Patent Publication Number: US-2022226509-A1

Title: High-brightness nanodot fluorophores by covalent functionalization

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Patent Application Ser. No. 62/855,121 filed May 31, 2019, which is hereby incorporated herein in its entirety. This application is a continuation-in-part of U.S. patent application Ser. No. 15/953,200, filed Apr. 13, 2018, which claims priority to U.S. Provisional Patent Application Ser. No. 62/485,379, filed Apr. 13, 2017, both of which are hereby incorporated herein in their entireties. 
    
    
     STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH 
     The inventions described herein were made with government support under Grant #1261910, Grant #1521057 and Grant #1738466 awarded by the National Science Foundation. The Government has certain rights in this invention. 
    
    
     BACKGROUND 
     Fluorophores are compounds with fluorescent properties that have biomedical applications. For example, fluorophores can be used as tracers or dyes for specific staining of certain molecules or structures. More particularly, fluorophores can be used to stain tissues, cells, or materials in a variety of analytical methods, such as fluorescent imaging and spectroscopy. 
     For the purpose of specific staining, fluorophores should be conjugated with biomolecules such as antibodies. However, reliable tracking and quantification of the fluorophores is challenging due to the low brightness and low photostability of commercial fluorophores. Therefore, a need exists for improved carrier molecules to carry fluorescent entities for biological and other applications. Other biological molecules may also benefit from improved carriers. 
     SUMMARY 
     A example compound according to the present disclosure includes, among other possible things, a nanodot carrier, a moiety, and a linker having first and second functional groups, wherein the first functional group is covalently linked to the nanodot carrier, and the second functional group is covalently linked to the moiety. 
     An example method of making a nanodot carrier according to the present disclosure includes, among other possible things, mechanically processing nanodots in polar liquid to create imperfections on the nanodots, and treating the nanodots to provide polar groups at the imperfections. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  schematically shows an example compound with a nanodot carrier. 
         FIGS. 1B-1C  schematically show synthesis of an example compound like the compound of  FIG. 1A  from a BN nanodot carrier. 
         FIG. 2A  shows SEM (scanning electron microscopy) images of h-BN bulk powder. 
         FIG. 2B  shows SEM images of h-BN powder after mechanical processing, in this example, treatment with a homogenizer. 
         FIG. 2C  shows TEM (transmission electron microscopy) images of example boron nitride (BN) nanodot carriers. 
         FIG. 2D  shows the excitation-dependent autofluorescence of example BN nanodot carriers and the fluorescence image (inset) under UV lamp. 
         FIGS. 3A-B  show Fourier Transform Infrared Spectroscopy (FITR) results for pristine BN nanodot carriers and processed BN nanodot carriers. 
         FIG. 4  shows the absorbance spectra of the example fluorophore, pristine carriers, and processed carriers of  FIG. 1B . 
         FIG. 5  shows fluorescence intensity of the example fluorophore, pristine carriers, and processed carriers, and processed carriers with linkers (i.e., functionalized carriers) of  FIG. 1B . 
     
    
    
     DETAILED DESCRIPTION 
     Very generally, high-brightness fluorophores contain a carrier element, a fluorescent element, and a linker linking the carrier element to the fluorescent element. For biomedical applications, each of the carrier element, the linker, and the fluorescent element must be biocompatible (though the requirements for biocompatibility will vary with the particular application). 
     One example carrier element is a processed nanomaterial, such as carbon nanotubes (CNT) and boron nitride nanotubes (BNNTs), both of which can be used for biomedical applications such as cellular drug delivery and spectroscopy applications. However, it has been shown that fluorescent elements linked to carbon nanotubes exhibit quenching, or a reduction in the brightness of the fluorescence. 
     It has been discovered that certain fluorophores having nanomaterial carriers not only do not exhibit the quenching effect, but also exhibit brightness several orders of magnitude higher than other known fluorophores, as will be discussed herein. 
     Referring now to  FIG. 1A , fluorophore compounds  20  are shown. The compounds  20  generally comprise an inorganic nano-scale (“nanomaterial”) carrier  22 , a linker  24 , and a moiety  26 . In some examples, the compound  20  includes more than one linker  24 , and more than one moiety  26 . 
     The carrier  22  is, in one example, a processed BNNT or CNT carrier. In the example of  FIG. 1A , the carrier  22  is a zero-dimensional BN “dot” (e.g., the size of the dot in all three dimensions is on the nano-scale, or less than about 100 nm), though carbon dots could also be used. In a more particular example, all three dimensions of a dot carrier are less than about 20 nm. Other example carriers  22  are multi-walled BNNT or CNT carriers, where each BNNT or CNT has multiple co-axial shells of hexagonal boron nitride (h-BN for BNNTs) or graphene (for CNTs), with a typical external diameter of more than about 0.4 nm but less than about 100 nm. The length of these BNNTs and CNTs is between about 1-100 nm. In other examples, the carrier  22  can be another nano-scale inorganic material, such as hexagonal boron nitride (h-BN) nanosheets/nanoparticles, graphene/graphite nanosheets/nanoparticles, molybdenum disulfide (MoS 2 ) nanosheets/nanoparticles, any transition metal dichalcogenide (TMDCs) nanosheets/nanoparticles, and any nanosheets/nanoparticles of layered materials (materials with covalent layered structures that bond with van der Waals forces between layers). 
     The linker  24  has two or more functional groups R and R′, as shown in  FIGS. 1A-B . The functional groups R and R′ are reactive groups that facilitate covalent bonding of the linker to other structures by any known chemistry. R and R′ can be the same or different functional groups. For example, R and R′ can be ethoxsilane and azide, respectively. R and R′ can be any known functional groups such as amine groups, carboxylic acid, isothiocyanate, maleimide, an alkyne group, a hydroxyl group, a thiol group, monosulfone, or an ester group such as a succinimidyl, sulfodichlorophenol, pentafluorophenyl or tetrafluorophenyl. The linker  24  can be any type of molecule that has two or more functional groups R and R′. One example linker  24  is a linear or branched polymeric 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 between each other in series. 
     One functional group R interacts covalently with the carrier  22 . A carrier  22  with a linker  24  is known as a “functionalized” carrier  220  as shown in  FIG. 1B . That is, when covalently linked to linker  24 , the carrier  22 /linker  24  structure has a functional group R′ which facilitates covalent bonding of the carrier  22 /linker  24  to another moiety  26 . 
     The moiety  26  is, in one example, a fluorescent entity. In this example, the molecule  20  is a fluorophore. The fluorescent entity is any fluorescent dye that is known in the art, including but not limited to coumarins, benzoxadiazoles, acridones, acridines, bisbenzimides, 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 which have two different dyes connected and which interact via FRET (fluorescence resonance energy transfer). The fluorescent entity covalently interacts with the functional group R′ of linker  24  as discussed above. 
     In other examples, moiety  26  is a labelling moiety or other moieties to be delivered to a human body by the carrier  22 , such as antibodies, peptides, DNAs, RNAs, oligonucleotides, or the like. 
     The moiety  26 , in other examples, can be molecules and chelating agents with radioactive isotopes, ferromagnetic, and/or magnetic elements. In these examples, the compound  20  can be used as a contrast agent for medical imaging such as PET, SPECT, CT, MRI, etc. 
     In another example, the moiety  26  can include combinations of any of the example moieties  26  discussed above. In this example, the compound  20  can be used as a heterogeneous probe for biomedical detection and sensing. 
     Some nanomaterial carriers  22 , and in particular, 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, it has been discovered that carriers  22  such as the BN dot carrier shown in  FIGS. 1A-C  that have been subject to mechanical processing in solution or solvent, such as agitation, exhibit increased propensity to covalently interact with functional groups, such as functional group R on linker  24 . The solution/solvent can be the same solution/solvent in which source material is treated to form nanodots as discussed in more detail below, or a different solution/solvent. Furthermore, it has been discovered that mechanical processing of nanomaterial carriers improves the solubility of the nanomaterial carriers in aqueous solutions, which can improve biocompatibility. Additionally, mechanical processing cuts carrier material into smaller pieces which can be desirable when forming dots, for example. Agitation can be accomplished by homogenizer and/or sonication, such as tip sonication or bath sonication, for instance. 
     Referring now to  FIG. 1B , mechanical processing results in carrier  22  with imperfections  23 . During mechanical processing in solution/solvent, imperfections  23  form on the carrier  22  such that localized polarities or charges are formed at the imperfections  23 . Polar or charged groups from the solution/solvent interact with the localized polarities or charges at the imperfections. For the example, in the example of  FIG. 1B , the carrier is an h-BN nanodot carrier  22 . In this particular example, imperfections  23  are disruptions in the hexagonal structure of the boron nitride material, which disruptions have localized polarity imbalances. For example, for certain solvents/solutions, hydroxyl groups from the solvent/solution may interact with the imperfections  23 , though other solvents/solutions may have other polar or charged groups that can interact with the localized imperfections  23 , such as amino, carboxylic acids, or aldehyde groups, depending on the processing and type of solvent/solution. 
     In one particular example method of making carriers  22 , h-BN powder is treated in dimethylformamide (DMF) or another polar solution/solvent for two to four hours by using a homogenizer. In one example, the treatment in polar solvent is solvothermal (e.g., 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 (e.g., diameter) is about 13 μm  FIG. 2A  shows images of example h-BN particles with average size of about 13 μm prior to treatment in DMF. The homogenizer causes the BN dot carriers  22  to become smaller and remain suspended in the DMF solution. In this example, after treatment in DMF solution, the BN dot carriers  22  become smaller, and the size is reduced to less than about 2-5 μm, as shown in  FIG. 2B . 
     After the DMF treatment, the BN dot carrier  22  suspension undergoes an agitation treatment, such as sonication. In a particular example, the suspension is treated by bath sonication for 20-30 hours. The size of the BN dot carrier  22  is reduced to about 1-3 μm after sonication. 
     After the agitation treatment, the DMF/BN dot carrier  22  suspension is heat treated. In a particular example, the suspension is heated at 150° C. for 7 to 12 hours while stirring with a magnetic stir bar. The stir bar ensures that the BN dot carriers  22  remain suspended in the DMF solution. 
     Agitation and heat treatment result in carriers  22  with imperfections  23 , as in the example of  FIG. 1B . 
     After the heat treatment, the carriers  22  suspension is centrifuged to precipitate 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 the suspension is about 2-10 nm after heat treatment and centrifugation, as confirmed by TEM (transmission electron microscopy) imaging shown in  FIG. 2C . Furthermore, the carriers  22  are nearly invisible using SEM imaging, confirming that the carriers  22  are very small and have dimensions in the nano-scale. Generally, the carriers  22  have less than about 30 layers of h-BN, which corresponds to a thickness dimension of less than about 100 nm. The length/width dimensions are also less than about 100 nm. In a particular example, the carriers  22  have between about 4-8 layers of h-BN and have dimensions of about 2-10 nm. 
     After the centrifugation, the carriers  22  suspension undergoes solvent exchange. That is, the solvent (DMF) is switched for another solvent, water. Carriers  22  suspended in water are ready for biological applications or linking with moieties  26  to be carried, as discussed herein. Solvent exchange is accomplished as follows. DMF is evaporated into air by heating the suspension. In a particular example, the suspension is heated to 150° C. until the DMF is evaporated. After heating, the remaining carriers  22  are placed into a water/ethanol mixture. In a particular example, the water/ethanol mixture is 50% water and 50% ethanol. The carriers  22 /water/ethanol mixture is then heated to evaporate the ethanol at an appropriate temperature as would be known in the art. In a particular example, DMF can be removed by vacuum treatment and then the carriers  22  can be suspended in water. 
     It has been discovered that making carriers  22  according to the above-described method leads to a production yield orders of magnitude higher than prior art methods. For example, for the method performed with 20-30 minutes of bath sonication, heat treatment for 7 to 12 hours while stirring with a magnetic stir bar, and centrifugation at 10,000 rpm for 10 minutes, the production yield is about 47%, as compared to the reported 1-26% for prior methods. Production yield is the weight percentage of h-BN bulk powder that become carriers  22  after the evaporation step discussed above. 
     For the example DMF solution, hydrocarbon groups or fragments from the solution interact with the localized polarities at the imperfections  23  of carriers  22 , though other solutions may have other polar groups that can interact with the localized polarities, such as amino, carboxylic acids, aldehyde, etc. The carriers  22  can then undergo acid treatment according to any known method, which replaces the hydrocarbon groups or fragments with hydroxyl groups (—OH groups) at the imperfections  23  of carrier  22 , which result in processed carriers (discussed in more detail below). Acid treatment also removes other contamination from the carriers  22 , such as the hydrocarbon fragments of DMF. The processed carriers can then be linked to linkers  24  by any known chemistry that causes the R group of linker  24  to link covalently with the hydroxyl groups, to form functionalized carriers  220 . 
     Carriers  22  made according to the above method are autofluorescent. That is, the carriers  22  have a measurable intrinsic fluorescence.  FIG. 2D  shows fluorescence intensity of the carriers  22  shown in  FIGS. 2A-C  formed by the above method. Without being bound by any particular theory, the autofluorescence may be related to imperfections  23  formed on the surfaces and edges of the carriers  22  during the above method. The imperfections  23  may bond with hydrocarbon fragments of DMF, including carbon-substituted N vacancy point defects, carbene structure at zigzag edges and BO 2   −  and BO −  species. These imperfections  23  are expected to create a series of energy states near the edges of the valence and conduction bands of h-BN material. 
       FIGS. 1B-1C  show synthesis of a compound  20 . In this example, the carrier is an h-BN carrier  22  made by treating h-BN powder in a polar organic solvent which facilitates arrangement of the h-BN into a nanodot. Example polar organic solvents are dimethylformamide (DMF), N-Methyl-2-pyrrolidone (NMP), and ethanol. In a particular example, the carriers  22  are made according to the method described above. 
     In the example of  FIG. 1B , h-BN dot carriers  22  made according to the method described above are treated with acid, here, nitric acid (HNO 3 ) to provide processed carriers  210 . Acid treatment causes the attachment of —OH (hydroxyl) groups to the imperfections  23 , which, as discussed above, have imbalanced polarities that are attracted to the —OH groups.  FIGS. 3A-B  show FTIR (Fourier Transform Infrared Spectroscopy) spectra for processed carriers  210  and non-functionalized (“pristine”) h-BN dot carriers  22 . As shown in  FIG. 3A , C—H stretching from DMF fragments at 2950 cm-1 of the pristine h-BN dot carriers  22  disappeared after the nitric acid treatment. A broad IR (infrared) band at 3100 cm −1  is detected from the treated sample, which indicates that hydroxyl groups were introduced after acid treatment. There is a red shift on the —OH band due to the slight energy band change of zigzag edges of processed carriers  210  after DMF and contaminations were removed. The removal of these DMF fragments is also supported by the disappearance B—O (˜1255 cm-1), B—C or C—N (˜1150 cm-1) bonds shown in  FIG. 3B . In other words, this FTIR analysis confirms the presence of hydroxyl groups on the processed carriers  210  by the presence of the expected peaks in the spectra. 
     The —OH groups attached to the imperfections  23  are themselves polar/charged. Turning again to  FIG. 1B , the polar or charged groups (e.g., —OH groups, in this example) facilitate covalent interactions between the processed carrier  210  and the functional group R on linker  24 . The polar groups also increase the hydrophilicity of the processed carrier  210  by facilitating polar or ionic interactions with water molecules or ions in the water. Therefore, the functionalized carrier  220  exhibits improved solubility dispersion in aqueous solution as compared to other carriers that do not include the processed carriers  210 . 
     The processed carriers  210  have increased capacity for attaching to linkers  24  and thus moieties  26  due to the polar or charged groups as compared to non-functionalized carriers. More specifically, the polar or charged groups act as reactive sites for covalently linking the processed carrier  210  to linker  24  via functional group R. Accordingly, the brightness of the fluorophore  20  having a functionalized carrier  220  and a fluorescent entity  26  is higher than prior art fluorophores because the functionalized carrier  220  can be linked to multiple fluorescent entities  26 . More generally, the functionalized carriers  220  can be linked to more moieties  26  than non-processed carriers. 
     In a particular example, the BN dot carriers  22  that are processed to form processed carriers  210  as discussed above have 4 layers of h-BN that are each about 2.5 nm in diameter. Each layer can bond to 10 or more linkers  24  and fluorescent entities  26  or other moieties  26  after processing as discussed above. Thus, the example processed carriers  210  can bond to 40 or more linkers  24  and fluorescent entities  26  to form a fluorophore. The fluorophore  20  is thus 40 or more times brighter than a carrier with a single fluorescent entity. For branched linkers (n branches), the intensity will be as larger as 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 linked to the processed carrier  210 . In this example, the R group is a ethoxy silane group and the R′ group is an azide group. As shown in  FIG. 1B , the R group is reactive with processed carrier  210  at imperfections  23  (and in particular, the polar-charged groups at imperfections  23 ) and the R′ group is reactive with moiety  26 . 
     In other examples, the linker  24  is an amino-silane linkers. Other linkers  24  might have a variety of functional groups such as amino, carboxylic acid, succinimdyl ester, maleimide, carboimide, pyridyldithiol, haloacetyl, aryl azide, azide, alkyne, hydrazide and monosulfone groups. Those groups could be used for the conjugation of carriers  22  to dye, drug, or any targeting material. Cross-linkers which contain dual functional group can also be used to obtain functional group to conjugate linkers  24  to other entities such as dye, peptide, oligonucleotide, DNA, RNA, antibody, proteins, drugs or other nanoparticles. Those cross-linkers might be SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC ((sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), AMAS (N-α-maleimidoacet-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 where n=2,4,6,8,12,24 (PEGylated SMCC cross-linker), SPDP (succinimidyl 3-(2-pyridyldithio)propionate), LC-SPDP, sulfo-LC-SPDP, SMPT (4-succinimidyloxycarbonyl- alpha-methyl-α(2-pyridyldithio)toluene), PEGn-SPDP (where 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′-azipentanoate), 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 known linkers. 
     In the example of  FIG. 1B , the moiety  26  is a fluorescent entity, and in particular, is FITC (fluorescein isothiocyanate), which is a green dye. FITC can be conjugated to the linker  24  at R′ by any known chemistry. For instance, for the azide-silane linker  24  of  FIG. 1B , a copper(I)-induced click reaction can be performed to covalently bond the R′ group of the linker  24  to an alkyne group of FITC. 
       FIG. 4  shows the absorbance spectra of the example fluorophore  20  of  FIG. 1B . The characteristic absorbance signal of FITC at around 490 nm and the peak at 280 nm attributed to aromatic triazole (shown at the arrows) is present in the fluorophores  20 , confirming conjugation of the processed carrier  210  with the linker  24  and FITC entity  26 .  FIG. 4  also shows the absorbance spectra of pristine carriers  22  and processed carriers  210  for comparison. 
       FIG. 5  shows fluorescence intensity of the example fluorophore  20  of  FIG. 1B  after excitation with 492 nm irradiation. The fluorophore  20  emits at 515 nm, the characteristic emission signal of FITC molecules. This confirms that FITC molecules are covalently conjugated on the fluorophores  20 . Fluorescence intensity of pristine carriers  22 , processed carriers  210 , and processed carriers  210  with linkers  24  (i.e., functionalized carrier  220 ) is also shown for comparison. 
     The same chemistry (e.g., copper (I)-induced click reaction discussed above) or other known chemistries can be applied to conjugate various fluorescent entities  26  that contain alkyne functional group such as sulforhodamine alkyne, sulfo-cy5.5 alkyne, etc. to the processed carrier  210  via linkers  24 . Other moieties  26  such as alkyne-polyethylene glycol, alkyne antibodies, etc. can also be conjugated to the processed carrier  210  via linkers  24  using the same chemistry or other known chemistries. For example, alkyl antibodies can made by reducing an antibody using DTT (Dithiothreitol), which results in reduced sulfuhydryl groups, which can then be connected to with maleimide-PEG4-alkyne or another alkyne-containing moiety according to known procedure. Other small molecules such as sugars, nitroxides, biotin, drugs, etc. or macromolecules, peptides, DNA, RNA sequences, proteins such as SA (streptavidin and its derivatives) can also be covalently connected to the functionalized BN carrier  210 /linker  24  according to known methods. 
     Though the preceding description of processed carrier  210  is made with respect to h-BN dots, carbon dots, and other nanodots of layered materials (TMDCs, etc. as discussed above) can be linked to linkers  24  by chemical means, such as by acid treatment, and then linked to moieties  26 , as discussed above. 
     Example Experimental Method 
     1. Synthesis of BN QDs 
     BN powder was firstly exfoliated to nanosheets through a solvent exfoliation method as reported previously. Typically, 51.3 mg of BN powder and 30 mL of DMF were homogenized for 3 hours under stirring. Then it was kept under sonication at least for 24 h and then heated with stir bar for 9 hour at 150° C. Afterwards, the resulting suspension was centrifuged for 10 min at 10000 rpm to separate the centrifuge and supernatant. The faint yellow supernatant was the BN dots (average size 2-10 nm) dispersion confirmed with TEM. DMF was removed by using high temperature the furnace under vacuum. The BN dots were stirred overnight in concentrated HNO 3 . Afterwards, it the mixture was neutralized by sodium hydroxide solution. It was purified through dialysis (by using MWCO 1 KDa dialysis bag). Then the sample was collected by freeze-drying. 
     2. Covalent Functionalization of BN Dots with 3-(Azidopropyl)triethoxysilane) 
     Freeze-dried powder was dispersed in ethanol and toluene. Afterwards, 3-(Azidopropyl)triethoxysilane) (60 μl) was added in mixture. The mixture was heated to reflux and stirred under nitrogen overnight. The solvent was removed through rotation evaporation and the residue was dispersed in 70% ethanol (RE dialysis tubing 1 kDa). After dialysis, azide-silane functionalized BN dots were obtained. The sample was used directly without removing solvent. 
     3. Connection BN Dots with FITC 
     The functionalized BN dots was mixed with FITC alkyne (10 nM), sodium ascorbate (7.2 μM) and copper sulfate (7.2 μM). The reaction was processing under room temperature overnight. The solvent was removed through rotation evaporation and was dispersed in 70% ethanol for dialysis purification (RE dialysis tubing 1 kDa). The sample was stored 4° C. for analysis. 
     The preceding description is exemplary rather than limiting in nature. Variations and modifications to the disclosed examples may 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.