JP2022534285A - High-Brightness Nanodot Fluorescent Dyes with Covalent Functionalization - Google Patents

Abstract

Exemplary compounds according to the present disclosure include, among other things, a nanodot carrier, a moiety, and a linker having first and second functional groups, wherein the first functional group covalently bonds to the nanodot carrier and the second functional group covalently bonds to the moiety. Exemplary methods of manufacturing nanodot carriers are also disclosed.

Description

(Cross reference to related applications)
This application claims priority to US Provisional Patent Application No. 62/855,121, filed May 31, 2019, which is incorporated herein in its entirety. This application is a continuation-in-part of U.S. patent application Ser. , 379, which is hereby incorporated herein in its entirety.

(Statement on Federally Sponsored Research)
The invention described herein was made with government support under Grant #1261910, Grant #1521057 and Grant #1738466 awarded by the National Science Foundation. The United States Government has certain rights in this invention.

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

For the purpose of specific staining, fluorochromes need to be conjugated with biomolecules such as antibodies. However, the low brightness and low photostability of commercially available fluorochromes make reliable tracking and quantification of fluorochromes difficult. Therefore, there is a need for improved carrier molecules for carrying fluorescent entities for biological and other applications. Other biological molecules may also benefit from improved carriers.

Exemplary compounds according to the present disclosure include, among other things, a nanodot carrier, a moiety, and a linker having first and second functional groups, the first functional group covalently bonding to the nanodot carrier and the second functional group covalently bonds to the moiety.

An exemplary method of making nanodot carriers according to the present disclosure includes, among other things, mechanically treating the nanodots in a polar liquid to create defects on the nanodots and treating the nanodots to provide polar groups in the defects. Including.

1 is a schematic diagram of an example compound with nanodot carriers. FIG. 1B is a schematic representation of the synthesis of an exemplary compound, such as that of FIG. 1A, from BN nanodot supports. FIG. 1B is a schematic representation of the synthesis of an exemplary compound, such as that of FIG. 1A, from BN nanodot supports. FIG. FIG. 4 shows SEM (Scanning Electron Microscope) images of h-BN bulk powder. FIG. 4 shows an SEM image of h-BN powder after mechanical treatment, in this example treatment with a homogenizer. FIG. 3 shows a TEM (transmission electron microscope) image of an exemplary boron nitride (BN) nanodot carrier. Excitation-dependent autofluorescence and fluorescence images under a UV lamp (inset) of an exemplary BN nanodot carrier. FIG. 4 shows Fourier transform infrared spectroscopy (FITR) results for pristine BN nanodot carriers and treated BN nanodot carriers. FIG. 4 shows Fourier transform infrared spectroscopy (FITR) results for pristine BN nanodot carriers and treated BN nanodot carriers. 1B shows the absorbance spectra of the exemplary fluorochrome of FIG. 1B, the original carrier, and the treated carrier; FIG. FIG. 1B shows the fluorescence intensity of the exemplary fluorochromes of FIG. 1B, the original carrier, the treated carrier, and the treated carrier with a linker (ie, functionalized carrier).

Very generally, a bright fluorescent dye comprises a carrier element, a fluorescent element, and a linker connecting the carrier element to the fluorescent element. For biomedical applications, each of the carrier element, linker, and fluorescent element should be biocompatible (although biocompatibility requirements vary depending on the particular application).

One example carrier element is engineered nanomaterials such as carbon nanotubes (CNT) and boron nitride nanotubes (BNNT), both of which can be used in biomedical applications such as cellular drug delivery and spectroscopy applications. can be done. However, fluorescent elements attached to carbon nanotubes have been shown to exhibit quenching, or a decrease in fluorescence brightness.

It has been found that certain fluorochromes with nanomaterial carriers not only exhibit no quenching effect, but also exhibit brightness several orders of magnitude higher than other known fluorochromes, as discussed herein.

Referring now to FIG. 1A, a fluorochrome compound 20 is shown. Compound 20 generally includes inorganic nanoscale (“nanomaterial”) carrier 22 , linker 24 , and moiety 26 . In some examples, compound 20 includes two or more linkers 24 and two or more moieties 26 .

Support 22 is, in one example, a treated BNNT or CNT support. In the example of FIG. 1A, the carriers 22 are zero-dimensional BN "dots" (eg, dots in all three dimensions are nanoscale, ie, less than about 100 nm in size), but carbon dots may also be used. In a more specific example, all three dimensions of the dot carrier are less than about 20 nm. Other exemplary supports 22 are multilayer BNNT or CNT supports, each BNNT or CNT having multiple coaxial shells of hexagonal boron nitride (h-BN for BNNTs) or graphene (for CNTs). and a typical outer diameter greater than about 0.4 nm but less than about 100 nm. The length of these BNNTs and CNTs is about 1-100 nm. In other examples, the support 22 includes hexagonal boron nitride (h-BN) nanosheets/nanoparticles, graphene/graphite nanosheets/nanoparticles, molybdenum disulfide (MoS ₂ ) nanosheets/nanoparticles, any transition metal dichalcogenide ( TMDC) nanosheets/nanoparticles, and any nanosheets/nanoparticles of layered materials (materials with a covalent layered structure that couples van der Waals forces between layers).

Linker 24 has two or more functional groups R and R', as shown in Figures 1A-B. Functional groups R and R' are reactive groups that facilitate covalent attachment of the linker to other structures by known chemistries. R and R' are the same or different functional groups. For example, R and R' are ethoxysilane and azide, respectively. R and R' are amine groups, carboxylic acid groups, isothiocyanate groups, maleimide groups, alkyne groups, hydroxyl groups, thiol groups, monosulfone groups, or ester groups such as succinimidyl, sulfodichlorophenol, pentafluorophenyl or tetrafluorophenyl. , may be any known functional group. Linker 24 is any type of molecule having two or more functional groups R and R'. An example 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 together in series.

One functional group R covalently interacts with the carrier 22 . A carrier 22 with linkers 24 is known as a “functionalized” carrier 220, as shown in FIG. 1B. That is, when covalently attached to linker 24 , carrier 22 /linker 24 structure has a functional group R′ that facilitates covalent attachment of carrier 22 /linker 24 to another moiety 26 .

Portion 26, in one example, is a fluorescent entity. In this example, molecule 20 is a fluorescent dye. Fluorescent entities include coumarins, benzoxadiazoles, acridones, acridines, bisbenzimides, indoles, benzoisoquinolines, naphthalenes, anthracenes, xanthenes, pyrenes, porphyrins, fluoresceins, rhodamines, boron-dipyrromethene (BODIPY), and cyanine derivatives, but Any fluorescent dye known in the art, including but not limited to. Many such fluorescent dyes are commercially available. Fluorescent entities can also include tandem dyes that have two different dyes attached and interact via FRET (Fluorescence Resonance Energy Transfer). The fluorescent entity covalently interacts with functional group R' of linker 24 as described above.

In other examples, moieties 26 are label moieties or other moieties delivered to the human body by carrier 22, such as antibodies, peptides, DNA, RNA, oligonucleotides, and the like.

Moiety 26 is, in other examples, molecules and chelators with radioactive isotopes, ferromagnetic and/or magnetic elements. In these examples, compound 20 can be used as a contrast agent for medical imaging such as PET, SPECT, CT, MRI.

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

Some nanomaterial carriers 22, particularly boron nitride (BN) based nanomaterials, are known to be chemically inert. Therefore, it has been difficult to functionalize prior art nanomaterial carriers for covalent interactions with other structures. However, a support 22, such as the BN dot support shown in FIGS. It was found to show an increased propensity to interact. The solution/solvent may be the same solution/solvent in which the raw material is processed to form nanodots, or a different solution/solvent, as described in more detail below. Furthermore, it has been found that mechanical treatment of nanomaterial carriers improves the solubility of nanomaterial carriers in aqueous solutions, which can improve biocompatibility. In addition, mechanical processing cuts the carrier material into smaller pieces, which may be desirable, for example, when forming dots. Agitation can be accomplished by a homogenizer and/or sonication, eg, tip or bath sonication.

Referring now to FIG. 1B, mechanical treatment results in carrier 22 having imperfections 23 . During mechanical processing in solutions/solvents, defects 23 are formed on the carrier 22 and local polarities or charges are formed in the defects 23 . Polar or charged groups from the solution/solvent interact with local polarity or charge at the defect. For example, in the example of FIG. 1B, the carrier is an h-BN nanodot carrier 22 . In this particular example, defect 23 is a disruption of the hexagonal structure of the boron nitride material, which has a localized polarity imbalance. For example, for certain solvents/solutions, hydroxyl groups from the solvent/solution may interact with defect 23, whereas other solvents/solutions may interact with amino acids, depending on the treatment and type of solvent/solution. It may have other polar or charged groups that can interact with local defects 23, such as groups, carboxylic acid groups, or aldehyde groups.

In one particular exemplary method of making carrier 22, h-BN powder is treated in dimethylformamide (DMF) or another polar solution/solvent for 2-4 hours by using a homogenizer. In one example, processing in polar solvents is solvothermal (eg, the solvent/solution is heated). In one example, the h-BN powder has an average particle size of between about 10-20 μm. In a particular example, the average particle size (eg diameter) is about 13 μm. FIG. 2A shows an image of an example of h-BN particles with an average size of about 13 μm before treatment with DMF. The homogenizer makes the BN dot carriers 22 smaller and leaves them suspended in the DMF solution. In this example, after treatment with the DMF solution, the BN dot carriers 22 become smaller, shrinking in size to less than about 2-5 μm, as shown in FIG. 2B.

After the DMF treatment, the BN dot carrier 22 suspension undergoes a stirring treatment such as ultrasonic treatment. In a particular example, the suspension is treated by bath sonication for 20-30 hours. The size of the BN dot carriers 22 is reduced to about 1-3 μm after sonication.

After stirring, the DMF/BN dot carrier 22 suspension is heat treated. In a particular example, the suspension is heated at 150° C. for 7-12 hours while being stirred with a magnetic stirrer. A stir bar ensures that the BN dot carriers 22 remain suspended in the DMF solution.

Stirring and heat treatment, as in the example of FIG. 1B, results in a carrier 22 having defects 23 .

After heat treatment, the carrier 22 suspension is centrifuged to sediment large particles. In a particular example, the suspension is centrifuged at 10,000 rpm for 10 minutes. In this example, the size of the carriers 22 in suspension is about 2-10 nm after heat treatment and centrifugation, as confirmed by the TEM (transmission electron microscope) image shown in FIG. 2C. Moreover, the carrier 22 is barely visible using SEM images, confirming that the carrier 22 is very small and has nanoscale dimensions. Generally, carrier 22 has less than about 30 layers of h-BN, corresponding to a thickness dimension of less than about 100 nm. The length/width dimension is also less than about 100 nm. In a particular example, carrier 22 has about 4-8 layers of h-BN and has dimensions of about 2-10 nm.

After centrifugation, the carrier 22 suspension undergoes solvent exchange. That is, the solvent (DMF) is exchanged for another solvent, water. As discussed herein, the carrier 22 suspended in water is ready for biological use or for binding with the moieties 26 to be carried. Solvent exchange is performed as follows. The DMF is evaporated into the air by heating the suspension. In a particular example, the suspension is heated to 150°C until the DMF evaporates. After heating, the remaining carrier 22 is placed in the water/ethanol mixture. In a particular example, 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 a particular example, DMF can be removed by vacuum treatment and then carrier 22 can be suspended in water.

It has been found that manufacturing the carrier 22 according to the above method results in a production yield that is orders of magnitude higher than prior art methods. For example, for a method of bath sonication for 20-30 minutes, heat treatment for 7-12 hours while stirring with a magnetic stirrer, and centrifugation at 10,000 rpm for 10 minutes, the production yield is lower than that of the conventional method. about 47%, compared to 1-26% reported in . Production yield is the weight percent of h-BN bulk powder that becomes carrier 22 after the evaporation step described above.

For exemplary DMF solutions, hydrocarbon groups or fragments from the solution interact with localized polarities at defects 23 of support 22, whereas other solutions interact with localized polarities such as amino, carboxylic acid, aldehyde, etc. may have other polar groups that can interact with Support 22 is then subjected to acid treatment according to any known method, which replaces hydrocarbon groups or fragments with hydroxyl groups (—OH groups) at defects 23 of support 22, resulting in (discussed in more detail below). Acid treatment also removes other contaminants from carrier 22, such as hydrocarbon fragments of DMF. The treated carrier is then linked to linker 24 by any known chemistry that covalently bonds the hydroxyl groups with the R groups of linker 24 to form functionalized carrier 220 .

Carriers 22 made according to the above method are autofluorescent. That is, carrier 22 has a measurable intrinsic fluorescence. FIG. 2D shows the fluorescence intensity of the carrier 22 shown in FIGS. 2A-C formed by the method described above. Without being bound by a particular theory, autofluorescence may be related to defects 23 formed in the surface and edges of carrier 22 during the above method. Defects 23 can combine with hydrocarbon fragments of DMF, including carbon-substituted N-vacancy defects, carbene structures at the zigzag edges, and BO ₂ ⁻ and BO ⁻ species. These defects 23 are expected to create a series of energy states near the valence and conduction band edges of the h-BN material.

1B-1C show the synthesis of compound 20. FIG. In this example, the support is h-BN support 22 made by treating h-BN powder in a polar organic solvent that facilitates the placement of h-BN into nanodots. Examples of polar organic solvents are dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), and ethanol. In a particular example, carrier 22 is made according to the methods described above.

In the example of FIG. 1B, the h-BN dot carrier 22 made according to the method described above is treated with an acid, here nitric acid (HNO ₃ ) to provide a treated carrier 210 . The acid treatment results in the attachment of —OH (hydroxyl) groups to defects 23, which, as mentioned above, have an unbalanced polarity that is attracted to —OH groups. 3A-B show FTIR (Fourier Transform Infrared Spectroscopy) spectra of treated support 210 and unfunctionalized (“pristine”) h-BN dot support 22. FIG. As shown in FIG. 3A, the C—H stretching from the DMF fragment at 2950 cm ⁻¹ of 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 DMF and contaminants are removed, there is a red shift in the —OH band due to the slight energy band change of the zigzag edges of the treated carrier 210 . Elimination of these DMF fragments is also supported by the disappearance of the BO (~1255 cm ^-1 ), BC, or CN (~1150 cm ^-1 ) bonds 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.

The —OH group attached to defect 23 is itself polar/charged. Returning again to FIG. 1B, polar or charged groups (eg, —OH groups in this example) promote covalent interactions between the treated support 210 and functional groups R on linker 24 . The polar groups also increase the hydrophilicity of the treated carrier 210 by promoting polar or ionic interactions with water molecules or ions in water. Thus, functionalized carrier 220 exhibits improved solubility distribution in aqueous solutions compared to other carriers without treated carrier 210 .

The treated support 210 has increased capacity for attaching linkers 24 and thus moieties 26 due to polar or charged groups compared to non-functionalized supports. More specifically, the polar or charged groups act as reactive sites for covalent attachment of the treated carrier 210 to the linker 24 via the functional group R. Therefore, since the functionalized carrier 220 can be linked to multiple fluorescent entities 26, the brightness of the fluorescent dye 20 with the functionalized carrier 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 moieties 26 than the untreated support.

In a particular example, a BN dot carrier 22 treated to form a treated carrier 210 as described above has four layers of h-BN, each approximately 2.5 nm in diameter. Each layer can be attached to ten or more linkers 24 and fluorescent entities 26 or other moieties 26 after being treated as described above. Accordingly, an exemplary engineered carrier 210 can be attached to 40 or more linkers 24 and fluorescent entities 26 to form a fluorescent dye. The fluorochrome 20 is thus more than 40 times brighter than a carrier with a single fluorescent entity. In the case of branched linkers (n-branch), the intensity is 40n times that of a carrier with a single fluorescent entity.

Turning again to FIG. 1B, an example triethoxysilane linker 24 , which in this particular example is 3-(azidopropyl)triethoxysilane, is attached to the treated support 210 . In this example, the R group is an ethoxysilane group and the R' group is an azide group. As shown in FIG. 1B, the R groups are reactive with the treated support 210 at defects 23 (particularly the polar charged groups of defects 23) and the R' groups are reactive with moieties 26.

In another example, linker 24 is an aminosilane linker. Other linkers 24 can 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 attach the carrier 22 to dyes, drugs, or any target substance. A crosslinker containing dual functional groups is used to provide functional groups that attach the linker 24 to other entities such as dyes, peptides, oligonucleotides, DNA, RNA, antibodies, proteins, drugs, or other nanoparticles. be able to. Their 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-malemidocaproyl-oxysuccinimide ester), sulfo-EMCS, SMPB (succinimidyl 4-(p-maleimidophenyl) butyrate), sulfo- SMPB, SMPH (succinimidyl 6-((beta-maleimidopropionamido)hexanoate), LC-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), sulfo-KMUS ( N-κ-maleimidoundecanoyl-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'-adipentanamido)ethyl)-1,3'-dithiopropionate), sulfo- SDAD, DCC (N,N'-dicyclo hexylcarbodiimide), 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 known linkers.

In the example of FIG. 1B, moieties 26 are fluorescent entities, in particular the green dye FITC (fluorescein isothiocyanate). FITC can be conjugated to linker 24 at R' by any known chemistry. For example, in the case of the azido-silane linker 24 of FIG. 1B, a copper(I)-induced click reaction can be performed to covalently bond the R' group of linker 24 to the alkyne group of FITC.

FIG. 4 shows the absorbance spectrum of the exemplary fluorochrome 20 of FIG. 1B. A characteristic absorbance signal of FITC at about 490 nm (indicated by an arrow) and a peak at 280 nm due to the aromatic triazole are present in the fluorochrome 20, the treated carrier 210 and the linker 24 and the FITC entity. 26 confirms binding. FIG. 4 also shows the absorbance spectra of original carrier 22 and treated carrier 210 for comparison.

FIG. 5 shows the fluorescence intensity of the exemplary fluorochrome 20 of FIG. 1B after excitation with 492 nm illumination. The fluorochrome 20 emits at 515 nm, which is the characteristic emission signal of FITC molecules. This confirms that the FITC molecule is covalently attached onto the fluorochrome 20 . Fluorescence intensities of pristine carrier 22, treated carrier 210, and treated carrier 210 with linkers 24 (ie, functionalized carrier 220) are also shown for comparison.

Applying the same chemistry (eg, the copper(I)-induced click reaction described above) or other known chemistries, various fluorescent entities 26 containing alkyne functional groups such as sulforhodamine alkynes, sulfo-cy5.5 alkynes, etc. can be attached to the treated support 210 via linkers 24 . Other moieties 26, such as alkyne-polyethylene glycol, alkyne antibodies, etc., can also be attached via linkers 24 to the treated carrier 210 using the same or other known chemistries. For example, alkyl antibodies are made by reducing antibodies using DTT (dithiothreitol), which reduces sulfhydryl groups to maleimido-PEG4-alkyne or another alkyne-containing moiety according to known procedures. combined. Other small molecules such as sugars, nitroxides, biotin, drugs, or proteins such as macromolecules, peptides, DNA, RNA sequences, SA (streptavidin and its derivatives) may also be functionalized BN carriers 210 by known methods. / can be covalently attached to the linker 24 .

Although the foregoing description of the treated support 210 is in terms of h-BN dots, carbon dots and other nanodots of layered materials (such as TMDC above) can be treated by chemical means such as by acid treatment as described above. is linked to linker 24 by and then to portion 26 .

Example of experimental method 1 . Synthesis of BN QDs BN powder was first exfoliated into nanosheets by a solvent exfoliation method as previously reported. Generally, 51.3 mg of BN powder and 30 mL of DMF were homogenized with stirring for 3 hours. It was then placed under ultrasonic treatment for at least 24 hours and then heated at 150° C. for 9 hours while stirring with a stir bar. The resulting suspension was then centrifuged at 10000 rpm for 10 minutes to separate the centrifuge and the supernatant. The faint yellow supernatant was a dispersion of BN dots (average size 2-10 nm) confirmed by TEM. DMF was removed by using a high temperature furnace under vacuum. BN dots were stirred overnight in concentrated _HNO3 . The mixture was then neutralized with sodium hydroxide solution. It was purified by dialysis (using MWCO 1 KDa dialysis bags). Samples were then collected by freeze-drying.

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

3. Connection of BN dots with FITC Functionalized BN dots were mixed with FITC alkyne (10 nM), sodium ascorbate (7.2 μM) and copper sulfate (7.2 μM). The reaction was allowed to proceed overnight at room temperature. Solvent was removed by rotary evaporation and dispersed in 70% ethanol for dialysis purification (RE dialysis tubing 1 kDa). Samples were stored at 4°C for analysis.

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

Claims (20)

a nanodot carrier;
part and
a linker having first and second functional groups, wherein the first functional group is covalently attached to the nanodot carrier and the second functional group is covalently attached to the moiety;
A compound with The compound according to claim 1, characterized in that said nanodot carriers are h-BN nanodot carriers. A compound according to claim 2, characterized in that said nanodot carriers have a dimension of 2-10 nm. 4. The compound of claim 3, wherein the nanodot carrier comprises less than 30 layers of h-BN. 5. The compound of claim 4, wherein the nanodot carrier comprises 4-8 layers of h-BN. Further comprising a plurality of linkers and a plurality of moieties, wherein each layer of the nanodot carrier is linked to 10 or more linkers of the plurality of linkers, and each linker is linked to one portion of the plurality of moieties. 5. The compound of claim 4, characterized in that: 2. The compound of claim 1, wherein the nanodot carrier has at least one polar group, and the first functional group is covalently bonded to the nanodot carrier at the at least one polar group. 8. The compound of claim 7, wherein said at least one polar group is a hydroxyl (--OH) group. 2. The compound of Claim 1, wherein said moiety comprises at least one of a fluorescent entity, a biomolecule, a chelating agent, and combinations thereof. mechanically treating the nanodots in a polar liquid to form defects on the nanodots;
treating the nanodots to provide polar groups in the defects;
A method for producing a nanodot carrier, comprising: 4. The claim further comprising covalently attaching a linker to the nanodot, wherein the linker has first and second functional groups, the first functional group covalently attaching to the polar group. 10. The method according to 10. 12. The method of claim 11, further comprising covalently attaching a moiety to said second functional group. 13. The method of claim 12, wherein said moieties are fluorescent entities. 11. The method of claim 10, wherein said mechanically treating includes agitation. 15. The method of claim 14, wherein said agitation is accomplished by sonication or a homogenizer. 11. The method of claim 10, wherein treating the nanodots is acid treatment, and the polar groups are hydroxyl (-OH) groups. 11. The method of claim 10, wherein said polar liquid is dimethylformamide (DMF). 11. The method of claim 10, further comprising precipitating the nanodot carriers by centrifuging the nanodot carriers and the polar liquid after the mechanical treatment. 19. The method of claim 18, further comprising exchanging the polar liquid for water after said centrifugation. 20. The method of claim 19, wherein treating said nanodots is an acid treatment, said treatment being performed after said exchange.

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