Patent Description:
Fluorophores may be attached to other molecules for delivery to certain tissues, cells or materials. When attached to these other delivery molecules, fluorophores can exhibit quenching, which is a reduction in the brightness of the fluorescence of the fluorophore.

An example fluorophore according to the present application includes a carrier which is a boron nitride nanotube, at least one fluorescent entity, and an amphiphilic linker linking each of the at last one fluorescent entities to the carrier. The linker has a molecular weight greater than <NUM> Da and/or a stretched linker length greater than <NUM> nanometers. The linker comprises at least one hydrophilic portion and a hydrophobic portion, wherein the hydrophobic portion is non-covalently bonded to the carrier and the hydrophilic portion is covalently bonded to the fluorescent entity.

Any references in the description to carbon nanotubes (CNT) are for reference purposes only and do not form part of the invention.

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 nanomaterial, such as carbon nanotubes (CNT) and boron nitride nanotubes (BNNTs), both of which are recognized as biologically compatible nanomaterials for biomedical applications such as cellular drug delivery and spectroscopy applications. However, it has been shown that fluorescent elements linked to 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>, fluorophores <NUM> are schematically shown. Fluorophores <NUM> generally comprise an inorganic nano-scale carrier <NUM>, a linker <NUM>, and a fluorescent entity <NUM>.

The carrier <NUM> is a BNNT carrier. In a particular example, the carrier <NUM> is a multi-walled BNNT carrier, where each BNNT has multiple co-axial shells of hexagonal boron nitride (h-BN for BNNTs), with a typical external diameter of more than about <NUM> but less than about <NUM>. The length of these BNNTs may be between about <NUM>-<NUM>. The carrier <NUM> can be fabricated by any known method.

The linker <NUM> is an amphiphilic polymeric linker, linking each of the at least one fluorescent entities to the carrier, the linker having a molecular weight greater than <NUM> Da and/or a stretched linker length greater than <NUM> nanometers. The linker <NUM> includes a hydrophobic region <NUM> and a hydrophilic region <NUM>. The hydrophobic region <NUM> non-covalently bonds to the nanotube carrier <NUM>, while the hydrophilic region is covalently bonded to the fluorescent entity <NUM>. One example linker <NUM> is DSPE-PEGn (<NUM>,<NUM>-distearoyl-sn-glycero-<NUM>-phosphoethanolamine-N-[(polyethylene glycol)n]), where n is a number of polyethylene glycol (PEG) molecules in a PEG chain. Other linkers <NUM> can similarly include a PEG chain (or a different chain) which varies in length.

In addition to the DSPE-PEG linkers <NUM> discussed above, many other potential linkers are known in the art. For example, a linker <NUM> may comprise one or more groups selected from -CH<NUM>-, -CH=, -C=, -NH-, -N=, O-, -NH<NUM>-, -N<NUM>-, -S-, -C(O)-, -C(O)<NUM>-, - C(S)-, -S(O)-, -S(<NUM>)<NUM>-, or any combination thereof. It will be appreciated that a linker comprising more than one of the above groups will be selected such that the linker <NUM> is stable; for example, a linker <NUM> may not include two adjacent -O- groups, which would generate an unstable peroxide linkage. The linker <NUM> may be a straight chain, a branched chain, or may include one or more ring systems. Non-limiting exemplary linkers include a hydrophobic area which can be fatty acids, phospholipids, sphingolipids, phosphosphingolipids [such as DSPE, <NUM>-O-hexadecanyl-<NUM>-O-(9Z-octadecenyl)-sn-glycero-<NUM>-phospho-(<NUM>'-rac-glycerol) (ammonium salt), N-octanoyl-sphingosine-<NUM>-{succinyl[methoxy(polyethylene glycol)<NUM>, D-erythro-sphingosyl phosphoethanolamine, <NUM>,<NUM>-diphytanoyl-sn-glycero-<NUM>-phospho-L-serine, <NUM>-sn-phosphatidyl-L-serine (PS), glycosylphosphatidylinositol,<NUM>,<NUM>-dioleoyl-sn-glycero-<NUM>-phosphoethanoamine but not limited). The hydrophobic unit can be used to conjugate with water soluble polymeric chains such as PEG (or PEO polyethyleneoxide), PMO (poly methyl oxazoline), PEI (polyethyleneimine), polyvinyl alcohol, polyvinylpyrolidone, polyacrylamide, polypeptide, carbohydrate anchors. The water soluble polymeric chains are attached to the linkers at one end, and attached to the fluorescent entity at a second end. These hydrophobic and hydrophilic units must have reactive groups as mentioned above and such that the groups conjugate together into amphiphilic linkers.

The fluorescent entity <NUM> is any know fluorescent dye, 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 <NUM> is bonded to the linker <NUM> by any appropriate method.

Generally, the brightness of the fluorophore <NUM> is directly related to the number of fluorescent entities <NUM> on the fluorophore <NUM>. That is, a fluorophore <NUM> with less fluorescent entities <NUM> will exhibit a lower brightness than a fluorophore <NUM> with more fluorescent entities <NUM>. However, it has also been discovered that linker <NUM> length also affects the brightness of the fluorophore <NUM>. In the particular example DSPE-PEGn linker <NUM> discussed above, varying the number of PEG molecules in the PEG chain (n) varies the length of the linker <NUM>, and thus the brightness of the fluorophore <NUM>. It will be appreciated that varying linker lengths of the other types of linkers discussed above can also be achieved. More particularly, it has been discovered that fluorophores <NUM> having linker <NUM> molecular weight of greater than about <NUM> Da (which corresponds to a stretched linker length of about <NUM>-<NUM> for a linker <NUM> with a PEG chain) exhibit a nonlinear quenching effect, which is unexpected. Accordingly, the fluorophores <NUM> described herein exhibit brightness several orders of magnitude higher than prior art fluorophores. Furthermore, it has been discovered that fluorophores <NUM> with different fluorescent entities <NUM> may have a different relationship between their fluorescent properties and linker <NUM> length.

In one example, the linker <NUM> can include a functional group R. The functional group R is a reactive group that facilitates covalent bonding of the linker <NUM> to the fluorescent entity <NUM> by know chemistry. An example functional group R is an amine group. Other example functional groups are carboxylic acid, isothiocyanate, maleimide, an alkynyl group, an azide group, a thiol group, monosulfone, or an ester group such as a succinimidyl, sulfodichlorophenol, pentafluorophenyl or tetrafluorophenyl. The functionalized linker <NUM> (that is, a linker <NUM> with a functional group R) may be commercially available, or may be synthesized according to methods described herein or other methods known to those skilled in the art.

<FIG> show an example red fluorophore <NUM>. The example red fluorophore <NUM> includes a BNNT carrier <NUM>, an amide-functionalized DSPE-PEGn-NH<NUM> linker <NUM> (that is, a linker as discussed above with an amide functional group, NH<NUM>), and a sulforhodamine B (RhB, red) fluorescent entity <NUM>.

The RhB fluorescent dye entity <NUM> is covalently bonded to the DSPE-PEGn-NH<NUM> linker <NUM> by any method to form a dye-linker structure <NUM>, <NUM> as shown in <FIG>. For example, the RhB fluorescent entity <NUM> is combined with DSPE-PEGn-NH<NUM> linker <NUM> in an ice bath under nitrogen in anhydrous dichloromethane (DCM), and then purified by flash chromatography or another purification method.

The use of DSPE-PEGn-NH<NUM> linker <NUM> with various molecular weight (MW) PEG chains (that is, with various n values) causes dye-linker structure <NUM>, <NUM> to emit at different fluorescence intensity and fluorescence quantum yield (QY). Since the dye entity <NUM> is at the end of the PEG chain of the linker <NUM> opposite the linker <NUM> connection to the BNNT carrier <NUM>, higher MW of the PEG chain (and higher n values) means the linker <NUM> is longer, and thus that the dye entity <NUM> would be further from the BNNT carrier <NUM>. Though the below description is made with respect to the particular example red fluorophore <NUM>, it should be understood that it is also applicable to fluorophores <NUM> including other linkers, carriers, or fluorescent entities, as discussed above.

<FIG> shows the QY for the dye-linker structure <NUM>, <NUM> with various PEG MW (<NUM>, <NUM>, <NUM>, <NUM>, and <NUM>,<NUM> Da, corresponding to n= <NUM>, <NUM>, <NUM>, <NUM>, <NUM> PEG molecules, respectively). The length of a fully stretched PEGn chain can be estimated because the known length of one PEG entity is <NUM>. For example, a fully stretched <NUM> MW PEG chain is calculated as <NUM>,<NUM>/<NUM> × <NUM> = <NUM>. The fully stretched linker lengths for PEG chains with MW of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM> are estimated to be <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, and <NUM>-<NUM>, respectively. In reality, the PEG chains may be coiled about one another or themselves, and may not retain their fully stretched state.

The relative fluorescence QY for each dye-linker sample was calculated by QY (sample) = QY (Reference) x [Slope(sample)/Slope(reference)] x [r(sample)/r(reference)], where r is the refractive index. As indicated by the equation, the relative QY was independent of the dye concentration of the samples as it was calculated by fluorescence/absorbance slope ratio of the samples and reference which were both linearly scaled to concentration as shown in <FIG>.

As shown in <FIG>, it is surprising to see that the QY changes for various linker <NUM> MW (e.g., various linker <NUM> length). In particular, for the example fluorophore <NUM>, the QY increases in a nonlinear manner with the linker length for MW of <NUM> to <NUM> but is saturated of decreasing at MW by <NUM>,<NUM> (that is at MW <NUM>, a longer linker does not cause a higher QY). It is also unexpected to see that the QY at MW of <NUM> was higher than the QY at MW of <NUM> and <NUM>. The maximum QY detected in the case of MW=<NUM> was also close to the standard QY free RhB (~<NUM> in distilled water), which indicates that fluorescence quenching is absent.

For example fluorophore <NUM>, the dye-linker structure <NUM>, <NUM> (DSPE-PEGn-NH<NUM>-RhB) is non-covalently labeled on the BNNT carrier <NUM> as shown in <FIG> by any method. The BNNT carrier <NUM> is fabricated and cut by any known method to a desired length. For example, the BNNT carrier <NUM> is between about <NUM> and <NUM>, more particularly, between about <NUM> and <NUM>. The BNNT carrier <NUM> is exposed to the dye-linker structure <NUM>, <NUM> so that the dye-linker structure <NUM>, <NUM> non-covalently bonds to the BNNT carrier <NUM> by any method. Optionally, the BNNT carrier <NUM>/dye-linker structure <NUM>-<NUM> solution can be distilled or filtered to remove excess unbonded dye-linker structures <NUM>, <NUM>.

As shown in <FIG>, the alkyl chain (-C(O)(CH<NUM>)<NUM>) of the linker <NUM> is non-covalently adsorbed on the surface of BNNT carrier <NUM> while the PEGn-NH<NUM>-RhB of the dye-linker structure <NUM>, <NUM> extends away from the BNNT carrier <NUM>. For other examples, the hydrophobic end of the linker <NUM> non-covalently bonds to the carrier <NUM> while the free hydrophilic end is covalently bonded to a fluorescent entity <NUM>, as discussed above.

It is noted that the surface area of one DSPE-PEGn-NH<NUM> linker <NUM> molecule adsorb on a BNNT is <NUM><NUM>. This means there can be as many as <NUM>×<NUM><NUM> DSPE-PEGn-NH<NUM>-RhB dye-linker structures <NUM>, <NUM> on a single BNNT carrier <NUM> that is <NUM> long and <NUM> in diameter if all the dye-linker structures <NUM>, <NUM> are lined up in a straight line. Accordingly, the fluorophore <NUM> is estimated to have <NUM> orders of magnitude more fluorescent entities than prior art fluorophores that consist of only <NUM>-<NUM> florescent entities. More generally, it is estimated that the fluorophores <NUM> described herein include at least <NUM>, and more particularly at least <NUM> dye-linker structures <NUM>, <NUM>.

<FIG> shows the QY of the fluorophores <NUM> with various dye-linker structures <NUM>, <NUM> as discussed above. As shown, the trend of the QY is quite similar to that of QY for the dye-linker structures <NUM>, <NUM> alone, as illustrated in <FIG>.

<FIG> shows the labeling efficiency of the various dye-linker structures <NUM>, <NUM> discussed above on BNNT carriers <NUM>. The labelling efficiency was calculated by determining the concentrations of dye-linker structures <NUM>, <NUM> after being labeled on carrier BNNTs <NUM>. This actual concentration of dye-linker structures <NUM>, <NUM> was then compared to initial dye concentration being used for each labeling process to determine the labeling efficiency.

It is surprising to see that for the example fluorophore <NUM>, labeling efficiencies for small (MW=<NUM>) and large (MW=<NUM>) linkers are significantly low (<<NUM>%). The labeling efficiency for linkers with intermediate MW (<NUM>, <NUM>, <NUM>) are quite similar in labeling efficiency (<NUM>-<NUM>%).

The fluorescence brightness of the fluorophore <NUM> is also related to the concentration of dye-linker structures <NUM>, <NUM> that the BNNT carriers <NUM> are exposed to. This in turn affects the labelling efficiency, discussed above, and ultimately the number of fluorescent dye entities <NUM> on each BNNT carrier <NUM>. <FIG> shows a plot of fluorescence intensity versus concentration of dye-linker structures <NUM>, <NUM> themselves, while <FIG> shows a plot of fluorescence intensity versus concentration of dye-linker structures <NUM>, <NUM> for fluorophores <NUM>.

As shown in <FIG>, it is unexpected to see that large quantity of dye-linker structures <NUM>, <NUM>, e.g., at high concentrations, can be labeled on BNNT carriers <NUM> without noticeable decrease in fluorescence intensity. This means, stable and non-covalent bonding between BNNT carrier <NUM> and the dye-linker structures <NUM>, <NUM> can prevent aggregation and collisional quenching and therefore lead to controllable and enhanced fluorescence brightness by using more concentrated dye-linkers for the labeling.

Brightness of fluorophores is defined as product of quantum yield (QY) and extinction coefficient (ε). Since a single BNNT carrier <NUM> could be loaded as many as <NUM>×<NUM><NUM> fluorescent entities, as discussed above, the brightness of each of the example fluorophores <NUM> is several orders of magnitude brighter than prior art fluorophores which have only a few fluorescent dye entities on each fluorophore (e.g., <NUM>-<NUM>, as discussed above). In fact, the extinction coefficient for the example fluorophores <NUM> with various molecular weight linkers <NUM> are in the range of <NUM>×<NUM><NUM> to <NUM>×<NUM><NUM> M-<NUM>cm-<NUM> (as shown in <FIG>), which is much higher than the extinction coefficient of brightest commercial dye (phycoerythrin (PE)) with an extinction coefficient of about <NUM>×<NUM><NUM> M-<NUM>cm-<NUM>. <FIG> shows the brightness of the fluorophores <NUM>. <FIG> show the extinction coefficient and brightness of the dye-linker structures <NUM>, <NUM>.

As shown in <FIG>, the brightness of the example fluorophores <NUM> are much higher ~<NUM><NUM> than those of the dye-linker structures <NUM>, <NUM> shown in <FIG>. This is due to the high extinction coefficients of the fluorophores <NUM> for all linker <NUM> lengths, as compared to those of the free dye-linker structures <NUM>, <NUM>. The extinction coefficient is dependent on the labeling efficiency of the dye-linker structures <NUM>, <NUM> onto the BNNT carriers <NUM> (<FIG>). Therefore, the brightness are highest for the linkers with MW = <NUM> and <NUM> Da. In any case, the extinction coefficients for the example fluorophores <NUM> for all linker <NUM> lengths are several orders of magnitudes higher than those of existing commercial fluorophores.

Another example green fluorophore <NUM>, shown in <FIG> includes a nanotube carrier <NUM> and a DSPE-PEG-NH<NUM> linker <NUM>, as in the previous example, but includes a fluorescein isothiocyanate (FITC, green) fluorescent entity <NUM> instead of RhB as in the previous example. <FIG> shows QY for dye-linker structures <NUM>, <NUM> for the same molecular weight linkers <NUM> as in the previous example. As shown in <FIG>, the dye-linker structures <NUM>, <NUM> exhibit a non-linear trend with linker <NUM> molecular weight.

<FIG> shows QY of dye-linker structures <NUM>, <NUM> labelled onto two types of nanotube carriers <NUM>, CNTs (reference) and BNNTs. As shown, there is a nominal difference in QY between CNT and BNNT carriers. Also, there is generally a linear trend between linker molecular weight and QY for both CNT and BNNT carriers. The fluorophores <NUM> exhibited lower QY than laser grade fluorescein was used as reference which is known to have QY of <NUM> in phosphate-buffered saline (PBS) solution. Therefore, the fluorophores <NUM> exhibited quenching. This could be due to the relatively low labelling efficiency of the dye-linker structures <NUM>, <NUM> onto the nanotube carriers <NUM> as compared to the first example fluorophores <NUM>, especially for low molecular weight linkers <NUM> (shown in <FIG> and <FIG>, respectively). It should be noted that FITC is a pH sensitive dye and the low labeling efficiency of these short-length dye-linker structures <NUM>, <NUM> is affected by the molecular structure of dye-linker structures <NUM>, <NUM>.

Another example far-red fluorophore <NUM>, shown in <FIG> includes a nanotube carrier <NUM> and a DSPE-PEG-NH2 linker <NUM>, as in the previous example, but includes a sulfoCy5 (far-red) fluorescent entity <NUM> instead of RhB or FITC as in the previous examples. <FIG> shows QY for dye-linker structures <NUM>, <NUM> for the same molecular weight linkers <NUM> as in the previous example. As shown in <FIG>, the dye-linker structures <NUM>, <NUM> exhibit a non-linear trend with linker <NUM> molecular weight.

<FIG> shows QY of dye-linker structures <NUM>, <NUM> labelled onto two types of nanotube carriers <NUM>, CNTs (reference) and BNNTs. As shown, there is a nominal difference in QY between CNT and BNNT carriers. Also, there is generally a linear trend between linker molecular weight and QY for both CNT and BNNT carriers. The fluorophores <NUM> exhibited lower QY than a reference dye (<NUM>,<NUM>'Diethythiadicarbobynine iodine, which is known to have QY of <NUM> in EtOH). Therefore, the fluorophores <NUM> exhibited quenching, especially for linker <NUM> MW below <NUM>. This could be due to the relatively low labelling efficiency of the dye-linker structures <NUM>, <NUM> onto the nanotube carriers <NUM> as compared to the first example fluorophores <NUM>, especially for low molecular weight linkers <NUM> (shown in <FIG> and <FIG>, respectively). It should be noted that sulfoCy5 is a small molecule and that Cy5 dyes are known to quench and aggregate at high concentrations.

There were no noticeable spectral peak shift in the absorption spectra of the red fluorophores <NUM>, the green fluorophores <NUM>, or the far-red fluorophores <NUM>. This means, the non-covalent bonding of these dye-linkers structures to the carrier were stable to prevent dye aggregation and collisional quenching and therefore led to the enhanced fluorescence intensity when higher dye-linker concentrations were used in the labeling process. However, this was not the case, when dye-linker length below <NUM> for the far-red fluorophores <NUM>. There was significant aggregation.

The green fluorophores <NUM> and far-red fluorophores <NUM> exhibited high extinction coefficients within linkers of molecular weight <NUM>, as estimated from the slope of the absorbance of the FITC entity <NUM> as a function of dye concentration (which in turn is related to the number of dye-linker structures on each nanotube carrier. <FIG> shows the extinction coefficient of fluorophores <NUM> versus number of dye-linker structures <NUM>, <NUM> per BNNT carrier <NUM>. As shown, the extinction coefficient of these green fluorophores <NUM> can be as high as <NUM>×<NUM><NUM> to <NUM>×<NUM><NUM> M-<NUM>cm-<NUM>. The fluorescence intensity continues to increase linearly with the number of dye-linker structures <NUM>, <NUM> labeled on BNNT carriers <NUM> (at the range of <NUM>×<NUM><NUM> to <NUM>×<NUM><NUM> dye-linker structures <NUM>, <NUM> per BNNT carrier <NUM>). This is unexpected as in prior art fluorophores, quenching occurred where more than a few dyes molecules were conjugated in close proximity.

<FIG> and <FIG> show brightness and extinction coefficients of green and far-red fluorophores <NUM>, <NUM> respectively, for both CNT (reference) and BNNT carriers. As shown, the extinction coefficients for green fluorophores <NUM> increase with linker MW up to about <NUM>×<NUM><NUM> M-<NUM>cm-<NUM> while brightness ranges between about <NUM>×<NUM><NUM> and <NUM>×<NUM><NUM>. For the far-red fluorophore <NUM>, the extinction coefficients increase with linker MW up to about <NUM>×<NUM><NUM> M-<NUM>cm-<NUM> and brightness ranges between about <NUM>×<NUM><NUM> and <NUM>×<NUM><NUM>.

As discussed above, BNNT and CNT are structurally similar. However, CNTs are electrically conductive, while BNNTs are electrically insulating. When similar sized BNNTs and CNTs are labelled with the same red dye-linker structure, for instance, the example red dye linker structure <NUM>, <NUM> discussed above, the BNNT fluorophores exhibit a fluorescence intensity about <NUM> times larger than that for similar CNTs. This result suggests that the relative QY of red-fluorophores using BNNTs as the carrier can be <NUM>-times higher than the QY of red-fluorophores using CNTs as the carrier.

One explanation for the difference is as follows. It is understood that fluorescent entities in physical contact with an electrically insulating matter will subjected to lower fluorescence quenching as compared to the case when the fluorescence particles are in contact with an electrically conducting matter. However, the fluorescent entity <NUM> used herein is connected to the carrier <NUM> through a long polymeric linker <NUM> e.g., one that is electrically insulating such as the DSPE-PEG linker discussed above. Accordingly, it is expected the linker <NUM> insulates the fluorescent entity <NUM> from any effect of the electrical conductivity of a CNT carrier <NUM>. Unexpectedly, the discovered different fluorescent intensities between fluorophores with CNT carriers and BNNT carries implies characteristics of the carrier do affect the fluorescence of a fluorophore. The result also suggests that electrically insulating nanomaterials form higher-brightness fluorophores with high QY than prior art fluorophores. Other electrically insulating nanomaterials that can be used as carrier <NUM> include BN nanosheets, BN nanoparticles, silica particles, alumina particles, nanowires or nanorods of Si, Ge, ZnO, etc..

Nonetheless, although the relative QY of fluorophores made by using CNTs are <NUM>. 5x lower than those made by using BNNTs, the number of dye-linkers per CNT and per BNNT can be identical, as discussed above. Therefore, the extinction coefficients of fluorophores prepared by using CNTs would be the same order of magnitude as those prepared by using BNNTs. Accordingly, high-brightness fluorophores with CNT carriers are still much brighter than other prior art fluorophores.

For the cases of green and far-red labelled fluorophores such as the example fluorophores <NUM>, <NUM> discussed above, there was no significant QY difference when these dye-linker structures are labelled on BNNT versus and CNT as shown in <FIG> and <FIG>. Apparently, the electrically insulating or conducting nature of the nanomaterials (BNNTs and CNTs here) did not affect the QY of FITC and Cy5 fluorescent entities as they did on RhB fluorescent entities. This means, the lengths of linkers (e.g., linkers with MW <NUM> or higher) are sufficient to prevent FITC and Cy5 from significant quenching to the nanomaterials of the carrier. All these green and far-red fluorophores are much brighter than commercial dyes due to their high extinction coefficients, as discussed above.

The high-brightness fluorophores described here are photostable even under the irradiation of tightly focused laser under a confocal fluorescence microscopy. For example, red fluorophores <NUM> in HeLa cells were monitored for five days and did not indicate visible reduction in fluorescence intensity as examined using the same microscopy setting (same focus ratio, same light source power, same gain and same excitation wavelength). This indicates the example fluorophores described herein are more photostable than prior art stains regularly used for cell microscopy imaging. Furthermore, proliferation and signal stability were observed in daughter cells. This result suggests that the structure of the fluorophores described herein is stable and biological compatible such that it can also be used as a photostable stain in vitro and in vivo for tracking.

Additionally, cells incubated with the red fluorophores <NUM> described above exhibited a relationship between concentration of dye-linkers and fluorescence intensity. This means fluorescence intensity described above for red, green, and far-red fluorophores has the same trend inside cells and is therefore applicable for in vitro and in vivo cell tracking application.

Though the example fluorophores described above include only one type of fluorescent entity <NUM>, fluorophores <NUM> including multiple types of fluorescent entities <NUM> linked to a single carrier <NUM> are also contemplated.

Furthermore, any of the fluorophores <NUM> described herein can also be conjugated with biological molecules such as antibodies in addition to fluorescent entities <NUM> using the same or different linkers <NUM>. This allows the fluorophores <NUM> to be simultaneously functionalized with biological molecules for specific biological labeling on cell membranes or other structures inside cells. Antibodies or other biological molecules can be attached to linkers <NUM> by known methods and chemistries. As discussed above, linkers <NUM> include R functional groups to facilitate conjugation to other molecules. The R group can be selected according to the biological molecule to be attached to the linker <NUM>. For instance, a monosulfone-thiol reaction can be used to conjugate an antibody to a monosulfone R-group. Malimide R groups can also be used.

In one example, the green fluorophore <NUM> with <NUM> MW linker <NUM> discussed above was co-labelled anti-human CD4 on BNNT carriers <NUM>. Absorption spectra indicated that the antibody concentration on fluorophore <NUM> was comparable to that of the commercial FITC fluorophores with CD4. The fluorophore <NUM> with CD4 had 5X higher fluorescence intensity than the commercial anti-human CD4 FITC compound at 4X lower concentration.

In addition to or instead of antibodies, fluorophores <NUM> can also be labelled with peptides, oligonucleotides or other macromolecules such as DNA, RNA, antibodies via a linker <NUM> in the same manner discussed above.

Claim 1:
A fluorophore, comprising:
a carrier which is a boron nitride nanotube;
at least one fluorescent entity; and
an amphiphilic linker linking each of the at last one fluorescent entities to the carrier, the linker having a molecular weight greater than <NUM> Da and/or a stretched linker length greater than <NUM> nanometers, wherein the linker comprises at least one hydrophilic portion and a hydrophobic portion, and wherein the hydrophobic portion is non-covalently bonded to the carrier and the hydrophilic portion is covalently bonded to the fluorescent entity.