Bright nanotube fluorescent dyes via covalent functionalization

Abstract

An exemplary compound according to examples of the present disclosure includes, among other things, a nanotube support, a moiety, and a linker having first and second functional groups, where the first functional group is covalently attached to the nanotube support and the second functional group is covalently attached to the moiety. Also disclosed are exemplary methods of making nanotube supports according to the present disclosure.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 62 / 855,128, filed May 31, 2019, which is hereby incorporated in its entirety. This application is a continuation-in-part of U.S. Provisional Application No. 15 / 953,200, filed April 13, 2018, which claims priority to U.S. Provisional Application No. 62 / 485,379, filed April 13, 2017, which is hereby incorporated in its entirety.

[0002] (Statement regarding federally sponsored research) The invention described herein was made with government support under Grants #1261910, #1521057 and #1738466 awarded by the National Science Foundation. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Fluorophores are compounds with fluorescent properties that have biomedical applications. For example, fluorescent dyes can be used as tracers or dyes to specifically stain certain molecules or structures. More specifically, fluorescent dyes are used to stain tissues, cells, or materials in various analytical methods such as fluorescence imaging and spectroscopy.

[0004] For the purpose of specific staining, fluorescent dyes can be conjugated with biological molecules such as antibodies. However, reliable tracking and quantification of fluorescent dyes is difficult due to the low brightness and poor photostability of commercially available fluorescent dyes. Thus, there is a need for improved carrier molecules to carry fluorescent entities for biological and other applications. Other biological molecules could also benefit from improved carriers. Summary of the Invention [Means for solving the problem]

[0005] One exemplary compound according to the present disclosure includes, inter alia, a nanotube support, a moiety, and a linker having first and second functional groups, where the first functional group is covalently attached to the nanotube support and the second functional group is covalently attached to the moiety.

[0006] An exemplary method of making nanotube compounds according to the present disclosure includes, among other things, mechanically treating nanotubes in a polar liquid, whereby the mechanical treatment forms imperfections on the nanotubes and provides polar groups at the imperfections to covalently bond a linker to the nanotubes, the linker having a first and a second functional group, the first functional group covalently bonded to the polar group. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram of a compound having a nanomaterial support. [Figure 2A] FIG. 2 shows an image of a nanomaterial carrier of the compound of FIG. 1. [Figure 2B] For example, FIG. 2 shows the results of Fourier transform infrared spectroscopy for a nanomaterial carrier of the compound of FIG. [Diagram 3] FIG. 1 shows the excitation spectra between 245 and 270 nm of a different group of compounds with nanomaterial carriers. [Figure 4] FIG. 1 shows the excitation spectra between 440 and 520 nm of a different group of compounds with nanomaterial carriers. [Diagram 5] FIG. 2 shows XPS (X-ray photon spectroscopy) spectra of examples of compounds as in FIG. 1 with an azido linker. [Figure 6] FIG. 2 shows the fluorescence intensity of a compound such as that of FIG. 1 having an azido linker and a FITC fluorescent entity. [Figure 7A] FIG. 2 shows the fluorescence intensity of a compound such as that of FIG. 1 having an azide-PEG linker and a FITC fluorescent entity. [Figure 7B]FIG. 2 shows the fluorescence intensity of a compound such as that of FIG. 1 having an azide-PEG linker and a sulforhodamine fluorescent entity. [Figure 7C] FIG. 2 shows the fluorescence intensity of a compound such as that of FIG. 1 having an azide-PEG linker and a sulfo-Cy5.5 fluorescent entity. [Figure 8A] FIG. 2 shows the fluorescence intensity of a compound such as that of FIG. 1 having an amino linker and a FITC fluorescent entity. [Figure 8B] FIG. 2 shows the fluorescence intensity of compounds with an amino linker and a sulforhodamine fluorescent entity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Most commonly, a bright fluorescent dye comprises a carrier element, a fluorescent element, and a linker that connects the carrier element to the fluorescent element. For biomedical applications, each of the carrier element, the linker, and the fluorescent element must be biocompatible (although biocompatibility requirements vary depending on the particular application).

[0009] Exemplary carrier elements are nanomaterials such as carbon nanotubes (CNTs) and boron nitride nanotubes (BNNTs), both of which can be used in biomedical applications such as cellular drug delivery and spectroscopy applications. However, fluorescent elements bound to certain nanotubes have been shown to exhibit quenching, or a decrease in fluorescence brightness.

[0010] Certain fluorescent dyes with nanomaterial carriers have been found to not only exhibit no quenching effect, but also exhibit brightness several orders of magnitude higher than other known fluorescent dyes, as discussed herein.

[0011] 1, compound 20 is shown. Compound 20 generally comprises an inorganic nanoscale ("nanomaterial") support 22, a linker 24, and a moiety 26. In some examples, compound 20 comprises two or more linkers 24 and two or more moieties 26.

[0012] The support 22, in one example, is a BNNT or CNT support. In a particular example, the support 22 is a multi-walled BNNT or CNT support, each BNNT or CNT having multiple concentric shells of hexagonal boron nitride (h-BN in the case of BNNT) or graphene (in the case of CNT), with a typical outer diameter greater than about 0.4 nm but less than about 80 nm. The length of these BNNTs and CNTs is about 100-2000 nm. In other examples, the support 22 may be another nanoscale inorganic material, such as hexagonal boron nitride (h-BN) nanosheets / nanoparticles and graphene / graphite nanosheets / nanoparticles, or zero-dimensional nanomaterials ("dots").

[0013] The linker 24 has two or more functional groups R and R', as shown in FIG. 1. The functional groups R and R' are reactive groups that facilitate covalent attachment of the linker to other structures by known chemistry. R and R' can be the same or different types of functional groups. The linker 24 is any type of molecule that has two or more functional groups R and R'. An example linker 24 is a linear or branched polymer molecule. In some examples, the linker 24 has a length of less than about 200 nm. In some examples, multiple linkers 24 can be connected to each other in series, for example, a first linker 24 can be connected to a second linker 24 at the R' group.

[0014] One functional group R covalently interacts with the support 22. A support 22 having a linker 24 is known as a "functionalized" support 22. That is, when covalently bonded to the linker 24, the support 22 / linker 24 structure has a functional group R' that facilitates covalent attachment of the support 22 / linker 24 to another moiety 26. An exemplary functional group R is a hydroxyl group, and an exemplary functional group R' is an azide or amine group. However, R and R' may be any well-known functional group, such as an amine group, a carboxylic acid, an isothiocyanate, a maleimide, an alkyne group, an azide group, a thiol group, a monosulfone, or an ester group, such as succinimidyl, sulfodichlorophenol, pentafluorophenyl, or tetrafluorophenyl.

[0015] In some instances, multiple linkers 24 can be connected to one another in series. For example, a second linker 24 can be connected to a functional group R' of a first linker 24, with the second linker 24 having its own R' for covalently bonding to a moiety 26.

[0016] Moiety 26, in one example, is a fluorescent entity. In this example, compound 20 is a fluorescent dye. The fluorescent entity may be any fluorescent dye known in the art, including, but not limited to, coumarin, benzoxadiazole, acridone, acridine, bisbenzimide, indole, benzoisoquinoline, naphthalene, anthracene, xanthene, pyrene, porphyrin, fluorescein, rhodamine, boron-dipyrromethene (BODIPY), and cyanine derivatives. Many such fluorescent dyes are commercially available. The fluorescent entity may also include tandem dyes, which have two different dyes connected and interact via FRET (fluorescence resonance energy transfer).

[0017] In other examples, moiety 26 is a labeling moiety or other moiety delivered to the body by carrier 22, such as an antibody, a peptide, DNA, RNA, an oligonucleotide, etc.

[0018] Moiety 26, in other examples, is a radioisotope, a ferromagnetic, and / or a molecule having a magnetic element, and a chelator. In these examples, compound 20 can be used as a contrast agent for medical imaging such as PET, SPECT, CT, MRI, etc.

[0019] In another example, moiety 26 can include any combination of the exemplary moieties 26 discussed above. In this example, compound 20 can be used as a heterogeneous probe for biomedical detection and sensing.

[0020] Some nanomaterial carriers 22, especially boron nitride-based nanomaterials, are known to be chemically inert. Thus, it has been difficult to functionalize prior art nanomaterial carriers. However, it has been found that nanomaterial carriers subjected to mechanical treatment in polar liquids (e.g., surfactant solutions or solvents) have an increased tendency to covalently interact with functional groups, such as functional group R of the linker 24. The mechanical treatment is, for example, stirring. Furthermore, it has been found that mechanical treatment of the nanomaterial carrier improves the solubility of the nanomaterial carrier in aqueous solutions, which may aid in biocompatibility. Mechanical treatment can also cut the nanomaterial carriers 22, such as nanotubes, to desired lengths after fabrication by any known method. Certain nanotube lengths, such as BNNTs with lengths of about 100-2000 nm, have advantages in terms of biocompatibility, improved fluorescence when used as fluorescent dyes, improved solubility in water, and / or other advantages that are described in more detail below.

[0021] Nanotubes can be fabricated according to any known method with lengths of about 10,000-500,000 nm and then cut to the desired length. In a particular example, nanotubes are cut to lengths of about 100-2000 nm by mechanical processing. On the other hand, longer nanotubes, or chunks of nanotubes, can be used as carriers 22, as long as they can be functionalized and dispersed in a biocompatible aqueous solution.

[0022] Thus, mechanical treatment cuts the nanotube supports 22 (such as BNNTs or CNTs) to a desired length, leaving them ready for functionalization with linkers 24, and improving their solubility in aqueous solutions. One example method of mechanical treatment is agitation. Agitation can be achieved, for example, by sonication, such as tip sonication or bath sonication.

[0023] During mechanical treatment in a polar liquid (such as an aqueous solution of a surfactant), imperfections 23 (FIG. 1) are formed in the nanotube support 22. If the nanotube support 22 is a tube (e.g., BNNT), the imperfections 23 are formed both at the cut ends of the support 22, e.g., where the tube is cut into shorter tubes during mechanical treatment in solution, and along the length of the tube 22. The imperfections 23 are disrupted or altered parts of the nanotube support 22 structure, thereby exhibiting a local polarity or charge at the imperfections 23. Polar or charged groups from the polar liquid interact with the local polarity or charge at the imperfections. For example, for certain solutions, hydroxyl (-OH) groups from the solution may interact with the imperfections 23, while other solutions may have other polar or charged groups that can interact with the local imperfections 23, such as amino, carboxylic acid, or aldehyde groups.

[0024] The polar or charged groups are themselves polar / charged and thus promote covalent interactions between the nanomaterial support 22 and the functional group R on the linker 24. The polar / charged groups also increase the hydrophilicity of the nanotube support 22 by promoting polar or ionic interactions with water molecules or ions in water. Thus, the nanotube support 22 exhibits improved solubility or dispersion in aqueous solutions after mechanical treatment in the solution.

[0025] After mechanical treatment, the nanotube support 22 has a plurality of defects 23 and associated polar or ionic groups, as described above. Each of those sites is available to covalently interact with a functional group R of the linker 24. Because there are a plurality of such sites on each nanomaterial support 22, a plurality of linkers 24 covalently interact with each nanomaterial support 22. Each linker 24 can be covalently attached to a moiety 26, such as a fluorescent entity, via the functional group R'. Thus, in examples where the moiety 26 is a fluorescent entity, the resulting fluorescent dye 20 has a plurality of fluorescent entities 26 whose fluorescence is cumulative. Thus, the resulting fluorescent dye 20 has an order of magnitude higher fluorescence than prior art fluorescent dyes. In other examples, as described in more detail below, moieties other than the fluorescent entity 26 can be covalently attached to the linker 24 via the functional group R'. In these examples, the nanomaterial support 22 can carry a plurality of moieties.

[0026] In one example method, the BNNT support 22 is placed in a polar liquid. The BNNT support 22 in the solution is then subjected to mechanical treatment in the form of agitation. In this example, the agitation is performed by ultrasonication, although a homogenizer or other methods could also be used. The ultrasonication results in shearing of the BNNT support 22. After ultrasonication, the nanotube support 22 is washed to remove excess solution. For example, washing can be performed with deionized water in a centrifugal filter unit. In this example, the solution is an aqueous solution of sodium cholate, which results in hydroxylation of the BNNT support 22 (addition of hydroxyl functional groups to the support at defects 23), although other solutions may be used.

[0027] After ultrasonic treatment and washing, the BNNT support 22 has hydroxyl groups. This was confirmed by Fourier transform infrared spectroscopy (FTIR) analysis, the results of which are shown in Figure 2B. In Figure 2B, the 3000-3500 cm -1 There is a peak between 0.01 and 0.1, confirming the presence of hydroxyl (-OH) groups. Depending on the solution and functionalization process, other chemical groups may be present after sonication and washing.

[0028] Furthermore, the BNNT support 22 stirred in the surfactant solution exhibits improved solubility in water after stirring. It has also been found that the solubility varies inversely with the length of the BNNT support 22. That is, shorter BNNT supports 22, for example, BNNT supports having lengths of about 100 to 350 nm, exhibit better dispersion (e.g., solubility) than longer BNNT supports 22, for example, BNNT supports having lengths of about 500 nm to 2000 nm.

[0029] It was also found that the BNNT support 22 subjected to tip ultrasonic treatment exhibited autofluorescence (i.e., the BNNT support 22 itself emits fluorescence without binding to the fluorescent entity 26). Figures 3-4 show a group of exemplary excitation graphs of the BNNT support 22 stirred as described above. The functionalized BNNT support 22 formed by the above method exhibited approximately 2 x 10 fluorescence at approximately 250-265 nm, as shown in Figures 3-4. 7 ~3×10 7 and at about 510-520 nm, about 3.5 × 10 6 ~4.5×10 6 The uncut BNNT carrier that has not been subjected to mechanical treatment emits no fluorescence at all.

[0030] In one particular example, the linker 24 is an azido linker. An exemplary azido linker 24 is 3-azidopropyl-triethoxysilane, although other azido linkers 24 are also contemplated. After the nanomaterial carrier 22 has undergone mechanical treatment, the 3-azidopropyl-triethoxysilane can be added to the nanomaterial carrier 22 as shown in the following formula 1:

[0031] [ka]

[0032] As shown in formula 1, the hydroxyl group on the BNNT support 22 is covalently bonded to the silicon atom of the azido linker 24 to form the intermediate support 22 / linker 24 (although in other examples, the functionalized support 22 may have different functional groups available for covalent bonding, as described above). The azido (N3) group in this example is the functional group R' of the linker 24. The intermediate support 22 / linker 24 is then covalently bonded to the moiety R2 by any known chemistry. Figure 5 shows the XPS (X-ray photoelectron spectroscopy) spectrum of the functionalized support 22 with the 3-azidopropyl-triethyoxysilane linker 24, confirming successful covalent functionalization with the BNNT support 22 with the expected peaks.

[0033] In a particular example, the moiety R2 in formula 1 is FITC, a green fluorescent entity 26, and the resulting compound is a green fluorescent dye 20. Due to the mechanical treatment of the nanomaterial support 22 and the availability of functional groups for covalent attachment to the linkers 24, the functionalized support 22 binds more linkers 24, and therefore more fluorescent entities 26, than conventional nanomaterial supports, as described above. Thus, the resulting green fluorescent dye 20 exhibits a fluorescence intensity several orders of magnitude higher than that of the prior art fluorescent dyes, when compared at the same concentration of fluorescent dye units. As shown in FIG. 6, at about 520 nm, the green fluorescent dye 20 has a fluorescence intensity of about 2.5×10 6 These samples have approximately 10 8 to 10 12 fluorescent dyes 20.

[0034] In another specific example, the nanomaterial carrier 22 / azide linker 24 is further functionalized with a second linker 24 comprising a PEG (polyethylene glycol) chain, such as alkyne-PEG5K-amino, where the amino (NH2) group is the functional group R' in formula 2 below. The PEG group improves the water solubility of the resulting carrier 22 / linker 24, making it easier to disperse in aqueous solutions such as PBS, plasma, etc. Water solubility may be desirable for certain biological applications. The length, composition, and degree of branching of the PEG chains can be selected to obtain the desired solubility. It should also be understood that other organic chains may be used in place of PEG to achieve the same effect. The intermediate carrier 22 / linker 24 is covalently attached to the moiety R2 by any well-known chemistry.

[0035] [ka]

[0036] 7A-C show the fluorescence intensity of fluorescent dyes 20 made by appending FITC (green), sulforhodamine (red), and sulfoCy5.5 (far red) fluorescent entities 26, respectively, to a carrier 22 / linker 24 shown in Formula 2, whereby the fluorescent entities 26 are covalently attached to the functional groups of the linker 24 by any well-known chemistry. As shown, the green fluorescent dye 20 has an intensity of approximately 1.2×10 at 520 nm. 6 The red fluorescent dye has a fluorescence intensity of about 7.8 × 10 at about 580 nm. 6 and the far-red fluorescent dye has a fluorescence intensity of about 3.5 × 10 at about 685 nm. 5 The samples have approximately 10 to 10 fluorescent dyes 20, and the fluorescence intensity per fluorescent dye 20 is several hundred to several thousand times higher than that of the fluorescent dyes of the prior art due to the different numbers of covalently attached fluorescent entities 26.

[0037] The length of the PEG or other organic chain in the above examples may affect the brightness of the resulting fluorescent dye 20 in some cases. For example, a fluorescent dye 20 without PEG or organic chains prepared as shown in formula 1 above ("direct bonding fluorescent dye") is less bright than a fluorescent dye with PEG or organic chains prepared as shown in formula 2 above ("chain fluorescent dye") after adjustment for sample size (e.g., the number of fluorescent dyes in the sample used to measure the fluorescence intensity, 20, as shown in Figures 6 and 7A, respectively). It was observed that the direct bonding fluorescent dye prepared as shown in formula 1 above exhibited only 2-fold higher fluorescence intensity than the chain fluorescent dye prepared as shown in formula 2 above, even though the sample of the direct bonding fluorescent dye had 571-fold higher fluorescent dye concentration than the sample of the chain fluorescent dye. This suggests that the use of a longer linker 24 (e.g., a linker 24 with PEG or other organic chains) can increase the solubility and brightness of the fluorescent dye 20. Without being bound to a particular theory, this may be due to a reduction in the quenching effect discussed above.

[0038] In another specific example, an amine linker 24 is added to the nanomaterial support 22. One example amine linker is 3-aminopropyltrimethoxysilane. As described above, the silicon atom of 3-aminopropyltrimethoxysilane is covalently bonded to a functional group (e.g., a hydroxyl group) of the functionalized support 22 to form the intermediate support 22 / linker 24. Figures 8A-B show the fluorescence intensity of fluorescent dyes 20 made by adding FITC (green) and sulforhodamine (red) fluorescent entities 26 to the support 22 / linker 24, respectively, resulting in covalent attachment of the fluorescent entity 26 to the functional group R' of the linker 24, which in this example is an amine. As shown, the green fluorescent dye 20 has a fluorescence intensity of about 3.7 x 10 at 520 nm. 6 The red fluorescent dye has a fluorescence intensity of about 2 × 10 at about 580 nm. 5 These samples (fluorescent dyes 20) have different numbers of added dyes, and therefore have approximately 10 8 to 10 12 fluorescent entities 26.

[0039] In addition to the azide and amine exemplary linkers 24 described above, other linkers with other functional groups may be used. For example, a linker 24 containing alkyne-NHS may be used according to known functionalization practices and covalently attached to the amino group of the Fc group of an antibody. Other small molecules (such as sugars, nitroxides, biotin, drugs, etc.), macromolecules, peptides, DNA, RNA sequences, proteins such as biotin, SA (streptavidin and its derivatives) may be used as targeting moieties 26.

[0040] Furthermore, the linking of the functionalized carrier 22 is not limited to the aminosilane linker 24. Other linkers 24 may have various functional groups such as amino, carboxylic acid, succinimidyl ester, maleimide, carboimide, pyridyldithiol, haloacetyl, arylazide, azide, alkyne, hydrazide, and monosulfone groups. These chemical groups can be used to bind the carrier 22 to dyes, drugs, or any targeting substance. Crosslinkers containing dual functional groups can be used to obtain functional groups that allow the linker 24 to be attached to other entities such as dyes, peptides, oligonucleotides, DNA, RNA, antibodies, proteins, drugs, or other nanoparticles. The crosslinkers are SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC (sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), AMAS (N-α-maleimidoaceto-oxysuccinimide ester), BMPS (N-β-maleimidopropyl-oxysuccinimide ester), GMBS (N-γ-maleimidobutyryl-oxysuccinimide ester), sulfo-GMBS, MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), sulfo-MBS, EMCS (N-ε-maleimidocaproyl-oxysuccinimide ester), sulfo-EMCS, SMPB (succinimidyl 4-(p-maleimidomethyl)cyclohexane-1-carboxylate), and BMPS (N-β-maleimidopropyl-oxysuccinimide ester). dophenyl)butyrate), Sulfo-SMPB, SMPH (Succinimidyl 6-((beta-maleimidopropionamido)hexanoate), LC-SMCC (Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), Sulfo-KMUS (N-κ-maleimido undecanoyl-oxysulfosuccinimide ester), SM(PEG)n (n=2,4,6,8,12,24) (PEGylated SMCC crosslinker), SPDP (Succinimidyl 3-(2-pyridyldithio)propionate), LC-SPDP, Sulfo-LC-SPDP, SMPT (4-Succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene), PEGn-SPDP (n=2,4,12,24), SIA (succinimidyl iodoacetate), SBAP (succinimidyl 3-(bromoacetamido)propionate), SIAP (succinimidyl (4-iodoacetyl)aminobenzoate), sulfo-SIAP, ANB-NOS (N-5-azido-2-nitrobenzoyloxysuccinimide), sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate), SDA (succinimidyl 4,4'-adipentanoate), sulfo-SDA, LC-SDA, sulfo-LC-SDA, SDAD (succinimidyl 2-(( 4,4'-azipentanamido)ethyl)-1,3'-dithiopropionate), sulfo-SDAD, DCC (N,N'-dicyclohexylcarbodiimide), EMCH (N-ε-maleimidocaproic acid hydrazide), MPBH (4-(4-N-maleimidophenyl)butyric acid hydrazide), KMUH (N-κ-maleimidoundecanoic acid hydrazide), PDPH (3-(2-pyridyldithio)propionyl hydrazide), PMPI (p-maleimidophenyl isocyanate), SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate), or other well-known crosslinkers.

[0041] Although the above description has been made with respect to BNNTs, CNTs can be similarly functionalized to serve as the support 22. CNTs can be functionalized by agitation as described above with respect to BNNTs. CNTs are also conventionally known to respond to functionalization by chemical means such as acid treatment. Like the functionalized BNNT support 22, the functionalized CNT support 22 exhibits autofluorescence and an improved ability to link to fluorescent entities. Thus, the fluorescent dye 20 with the CNT support 22 exhibits increased fluorescence compared to prior art fluorescent dyes with non-functionalized supports. EXAMPLES

[0042] Synthesis of BNNT-Si-azide: The sodium cholate cleaved BNNTs were washed in excess with distilled water for 5 min approximately 5-6 times by using 10 kDa at 4000 g. This process also enriches the BNNT samples. Metal contamination was then washed by acid treatment using HCl or HNO3. After acid treatment, the BNNTs were neutralized and washed in excess with distilled water during vacuum filtration from anodic membranes (pore size: 20 nm). The collected BNNT-OH was resuspended in EtOH using a sonication bath. Then, the ethanol was evaporated and the BNNTs (2.5 mg) were suspended in 5 ml of toluene and 1 ml of ethanol. Then, 20 μl of (3-azidopropyl)triethoxysilane was added and stirred overnight at 110 °C under nitrogen. The next day, the solvent was evaporated and resuspended in EtOH, and the BNNTs were washed and collected via vacuum filtration on anodic membranes (pore size: 20 nm). The BNNTs were analyzed by XPS to confirm the presence of Si.

[0043] Synthesis of BNNT-Si-triazole-FITC: BNNT-Si-azide (500 μl) was mixed with 20 μl of sodium ascorbate (0.12 M) and 20 μl of copper sulfate (0.12 M). Then, 2 μl of FITC-alkyne (3190 nM) was added from a stock solution in EtOH / DMSO. After stirring overnight, the BNNTs were washed with distilled water and washed excessively with EtOH (70%) through an anodic membrane via vacuum filtration. Afterwards, the BNNTs were collected in a glass vial by resuspension in EtOH. The solvent was evaporated and dried overnight. The BNNT-Si-FITC was then resuspended in 1 ml of PBS and the fluorescence intensity was measured via a Horiba Fluoromax-4.

[0044] Synthesis of BNNT-Si-amine: BNNT-OH (1 mg) was dispersed in 5 mL of toluene and 50 μl of (3-aminopropyl)triethoxysilane (APTES) was added. The reaction was heated to reflux and stirred overnight under nitrogen. The solvent was then removed by evaporation under reduced pressure. The residue was dispersed in 70% ethanol and washed through the anodic membrane with 70% ethanol and water. The precipitant was collected by rinsing the membrane with ethanol and concentrated solvent. The product was then dispersed in 2 mL of anhydrous DMSO and kept at 4 °C under nitrogen.

[0045] Synthesis of BNNT-Si-amine sulforhodamine: BNNT-silicone amine (500 μl in DMSO) was removed from the stock solution and mixed with 5 μl of sulforhodamine acid B chloride (15 mM in DMSO), 1 drop of triethylamine and 1 ml of chloroform. The reaction was allowed to proceed overnight under nitrogen. The chloroform was removed by evaporation and the residue was then diluted with 5 ml of water, keeping the DMSO below 10%. The product was collected by filtration from the anodic membrane.

[0046] Synthesis of BNNT-Si-amine FITC: The procedure was the same as for sulforhodamine: Fluorescein isothiocyanate (3.2 mM in EtOH+DMSO) was used as the starting material.

[0047] Synthesis of BNNT-Si-triazole-PEG5K-azide: BNNT-Si-N3 (0.3 mg in 1 ml distilled water) was mixed with 20 μl CuSO4 (0.12 M) and 20 μl sodium ascorbate (0.12 M). Then, 5.3 mg alkyne-PEG5K-amino was added. The mixture was stirred at room temperature for 2 h, then overnight, and then extracted with chloroform. The mixture was then evaporated, dispersed in saturated sodium bicarbonate, and 45 mg CuSO4 was added. Then, a stock solution of trifluoromethanesulfonic acid azide was added. Trifluoromethanesulfonic acid azide was synthesized according to well-known procedures. Sodium azide (0.4 g) was dissolved in 1 ml distilled water and 1 ml toluene in an ice bath. Then, 0.6 g trifluoromethanesulfonic anhydride was added dropwise and stirred for 2 h. Then, it was extracted with 6 ml toluene. This stock solution was used to convert the amino functionality to azide. After adding trifluoromethanesulfonic acid azide to the BNNT-Si-triazole-PEG5K-azide and stirring overnight, the BNNTs were extracted with chloroform. After evaporation, they were dispersed in PBS. The transmittance was measured to determine the concentration.

[0048] Synthesis of BNNT-Si-triazole-PEG5K-triazole-dye: BNNT-Si-triazole-PEG5K-azide (500 μl, at a concentration of 83 μg / ml) was already dispersed in PBS. CuSO4 (20 μl, 0.12 M) and sodium ascorbate (20 μl, 0.12 M) were added to the BNNT-azide, followed by 20 μl of FITC-alkyne (3.2 mM) and stirring overnight in a refrigerator. The next day, it was filtered through an anodic membrane and washed excessively with water and 70% ethanol. It was then dispersed in PBS and fluorescence was measured with a Horiba Fluoromax-4. Similarly, for sulforhodamine, 20 μl of sulforhodamine alkyne (15 mM) was used, and for sulfocyanine dye synthesis, 20 μl of sulfocyanine 5 alkyne (3.5 mM, dissolved in DMSO:water (1:1)) was used. Excess dye was removed by washing excessively with distilled water and 70% EtOH.

[0049] Synthesis of antibody alkyne: Anti-human CD4 (200 μg) was washed three times with PBS to remove sodium azide, and the sample was concentrated to 80 μl using a 30 KDa filter unit. Then, 20 μl of DTT from a 100 mM stock solution was added and dispersed in distilled water. After 30 min, the antibody was washed about five times with PBS to remove DTT. After concentrating the reduced antibody, it was mixed with 100 μl of maleimide-PEG4-alkyne (10.2 mg / ml in PBS). It was then stirred overnight and purified using a 30 KDa filter unit. Product formation was confirmed by SDS non-reducing gel at 150 V for 70 min.

[0050] The foregoing description is exemplary rather than limiting in nature. Variations and modifications of the disclosed examples will become apparent to those skilled in the art that do not necessarily depart from the essence of this invention. The scope of legal protection given to this invention can only be determined by studying the following claims.

Claims

1. A nanotube support; Part and a linker having a first and a second functional group, the first functional group covalently attached to the nanotube support and the second functional group covalently attached to the moiety; Equipped with the nanotube support is a boron nitride nanotube (BNNT); the boron nitride nanotubes have a length of 100 to 2000 nm; A compound, wherein the outer diameter of said nanotube support is greater than 0.4 nm and less than 80 nm.

2. 2. The compound of claim 1, wherein the nanotube support is a multi-walled nanotube support.

3. 2. The compound of claim 1, further comprising a plurality of linkers covalently attached to the nanotube support and a plurality of moieties, each linker being linked to one of the plurality of moieties.

4. 2. The compound of claim 1, wherein the nanotube support has at least one polar group, and the first functional group is covalently bonded to the nanotube support at the at least one polar group.

5. 5. The compound of claim 4, wherein the at least one polar group is a hydroxyl (-OH) group.

6. 2. The compound of claim 1, wherein the moiety comprises at least one of a fluorescent entity, a biomolecule, a chelator, and combinations thereof.

7. 2. The compound of claim 1, wherein the linker comprises a first linker and a second linker, the first linker having the first and third functional groups, the second linker having the fourth and second functional groups, the first linker is covalently attached to the nanotube support via the first functional group, the second linker is covalently attached to the first linker via a covalent interaction between the fourth functional group and the third functional group, and the moiety is covalently attached to the second functional group.

8. mechanically treating the nanotubes in a polar liquid, thereby creating defects on said nanotubes and providing polar groups to the defects; covalently attaching a linker to the nanotube; Prepare for this. the linker has a first and a second functional group, the first functional group being covalently bonded to the polar group; said mechanical treatment results in severing of said nanotubes; the nanotubes have a length of 100-2000 nm after the mechanical treatment; The method for producing a nanotube support, wherein the outer diameter of the nanotube is greater than 0.4 nm and less than 80 nm.

9. 9. The method of claim 8, wherein the moiety covalently attached to the second functional group is a fluorescent entity.

10. 9. The method of claim 8, wherein the mechanical treatment comprises stirring.

11. 11. The method of claim 10, wherein the stirring is performed by sonication or a homogenizer.

12. 9. The method of claim 8, wherein the polar group is a hydroxyl (-OH) group.

13. 9. The method of claim 8, wherein the nanotubes are boron nitride nanotubes (BNNTs).

14. 9. The method of claim 8, wherein the nanotubes are carbon nanotubes (CNTs).

15. 9. The method of claim 8, wherein the nanotubes are multi-walled nanotubes.

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