High-brightness nanodot fluorescent dyes achieved through covalent functionalization

Abstract

Commercially available fluorescent dyes have low brightness and poor photostability, making reliable tracking and quantification of these dyes difficult. Therefore, we provide improved carrier molecules for delivering fluorescent entities for biological and other applications. [Solution] Exemplary compounds according to this disclosure include, among other things, a nanodot carrier, a moiety, and a linker having first and second functional groups, the first functional group being covalently bonded to the nanodot carrier and the second functional group being covalently bonded to the moiety. Exemplary methods for producing the nanodot carrier are also disclosed.

Description

Technical Field

[0001] (Cross-Reference to Related Applications) This application claims the priority benefit of U.S. Provisional Patent Application No. 62 / 855,121, filed on May 31, 2019, which is incorporated herein by reference in its entirety. This application is a continuation-in-part of U.S. Patent Application No. 15 / 953,200, filed on April 13, 2018, which claims the priority benefit of U.S. Provisional Patent Application No. 62 / 485,379, filed on April 13, 2017, which is also incorporated herein by reference in its entirety.

[0002] (Statement on Federally Sponsored Research) The invention described herein was made with government support under Grant No. 1261910, Grant No. 1521057 and Grant No. 1738466 awarded by the National Science Foundation. The United States Government has certain rights in this invention.

Background Art

[0003] Fluorophores are compounds with fluorescent properties that have biomedical applications. For example, fluorophores can be used as tracers or dyes to specifically stain specific molecules or structures. More specifically, fluorophores 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, fluorophores need to be conjugated to biomolecules such as antibodies. However, commercially available fluorophores have low brightness and poor photostability, making reliable tracking and quantification of fluorophores difficult. Accordingly, there is a need for improved carrier molecules for carrying fluorescent entities for biological and other applications. Other biological molecules can also benefit from improved carriers.

Summary of the Invention

Means for Solving the Problems

[0005] The exemplary compounds described herein include, among other things, a nanodot support, a moiety, and a linker having first and second functional groups, the first of which is covalently bonded to the nanodot support, and the second of which is covalently bonded to the moiety.

[0006] Exemplary methods for fabricating nanodot supports according to the present disclosure include, in particular, mechanically treating nanodots in a polar liquid to create defects on the nanodots, and treating the nanodots to provide polar groups in the defects. [Brief explanation of the drawing]

[0007] [Figure 1A] This is a schematic diagram of an example compound having a nanodot support. [Figure 1B] This is a schematic diagram of the synthesis of exemplary compounds, such as the compound shown in Figure 1A, from a BN nanodot support. [Figure 1C] This is a schematic diagram of the synthesis of exemplary compounds, such as the compound shown in Figure 1A, from a BN nanodot support. [Figure 2A] This figure shows a scanning electron microscope (SEM) image of h-BN bulk powder. [Figure 2B] This figure shows an SEM image of h-BN powder after mechanical processing, in this example, processing with a homogenizer. [Figure 2C] This figure shows a TEM (transmission electron microscope) image of an exemplary boron nitride (BN) nanodot support. [Figure 2D] This figure shows excitation-dependent autofluorescence and fluorescence images under a UV lamp (inset) of an exemplary BN nanodot support. [Figure 3A] This figure shows the Fourier transform infrared spectroscopy (FITR) results for the original BN nanodot support and the treated BN nanodot support. [Figure 3B] This figure shows the Fourier transform infrared spectroscopy (FITR) results for the original BN nanodot support and the treated BN nanodot support. [Figure 4]Figure 1B shows the absorbance spectra of an exemplary fluorescent dye, the original carrier, and the treated carrier. [Figure 5] Figure 1B shows the fluorescence intensities of an exemplary fluorescent dye, the original carrier, the treated carrier, and the treated carrier with a linker (i.e., a functionalized carrier). [Modes for carrying out the invention]

[0008] Generally, high-brightness fluorescent dyes consist of a carrier element, a fluorescent element, and a linker that connects the carrier element to the fluorescent element. For biomedical applications, the carrier element, linker, and fluorescent element must each be biocompatible (although the biocompatibility requirements vary depending on the specific application).

[0009] One example of a support element is a treated nanomaterial such as carbon nanotubes (CNTs) and boron nitride nanotubes (BNNTs), both of which can be used in biomedical applications such as cell-drug delivery and spectroscopic applications. However, fluorescent elements bound to carbon nanotubes have been shown to exhibit quenching, or a decrease in fluorescence brightness.

[0010] Certain fluorescent dyes with nanomaterial supports not only do not exhibit quenching effects, but also, as discussed herein, exhibit brightness several orders of magnitude higher than other known fluorescent dyes.

[0011] Referring here to Figure 1A, a fluorescent dye 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 is, in one example, a processed BNNT or CNT support. In the example in Figure 1A, the support 22 is a zero-dimensional BN "dot" (e.g., the size of all three dimensions of the dots is nanoscale, i.e., less than about 100 nm), but carbon dots may also be used. In a more specific example, all three dimensions of the dot support are less than about 20 nm. Another exemplary support 22 is a multilayer BNNT or CNT support, where each BNNT or CNT has multiple coaxial shells of hexagonal boron nitride (h-BN in the case of BNNTs) or graphene (in the case of CNTs), with a typical outer diameter greater than about 0.4 nm but less than about 100 nm. The lengths of these BNNTs and CNTs are about 1–100 nm. In other examples, the support 22 may be another nanoscale inorganic material, such as hexagonal boron nitride (h-BN) nanosheets / nanoparticles, graphene / graphite nanosheets / nanoparticles, molybdenum disulfide (MoS2) nanosheets / nanoparticles, any transition metal dichalcogenide (TMDC) nanosheets / nanoparticles, and any nanosheets / nanoparticles of layered materials (materials having a covalent layer structure bonded by van der Waals forces between layers).

[0013] Linker 24 has two or more functional groups R and R', as shown in Figures 1A and B. Functional groups R and R' are reactive groups that promote the covalent bonding of the linker to other structures through known chemical actions. R and R' are the same or different functional groups. For example, R and R' are ethoxysilane and azide, respectively. R and R' may be any known functional group such as an amine group, carboxylic acid, isothiocyanate, maleimide, alkyne group, hydroxyl group, thiol group, monosulfone, or ester group such as succinimidyl, sulfodichlorophenol, pentafluorophenyl, or tetrafluorophenyl. Linker 24 is any type of molecule having two or more functional groups R and R'. One example of linker 24 is a linear or branched polymer molecule. In some examples, linker 24 has a length of less than about 200 nm. In some examples, multiple linkers 24 can be connected in series with each other.

[0014] One functional group R interacts covalently with the carrier 22. The carrier 22 having the linker 24 is known as a "functionalized" carrier 220, as shown in FIG. 1B. That is, when covalently bonded to the linker 24, the carrier 22 / linker 24 structure has a functional group R' that promotes covalent bonding of the carrier 22 / linker 24 to another moiety 26.

[0015] In one example, the moiety 26 is a fluorescent entity. In this example, the molecule 20 is a fluorescent dye. The fluorescent entity is 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 can also include tandem dyes that have two different dyes connected and interact via FRET (fluorescence resonance energy transfer). The fluorescent entity interacts covalently with the functional group R' of the linker 24 as described above.

[0016] In other examples, the moiety 26 is a labeling moiety or other moiety delivered to the human body by the carrier 22, such as an antibody, peptide, DNA, RNA, or oligonucleotide.

[0017] In other examples, the moiety 26 is a molecule or chelating agent having a radioisotope, a ferromagnetic element, and / or a magnetic element. In these examples, the compound 20 can be used as a contrast agent for medical imaging such as PET, SPECT, CT, and MRI.

[0018] In another example, the moiety 26 can comprise a combination of any of the exemplary moieties 26 discussed above. In this example, the compound 20 can be used as a heterogeneous probe for biomedical detection and sensing.

[0019] Some nanomaterial carriers 22, especially boron nitride (BN)-based nanomaterials, are known to be chemically inert. Accordingly, it has been difficult to functionalize prior art nanomaterial carriers for covalent interaction with other structures. However, it has been found that carriers 22 such as the BN dot carrier shown in FIGS. 1A to 1C, which have been subjected to mechanical treatment in a solution or solvent such as stirring, exhibit an increased tendency to interact covalently with functional groups such as the functional group R on the linker 24. The solution / solvent may be the same solution / solvent in which the raw material is processed to form nanodots, as described in more detail below, or may be a different solution / solvent. Furthermore, it has been found that mechanical treatment of the nanomaterial carrier improves the solubility of the nanomaterial carrier in an aqueous solution, which can improve biocompatibility. Furthermore, mechanical treatment cleaves the carrier material into smaller fragments, which may be desirable, for example, when forming dots. Stirring can be achieved by a homogenizer and / or sonication, for example, tip sonication or bath sonication.

[0020] Referring now to FIG. 1B, mechanical treatment results in the carrier 22 having imperfections 23. During mechanical treatment in the solution / solvent, imperfections 23 are formed on the carrier 22, and local polarity or charge is formed at the imperfections 23. Polar or charged groups from the solution / solvent interact with the local polarity or charge at the imperfections. For example, in the example of FIG. 1B, the carrier is an h-BN nanodot carrier 22. In this specific example, the imperfection 23 is a break in the hexagonal structure of the boron nitride material, and the break has a local polarity imbalance. For example, for a particular solvent / solution, hydroxyl groups from the solvent / solution may interact with the imperfections 23, while other solvents / solutions may have other polar or charged groups capable of interacting with the local imperfections 23, such as amino groups, carboxylic acid groups, or aldehyde groups, depending on the treatment and type of the solvent / solution.

[0021] In one particular exemplary method for preparing the carrier 22, the h-BN powder is treated in dimethylformamide (DMF) or another polar solution / solvent for 2–4 hours by using a homogenizer. In one example, the treatment in a polar solvent is solvothermal (e.g., the solvent / solution is heated). In one example, the h-BN powder has an average particle size between approximately 10–20 μm. In a specific example, the average particle size (e.g., diameter) is approximately 13 μm. Figure 2A shows an image of an example of h-BN particles with an average size of approximately 13 μm before treatment with DMF. The homogenizer makes the BN dot carrier 22 smaller and leaves it suspended in the DMF solution. In this example, after treatment with the DMF solution, the BN dot carrier 22 becomes smaller, and its size is reduced to less than approximately 2–5 μm, as shown in Figure 2B.

[0022] After DMF treatment, the BN dot carrier 22 suspension undergoes agitation treatment such as sonication. In certain cases, the suspension is treated by bath sonication for 20-30 hours. The size of the BN dot carrier 22 is reduced to approximately 1-3 μm after sonication.

[0023] After stirring, the DMF / BN dot carrier 22 suspension is heat-treated. In certain cases, the suspension is heated at 150°C for 7–12 hours while being stirred with an electromagnetic stirrer. The stirring bar ensures that the BN dot carrier 22 remains suspended in the DMF solution.

[0024] As shown in the example in Figure 1B, stirring and heat treatment result in a carrier 22 having defects 23.

[0025] After heat treatment, the support 22 suspension is centrifuged to precipitate larger particles. In a specific example, the suspension is centrifuged at 10,000 rpm for 10 minutes. In this example, the size of the support 22 in the suspension is approximately 2–10 nm after heat treatment and centrifugation, as confirmed by the TEM (transmission electron microscope) image shown in Figure 2C. Furthermore, the support 22 is barely visible using SEM images, confirming that the support 22 is very small and has nanoscale dimensions. Generally, the support 22 has fewer than 30 layers of h-BN, corresponding to a thickness dimension of less than approximately 100 nm. The length / width dimension is also less than approximately 100 nm. In a specific example, the support 22 has approximately 4–8 layers of h-BN and dimensions of approximately 2–10 nm.

[0026] After centrifugation, the carrier 22 suspension undergoes solvent exchange; that is, the solvent (DMF) is replaced with another solvent, water. As discussed herein, the carrier 22 suspended in water is ready for biological applications or binding with the portion 26 to be carried. Solvent exchange is carried out as follows: The DMF is evaporated into the air by heating the suspension. In certain examples, the suspension is heated to 150°C until the DMF evaporates. After heating, the remaining carrier 22 is placed in a water / ethanol mixture. In certain examples, the water / ethanol mixture is 50% water and 50% ethanol. The carrier 22 / water / ethanol mixture is then heated to evaporate the ethanol at a suitable temperature as known in the art. In certain examples, the DMF can be removed by vacuum treatment, and the carrier 22 can then be suspended in water.

[0027] It has been found that producing the carrier 22 according to the above method yields a production yield that is orders of magnitude higher than that of conventional methods. For example, in the case of a method involving 20-30 minutes of bath ultrasonic treatment, 7-12 hours of heat treatment with stirring in an electromagnetic stirrer, and 10 minutes of centrifugation at 10,000 rpm, the production yield is approximately 47%, compared to 1-26% reported by conventional methods. The production yield is the weight percentage of the h-BN bulk powder that becomes the carrier 22 after the evaporation step described above.

[0028] In the case of an exemplary DMF solution, hydrocarbon groups or fragments from the solution interact with localization polarity at defects 23 of the support 22, while other solutions may have other polar groups that can interact with localization polarity, such as aminos, carboxylic acids, and aldehydes. The support 22 is then subjected to acid treatment according to any known method, which replaces the hydrocarbon groups or fragments at defects 23 of the support 22 with hydroxyl groups (-OH groups), resulting in a treated support (described in more detail below). The acid treatment also removes other contaminants from the support 22, such as hydrocarbon fragments of DMF. The treated support is then linked to the linker 24 by any known chemical action that covalently bonds the hydroxyl groups to the R groups of the linker 24 to form a functionalized support 220.

[0029] The support 22 prepared according to the above method is autofluorescent; that is, the support 22 has a measurable intrinsic fluorescence. Figure 2D shows the fluorescence intensity of the support 22 shown in Figures 2A-C, formed by the above method. Without being constrained by any particular theory, the autofluorescence may be related to defects 23 formed on the surface and edges of the support 22 during the above method. The defects 23 can be coupled with hydrocarbon fragments of DMF, including carbon-substituted N vacancy defects, carbene structures at the zigzag edges, and BO2- and BO- species. These defects 23 are expected to create a series of energy states near the edges of the valence and conduction bands of the h-BN material.

[0030] Figures 1B-1C show the synthesis of compound 20. In this example, the support is an h-BN support 22 prepared by treating h-BN powder in a polar organic solvent to facilitate the placement of h-BN onto nanodots. Examples of polar organic solvents are dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), and ethanol. In a specific example, the support 22 is prepared according to the method described above.

[0031] In the example in Figure 1B, the h-BN dot carrier 22 prepared according to the method described above is treated with an acid, in this case nitric acid (HNO3), to provide a treated carrier 210. The acid treatment results in the attachment of -OH (hydroxyl) groups to defects 23, which have an unbalanced polarity attracted to the -OH groups as described above. Figures 3A-B show the FTIR (Fourier transform infrared spectroscopy) spectra of the treated carrier 210 and the unfunctionalized ("original") h-BN dot carrier 22. As shown in Figure 3A, the CH stretching from the DMF fragment at 2950 cm⁻¹ in the original h-BN dot carrier 22 disappeared after nitric acid treatment. A broad IR (infrared) band at 3100 cm⁻¹ was detected from the treated sample, indicating the introduction of hydroxyl groups after acid treatment. After the removal of DMF and contaminants, there is a redshift in the -OH band due to a slight energy band change at the zigzag edge of the treated carrier 210. The removal of these DMF fragments is also supported by the disappearance of BO (~1255 cm-1), BC, or CN (~1150 cm-1) bonds, as shown in Figure 3B. In other words, this FTIR analysis confirms the presence of hydroxyl groups on the treated support 210 by the presence of expected peaks in the spectrum.

[0032] The -OH group bonded to defect 23 is itself polar / charged. Returning to Figure 1B, the polar or charged group (e.g., the -OH group in this example) facilitates the covalent interaction between the treated support 210 and the functional group R on the linker 24. The polar group also increases the hydrophilicity of the treated support 210 by facilitating polar or ionic interactions with water molecules or ions in water. Thus, the functionalized support 220 exhibits improved solubility dispersion in aqueous solution compared to other supports that do not contain the treated support 210.

[0033] The treated support 210 has an increased capacity for attachment to the linker 24 and therefore to the portion 26 compared to the unfunctionalized support due to the polar or charged groups. More specifically, the polar or charged groups act as reaction sites for covalently bonding the treated support 210 to the linker 24 via the functional group R. Therefore, since the functionalized support 220 can be linked to multiple fluorescent entities 26, the brightness of the fluorescent dye 20 having the functionalized support 220 and the fluorescent entities 26 is higher than that of conventional fluorescent dyes. More generally, the functionalized support 220 can be linked to more portions 26 than the untreated support.

[0034] In a specific example, a BN dot carrier 22 processed to form a carrier 210 processed as described above has four layers of h-BN, each with a diameter of approximately 2.5 nm. Each layer, after processing as described above, can be bound to 10 or more linkers 24 and fluorescent entities 26 or other parts 26. Thus, the exemplary processed carrier 210 can be bound to 40 or more linkers 24 and fluorescent entities 26 to form a fluorescent dye. Therefore, the fluorescent dye 20 is more than 40 times brighter than a carrier with a single fluorescent entity. In the case of branched linkers (n-branched), the intensity is 40n times the intensity of a carrier with a single fluorescent entity.

[0035] Turning back to Figure 1B, an example triethoxysilane linker 24, which in this particular example is 3-(azidopropyl)triethoxysilane, is linked to the treated carrier 210. In this example, the R group is an ethoxysilane group and the R' group is an azide group. As shown in Figure 1B, the R group is reactive with the treated carrier 210 at defect 23 (particularly the polar charged group of defect 23), and the R' group is reactive with portion 26.

[0036] In another example, linker 24 is an aminosilane linker. Other linker 24s may have a variety of functional groups, including amino, carboxylic acid, succinimudyl ester, maleimide, carboimide, pyridyldithiol, haloacetyl, aryl azide, azide, alkyne, hydrazide, and monosulfone groups. These chemical groups can be used to link the support 22 to a dye, drug, or any target substance. Crosslinking agents containing double functional groups can be used to obtain functional groups that link linker 24 to other entities such as dyes, peptides, oligonucleotides, DNA, RNA, antibodies, proteins, drugs, or other nanoparticles. These crosslinking agents include SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC ((succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), AMAS (N-α-maleimoidaceto-oxysuccinimidester), BMPS (N-β-maleimidopropyl-oxysuccinimidester), GMBS (N-γ-maleimidobutyryl-oxysuccinimidester), sulfo-GMBS, MBS (m-maleimidobenzoyl-N-hydroxysuccinimidester), sulfo-MBS, EMCS (N-ε-maleidocaproyl-oxysuccinimidester), sulfo-EMCS, and SMPB (succinimidyl 4-(p-maleimidomethyl) (dophenyl)butyrate), sulfo-SMPB, SMPH (succinimidyl 6-((beta-maleimidopropionamide)hexanoate), LC-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidecaproate)), sulfo-KMUS (N-κ-maleimidoundecanoyl-oxysulfosuccinimid ester), SM(PEG)n (n=2,4,6,8,12,24) (PEGylated SMCC crosslinking agent), 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 (succinimidyliodoacetate), SBAP (succinimidyl 3-(bromoacetamide)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'-azipentanoate), sulfo-SDA, LC-SDA, sulfo-LC-SDA, SDAD (succinimidyl 2-( (4,4'-adipentanamide)ethyl)-1,3'-dithiopropionate), sulfo-SDAD, DCC (N,N'-dicyclohexylcarbodiimide), EMCH (N-ε-maleimidocaproate hydrazide), MPBH (4-(4-N-maleimidophenyl)butyrate hydrazide), KMUH (N-κ-maleimidoundecanoate hydrazide), PDPH (3-(2-pyridyldithio)propionyl hydrazide), PMPI (p-maleimidophenyl isocyanate), SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate), or other known linkers.

[0037] In the example in Figure 1B, portion 26 is a fluorescent entity, specifically FITC (fluorescein isothiocyanate), a green dye. FITC can be conjugated to linker 24 at R' by any known chemical reaction. For example, in the case of the azido-silane linker 24 in Figure 1B, a copper(I)-induced click reaction can be used to covalently bond the R' group of linker 24 to the alkyne group of FITC.

[0038] Figure 4 shows the absorbance spectrum of the exemplary fluorescent dye 20 in Figure 1B. The characteristic absorbance signal of FITC at approximately 490 nm (indicated by the arrow) and the peak at 280 nm, attributed to the aromatic triazole, confirm the presence of the fluorescent dye 20 and its binding to the treated support 210, linker 24, and FITC entity 26. Figure 4 also shows the absorbance spectra of the original support 22 and the treated support 210 for comparison.

[0039] Figure 5 shows the fluorescence intensity of the exemplary fluorescent dye 20 in Figure 1B after excitation by irradiation at 492 nm. The fluorescent dye 20 emits light at 515 nm, which is a characteristic emission signal of the FITC molecule. This confirms that the FITC molecule is covalently bonded to the fluorescent dye 20. The fluorescence intensities of the original support 22, the treated support 210, and the treated support 210 with linker 24 (i.e., the functionalized support 220) are also shown for comparison.

[0040] By applying the same chemical reaction (e.g., the copper(I)-induced click reaction described above) or other known chemical reactions, various fluorescent entities 26 containing alkyne functional groups, such as sulforhodamine alkynes and sulfo-cy5.5 alkynes, can be conjugated to the treated carrier 210 via the linker 24. Other moieties 26, such as alkyne-polyethylene glycol and alkyne antibodies, can also be conjugated to the treated carrier 210 via the linker 24 using the same chemical reaction or other known chemical reactions. For example, alkyl antibodies can be created by reducing the antibody using DTT (dithiothreitol), thereby reducing the sulfhydryl group and conjugating it to maleimide-PEG4-alkyne or another alkyne-containing moiety according to known procedures. Other small molecules such as sugars, nitroxides, biotin, and drugs, or macromolecules, peptides, DNA, RNA sequences, and proteins such as SA (streptavidin and its derivatives), can also be covalently conjugated to the functionalized BN carrier 210 / linker 24 by known methods.

[0041] The above description of the processed carrier 210 has been made with respect to h-BN dots, but carbon dots and other nanodots of the layered material (such as TMDC above) are linked to the linker 24 by chemical means such as acid treatment as described above, and then linked to the portion 26.

[0042] Examples of experimental methods 1. Synthesis of BN QD The BN powder was first exfoliated into nanosheets by solvent exfoliation, as previously reported. Generally, 51.3 mg of BN powder and 30 mL of DMF were homogenized with stirring for 3 hours. This was then subjected to sonication for at least 24 hours, followed by heating at 150°C for 9 hours with stirring using a stirring bar. The resulting suspension was then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was separated from the centrifuge. The faintly yellow supernatant was a dispersion of BN dots (average size 2–10 nm) confirmed by TEM. The DMF was removed by using a high-temperature furnace under vacuum. The BN dots were stirred overnight in concentrated HNO3. The mixture was then neutralized with a sodium hydroxide solution. This was purified by dialysis (using an MWCO 1 KDa dialysis bag). The sample was then collected by lyophilization.

[0043] 2. Covalent functionalization of BN dots with 3-(azidopropyl)triethoxysilane The lyophilized powder was dispersed in ethanol and toluene. Then, 60 μl of 3-(azidopropyl)triethoxysilane was added to the mixture. The mixture was heated under reflux and stirred overnight under nitrogen. The solvent was removed by rotary evaporation, and the residue was dispersed in 70% ethanol (RE dialysis tube 1 kDa). After dialysis, azidosilane-functionalized BN dots were obtained. The sample was used directly without solvent removal.

[0044] 3. Connecting BNdot to FITC Functionalized BN dots were mixed with FITC alkynes (10 nM), sodium ascorbate (7.2 μM), and copper sulfate (7.2 μM). The reaction was allowed to proceed overnight at room temperature. The solvent was removed by rotary evaporation, and the mixture was dispersed in 70% ethanol for dialysis purification (RE dialysis tube 1 kDa). The samples were stored at 4°C for analysis.

[0045] The foregoing description is illustrative and not restrictive in nature. Variations and modifications of the disclosed embodiments that do not necessarily depart from the essence of the invention will be apparent to those skilled in the art. The scope of legal protection granted to the invention can only be determined by considering the following claims.

Claims

1. Nanodot carrier and parts and, A linker having first and second functional groups, wherein the first functional group is covalently bonded to the nanodot support and the second functional group is covalently bonded to the portion thereof, A compound possessing the following characteristics.

2. The compound according to claim 1, characterized in that the nanodot carrier is an h-BN nanodot carrier.

3. The compound according to claim 2, characterized in that the nanodot carrier has dimensions of 2 to 10 nm.

4. The compound according to claim 3, characterized in that the nanodot carrier comprises less than 30 layers of h-BN.

5. The compound according to claim 4, characterized in that the nanodot support comprises 4 to 8 layers of h-BN.

6. The compound according to claim 4, further comprising a plurality of linkers and a plurality of parts, wherein each layer of the nanodot carrier is connected to 10 or more linkers of the plurality of linkers, and each linker is connected to one of the plurality of parts.

7. The compound according to claim 1, characterized in that the nanodot support has at least one polar group, and the first functional group is covalently bonded to the nanodot support at the at least one polar group.

8. The compound according to claim 7, characterized in that the at least one polar group is a hydroxyl (-OH) group.

9. The compound according to claim 1, characterized in that the aforementioned portion comprises at least one of a fluorescent entity, a biomolecule, a chelating agent, and a combination thereof.

10. The nanodots are mechanically treated in a polar liquid to form defects on them. The nanodots are treated so that polar groups are provided in the defects. A method for manufacturing nanodot carriers, which includes the following features.

11. The method according to 10, further comprising covalently bonding a linker to the nanodot, wherein the linker has first and second functional groups, and the first functional group is covalently bonded to the polar group.

12. The method according to 11, further comprising covalently bonding a portion to the second functional group.

13. The method according to 12, characterized in that the aforementioned portion is a fluorescent substance.

14. The method according to 10, characterized in that the mechanical processing includes stirring.

15. The method according to 14, characterized in that the stirring is achieved by ultrasonic treatment or homogenization.

16. The method according to 10, characterized in that the treatment of the nanodots is an acid treatment, and the polar group is a hydroxyl (-OH) group.

17. The method according to 10, characterized in that the polar liquid is dimethylformamide (DMF).

18. The method according to 10, further comprising precipitating the nanodot carrier by centrifuging the nanodot carrier and the polar liquid after the mechanical treatment.

19. The method according to 18, further comprising replacing the polar liquid with water after the centrifugation.

20. The method according to 19, characterized in that the treatment of the nanodots is an acid treatment, and the treatment is performed after the replacement.

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