, labelled carbon-nanotubes using UV/vis spectroscopy or Raman Spectroscopy.

“Methods to Separate Carbon Nanotubes”

The present disclosure describes methods to separate carbon nanotubes in certain aspects. These methods include labelling each carbon nanotube with a biological moiety to produce labelled nanotubes. Gel electrophoresis is used to create an electrophoresis solution that contains the labelled nanotubes. Then, the electrophoresis mix is removed from which are stained, or ranged, target parameters. Finally, the separated carbon nanotubes are extracted from the gel. The method may also include measuring the average length of stained, labelled carbon-nanotubes using UV/vis spectroscopy or Raman Spectroscopy in certain embodiments.

“In some embodiments the nanotubes may be commercially prepared or laboratory-prepared. The nanotubes could be carbon nanotubes, for example. In some cases, however, the methods described here can also be applied to nanoparticles and nanotubes. Some carbon nanotubes have a single-walled carbon nanotube. Other embodiments of the carbon nanotubes include double-walled carbon Nanotubes. Other embodiments of the carbon nanotubes include multi-walled carbon Nanotubes (e.g. three-walled or quadruple-walled; quintuple-walled). “Carbon nanotubes” is used hereinafter, except where otherwise noted. It can refer to any combination of single-walled, doublewalled carbon nanotubes or multi-walled nanotubes.

“In some embodiments of the current methods, the carbon nanotubes that are to be separated can be labelled with a biological moiety. In some cases, carbon nanotubes may be labeled with a protein. Any soluble, structural, linear, or globular protein that is suitable for the present methods can be used as a protein. Examples of readily available proteins are those that have been isolated from milk and other biological systems. You can also prepare or isolate the protein from any source. The protein may be an enzyme, a domain of an enzyme, a bioactive protein peptide, an anti-hormone or a hormone in certain embodiments. The protein may be any soluble or structural, linear, or globular protein in some embodiments.

“In some embodiments the enzyme is an oxygenoreductase. Other embodiments of the enzyme are a transferase. The enzyme can also be a hydrolase in certain embodiments. The enzyme may also be called an isomerase in some embodiments. The enzyme can also be a ligase in certain embodiments. The enzyme can also be a lyase in some embodiments. The enzyme may be an egg white lysozyme in some embodiments. Other embodiments allow the biological moiety to be a peptide, peptide fragment or enzyme. Any peptide fragment that contains 2-10, 2-100 or more amino acids can be considered suitable. You can choose to use natural, unnatural amino acids, or derivatives of them. The biological label may include chicken egg white Lysozyme in some instances.

“In some embodiments, the antimicrobial peptide may be a Tachykinin or vasoactive intestinal protein, a Tachykinin or vasoactive intestinal polypeptide-related, an opioid peptide or any other peptide such a B-type Natriuretic Peptide (BNP), or a lactotripeptides or a traditional Chinese medicine Colla Coni Asini component in hematopoiesis. The carbon nanotubes may be labeled with a protein domain in some embodiments. The carbon nanotubes may be labeled with a bioactive protein in some cases. The protein can be an antibody in some embodiments. The protein may also be a hormone in some embodiments. You can use hormones that are found in multicellular organisms (plants and animals, fungi brown algae, red algae, and fungi) or unicellular organisms.

The present methods may include labelling carbon nanotubes with a biological moiety. To label carbon nanotubes, it is possible to react the carbon nanotubes with the biological moiety using a linking agent. The linking reagent may react with functional groups on carbon nanotubes’ surfaces in some instances. Other embodiments allow the linking agent to react with functional moiety functional groups. Proteins, enzymes, and peptides are typically composed of amino acid residues that can include functional groups such as alcohols or thioethers. Carboxamides, amines. amides. thiols. In some cases, the linking agent reacts with the functional group of the biological moiety. Some embodiments use a peptide-forming agent as the linking reagent.

“In some embodiments, the linking agent covalently links carbon nanotubes with the biological moiety. Other embodiments allow the carbon nanotubes and biological moiety to be covalently linked. Combining a biological moiety and an affinity for carbon nanotubes can result in non-covalent linking. Hydrogen bonding interactions can be used to link the carbon nanotubes’ surfaces with the biological moiety. Other embodiments of non-covalent linking are the result Van der Waals interactions between carbon nanotubes & the biological moiety. The non-covalent linking in certain embodiments is caused by hydrogen bonding interactions or electrostatic interactions. interactions or hydrophobic effects.

“In some embodiments, the linking agent can be a peptide-coupling agent. The linking agent in some embodiments is a carbodiimide-reagent. These carbodiimide agents can form amide bonds between the biological moiety of the carbon nanotubes. In some embodiments, the carbodiimide reagent is N-ethyl-N?-(3-(dimethylamino)propyl)carbodiimide. In other embodiments, the linking reagent can be dicyclohexylcarbodiimide. In certain embodiments, the linking reagent can be diisopropylcarbodiimide. Any peptide coupling agent that links the carbon nanotubes and the biological moiety is possible. In some embodiments, the linking agent is a carbodiimide reagent, a polymer phospholipid-polyethylene glycol (PL-PEG-NH2), a polystyrene-block-polyacrylic acid (PS-b-PAA), an imidazolium salt based ionic liquid (IS-IL), or an ethylenediamine. The present methods also allow for the separation of labelled carbon nanotubes. In some cases, the labelled carbon nanotubes can be loaded into gels and separated by electrophoresis. Gel electrophoresis, which is a technique used to separate macromolecules like DNA, RNA and proteins, is well-known in the art. The present methods use the advantages of biological moieties and the high resolution power of gel electrophoresis to separate the labelled carbon-nucleotides. Electrophoresis may include sodium dodecyl-sulfate polyacrylamide electrophoresis, (SDS?PAGE) in some embodiments. Other embodiments use agarose gel. The gel can also be a starch gel in certain embodiments. In some embodiments, electrophoresis takes place under denaturing conditions. Other embodiments allow for electrophoresis to be conducted under non-denaturing conditions. The electrophoresis is performed at a constant electrical field in certain embodiments. Other embodiments allow for the use of a pulsed, gradient, or inverted field to run the electrophoresis procedure.

After the electrophoresis gel has separated the carbon nanotubes, the gel can be stained to produce stained carbon nanotubes. This staining shows the bands and separated populations of carbon nanotubes within the gel. You can use any staining agent to visualize the labelled carbon-nanotubes. To satinize the nanotubes, you can use ethidium bromide in certain embodiments. Other embodiments use coomassie to make stained carbon nanotubes. In some embodiments, the visualizing agents are silver nitrate and silver-ammonia complex solutions. The gel containing the separated and labelled carbon nanotubes can be stained by contact with the staining agent.

“The present methods can also be used to effect separation by determining a target parameter value, or range of the carbon nanotubes. The target parameter could be the length, shape, diameter, number of walls or any combination thereof. In some embodiments, the carbon nanotubes labelled are separated by their length. Other embodiments allow the labelled carbon nanotubes to be separated by their shape. The visual intensity of the stained, labeled carbon nanotubes can also be used to determine the target parameter’s value or range. This can be done in one or more sections of the electrophoresis Gel. In some embodiments, the parameter can be length. To determine this, you will need to calculate the length of the stained, labeled carbon nanotubes using the following formula:

“L = d? ? exp [ 3 ? ? ? ? ? ? ? ? q ? ( d) e – 2 ? ? ? 2 + 1]nwhere L is the length calculated, d is the mean diameter, and? is the optimum concentration. is the solvent viscosity. q(d), is persistence length. e is electron charge. The electrophoretic mobility is calculated using Usrey’s equation, Usrey and al., 2007, [34]. The formula below can be used to determine the relationship between the length of the carbon nanotubes and the visual intensity of the stain-labeled populations in gel.

“Some embodiments of the carbon nanotubes that can be separated using the present methods have a length between 10 nm and about 1 cm. In some embodiments, carbon nanotubes can have lengths of approximately 10 nm to 10 m, about one m to around 100 m, about one m to about one mm, 100 m to roughly 1 mm, 100 m to approximately 1 cm or about one mm to about a cm or any other value. The resolution of separation achieved by the present methods may be approximately 1 nm to about 10 nm or about 100 nm or about any other value or range thereof.

“In some embodiments, carbon nanotubes that are separated using the present methods have a diameter of approximately 0.5 nm to 100 nm. In certain embodiments, for example, the diameter of carbon nanotubes can be as low as 0.5 nm in some cases, and as high at about 10 nm in others.

“In certain embodiments, carbon nanotubes can be surface functionalized carbon nanotubes. Any surface functionalization compatible to the present methods may be used. Some embodiments of the carbon nanotubes’ surface are functionalized with a carboxylic acids moiety. Some embodiments use a carboxylate salt as the carboxylic acid. Any cation that does not interfere in the electrophoresis process can be used as a suitable counterion. In some embodiments, the counterion could be an alkali metallic. The alkali metal in some embodiments can be either lithium, sodium, or potassium. Other embodiments use surface functionalized carbon nanotubes that are amine,amide, or thiol (??NH2SH). (CO)NH2, or?CONHCH2CH2SH functionalized carbon nanotubes. You can either have the amine as a base or an amine salt. Any anion that does not interfere in the electrophoresis can be used as a suitable counterion. In some embodiments, the counterion may be a halogen-ion. The halogen ion in certain embodiments can be F, Cl or Br. Other embodiments use carbon nanotubes that are? (CO)NH2 and?CONHCH2CH2SH are functionalized carbon nanotubes.

“Another aspect of the present methods is gel electrophoresis, which separates the labelled nanotubes into groups. Some embodiments of the labelled carbon-nanotubes found in groups of labelled nanotubes have a substantially identical length. The length of the labelled nanotubes can vary by as much as 10% from the average length of the group. Other embodiments allow the length of the carbon nanotubes labeled in the group to vary by +/-10% or +//8% or +//6% or +//1% or +//0.01% of the average length for the group of labelled nanotubes or any other value.

“Some embodiments of the present methods include measuring the average lengths of the carbon nanotubes that have been isolated, stained and labelled. In some embodiments, the average length can also be measured using one or more of UV, atomic force microscopy, (AFM), or Raman spectroscopy.

“Methods to Determine the Length Carbon Nanotubes”

“Another aspect of the disclosure is that it provides a method of determining the average length a sample labelled Carbon nanotubes. This involves: labelling each carbon nanotube with a biological moiety to produce labelled nanotubes; gel electrophoresis to create an electrophoresis Gel comprising the carbon nanotubes labelled with a visualizing agents to provide stained carbon tubes; and measuring the intensity of the stained carbon nanotubes at a plurality locations in the gel. The average length of their distance traveled in the gel.

“In some embodiments the nanotubes may be commercially prepared or laboratory-prepared. The nanotubes could be carbon nanotubes, for example. In some cases, however, the methods described here can also be applied to nanoparticles and nanotubes. Some carbon nanotubes have a single-walled carbon nanotube. Other embodiments of the carbon nanotubes include double-walled carbon Nanotubes. Other embodiments of the carbon nanotubes include multi-walled carbon Nanotubes (e.g. three-walled or quadruple-walled; quintuple-walled). “Carbon nanotubes” is used hereinafter, except where otherwise noted. It can refer to any combination of single-walled, doublewalled carbon nanotubes or multi-walled nanotubes.

“In some embodiments of the current methods, the carbon nanotubes that are to be separated can be labelled with a biological moiety. In some cases, carbon nanotubes may be labeled with a protein. Any soluble, structural, linear, or globular protein that is suitable for the present methods can be used as a protein. Examples of readily available proteins are those that have been isolated from milk and other biological systems. You can also prepare or isolate the protein from any source. The protein may be an enzyme, a domain of an enzyme, a bioactive protein peptide, an anti-hormone or a hormone in certain embodiments. The protein may be any soluble or structural, linear, or globular protein in some embodiments.

“In some embodiments the enzyme is an oxygenoreductase. Other embodiments of the enzyme are a transferase. The enzyme can also be a hydrolase in certain embodiments. The enzyme may also be called an isomerase in some embodiments. The enzyme can also be a ligase in certain embodiments. The enzyme can also be a lyase in some embodiments. The enzyme may be an egg white lysozyme in some embodiments. Other embodiments allow the biological moiety to be a peptide, peptide fragment or enzyme. Any peptide fragment that contains 2-10, 2-100 or more amino acids can be considered suitable. You can choose from natural, unnatural or derivative amino acids for your amino acid residues. The biological label may include chicken egg white Lysozyme in some instances.

“In some embodiments, the antimicrobial peptide may be a Tachykinin or vasoactive intestinal protein, a Tachykinin or vasoactive intestinal polypeptide-related, an opioid peptide or any other peptides such as a B type natriuretic (BNP), lactotripeptides or a traditional Chinese medicine Colla Corii Asini component in hematopoiesis. The carbon nanotubes may be labeled with a protein domain in some embodiments. The carbon nanotubes may be labeled with a bioactive protein in some cases. The protein can be an antibody in some embodiments. The protein may also be a hormone in some embodiments. You can use hormones that are found in multicellular organisms (plants and animals, fungi brown algae, red algae, and fungi) or in unicellular organisms.

The present methods may include labelling carbon nanotubes with a biological moiety. To label carbon nanotubes, it is possible to react the carbon nanotubes with the biological moiety using a linking agent. The linking reagent may react with functional groups on carbon nanotubes’ surfaces in some instances. Other embodiments allow the linking agent to react with functional moiety functional groups. Proteins, enzymes, and peptides are typically composed of amino acid residues that can include functional groups such as alcohols or thioethers. Carboxamides, amines. amides. thiols. In some cases, the linking agent reacts with the functional group of the biological moiety. Some embodiments use a peptide-forming agent as the linking reagent.

“In some embodiments, the linking agent covalently links carbon nanotubes with the biological moiety. Other embodiments allow the carbon nanotubes and biological moiety to be covalently linked. Combining a biological moiety and an affinity for carbon nanotubes can result in non-covalent linking. Hydrogen bonding interactions can be used to link the carbon nanotubes’ surfaces with the biological moiety. Other embodiments of non-covalent linking are the result Van der Waals interactions between carbon nanotubes & the biological moiety. The non-covalent linking in certain embodiments is caused by hydrogen bonding interactions or electrostatic interactions. interactions or hydrophobic effects.

“In some embodiments, the linking agent can be a peptide-coupling agent. The linking agent in some embodiments is a carbodiimide-reagent. These carbodiimide agents can form amide bonds between the biological moiety of the carbon nanotubes. In some embodiments, the carbodiimide reagent is N-ethyl-N?-(3-(dimethylamino)propyl)carbodiimide. In other embodiments, the linking reagent can be dicyclohexylcarbodiimide. In certain embodiments, the linking reagent can be diisopropylcarbodiimide. Any peptide coupling agent can link the carbon nanotubes and the biological moiety. In some embodiments, the linking agent is a carbodiimide reagent, a polymer phospholipid-polyethylene glycol (PL-PEG-NH2), a polystyrene-block-polyacrylic acid (PS-b-PAA), an imidazolium salt based ionic liquid (IS-IL), or an ethylenediamine.”

“In another aspect of the invention, the methods allow for the separation of labelled carbon nanotubes in order to determine their length. In some cases, the labelled carbon nanotubes can be loaded into gels and separated by electrophoresis. Gel electrophoresis, which is a technique used to separate macromolecules like DNA, RNA and proteins, is well-known in the art. The present methods use the advantages of biological moieties and the high resolution power of gel electrophoresis to separate the labelled carbon-nucleotides. Electrophoresis may include sodium dodecyl-sulfate polyacrylamide electrophoresis, (SDS?PAGE) in some embodiments. Other embodiments use agarose gel. The gel can also be a starch gel in certain embodiments. In some embodiments, electrophoresis takes place under denaturing conditions. Other embodiments allow for electrophoresis to be conducted under non-denaturing conditions. The electrophoresis is performed at a constant electrical field in certain embodiments. Other embodiments allow for the use of a pulsed, gradient, or inverted field to run the electrophoresis procedure.

After the labelled carbon-nanotubes have been separated from the electrophoresis gel they can be stained to produce stained, labelled carbon-nanotubes. This staining shows the bands and separated populations of carbon nanotubes within the gel. You can use any staining agent which visualizes the labelled nanotubes. To stain the carbon nanotubes labelled with ethidium bromide, one example is coomassie blue. To provide stained carbon nanotubes that are labelled, you can use coomassie bleu. The visualizing agent may contain silver nitrate, silver-ammonia complex solutions or both. You can stain the gel by contacting it with the staining agent.

“In another aspect, the methods described can be used to separate labeled carbon-nanotubes in order to determine their length. Separation to determine other parameters is possible, however. The target parameter could be length, shape or diameter of the carbon nanotubes, or even the number of walls. In some embodiments, the carbon nanotubes labelled are separated by their length. Other embodiments allow the labelled carbon nanotubes to be separated by their shape. The visual intensity of the stained, labeled carbon nanotubes can also be used to determine the target parameter’s value or range. This can be done in one or more sections of the electrophoresis Gel. In some embodiments, the parameter can be length. To determine this, you will need to calculate the length of the stained, labeled carbon nanotubes using the following formula:

“L = d? ? exp [ 3 ? ? ? ? ? ? ? ? q ? ( d) e – 2 ? ? ? 2 + 1]nL is the calculated length, and d is the mean diameter. 2 + 1 ]nwhere L is the calculated length, d is the mean diameter, and e is an electron charge. The electrophoretic mobility. The formula below can be used to determine the relationship between the visual intensity and the length of the carbon nanotubes stained and labelled in gel.

“Some embodiments of the carbon nanotubes that can be separated using the present methods have a length between 10 nm and about 1 cm. In some embodiments, carbon nanotubes can have lengths of approximately 10 nm to 10 m, about one m to around 100 m, about one m to about one mm, 100 m to roughly 1 mm, 100 m to approximately 1 cm or about one mm to about a cm or any other value. The resolution of separation achieved by the present methods may be approximately 1 nm to about 10 nm or about 100 nm or about any other value or range thereof.

“In some embodiments, carbon nanotubes that are separated using the present methods have a diameter of approximately 0.5 nm to 100 nm. In certain embodiments, for example, the diameter of carbon nanotubes can be as low as 0.5 nm in some cases, and as high at about 10 nm in others.

“In certain embodiments, carbon nanotubes can be surface functionalized carbon nanotubes. Any surface functionalization compatible to the present methods may be used. Some embodiments of the carbon nanotubes’ surface are functionalized with a carboxylic acids moiety. Some embodiments use a carboxylate salt as the carboxylic acid. Any cation that does not interfere in the electrophoresis process can be used as a suitable counterion. In some embodiments, the counterion could be an alkali metallic. The alkali metal in some embodiments can be either lithium, sodium, or potassium. Other embodiments use surface functionalized carbon nanotubes that are amine,amide, or thiol (??NH2SH). (CO)NH2, or?CONHCH2CH2SH functionalized carbon nanotubes. You can either have the amine as a base or an amine salt. Any anion that does not interfere in the electrophoresis can be used as a suitable counterion. In some embodiments, the counterion may be a halogen-ion. The halogen ion in certain embodiments can be F, Cl or Br. Other embodiments use carbon nanotubes that are? (CO)NH2 and?CONHCH2CH2SH are functionalized carbon nanotubes.

“Another aspect of the present methods is gel electrophoresis, which separates the labelled nanotubes into groups. Some embodiments of the labelled carbon-nanotubes found in groups of labelled nanotubes have a substantially identical length. The length of the nanotubes labeled in the group may vary by +/-10% from the average length of the group. Other embodiments allow the length of nanotubes to be labeled in the group to vary by +/-10% or +//8% or +//6% or +//1% or +//0.01% of the average length for the group of carbon nanotubes or any other value.

“Results of XRD patterns (FIGS. 1A, 1B, and 1C), FTIR(FIG. 2), and SEM micrographs. 3A, 3B, and 3C show that a biological moiety, such as lysozyme, can be disposed onto the SWCNTs surfaces during conjugation. In some embodiments, the biological moiety can be linked to about 1% of the surface functional groups of carbon nanotubes. The biological moiety can be linked to at most about 2%, 3%, 4%, 5%, 5%, 6%, 5%, 5%, 6%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 45%, 65%, 65%, at minimum about 70%, a least about 75% and 80% respectively.

“In embodiments in which the biological moiety has been covalently linked to carbon nanotubes’ surface, the presence covalently attached proteins may result in an intrinsic positive charge and a functionalization charge. These together make up the net charge of any particular nanotube or bundle of nanotubes in solution. High precision separation is possible because the length of carbon nanotubes (e.g. SWCNT) determines the amount of conjugated biological moiety. The net charge of carbon nanotubes is related to the amount linked biological moiety (e.g. lysozyme). Different lengths of carbon nanotubes (e.g. SWCNT) have different mobility in an electrophoretic field. According to the following formula, the mobility and velocity of charged CNT pieces depend on their electrical field (E, Volts/cm) as well as the net charge (q) on them. However, the friction between the molecules is inversely affected by the latter.

“V = Eq. fnwhere: f=frictional coefficient of the mass and form of the fragment, and V=velocity/mobility fragment [38]. CNTs smaller than the larger ones can pass through gel with greater mobility. The gel acts as a sieve, allowing smaller nanotubes to pass through while keeping larger ones in place. The friction coefficient refers to the ease with which a carbon nanotube fragment passes through the gel pores. The main determinant of SWCNT mobility in a gel matrix will be their length. Substituting length for the frictional coefficient results in: mobility=(voltage)(charge)/(length). This means that the charge/length ratio is the primary determinant of the mobility of a carbon-natube during gel electrophoresis.

“The length distribution for conjugated SWCNTs is represented by the distributions of the calculated lengths from Usrey?s equation versus visual intensity of bands of the stained, labeled carbon nanotubes (FIG. 7). You can view the intensity as a measure for the number of CNTs that have a particular length on the gel. The higher the visual intensity, then the more CNTs that have that length are in that group. FIG. FIG. 7. The main distribution of lengths for CNTs is between 38-50 microns. Another peak is at 60 microns, which corresponds with CNTs that were in or near the loading well.

“Methods to Adjust Carbon Nanotube Parameters in Manufacturing Processes.”

“Another aspect of the disclosure is that it provides methods for adjusting lengths of carbon-nanotubes made in a carbon tube manufacturing process. This includes: taking a sample of carbon, labelling each carbon, and then exposing the gel containing the labelled nanotubes to gel electrophoresis. The gel containing the labelled nanotubes can be treated with a visualizing agent, which will produce stained, labelled nanotubes.

The art reveals that there are many carbon nanotube manufacturing methods for commercial or laboratory scale production. In some embodiments, carbon nanotube manufacturing can include arc discharge, laser, chemical vapor deposition, beam wavelength, voltage, and carbon monoxide disproportionation at high pressure (HiPCO). One or more parameters are selected from the following group: temperature, pressure and raw material feed rate.

CNT-based nanobiotechnology and nanomedicine have one problem: the lack of uniform length in mass production of CNTs. It is crucial to distinguish CNTs according to their conductivity and length in these applications.

The present methods are a valuable tool to control the quality of carbon nanotubes manufactured in large quantities. In some embodiments, the disclosure offers a method to adjust parameters and control quality in the production carbon nanotubes. The method may include the collection of a sample from a population of carbon nanotubes using a nanotube manufacturing process and the separation of the carbon tubes, as described in this document. Other embodiments include measuring the length of a sample of a carbon-nanotube population using a nanotube manufacturing method and, where necessary, changing one or more parameters.

“For the purposes of this invention, the chemical element are identified according to the Periodic Table of the Elements CAS version Handbook of Chemistry and Physics, the 67th edition, 1986-87 inside cover.”

“One of ordinary skill in relevant arts will understand that there are other modifications and adaptations possible to the compositions or methods described herein. This is in view of information available to the ordinarily skilled artist and it can be done without departing from any embodiment of the invention.”

“EXAMPLES”

“Having described the invention in detail, it will become clearer by referring to the following examples. These are provided for illustration purposes only and are not meant to limit the invention.”

“Materials & Methods”

“Lyophilized chicken eggs white lysozyme, EC 3.2.1.17 was purchased from Inovatech, Inc., Abbotsford, BC, Canada and Micrococcus lysodeikticus cell, Sigma-Aldrich Corporation, St. Louis, Mo. They were used as dry powder and salt-free. MKnano, Canada purchased carboxyl single-walled carbon Nanotubes (SWCNT?COOH) with an outer diameter of 1-2 nm. MES [2-(N-morpholino) ethane sulfonic acid] buffer, N-ethyl-N?-(3-(dimethyl amino) propyl) carbodiimide hydrochloride (EDC), Tris-hydroxymethyl aminomethane (Tris), N,N methylenebisacrylamide (Bis), acrylamide, sodium dodecyl sulfate, ammonium persulfate, tetramethylethylenediamine (TEMED), 2-mercaptoethanol(2ME), 3,3-5,5 tetrabromophenolsulfonphthalein (Bromophenol Blue) and all other chemicals were purchased from Sigma-Aldrich Corporation and used as received.”

“Example 1?” Enzyme Attachment to SWCNTs

“Details on chemically bonding Lysozyme (to SWCNTs) are reported in literature [41]. A carbodiimide was used to conjugate lysozyme onto SWCNTs [42]. The activated SWCNTs in MES buffer were 50 mM, pH 6.2 (1 mg/mL), and then added to an equal volume 400 mM N.hydroxysuccinimide in MES buffer. For coupling of NHS to the carboxylic groups on the surface of nanotubes, 20 mM N-ethyl-N?-(3-(dimethylamino) propyl) carbodiimide hydrochloride (EDC) was added to the mixture. After stirring the mixture at 200 rpm for 30 minutes, it was subjected to sonication (MSE Ultrasonic disintegrators, 150W, England) for approximately 30 minutes. The mixture was centrifuged. To remove excess EDC or NHS, the centrifuge steps were repeated 3 times. The enzyme solution (10 mg/ml and 10 mMphosphate buffer, pH 8) was added to the nanotubes after they had been rinsed. It was sonicated for approximately. It took approximately 1 minute to re-disperse SWCNTs. During the conjugation process, the mixture was stirred in an orbital shaker at 200 RPM at room temperature. To remove any non-specifically adsorbed enzyme, the conjugated lysozyme/SWCNTs solution was centrifuged. The same process was used to prepare control enzyme-nanotube conjugates, but without NHS or EDC.

“Example 2?Conjugated Lysozyme-SWCNTs Characterization”

“The morphology and activity of conjugated lysozyme SWCNTs was compared to activated SWCNTs using time-scanning electron microscopy (SEM), D8, Advance, Bruker) at?=0.1542 nm and FTIR spectroscopy(Shimadzu FTIR-8300 spectrophotometer). These methods were used for characterizing conjugated lysozyme SWCNTs. Three time periods were used to sonicate conjugated lysozyme SWCNT samples: 3, 7, and 10, respectively.

“Example 3?” SDS-PAGE Electrophoresis, Silver Staining

“Acrylamide (29.2g) and Bis (8.8g) were mixed in 100 ml of water, and then filtered to make gel stock solution (30% m/v). Mixing 10.0ml gel stock solution with 10.0ml TrisHCl (1.5 mole L-1, pH 8.80), 200 -800?l NH4)2S2O8 (10%, m/v) along with 0.4g SDS was enough to prepare the separating gel solution. Finally, water was added to dilute to 40mL. 1.33 ml gel stock solution was added to 50 ml (NH4)2S2O8 (10% m/v) and 2.5 ml TrisHCl (6.80 mol L-1), pH 6.80. The gel stock solution was then mixed with 10.0 ml. The mixtures were then diluted with water to 10.0 mL, and finally 10 mL of TEMED was added. To remove any enzyme that may be physically attached, the samples were washed with phosphate buffer (10mM, pH 8), several times before electrophoresis. To prepare the electrophoresis buffer, Tris (15.14g), Glycine (72.5 g) and SDS (5g) were dissolved in 500 ml of distilled water. The pH of the solution was adjusted to 8.30. The vertical polyacrylamide gel system used consisted of stacking and separating (0.0%, m/v), gels. The sample loading volume was 15.?L. The gels were stained using Coomassie Brilliant Blue R-250. Blum method was used to stain gels with silver [43]. This involves fixing the gels with methanol, paraformaldehyde, and acetic acids, as well as washing them with ddH2O (50% and 30%), sensitizing with Na2S2O3.5H2O and ddH2O, washing them with ddH2O and impregnating with silver nanorate and paraformaldehyde, washing them with ddH2O and developing with Na2CO3, Na2S2O3.5H2O3.5H2O3.5H2O solutions and Na2O3.5H2O3.5H2O3.5H2O5H2O3.5H2O3.5H2O solutions, ending with acetic acid 12% and stopping the solution-methanol at acetic acid at acetic acid acetic acid acetic acid acetic acid acetic acid acetic acid acetic acid 12% and acetic acid acetic acid acetic acid acetic acid acetic acid 12% and acetic acid acetickick acid acetic acid acetic acid acetic and acetic 12%

“Example 4:Characterization Conjugated SWNTs”

Carbodiimide was used to bio-conjugate lysozyme onto the nanotube surface. Analyzing the interactions between free and lysozyme SWCNTs was done using SEM,XRD andFTIR. FIGS. 1A, 1B and 1C show the XRD patterns. FIGS. 1A, 1B, and 1C showed that enzyme attachment was confirmed to the surface of the oxidized SWCNTs. The strong peaks in the SWCNTs correspond with the (002) and (100) carbon planes. The characteristic peak locations for conjugated lysozyme are 20 of, 14.0 and 30.0 respectively. These peaks are identical to those for the free lysozyme pattern XRD. The results revealed that there was no significant difference between conjugated and free lysozymes in XRD patterns. This could indicate either the adsorption of lysozyme onto SWCNTs or the absorption. These results show that the conjugation lysozyme and SWCNTs doesn’t cause a phase shift in lysozyme. This indicates that the lysozyme was not structurally denaturated and maintained its activity throughout the conjugation.

“Example 5:Characterization and Conjugation Of Lysozyme With SWCNTs By FT-ER”

“FTIR analysis has revealed the mechanism of SWCNTs to lysozyme conjugation. FTIR fingerprint is a result of amide links between amino acids residues in proteins and polypeptides. The positions of the amide types I and II bands in FTIR spectra are indicators of conformational changes within the protein secondary structure. They have been used in studies to examine immobilized enzyme molecules. FIGS. 2A, 2B and 3C show the FTIR spectra of free lysozyme as well as SWCNTs (and conjugated lysozyme?SWCNT) respectively. 2A, 2B, and 2C. SWCNT activation was demonstrated by the formation of?COOH functional group in the SWCNT matrix, according to the absorption peak positions at 1627.8 cm/1 and 3440.8 cm/1. A strong and broad NH3 stretching band is a characteristic of amino acids in the 2950-2600cm?1 region of the lysozyme spectrum. The absorption is increased to approximately 2000 cm?1 by the addition of an overtone region. These overtone bands often contain a prominent band between 2222 and 2000 cm?1 that is due to a combination asymmetrical vibrations of NH3+ bending vibrations and torsional oscillations of the NH3+ group[37]. A weak asymmetrical NH3+ bending band can be seen near 1661 cm?1 while a strong symmetrical bending band can be found near 1529 cm?1. The stretching of N?H groups is represented by a peak at 3600 cm’1. A peak at 1230cm?1 represents the stretching C?N group within amine groups. These peaks are seen in the lysozyme SWCNTs spectrum as an indicator of amide bonds between amine groups and activated SWCNTs carboxyl groups. The absorption peak at 1650cm?1 is indicative of C?O’s stretching vibration mode. Peaks at 3800cm?1 and 1650cm?1 represent the stretching of N?H group in the amide. The FTIR spectrum of lysozyme SWCNTs confirmed that amide bonds were formed between the amine groups and the carboxyl groups activated SWCNTs.

“Example 6: Characterization of Conjugation Of Lysozyme With SWCNTs By SEM”

“FIGS. “FIGS. An indication of conjugation is a thicker sidewall of conjugated lysozyme SWCNTs, which can be between 89.5 and 95 nm.

“Example 7:Length-Dependent Separation of Bio-Conjugated SWCTs Using SDS?PAGE and Visualization With Silver Staining”

“Conjugation of Lysozyme with CNTs is of particular interest because of its ability to disperse CNTs. According to molecular modeling [38] and experiments, lysozyme can sort nanotubes by their diameter. This example shows how lysozyme was conjugated onto carboxyl functionalized carbon-nanotubes to create carbon nanotubes that are length specific. Silver staining was used to enable selective visualization of nanotube fragments within the acrylamide gel, which is a problem for many nanotech researchers. Silver staining was able to show high resolution CNTs length-based seperation of lysozyme SWCNT fragments, in contrast to coomassie Blue staining. FIG. FIG. 4B shows silver staining for free lysozyme (lane 1, conjugated lysozyme SWCNTs, and SWCNTs(lane 3).

“The coomassie dyes R-250 and G-252 are anionic dyes that stoichiometrically bond to proteins through ionic interaction between positive protein amine group and dye sulfonic acids groups, as well as Van der Waals attraction.”

“Coomassie Blue staining didn’t show the lysozyme SWCNTs due to amide bond formation among primary amines protein and carboxyl group of SWCNTs. Therefore, there would be no binding between coomassie and lysozyme. Silver staining revealed sharp bands. Sharp bands could be due to the stability and proportion of lysozyme molecules, based on nanotube lengths, and silver staining sensitivity towards proteins. Different lengths of conjugated lysozyme SWCNT fragments had different mobilities.

“Example 8″Length Measurements of Lysozyme SWCNT Fragments Using Image Analysis Techniques

ImageJ was used for calculating the color intensity of a narrow rectangle on each lane, from the well to its bottom. Similar results were obtained (see FIG. 6), which are consistent with the visual evaluations. Following analysis of gel images, experimental data was obtained in the form mobility distribution (numbers of nanotubes per unit of mobility). FIG. 6. shows that SWCNTs of different lengths are present in the population for each experimental electrophoretic mobility value. FIG. 6. shows that SWCNTs of different lengths are found in the population for each experimental electrophoretic mobile value. The color intensity was used to retrieve the image database.

The present disclosure uses bio-conjugation and SDS-PAGE in combination with staining to seperate CNTs. This allows for measurement of a parameter (e.g. length) using electrophoretic mobility values in an acrylamide gel. These methods are useful for process validation and quality control in nanotube manufacturing processes.

“REFERENCES”

“”

Summary for “Length-based seperation of carbon nanotubes

There are many ways to synthesize carbon nanotubes (CNTs). They come in different sizes, lengths, and shapes. When nanotubes are used in typical applications (e.g. conductive and high strength composites), probes and interconnects [1], sensors[2], energy storage devices and energy conversion devices], hydrogen storage [3-5], and nanotube transistors[6, 7], etc.], the structural parameters can have a significant impact on the properties (e.g. reactivity) carbon nanotubes.

“Any new method that aims to separate and purify nanotubes in an scalable, reproducible and simple way should measure certain morphological parameters (e.g. diameter and length) of CNTs. The length of nanotubes has been shown to correlate positively with electrical and thermal activities. This means that longer multi-wall carbon nanotubes, (MWCNTs), typically have higher thermal conductivities and electrical conductivities. Nanotube length can also impact transistor performance [10], electromagnetic interference shielding and the mechanical properties of CNT-based epoxy compounds [11].

“The length of nanotubes can have a significant impact on the biological response, human health, and the environment. It is becoming more common to recognize that the characterization of nanotubes is an important step towards assessing the potential toxic effects of nanomaterials within biological systems. Cheng [13] demonstrated that the length of carbon nanotubes plays a significant role in in-vivo toxicology of functionalized CNTs in Zebra Fish embryos in his study. A study of single-walled carbon tubes (SWCNTs), cytotoxicity, showed that cells’ cytotoxic response to functionalized CNTs is dependent upon their degree of functionalization [14]. To manage and decrease the toxicity of CNTs, it is crucial to measure length and control the measurement of length to maximize the green chemistry potential.

Accordingly, the length-based separation and measurement of CNTs has received particular attention. The measurement of basic parameters such as the diameter and length CNTs remains a challenge for nanotechnology researchers as well as commercial scale CNT production. There are several methods for size selection and purification. These include ultrafiltration (e.g., toluene and solvent) treatment of nanotubes, floculation in aqueous surfactants (17), oxidation and acid wash coupled with centrifugation [18, 19], chromatographic purification (22, 23), field-flow fractionation [24], size-exclusion analysis on nanotubes suspended in sodium sulfate [25, 26].

“Electrophoretic methods also allow nanomaterials purification [27-29] as well as characterization [30 and 31]. Separation based on parameters like size, shape and length [27, 31,] is possible. Electrophoresis techniques have been shown to be effective in separating CNTS by length-, diameter-, or curvature-based methods. These include AC electrophoresis with isopropyl alcohol [29], capillary electrodephoresis for SDS-coated SWCNTs (27], and agarose electrophoresis [32] to fractionate SWCNT/nucleic acids complexes. Capillary electrophoresis was also used to determine the complexity of DNA-suspended WCNTs by streptavidin/biotin bind [33]. Some efforts have been made to separate CNTs by length using a gel permeation column and an inhomogeneous magnet field.

Nanotubes can be made in many lengths, diameters, or structures. Because some properties of nanotubes are dependent upon their structure, much of the research has been focused on measuring these parameters accurately. Nanotube science can benefit from length-based separation. Accurate measurement of the length of nanotubes is crucial for understanding how they grow and cut in CNTs synthesis [39]. Franklin and his collaborators provided the first experimental evidence on the effects of contact length on nanotube transistors. They created sets of devices with different lengths of nanotubes [10]. The length-based separation (or length-based) of CNTs is a crucial step in enabling their use in biologically relevant settings, such as drug delivery.

“There have been over 200 papers published in the literature on the subject of the separation of SWNTs. They are classified according to their conductivity, diameter and handedness. Several chromatographic methods have been reported for length-based separation of CNT’s by Duesberg [25, 44, 45] using size-exclusion chromatography (SEC), Rinzler [46] using high performance liquid chromatography (HPLC) and Fotios Papadimitrakopolous’s group[47] using gel permeation chromatography. It is not possible to predict when a carbon nanotube will leave the chromatography column. This is due to the stochastic nature particle-pore interactions. The ability to extract carbon nanotubes with specific lengths from chromatographic separation processes is not yet known.

“Besides centrifugation[48], the length-based separations of carbon nanotubes based on their electrical properties is more often performed using capillary electrodephoresis (CE) or agarose gel electrophoresis[31?32]. There are no electrophoretic methods that can accurately and quickly separate CNTs along their length.

“The above methods, which are not precise and scaleable, were performed using the UV/vis, AFM and Raman Spectroscopy. These techniques can be complicated and costly. There are no pre-electrophoretic methods that can be used to quickly, cost-effectively, and precisely separate CNTs from their lengths. Therefore, there is a need for CNT characterization and length-based separation that is precise, quick, simple, and economical.

“Another aspect of the disclosure is methods for determining average lengths of labelled Carbon Nanotubes. These include: labelling a plurality carbon tubes with a biological moiety to produce labelled nanotubes; electrophoresis of the labelled nanotubes with an electrophoresis agent to create a gel; staining the electrophoresis Gel containing the labelled nanotubes with visualizing agents to provide stained, clearly labelled nanotubes; and measuring the intensity of the distance traveled in the gel.

The present disclosure is based on methods of separating carbon Nanotubes. This includes labelling a plurality carbon-nanotubes with a biological moiety to produce labelled nanotubes; using gel electrophoresis to treat the labelled nanotubes with an agent that can be used to visualize the target parameter to remove stained carbon nanotubes and then isolating them from the gel. The method may also include measuring the average length of stained, labelled carbon-nanotubes using UV/vis spectroscopy or Raman Spectroscopy.

“Methods to Separate Carbon Nanotubes”

The present disclosure describes methods to separate carbon nanotubes in certain aspects. These methods include labelling each carbon nanotube with a biological moiety to produce labelled nanotubes. Gel electrophoresis is used to create an electrophoresis solution that contains the labelled nanotubes. Then, the electrophoresis mix is removed from which are stained, or ranged, target parameters. Finally, the separated carbon nanotubes are extracted from the gel. The method may also include measuring the average length of stained, labelled carbon-nanotubes using UV/vis spectroscopy or Raman Spectroscopy in certain embodiments.

“In some embodiments the nanotubes may be commercially prepared or laboratory-prepared. The nanotubes could be carbon nanotubes, for example. In some cases, however, the methods described here can also be applied to nanoparticles and nanotubes. Some carbon nanotubes have a single-walled carbon nanotube. Other embodiments of the carbon nanotubes include double-walled carbon Nanotubes. Other embodiments of the carbon nanotubes include multi-walled carbon Nanotubes (e.g. three-walled or quadruple-walled; quintuple-walled). “Carbon nanotubes” is used hereinafter, except where otherwise noted. It can refer to any combination of single-walled, doublewalled carbon nanotubes or multi-walled nanotubes.

“In some embodiments of the current methods, the carbon nanotubes that are to be separated can be labelled with a biological moiety. In some cases, carbon nanotubes may be labeled with a protein. Any soluble, structural, linear, or globular protein that is suitable for the present methods can be used as a protein. Examples of readily available proteins are those that have been isolated from milk and other biological systems. You can also prepare or isolate the protein from any source. The protein may be an enzyme, a domain of an enzyme, a bioactive protein peptide, an anti-hormone or a hormone in certain embodiments. The protein may be any soluble or structural, linear, or globular protein in some embodiments.

“In some embodiments the enzyme is an oxygenoreductase. Other embodiments of the enzyme are a transferase. The enzyme can also be a hydrolase in certain embodiments. The enzyme may also be called an isomerase in some embodiments. The enzyme can also be a ligase in certain embodiments. The enzyme can also be a lyase in some embodiments. The enzyme may be an egg white lysozyme in some embodiments. Other embodiments allow the biological moiety to be a peptide, peptide fragment or enzyme. Any peptide fragment that contains 2-10, 2-100 or more amino acids can be considered suitable. You can choose to use natural, unnatural amino acids, or derivatives of them. The biological label may include chicken egg white Lysozyme in some instances.

“In some embodiments, the antimicrobial peptide may be a Tachykinin or vasoactive intestinal protein, a Tachykinin or vasoactive intestinal polypeptide-related, an opioid peptide or any other peptide such a B-type Natriuretic Peptide (BNP), or a lactotripeptides or a traditional Chinese medicine Colla Coni Asini component in hematopoiesis. The carbon nanotubes may be labeled with a protein domain in some embodiments. The carbon nanotubes may be labeled with a bioactive protein in some cases. The protein can be an antibody in some embodiments. The protein may also be a hormone in some embodiments. You can use hormones that are found in multicellular organisms (plants and animals, fungi brown algae, red algae, and fungi) or unicellular organisms.

The present methods may include labelling carbon nanotubes with a biological moiety. To label carbon nanotubes, it is possible to react the carbon nanotubes with the biological moiety using a linking agent. The linking reagent may react with functional groups on carbon nanotubes’ surfaces in some instances. Other embodiments allow the linking agent to react with functional moiety functional groups. Proteins, enzymes, and peptides are typically composed of amino acid residues that can include functional groups such as alcohols or thioethers. Carboxamides, amines. amides. thiols. In some cases, the linking agent reacts with the functional group of the biological moiety. Some embodiments use a peptide-forming agent as the linking reagent.

“In some embodiments, the linking agent covalently links carbon nanotubes with the biological moiety. Other embodiments allow the carbon nanotubes and biological moiety to be covalently linked. Combining a biological moiety and an affinity for carbon nanotubes can result in non-covalent linking. Hydrogen bonding interactions can be used to link the carbon nanotubes’ surfaces with the biological moiety. Other embodiments of non-covalent linking are the result Van der Waals interactions between carbon nanotubes & the biological moiety. The non-covalent linking in certain embodiments is caused by hydrogen bonding interactions or electrostatic interactions. interactions or hydrophobic effects.

“In some embodiments, the linking agent can be a peptide-coupling agent. The linking agent in some embodiments is a carbodiimide-reagent. These carbodiimide agents can form amide bonds between the biological moiety of the carbon nanotubes. In some embodiments, the carbodiimide reagent is N-ethyl-N?-(3-(dimethylamino)propyl)carbodiimide. In other embodiments, the linking reagent can be dicyclohexylcarbodiimide. In certain embodiments, the linking reagent can be diisopropylcarbodiimide. Any peptide coupling agent that links the carbon nanotubes and the biological moiety is possible. In some embodiments, the linking agent is a carbodiimide reagent, a polymer phospholipid-polyethylene glycol (PL-PEG-NH2), a polystyrene-block-polyacrylic acid (PS-b-PAA), an imidazolium salt based ionic liquid (IS-IL), or an ethylenediamine. The present methods also allow for the separation of labelled carbon nanotubes. In some cases, the labelled carbon nanotubes can be loaded into gels and separated by electrophoresis. Gel electrophoresis, which is a technique used to separate macromolecules like DNA, RNA and proteins, is well-known in the art. The present methods use the advantages of biological moieties and the high resolution power of gel electrophoresis to separate the labelled carbon-nucleotides. Electrophoresis may include sodium dodecyl-sulfate polyacrylamide electrophoresis, (SDS?PAGE) in some embodiments. Other embodiments use agarose gel. The gel can also be a starch gel in certain embodiments. In some embodiments, electrophoresis takes place under denaturing conditions. Other embodiments allow for electrophoresis to be conducted under non-denaturing conditions. The electrophoresis is performed at a constant electrical field in certain embodiments. Other embodiments allow for the use of a pulsed, gradient, or inverted field to run the electrophoresis procedure.

After the electrophoresis gel has separated the carbon nanotubes, the gel can be stained to produce stained carbon nanotubes. This staining shows the bands and separated populations of carbon nanotubes within the gel. You can use any staining agent to visualize the labelled carbon-nanotubes. To satinize the nanotubes, you can use ethidium bromide in certain embodiments. Other embodiments use coomassie to make stained carbon nanotubes. In some embodiments, the visualizing agents are silver nitrate and silver-ammonia complex solutions. The gel containing the separated and labelled carbon nanotubes can be stained by contact with the staining agent.

“The present methods can also be used to effect separation by determining a target parameter value, or range of the carbon nanotubes. The target parameter could be the length, shape, diameter, number of walls or any combination thereof. In some embodiments, the carbon nanotubes labelled are separated by their length. Other embodiments allow the labelled carbon nanotubes to be separated by their shape. The visual intensity of the stained, labeled carbon nanotubes can also be used to determine the target parameter’s value or range. This can be done in one or more sections of the electrophoresis Gel. In some embodiments, the parameter can be length. To determine this, you will need to calculate the length of the stained, labeled carbon nanotubes using the following formula:

“L = d? ? exp [ 3 ? ? ? ? ? ? ? ? q ? ( d) e – 2 ? ? ? 2 + 1]nwhere L is the length calculated, d is the mean diameter, and? is the optimum concentration. is the solvent viscosity. q(d), is persistence length. e is electron charge. The electrophoretic mobility is calculated using Usrey’s equation, Usrey and al., 2007, [34]. The formula below can be used to determine the relationship between the length of the carbon nanotubes and the visual intensity of the stain-labeled populations in gel.

“Some embodiments of the carbon nanotubes that can be separated using the present methods have a length between 10 nm and about 1 cm. In some embodiments, carbon nanotubes can have lengths of approximately 10 nm to 10 m, about one m to around 100 m, about one m to about one mm, 100 m to roughly 1 mm, 100 m to approximately 1 cm or about one mm to about a cm or any other value. The resolution of separation achieved by the present methods may be approximately 1 nm to about 10 nm or about 100 nm or about any other value or range thereof.

“In some embodiments, carbon nanotubes that are separated using the present methods have a diameter of approximately 0.5 nm to 100 nm. In certain embodiments, for example, the diameter of carbon nanotubes can be as low as 0.5 nm in some cases, and as high at about 10 nm in others.

“In certain embodiments, carbon nanotubes can be surface functionalized carbon nanotubes. Any surface functionalization compatible to the present methods may be used. Some embodiments of the carbon nanotubes’ surface are functionalized with a carboxylic acids moiety. Some embodiments use a carboxylate salt as the carboxylic acid. Any cation that does not interfere in the electrophoresis process can be used as a suitable counterion. In some embodiments, the counterion could be an alkali metallic. The alkali metal in some embodiments can be either lithium, sodium, or potassium. Other embodiments use surface functionalized carbon nanotubes that are amine,amide, or thiol (??NH2SH). (CO)NH2, or?CONHCH2CH2SH functionalized carbon nanotubes. You can either have the amine as a base or an amine salt. Any anion that does not interfere in the electrophoresis can be used as a suitable counterion. In some embodiments, the counterion may be a halogen-ion. The halogen ion in certain embodiments can be F, Cl or Br. Other embodiments use carbon nanotubes that are? (CO)NH2 and?CONHCH2CH2SH are functionalized carbon nanotubes.

“Another aspect of the present methods is gel electrophoresis, which separates the labelled nanotubes into groups. Some embodiments of the labelled carbon-nanotubes found in groups of labelled nanotubes have a substantially identical length. The length of the labelled nanotubes can vary by as much as 10% from the average length of the group. Other embodiments allow the length of the carbon nanotubes labeled in the group to vary by +/-10% or +//8% or +//6% or +//1% or +//0.01% of the average length for the group of labelled nanotubes or any other value.

“Some embodiments of the present methods include measuring the average lengths of the carbon nanotubes that have been isolated, stained and labelled. In some embodiments, the average length can also be measured using one or more of UV, atomic force microscopy, (AFM), or Raman spectroscopy.

“Methods to Determine the Length Carbon Nanotubes”

“Another aspect of the disclosure is that it provides a method of determining the average length a sample labelled Carbon nanotubes. This involves: labelling each carbon nanotube with a biological moiety to produce labelled nanotubes; gel electrophoresis to create an electrophoresis Gel comprising the carbon nanotubes labelled with a visualizing agents to provide stained carbon tubes; and measuring the intensity of the stained carbon nanotubes at a plurality locations in the gel. The average length of their distance traveled in the gel.

“In some embodiments the nanotubes may be commercially prepared or laboratory-prepared. The nanotubes could be carbon nanotubes, for example. In some cases, however, the methods described here can also be applied to nanoparticles and nanotubes. Some carbon nanotubes have a single-walled carbon nanotube. Other embodiments of the carbon nanotubes include double-walled carbon Nanotubes. Other embodiments of the carbon nanotubes include multi-walled carbon Nanotubes (e.g. three-walled or quadruple-walled; quintuple-walled). “Carbon nanotubes” is used hereinafter, except where otherwise noted. It can refer to any combination of single-walled, doublewalled carbon nanotubes or multi-walled nanotubes.

“In some embodiments of the current methods, the carbon nanotubes that are to be separated can be labelled with a biological moiety. In some cases, carbon nanotubes may be labeled with a protein. Any soluble, structural, linear, or globular protein that is suitable for the present methods can be used as a protein. Examples of readily available proteins are those that have been isolated from milk and other biological systems. You can also prepare or isolate the protein from any source. The protein may be an enzyme, a domain of an enzyme, a bioactive protein peptide, an anti-hormone or a hormone in certain embodiments. The protein may be any soluble or structural, linear, or globular protein in some embodiments.

“In some embodiments the enzyme is an oxygenoreductase. Other embodiments of the enzyme are a transferase. The enzyme can also be a hydrolase in certain embodiments. The enzyme may also be called an isomerase in some embodiments. The enzyme can also be a ligase in certain embodiments. The enzyme can also be a lyase in some embodiments. The enzyme may be an egg white lysozyme in some embodiments. Other embodiments allow the biological moiety to be a peptide, peptide fragment or enzyme. Any peptide fragment that contains 2-10, 2-100 or more amino acids can be considered suitable. You can choose from natural, unnatural or derivative amino acids for your amino acid residues. The biological label may include chicken egg white Lysozyme in some instances.

“In some embodiments, the antimicrobial peptide may be a Tachykinin or vasoactive intestinal protein, a Tachykinin or vasoactive intestinal polypeptide-related, an opioid peptide or any other peptides such as a B type natriuretic (BNP), lactotripeptides or a traditional Chinese medicine Colla Corii Asini component in hematopoiesis. The carbon nanotubes may be labeled with a protein domain in some embodiments. The carbon nanotubes may be labeled with a bioactive protein in some cases. The protein can be an antibody in some embodiments. The protein may also be a hormone in some embodiments. You can use hormones that are found in multicellular organisms (plants and animals, fungi brown algae, red algae, and fungi) or in unicellular organisms.

The present methods may include labelling carbon nanotubes with a biological moiety. To label carbon nanotubes, it is possible to react the carbon nanotubes with the biological moiety using a linking agent. The linking reagent may react with functional groups on carbon nanotubes’ surfaces in some instances. Other embodiments allow the linking agent to react with functional moiety functional groups. Proteins, enzymes, and peptides are typically composed of amino acid residues that can include functional groups such as alcohols or thioethers. Carboxamides, amines. amides. thiols. In some cases, the linking agent reacts with the functional group of the biological moiety. Some embodiments use a peptide-forming agent as the linking reagent.

“In some embodiments, the linking agent covalently links carbon nanotubes with the biological moiety. Other embodiments allow the carbon nanotubes and biological moiety to be covalently linked. Combining a biological moiety and an affinity for carbon nanotubes can result in non-covalent linking. Hydrogen bonding interactions can be used to link the carbon nanotubes’ surfaces with the biological moiety. Other embodiments of non-covalent linking are the result Van der Waals interactions between carbon nanotubes & the biological moiety. The non-covalent linking in certain embodiments is caused by hydrogen bonding interactions or electrostatic interactions. interactions or hydrophobic effects.

“In some embodiments, the linking agent can be a peptide-coupling agent. The linking agent in some embodiments is a carbodiimide-reagent. These carbodiimide agents can form amide bonds between the biological moiety of the carbon nanotubes. In some embodiments, the carbodiimide reagent is N-ethyl-N?-(3-(dimethylamino)propyl)carbodiimide. In other embodiments, the linking reagent can be dicyclohexylcarbodiimide. In certain embodiments, the linking reagent can be diisopropylcarbodiimide. Any peptide coupling agent can link the carbon nanotubes and the biological moiety. In some embodiments, the linking agent is a carbodiimide reagent, a polymer phospholipid-polyethylene glycol (PL-PEG-NH2), a polystyrene-block-polyacrylic acid (PS-b-PAA), an imidazolium salt based ionic liquid (IS-IL), or an ethylenediamine.”

“In another aspect of the invention, the methods allow for the separation of labelled carbon nanotubes in order to determine their length. In some cases, the labelled carbon nanotubes can be loaded into gels and separated by electrophoresis. Gel electrophoresis, which is a technique used to separate macromolecules like DNA, RNA and proteins, is well-known in the art. The present methods use the advantages of biological moieties and the high resolution power of gel electrophoresis to separate the labelled carbon-nucleotides. Electrophoresis may include sodium dodecyl-sulfate polyacrylamide electrophoresis, (SDS?PAGE) in some embodiments. Other embodiments use agarose gel. The gel can also be a starch gel in certain embodiments. In some embodiments, electrophoresis takes place under denaturing conditions. Other embodiments allow for electrophoresis to be conducted under non-denaturing conditions. The electrophoresis is performed at a constant electrical field in certain embodiments. Other embodiments allow for the use of a pulsed, gradient, or inverted field to run the electrophoresis procedure.

After the labelled carbon-nanotubes have been separated from the electrophoresis gel they can be stained to produce stained, labelled carbon-nanotubes. This staining shows the bands and separated populations of carbon nanotubes within the gel. You can use any staining agent which visualizes the labelled nanotubes. To stain the carbon nanotubes labelled with ethidium bromide, one example is coomassie blue. To provide stained carbon nanotubes that are labelled, you can use coomassie bleu. The visualizing agent may contain silver nitrate, silver-ammonia complex solutions or both. You can stain the gel by contacting it with the staining agent.

“In another aspect, the methods described can be used to separate labeled carbon-nanotubes in order to determine their length. Separation to determine other parameters is possible, however. The target parameter could be length, shape or diameter of the carbon nanotubes, or even the number of walls. In some embodiments, the carbon nanotubes labelled are separated by their length. Other embodiments allow the labelled carbon nanotubes to be separated by their shape. The visual intensity of the stained, labeled carbon nanotubes can also be used to determine the target parameter’s value or range. This can be done in one or more sections of the electrophoresis Gel. In some embodiments, the parameter can be length. To determine this, you will need to calculate the length of the stained, labeled carbon nanotubes using the following formula:

“L = d? ? exp [ 3 ? ? ? ? ? ? ? ? q ? ( d) e – 2 ? ? ? 2 + 1]nL is the calculated length, and d is the mean diameter. 2 + 1 ]nwhere L is the calculated length, d is the mean diameter, and e is an electron charge. The electrophoretic mobility. The formula below can be used to determine the relationship between the visual intensity and the length of the carbon nanotubes stained and labelled in gel.

“Some embodiments of the carbon nanotubes that can be separated using the present methods have a length between 10 nm and about 1 cm. In some embodiments, carbon nanotubes can have lengths of approximately 10 nm to 10 m, about one m to around 100 m, about one m to about one mm, 100 m to roughly 1 mm, 100 m to approximately 1 cm or about one mm to about a cm or any other value. The resolution of separation achieved by the present methods may be approximately 1 nm to about 10 nm or about 100 nm or about any other value or range thereof.

“In some embodiments, carbon nanotubes that are separated using the present methods have a diameter of approximately 0.5 nm to 100 nm. In certain embodiments, for example, the diameter of carbon nanotubes can be as low as 0.5 nm in some cases, and as high at about 10 nm in others.

“In certain embodiments, carbon nanotubes can be surface functionalized carbon nanotubes. Any surface functionalization compatible to the present methods may be used. Some embodiments of the carbon nanotubes’ surface are functionalized with a carboxylic acids moiety. Some embodiments use a carboxylate salt as the carboxylic acid. Any cation that does not interfere in the electrophoresis process can be used as a suitable counterion. In some embodiments, the counterion could be an alkali metallic. The alkali metal in some embodiments can be either lithium, sodium, or potassium. Other embodiments use surface functionalized carbon nanotubes that are amine,amide, or thiol (??NH2SH). (CO)NH2, or?CONHCH2CH2SH functionalized carbon nanotubes. You can either have the amine as a base or an amine salt. Any anion that does not interfere in the electrophoresis can be used as a suitable counterion. In some embodiments, the counterion may be a halogen-ion. The halogen ion in certain embodiments can be F, Cl or Br. Other embodiments use carbon nanotubes that are? (CO)NH2 and?CONHCH2CH2SH are functionalized carbon nanotubes.

“Another aspect of the present methods is gel electrophoresis, which separates the labelled nanotubes into groups. Some embodiments of the labelled carbon-nanotubes found in groups of labelled nanotubes have a substantially identical length. The length of the nanotubes labeled in the group may vary by +/-10% from the average length of the group. Other embodiments allow the length of nanotubes to be labeled in the group to vary by +/-10% or +//8% or +//6% or +//1% or +//0.01% of the average length for the group of carbon nanotubes or any other value.

“Results of XRD patterns (FIGS. 1A, 1B, and 1C), FTIR(FIG. 2), and SEM micrographs. 3A, 3B, and 3C show that a biological moiety, such as lysozyme, can be disposed onto the SWCNTs surfaces during conjugation. In some embodiments, the biological moiety can be linked to about 1% of the surface functional groups of carbon nanotubes. The biological moiety can be linked to at most about 2%, 3%, 4%, 5%, 5%, 6%, 5%, 5%, 6%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 45%, 65%, 65%, at minimum about 70%, a least about 75% and 80% respectively.

“In embodiments in which the biological moiety has been covalently linked to carbon nanotubes’ surface, the presence covalently attached proteins may result in an intrinsic positive charge and a functionalization charge. These together make up the net charge of any particular nanotube or bundle of nanotubes in solution. High precision separation is possible because the length of carbon nanotubes (e.g. SWCNT) determines the amount of conjugated biological moiety. The net charge of carbon nanotubes is related to the amount linked biological moiety (e.g. lysozyme). Different lengths of carbon nanotubes (e.g. SWCNT) have different mobility in an electrophoretic field. According to the following formula, the mobility and velocity of charged CNT pieces depend on their electrical field (E, Volts/cm) as well as the net charge (q) on them. However, the friction between the molecules is inversely affected by the latter.

“V = Eq. fnwhere: f=frictional coefficient of the mass and form of the fragment, and V=velocity/mobility fragment [38]. CNTs smaller than the larger ones can pass through gel with greater mobility. The gel acts as a sieve, allowing smaller nanotubes to pass through while keeping larger ones in place. The friction coefficient refers to the ease with which a carbon nanotube fragment passes through the gel pores. The main determinant of SWCNT mobility in a gel matrix will be their length. Substituting length for the frictional coefficient results in: mobility=(voltage)(charge)/(length). This means that the charge/length ratio is the primary determinant of the mobility of a carbon-natube during gel electrophoresis.

“The length distribution for conjugated SWCNTs is represented by the distributions of the calculated lengths from Usrey?s equation versus visual intensity of bands of the stained, labeled carbon nanotubes (FIG. 7). You can view the intensity as a measure for the number of CNTs that have a particular length on the gel. The higher the visual intensity, then the more CNTs that have that length are in that group. FIG. FIG. 7. The main distribution of lengths for CNTs is between 38-50 microns. Another peak is at 60 microns, which corresponds with CNTs that were in or near the loading well.

“Methods to Adjust Carbon Nanotube Parameters in Manufacturing Processes.”

“Another aspect of the disclosure is that it provides methods for adjusting lengths of carbon-nanotubes made in a carbon tube manufacturing process. This includes: taking a sample of carbon, labelling each carbon, and then exposing the gel containing the labelled nanotubes to gel electrophoresis. The gel containing the labelled nanotubes can be treated with a visualizing agent, which will produce stained, labelled nanotubes.

The art reveals that there are many carbon nanotube manufacturing methods for commercial or laboratory scale production. In some embodiments, carbon nanotube manufacturing can include arc discharge, laser, chemical vapor deposition, beam wavelength, voltage, and carbon monoxide disproportionation at high pressure (HiPCO). One or more parameters are selected from the following group: temperature, pressure and raw material feed rate.

CNT-based nanobiotechnology and nanomedicine have one problem: the lack of uniform length in mass production of CNTs. It is crucial to distinguish CNTs according to their conductivity and length in these applications.

The present methods are a valuable tool to control the quality of carbon nanotubes manufactured in large quantities. In some embodiments, the disclosure offers a method to adjust parameters and control quality in the production carbon nanotubes. The method may include the collection of a sample from a population of carbon nanotubes using a nanotube manufacturing process and the separation of the carbon tubes, as described in this document. Other embodiments include measuring the length of a sample of a carbon-nanotube population using a nanotube manufacturing method and, where necessary, changing one or more parameters.

“For the purposes of this invention, the chemical element are identified according to the Periodic Table of the Elements CAS version Handbook of Chemistry and Physics, the 67th edition, 1986-87 inside cover.”

“One of ordinary skill in relevant arts will understand that there are other modifications and adaptations possible to the compositions or methods described herein. This is in view of information available to the ordinarily skilled artist and it can be done without departing from any embodiment of the invention.”

“EXAMPLES”

“Having described the invention in detail, it will become clearer by referring to the following examples. These are provided for illustration purposes only and are not meant to limit the invention.”

“Materials & Methods”

“Lyophilized chicken eggs white lysozyme, EC 3.2.1.17 was purchased from Inovatech, Inc., Abbotsford, BC, Canada and Micrococcus lysodeikticus cell, Sigma-Aldrich Corporation, St. Louis, Mo. They were used as dry powder and salt-free. MKnano, Canada purchased carboxyl single-walled carbon Nanotubes (SWCNT?COOH) with an outer diameter of 1-2 nm. MES [2-(N-morpholino) ethane sulfonic acid] buffer, N-ethyl-N?-(3-(dimethyl amino) propyl) carbodiimide hydrochloride (EDC), Tris-hydroxymethyl aminomethane (Tris), N,N methylenebisacrylamide (Bis), acrylamide, sodium dodecyl sulfate, ammonium persulfate, tetramethylethylenediamine (TEMED), 2-mercaptoethanol(2ME), 3,3-5,5 tetrabromophenolsulfonphthalein (Bromophenol Blue) and all other chemicals were purchased from Sigma-Aldrich Corporation and used as received.”

“Example 1?” Enzyme Attachment to SWCNTs

“Details on chemically bonding Lysozyme (to SWCNTs) are reported in literature [41]. A carbodiimide was used to conjugate lysozyme onto SWCNTs [42]. The activated SWCNTs in MES buffer were 50 mM, pH 6.2 (1 mg/mL), and then added to an equal volume 400 mM N.hydroxysuccinimide in MES buffer. For coupling of NHS to the carboxylic groups on the surface of nanotubes, 20 mM N-ethyl-N?-(3-(dimethylamino) propyl) carbodiimide hydrochloride (EDC) was added to the mixture. After stirring the mixture at 200 rpm for 30 minutes, it was subjected to sonication (MSE Ultrasonic disintegrators, 150W, England) for approximately 30 minutes. The mixture was centrifuged. To remove excess EDC or NHS, the centrifuge steps were repeated 3 times. The enzyme solution (10 mg/ml and 10 mMphosphate buffer, pH 8) was added to the nanotubes after they had been rinsed. It was sonicated for approximately. It took approximately 1 minute to re-disperse SWCNTs. During the conjugation process, the mixture was stirred in an orbital shaker at 200 RPM at room temperature. To remove any non-specifically adsorbed enzyme, the conjugated lysozyme/SWCNTs solution was centrifuged. The same process was used to prepare control enzyme-nanotube conjugates, but without NHS or EDC.

“Example 2?Conjugated Lysozyme-SWCNTs Characterization”

“The morphology and activity of conjugated lysozyme SWCNTs was compared to activated SWCNTs using time-scanning electron microscopy (SEM), D8, Advance, Bruker) at?=0.1542 nm and FTIR spectroscopy(Shimadzu FTIR-8300 spectrophotometer). These methods were used for characterizing conjugated lysozyme SWCNTs. Three time periods were used to sonicate conjugated lysozyme SWCNT samples: 3, 7, and 10, respectively.

“Example 3?” SDS-PAGE Electrophoresis, Silver Staining

“Acrylamide (29.2g) and Bis (8.8g) were mixed in 100 ml of water, and then filtered to make gel stock solution (30% m/v). Mixing 10.0ml gel stock solution with 10.0ml TrisHCl (1.5 mole L-1, pH 8.80), 200 -800?l NH4)2S2O8 (10%, m/v) along with 0.4g SDS was enough to prepare the separating gel solution. Finally, water was added to dilute to 40mL. 1.33 ml gel stock solution was added to 50 ml (NH4)2S2O8 (10% m/v) and 2.5 ml TrisHCl (6.80 mol L-1), pH 6.80. The gel stock solution was then mixed with 10.0 ml. The mixtures were then diluted with water to 10.0 mL, and finally 10 mL of TEMED was added. To remove any enzyme that may be physically attached, the samples were washed with phosphate buffer (10mM, pH 8), several times before electrophoresis. To prepare the electrophoresis buffer, Tris (15.14g), Glycine (72.5 g) and SDS (5g) were dissolved in 500 ml of distilled water. The pH of the solution was adjusted to 8.30. The vertical polyacrylamide gel system used consisted of stacking and separating (0.0%, m/v), gels. The sample loading volume was 15.?L. The gels were stained using Coomassie Brilliant Blue R-250. Blum method was used to stain gels with silver [43]. This involves fixing the gels with methanol, paraformaldehyde, and acetic acids, as well as washing them with ddH2O (50% and 30%), sensitizing with Na2S2O3.5H2O and ddH2O, washing them with ddH2O and impregnating with silver nanorate and paraformaldehyde, washing them with ddH2O and developing with Na2CO3, Na2S2O3.5H2O3.5H2O3.5H2O solutions and Na2O3.5H2O3.5H2O3.5H2O5H2O3.5H2O3.5H2O solutions, ending with acetic acid 12% and stopping the solution-methanol at acetic acid at acetic acid acetic acid acetic acid acetic acid acetic acid acetic acid acetic acid 12% and acetic acid acetic acid acetic acid acetic acid acetic acid 12% and acetic acid acetickick acid acetic acid acetic acid acetic and acetic 12%

“Example 4:Characterization Conjugated SWNTs”

Carbodiimide was used to bio-conjugate lysozyme onto the nanotube surface. Analyzing the interactions between free and lysozyme SWCNTs was done using SEM,XRD andFTIR. FIGS. 1A, 1B and 1C show the XRD patterns. FIGS. 1A, 1B, and 1C showed that enzyme attachment was confirmed to the surface of the oxidized SWCNTs. The strong peaks in the SWCNTs correspond with the (002) and (100) carbon planes. The characteristic peak locations for conjugated lysozyme are 20 of, 14.0 and 30.0 respectively. These peaks are identical to those for the free lysozyme pattern XRD. The results revealed that there was no significant difference between conjugated and free lysozymes in XRD patterns. This could indicate either the adsorption of lysozyme onto SWCNTs or the absorption. These results show that the conjugation lysozyme and SWCNTs doesn’t cause a phase shift in lysozyme. This indicates that the lysozyme was not structurally denaturated and maintained its activity throughout the conjugation.

“Example 5:Characterization and Conjugation Of Lysozyme With SWCNTs By FT-ER”

“FTIR analysis has revealed the mechanism of SWCNTs to lysozyme conjugation. FTIR fingerprint is a result of amide links between amino acids residues in proteins and polypeptides. The positions of the amide types I and II bands in FTIR spectra are indicators of conformational changes within the protein secondary structure. They have been used in studies to examine immobilized enzyme molecules. FIGS. 2A, 2B and 3C show the FTIR spectra of free lysozyme as well as SWCNTs (and conjugated lysozyme?SWCNT) respectively. 2A, 2B, and 2C. SWCNT activation was demonstrated by the formation of?COOH functional group in the SWCNT matrix, according to the absorption peak positions at 1627.8 cm/1 and 3440.8 cm/1. A strong and broad NH3 stretching band is a characteristic of amino acids in the 2950-2600cm?1 region of the lysozyme spectrum. The absorption is increased to approximately 2000 cm?1 by the addition of an overtone region. These overtone bands often contain a prominent band between 2222 and 2000 cm?1 that is due to a combination asymmetrical vibrations of NH3+ bending vibrations and torsional oscillations of the NH3+ group[37]. A weak asymmetrical NH3+ bending band can be seen near 1661 cm?1 while a strong symmetrical bending band can be found near 1529 cm?1. The stretching of N?H groups is represented by a peak at 3600 cm’1. A peak at 1230cm?1 represents the stretching C?N group within amine groups. These peaks are seen in the lysozyme SWCNTs spectrum as an indicator of amide bonds between amine groups and activated SWCNTs carboxyl groups. The absorption peak at 1650cm?1 is indicative of C?O’s stretching vibration mode. Peaks at 3800cm?1 and 1650cm?1 represent the stretching of N?H group in the amide. The FTIR spectrum of lysozyme SWCNTs confirmed that amide bonds were formed between the amine groups and the carboxyl groups activated SWCNTs.

“Example 6: Characterization of Conjugation Of Lysozyme With SWCNTs By SEM”

“FIGS. “FIGS. An indication of conjugation is a thicker sidewall of conjugated lysozyme SWCNTs, which can be between 89.5 and 95 nm.

“Example 7:Length-Dependent Separation of Bio-Conjugated SWCTs Using SDS?PAGE and Visualization With Silver Staining”

“Conjugation of Lysozyme with CNTs is of particular interest because of its ability to disperse CNTs. According to molecular modeling [38] and experiments, lysozyme can sort nanotubes by their diameter. This example shows how lysozyme was conjugated onto carboxyl functionalized carbon-nanotubes to create carbon nanotubes that are length specific. Silver staining was used to enable selective visualization of nanotube fragments within the acrylamide gel, which is a problem for many nanotech researchers. Silver staining was able to show high resolution CNTs length-based seperation of lysozyme SWCNT fragments, in contrast to coomassie Blue staining. FIG. FIG. 4B shows silver staining for free lysozyme (lane 1, conjugated lysozyme SWCNTs, and SWCNTs(lane 3).

“The coomassie dyes R-250 and G-252 are anionic dyes that stoichiometrically bond to proteins through ionic interaction between positive protein amine group and dye sulfonic acids groups, as well as Van der Waals attraction.”

“Coomassie Blue staining didn’t show the lysozyme SWCNTs due to amide bond formation among primary amines protein and carboxyl group of SWCNTs. Therefore, there would be no binding between coomassie and lysozyme. Silver staining revealed sharp bands. Sharp bands could be due to the stability and proportion of lysozyme molecules, based on nanotube lengths, and silver staining sensitivity towards proteins. Different lengths of conjugated lysozyme SWCNT fragments had different mobilities.

“Example 8″Length Measurements of Lysozyme SWCNT Fragments Using Image Analysis Techniques

ImageJ was used for calculating the color intensity of a narrow rectangle on each lane, from the well to its bottom. Similar results were obtained (see FIG. 6), which are consistent with the visual evaluations. Following analysis of gel images, experimental data was obtained in the form mobility distribution (numbers of nanotubes per unit of mobility). FIG. 6. shows that SWCNTs of different lengths are present in the population for each experimental electrophoretic mobility value. FIG. 6. shows that SWCNTs of different lengths are found in the population for each experimental electrophoretic mobile value. The color intensity was used to retrieve the image database.

The present disclosure uses bio-conjugation and SDS-PAGE in combination with staining to seperate CNTs. This allows for measurement of a parameter (e.g. length) using electrophoretic mobility values in an acrylamide gel. These methods are useful for process validation and quality control in nanotube manufacturing processes.

“REFERENCES”

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