COMBINED BRIGHT FLUORESCENT DYES WITH CONTROLLABILITY SPECTRAL SHAPES AND METHODS FOR USE THEREOF - Patent application

Abstract

The compound includes a first linker having a first end connected to a carrier, a second linker having a first end connected to a carrier, a third linker having a first end connected to a carrier, a first fluorescent entity connected to the second end of the first linker, a second fluorescent entity different from the first fluorescent entity connected to the second end of the second linker, and a biomolecule connected to the second end of the third linker. The biomolecule is configured to connect to a biomarker. Methods for detecting biomarkers are also disclosed.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 62 / 908,023, filed September 30, 2019, International Application No. PCT / US2020 / 035568, filed June 1, 2020, and International Application No. PCT / US2020 / 035574, filed June 1, 2020. U.S. Provisional Application No. 62 / 908,023, International Application No. PCT / US2020 / 035568, and International Application No. PCT / US2020 / 035574 are incorporated herein by reference in their entireties.

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

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

[0004] Flow cytometry (FCM) can count and sort thousands of cells per second and can be used for phenotyping of biomarkers. In such applications, each type of biomarker is specifically tagged with a fluorescent dye. By tagging a specific biomarker, a population of specific biomarkers can be quantified by quantifying the fluorescent signal from the specific fluorescent entity tagged to the biomarker. Traditional FCM uses bandpass filters to continuously separate and detect signals only near the peak of the fluorescence wavelength range of each fluorescent entity. Spectral flow cytometry (Spectral FCM) uses a series of numerous detectors to collect continuous fluorescence spectra from one or more fluorescent dyes across the entire detection window. Thus, Spectral FCM can also distinguish signals by the shape of the fluorescence spectrum. Summary of the Invention [Means for solving the problem]

[0005] A compound according to an exemplary embodiment of the present disclosure includes, inter alia, a first linker having a first end connected to a carrier, a second linker having a first end connected to a carrier, a third linker having a first end connected to a carrier, a first fluorescent entity connected to the second end of the first linker, a second fluorescent entity different from the first fluorescent entity connected to the second end of the second linker, and a biomolecule connected to the second end of the third linker, where the biomolecule is configured to connect to a biomarker.

[0006] In a further example of the foregoing, the support is a boron nitride nanotube (BNNT) or a carbon nanotube (CNT).

[0007] In a further example of any of the foregoing, the carrier is a nanodot.

[0008] In a further example of any of the foregoing, a first end of at least one of the first, second, and third linkers is covalently attached to the carrier.

[0009] In a further example of any of the foregoing, a first end of at least one of the first, second, and third linkers includes a functional group that covalently bonds the linker to the support.

[0010] In a further example of any of the foregoing, a second end of at least one of the first, second and third linkers is covalently attached to one of the first and second fluorescent entities or a biomolecule via a functional group.

[0011] In a further example of any of the foregoing, a first end of at least one of the first, second, and third linkers is non-covalently attached to the support.

[0012] In a further example of the foregoing, at least one of the first, second and third linkers is amphipathic and includes a hydrophobic region and a hydrophilic region, the hydrophobic region non-covalently binding to the carrier.

[0013] In a further example of any of the foregoing, at least one of the first, second, and third linkers has a molecular weight of about 1000-10000 Da.

[0014] In a further example of any of the foregoing, the support is a boron nitride nanotube.

[0015] In a further example of any of the foregoing, at least one of the first, second, and third linkers is DSPE-PEG. n (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol) n ]), where n is the number of polyethylene glycol (PEG) molecules in the PEG chain.

[0016] A method for detecting biomarkers according to an exemplary embodiment of the present disclosure includes, among other things, providing a sample with a plurality of fluorescent dyes, each of which includes a biomolecule, a first fluorescent entity, and a second fluorescent entity connected to a carrier. The biomolecule is configured to interact with a plurality of biomarkers in the sample. The method also includes exciting the plurality of fluorescent dyes in the sample with a laser and detecting at least one of the identity and the amount of the plurality of biomarkers in the sample based on the fluorescence spectrum emitted by the excited plurality of fluorescent dyes.

[0017] In a further example of any of the foregoing, the first and second fluorescent entities and the biomolecule are connected to the support by first, second and third linkers, respectively, at least one of the first, second and third linkers being linked to the support via a covalent bond.

[0018] In a further example of any of the foregoing, the first and second fluorescent entities and the biomolecule are connected to the support by first, second and third linkers, respectively, at least one of the first, second and third linkers being linked to the support via a non-covalent bond.

[0019] In a further example of any of the foregoing, at least one of the first, second, and third linkers is amphipathic and includes a hydrophobic region and a hydrophilic region. The hydrophobic region non-covalently binds to the carrier.

[0020] In a further example of any of the foregoing, at least one of the first, second, and third linkers has a molecular weight of about 1000-10000 Da.

[0021] In a further example of any of the foregoing, the support is a boron nitride nanotube.

[0022] In a further example of any of the foregoing, the plurality of fluorescent dyes is a first plurality of fluorescent dyes, the biomarker is a first biomarker, and the biomolecule is a second biomolecule, further comprising providing a second plurality of fluorescent dyes to the sample, each of the second plurality of fluorescent dyes including a second biomolecule, a third fluorescent entity, and a fourth fluorescent entity connected to a carrier. The second biomolecule is configured to interact with a plurality of second biomarkers in the sample. The second fluorescent dye connects to the second biomarker.

[0023] In a further example of any of the foregoing, the method further comprises exciting the second plurality of fluorescent dyes with a laser and detecting at least one of the identity and amount of the second plurality of biomarkers in the sample based on a fluorescence spectrum emitted by the excited second plurality of fluorescent dyes. [Brief description of the drawings]

[0024] [Figure 1] (A) is a schematic diagram of an antibody-bound high-brightness fluorescent dye structure, (B) is a schematic diagram of a dye-linker structure, and (C) is a schematic diagram of an antibody-linker structure. [Diagram 2] (A) is a diagram showing simulated fluorescent signals from dye 1 and dye 3, which emit with the same fluorescent intensity. (B) is a diagram showing simulated fluorescent signals from a composite fluorescent dye consisting of dye 1 and dye 3. (C) is a diagram showing simulated fluorescent signals from dye 4 and dye 6, which emit with the same fluorescent intensity. (D) is a diagram showing simulated fluorescent signals from a composite fluorescent dye consisting of dye 4 and dye 6. [Diagram 3](A) is a diagram showing simulated fluorescent signals from dyes 1, 2, and 3 at the same fluorescent intensity. (B) is a diagram showing simulated fluorescent signals from a composite fluorescent dye consisting of dyes 1, 2, and 3. (C) is a diagram showing simulated fluorescent signals from dyes 4, 5, and 6 at the same fluorescent intensity. (D) is a diagram showing simulated fluorescent signals from a composite fluorescent dye consisting of dyes 4, 5, and 6. [Figure 4] (A) is a diagram showing a simulated fluorescent signal from a composite fluorescent dye consisting of dyes 1, 2, and 3 that emit light at an intensity ratio of 1:1:1.5. (B) is a diagram showing a simulated fluorescent signal from a composite fluorescent dye containing dyes 1, 2, and 3 that emit light at an intensity ratio of 1:1.5:1. (C) is a diagram showing a simulated fluorescent signal from a composite fluorescent dye containing dyes 1, 2, and 3 that emit light at an intensity ratio of 1.5:1:1. [Diagram 5] (A) shows the experimental absorption bands (solid curves) and fluorescence bands (dashed curves) of FITC, SRD, and Cy5 dyes. For illustration, all curves are normalized to the same scale. (B) shows the fluorescence signals of composite fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization with a dye-linker concentration ratio of 1:1:1. The solid, dotted, and dashed curves are the actual fluorescence signals from those composite fluorescent dyes excited at 492 nm, 568 nm, and 647 nm. (C) shows the fluorescence signals of composite fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization with a dye-linker concentration ratio of 1:1:2. The solid, dotted, and dashed curves are the actual fluorescence signals from those composite fluorescent dyes excited at 492 nm, 568 nm, and 647 nm. (D) Fluorescence signals of conjugated fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization with a dye-to-linker concentration ratio of 1:4:1. The solid, dotted, and dashed curves are the actual fluorescence signals from those conjugated fluorescent dyes excited at 492 nm, 568 nm, and 647 nm. [Figure 6](A) Comparison of the fluorescence signals of composite fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals are excited at 492 nm. (B) Comparison of the fluorescence signals of composite fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals are excited at 568 nm. (C) Comparison of the fluorescence signals of composite fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals are excited at 647 nm. [Figure 7] (A) Fluorescence signals of composite fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by covalent functionalization at a dye-linker concentration ratio of 1:1:1. The solid, dotted, and dashed curves are the actual fluorescence signals excited at 492 nm, 568 nm, and 647 nm. (B) Fluorescence signals of composite fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by covalent functionalization at a dye-linker concentration ratio of 2:2:1. The solid, dotted, and dashed curves are the actual fluorescence signals excited at 492 nm, 568 nm, and 647 nm. (C) Fluorescence signals of composite fluorescent dyes consisting of FITC, SRD, and Cy5 dyes prepared by covalent functionalization at a dye-linker concentration ratio of 2:1:1. The solid, dotted, and dashed curves are the actual fluorescence signals excited at 492 nm, 568 nm, and 647 nm. [Figure 8](A) Comparison of the fluorescence signals of composite fluorochromes consisting of FITC, SRD, and Cy5 dyes prepared by covalent functionalization using different dye concentration ratios. All signals are excited at 492 nm. (B) Comparison of the fluorescence signals of composite fluorochromes consisting of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals are excited at 568 nm. (C) Comparison of the fluorescence signals of composite fluorochromes consisting of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals are excited at 647 nm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Most commonly, a bright fluorescent dye comprises a carrier element, a fluorescent element, and a linker that connects the carrier element to the fluorescent element. For biomedical applications, each of the carrier element, the linker, and the fluorescent element must be biocompatible (although biocompatibility requirements vary depending on the particular application).

[0026] Exemplary carrier elements are nanomaterials such as carbon nanotubes (CNTs) and boron nitride nanotubes (BNNTs), both of which are recognized as biocompatible nanomaterials for biomedical applications such as cellular drug delivery and spectroscopy applications. However, fluorescent elements bound to nanotubes have been shown to exhibit quenching, or a decrease in fluorescence brightness.

[0027] As described in U.S. Patent Application No. 15 / 953200, filed April 13, 2018 and published as U.S. Patent Application Publication No. 2018 / 0296705, International Application No. PCT / US2020 / 035568, filed June 1, 2020, and International Application No. PCT / US2020 / 035574, certain fluorescent dyes with nanomaterial carriers have been found to not only exhibit no quenching effect, but also exhibit brightness several orders of magnitude higher than other known fluorescent dyes. U.S. Patent Application No. 15 / 953200, International Application No. PCT / US2020 / 035568, and International Application No. PCT / US2020 / 035574 are incorporated herein by reference in their entireties.

[0028] Referring now to FIG. 1A, a fluorescent dye 20 is shown in schematic form. The fluorescent dye 20 generally comprises an inorganic or organic nanoscale carrier 22, a linker 24, a fluorescent entity 26, and a biomolecule 28 (such as an antibody). The antibody / biomolecule 28 can be selected to interact with biomarkers, including cell walls, extracellular vesicles (e.g., exosomes), and the like. Examples of biomarkers include surface markers, such as CD9, CD127, and intracellular markers. This interaction connects the fluorescent dye 20 to the biomarker, such that the biomarker can be detected (and counted, identified, etc.) by detection of the shape of the fluorescence spectrum of the fluorescent dye 20, as described in more detail below.

[0029] In one example, the support 22 is a boron nitride nanotube (BNNT) or carbon nanotube (CNT) support. The support 22 can be manufactured by any known method.

[0030] In a particular example, the support 22 is a multi-walled BNNT or CNT support, each BNNT or CNT having multiple concentric shells of hexagonal boron nitride (h-BN in the case of BNNT) or graphene (in the case of CNT), with a typical outer diameter greater than about 1 nm and less than about 80 nm. The length of such BNNTs and CNTs is about 1-5000 nm. In another example, the support 22 may be another nanoscale inorganic material, such as boron nitride (h-BN) nanosheets / nanoparticles and graphene / graphite nanosheets / nanoparticles. Boron nitride nanodots and carbon nanodots are also contemplated. In one example, as described more fully in International Application No. PCT / US2020 / 035574, the nanodots are treated with mechanical agitation to promote the formation of defects in the nanostructure of the dots, which defects promote / enable binding to the linker 24, thereby allowing more linker 24, and therefore more fluorescent entity 26, to bind to the nanodot and improve the fluorescence of the resulting fluorescent dye 20.

[0031] 1B, linker 24, in one example, is an amphiphilic polymer linker. That is, linker 24 includes a hydrophobic region 25 and a hydrophilic region 27. Hydrophobic region 25 is non-covalently attached to nanotube support 22, while a functional group of hydrophilic region 27 is covalently attached to fluorescent entity 26 (or another entity, described below). One example linker 24 is DSPE-PEG. n -NH2(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) n ]), where n is the number of polyethylene glycol (PEG) molecules in the PEG chain. Other linkers 24 can similarly include PEG chains of varying lengths (or different chains).

[0032] 1C, the hydrophobic region 25 is non-covalently attached to the nanotube support 22, while the functional group of the hydrophilic region 27 is covalently attached to an antibody 28 (or another biomolecule such as a nucleic acid). n-Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) n ]), where n is the number of polyethylene glycol (PEG) molecules in the PEG chain. Other linkers 24 can similarly include PEG chains (or different chains) that can vary in length and have various functional groups.

[0033] In one example, as described more fully in U.S. Patent Application Serial No. 15 / 953,200, linker 24 has a molecular weight greater than about 1000 Da (corresponding to an extended linker length of about 5-10 nm for linker 24 with PEG chains) and less than about 10,000 Da, which can improve the fluorescence of the resulting fluorescent dye 20 compared to prior art fluorescent dyes. In a further example, the molecular weight of linker 24 is greater than about 2400 Da and less than about 10,000 Da.

[0034] In addition to the DSPE-PEG linker 24 discussed above, many other potential linkers are known in the art. For example, the linker 24 may include one or more groups selected from -CH2-, -CH=, -C≡, -NH-, -N=, O-, -NH2-, -N3-, -S-, -C(O)-, -C(O)2-, -C(S)-, -S(O)-, -S(O)2-, or any combination thereof. It will be understood that a linker containing two or more of the above groups is selected so that the linker 24 is stable, for example, so that the linker 24 does not contain two adjacent -O- groups that would generate an unstable peroxide bond. The linker 24 may be linear, branched, or include one or more ring systems. Non-limiting exemplary linkers include fatty acids, phospholipids, sphingolipids, sphingophospholipids (such as, but not limited to, DSPE, 1-O-hexadecanyl-2-O-(9Z-octadecenyl)-sn-glycero-3-phospho-(1'-rac-glycerol) (ammonium salt), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)5000, D-erythro sphingosylphosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine, 3-sn-phosphatidyl-L-serine (PS), glycosylphosphatidylinositol, 1,2-dioleoyl-sn-glycero-3-phosphoethanoamine) hydrophobic regions. The hydrophobic units are used to conjugate with water-soluble polymer chains, such as PEG (or PEO polyethylene oxide), PMO (polymethyloxazoline), PEI (polyethylenimine), polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polypeptides, carbohydrate anchors, etc. The water-soluble polymer chains are attached to a linker at one end and to a fluorescent entity (or another moiety, as described below) at the second end. These hydrophobic and hydrophilic units must have reactive groups, as described above, and such that the groups are attached together to the amphiphilic linker.

[0035] In another example, as described more fully in International Application PCT / US2020 / 035574, the fluorescent dye 20 can be created by covalent functionalization of a linker 24 to the carrier 22. In this example, the linker 24 includes a functional group "R" that interacts with the carrier 22 and a functional group "R" that interacts with other moieties attached to the carrier 22, such as a fluorescent dye molecule 26 and an antibody 28. This linker 24 in this example may be, but is not necessarily, amphiphilic (it may or may not have hydrophobic / hydrophilic regions 25 / 27). Furthermore, in some examples, multiple linkers 24 can be connected to each other in series. An exemplary functional group is an azide group, but any well-known functional group is contemplated. In a further example described in International Application PCT / US2020 / 035574, the support 22 is treated, such as by mechanical agitation in a polar liquid, to create defects in the nanostructure of the support 22, which defects promote / enable binding to the linker 24 via the functional group R.

[0036] The fluorescent entity 26 may be any of the well-known fluorescent dyes, 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 26 is attached to the linker 24 by any suitable method.

[0037] Any of the high brightness fluorophores 20 ("HBF") described herein can include more than one type of fluorescent entity 26 on each support 22 to create a "composite fluorophore." For example, Figure 2A shows the fluorescence spectra of two exemplary fluorophores 26, "Dye 1" and "Dye 3." The dyes have emission spectra similar to fluorescein isothiocyanate (FITC) and cyanine-5 (Cy5), with peak emissions at approximately 515 nm and 665 nm, respectively.

[0038] When "dye 1" and "dye 3" emit light with the same fluorescence intensity (1:1 ratio), the fluorescence spectrum of the composite fluorescent dye 20 having these dyes has two peaks as shown in FIG. 2B.

[0039] In another example, composite fluorescent dye 20 includes two other exemplary dyes, "Dye 4" and "Dye 6," which have a different fluorescence spectrum and a relatively narrower spacing between the spectral peaks compared to the above example, as shown in Figure 2C. In this example, "Dye 4" and "Dye 6" simulate the emission spectra of Alexa 555 and Alexa 568, with peak emissions at approximately 573 nm and 603 nm, respectively.

[0040] When "Dye 4" and "Dye 6" emit with the same fluorescence intensity (1:1 ratio), the fluorescence spectrum of the composite fluorescent dye 20 consisting of those dyes will be as shown in Figure 2D, which has two closer peaks compared to that shown in Figure 2B. In this case, the intensity of the second peak at 603 nm will be higher because the spectrum overlaps with the tail of the adjacent spectrum (see Figure 2C).

[0041] Thus, it has been discovered that composite fluorescent dyes 20 made from different types of dyes result in fluorescence spectra with different, distinguishable shapes, as shown in Figures 2B and 2D.

[0042] As another example, composite fluorescent dye 20 can include three types of dyes 1, 2, and 3 with different fluorescence spectra as shown in Figure 3 A. In this example, dyes 1, 2, and 3 simulate the emission spectra of fluorescein isothiocyanate (FITC), sulforhodamine B (SRD), and cyanine-5 (Cy5), with peak emissions at approximately 515 nm, 588 nm, and 665 nm, respectively.

[0043] When "Dye 1", "Dye 2", and "Dye 3" emit with the same fluorescence intensity (1:1:1 ratio), the fluorescence spectrum of the composite fluorescent dye 20 composed of those dyes has three peaks, as shown in Figure 3B. As shown, the intensity of the peak 588 nm is higher than that of the individual dyes 2 shown in Figure 3A because the spectrum overlaps with the tails of the neighboring spectra (Figure 3A).

[0044] As another example, the composite fluorescent dye 20 can include dyes 4, 5, and 6, which have different fluorescence spectra and narrower spacing between spectral peaks compared to the previous examples, as shown in Figure 3C. These dyes 4, 5, and 6 simulate the emission spectra of Alexa555, SRD, and Alexa568, with peak emissions at approximately 573 nm, 585 nm, and 603 nm, respectively. The spacing between their emission peaks is approximately 12 nm to 18 nm.

[0045] When "Dye 4," "Dye 5," and "Dye 6" emit with the same fluorescence intensity (1:1:1 ratio), the fluorescence spectrum of composite fluorescent dye 20 composed of these dyes is shown in Figure 3D. As shown, the individual emission peaks are closely spaced, so all three individual spectra merged into a broad spectrum with a unique shape.

[0046] As suggested by FIGS. 3B and 3D, the choice of dyes and the spacing between their emission peaks affects the fluorescence spectral shape of a composite fluorescent dye 20 that contains multiple dyes.

[0047] In another example, the composite fluorescent dye 20 may include dyes that emit with different fluorescence intensities from each other. FIG. 4A shows the integrated spectrum of dyes 1, 2, and 3 with a fluorescence intensity ratio of 1:1:1.5. FIG. 4B shows the integrated spectrum of dyes 1, 2, and 3 with a fluorescence intensity ratio of 1:1.5:1. FIG. 4C shows the integrated spectrum of dyes 1, 2, and 3 with a fluorescence intensity ratio of 1.5:1:1. As shown, even if the composite fluorescent dyes 20 include the same type of dyes, all the spectra obtained are unique in shape. The shape of the fluorescence spectrum of the composite fluorescent dye 20 depends on the fluorescence intensity ratio of the individual dyes under a specific excitation laser source / wavelength.

[0048] FIG. 5A shows the normalized absorption bands (solid lines) and fluorescence bands (dashed lines) of a composite fluorescent dye 20 with three dyes 26 on each carrier 22. In this example, the carrier 22 is a BNNT carrier. Spectra 1 and 2 are the absorption and fluorescence spectra of FITC, respectively. Spectra 3 and 4 are the absorption and fluorescence spectra of SRD, respectively. Spectra 5 and 6 are the absorption and fluorescence spectra of Cy5, respectively.

[0049] FIG. 5B shows the fluorescence signals of those composite fluorescent dyes 20 excited at 492 nm (solid line), 568 nm (dotted line), and 647 nm (dashed line), respectively.

[0050] Since the 492 nm laser is well within the absorption band of FITC (spectrum 1 in Figure 5A), strong fluorescence from FITC at 520 nm is detected as shown as a solid spectrum in Figure 5B, with a long tail given by the weak fluorescence of SRD (588 nm) and Cy5 (665 nm). The 492 nm laser is not expected to result in emission of SRD and Cy5, since the excitation laser is far away from their absorption bands (spectrums 3 and 5 in Figure 5A, respectively). Thus, the small fluorescence from SRD at 588 nm is excited by the fluorescence from FITC. Similarly, the small fluorescence from Cy5 at 665 nm is excited by the fluorescence from SRD.

[0051] The fluorescence signal of their composite fluorochrome 20 excited at 568 nm (dotted spectrum in Figure 5B) is explained as follows. The 568 nm laser excites SRD, which showed strong emission at 568 nm and a weak fluorescence tail of Cy5 at 665 nm. This 568 nm laser is not supposed to excite FITC and Cy5 because the excitation laser is far away from their absorption bands. Therefore, the small fluorescence from Cy5 at 665 nm is excited by the fluorescence from SRD.

[0052] The fluorescence signals of their composite fluorochromes 20 excited at 647 nm (dashed spectrum in FIG. 5B) are explained as follows: The 647 nm laser excited only the Cy5 dye, resulting in strong emission at 665 nm.

[0053] As shown in FIG. 5B, the shapes of the fluorescence spectra of the composite fluorescent dye 20 are different when excited by different lasers.

[0054] 5C and 5D summarize the effect of dye concentration used in the preparation of composite fluorescent dye 20. Dye concentration is defined as the number of dye molecules per unit volume of synthesis solution.

[0055] Figure 5C shows the fluorescence signals of composite HBF20 excited at 492 nm (solid line), 568 nm (dotted line), and 647 nm (dashed line), respectively. The composite HBF20 is produced by using a mixture of FITC, SRD, and Cy5 dye molecules in a concentration ratio of 1:1:2. The total dye concentrations used are the same as those described above for Figure 5B.

[0056] Figure 5D shows the fluorescence signals of composite HBF20 excited at 492 nm (solid line), 568 nm (dotted line), and 647 nm (dashed line), respectively. The composite HBF20 is produced by using a mixture of FITC, SRD, and Cy5 dye molecules in a concentration ratio of 1:4:1. The total dye concentrations used are the same as those described for Figures 5B and 5C.

[0057] As shown, the solid-state spectra in Figures 5C and 5D have different shapes excited by a laser at 492 nm due to the fact that their HBF20 have different dye concentration ratios, even though the total dye concentration is the same. For comparison, all spectra excited at 492 nm are shown together in Figure 6A. As shown, composite HBF20 with different dye concentration ratios (1:1:1, 1:1:2, 1:4:1) but the same total dye concentration emit fluorescence with different spectral shapes even when excited with the same laser wavelength. The same is true when comparing all dotted spectra (Figure 6B excited at 568 nm), or all dashed spectra (Figure 6C excited at 647 nm).

[0058] As described in U.S. Patent Application No. 15 / 953,200, filed April 13, 2018, and published as U.S. Patent Application Publication No. 2018 / 0296705, any of the above exemplary conjugated fluorescent dyes 20 can include BNNT as the carrier 22 and DSPE-PEG5000 as the linker 24.

[0059] In another example, the fluorescent dye 20 includes a linker 24 covalently attached to the carrier 22 and the fluorescent entity 26, as described above. Thus, the exemplary fluorescent dyes 20 are prepared by covalent functionalization. For example, the fluorescent dye 20 includes a mixture of three different types of dyes, namely FITC, SRD, and Cy5 dye molecules, in three concentration ratios (1:1:1, 2:2:1, 2:1:1), as shown in Figures 7A, 7B, and 7C, respectively. The total dye concentration used in each of the three composite HBFs 20 is the same.

[0060] As shown in Figure 7A, the use of different excitation lasers (492 nm, 568 nm, and 647 nm) results in different spectral shapes, as shown by the solid, dotted, and dashed spectra, respectively, for a 1:1:1 dye concentration ratio.

[0061] As shown in Figure 7B, the use of different excitation lasers (492 nm, 568 nm, and 647 nm) results in different spectral shapes, as shown by the solid, dotted, and dashed spectra, respectively, for a dye concentration ratio of 2:2:1.

[0062] As shown in Figure 7C, the use of different excitation lasers (492 nm, 568 nm, and 647 nm) results in different spectral shapes, as indicated by the solid, dotted, and dashed spectra, respectively, for a dye concentration ratio of 2:1:1.

[0063] 8A compares the spectral shapes of conjugated fluorescent dyes 20 prepared with dye concentration ratios of 1:1:1, 2:2:1, and 2:1:1 excited at 492 nm. As shown, the spectral shape of conjugated fluorescent dyes 20 with a dye-linker 24 / 26 concentration ratio of 1:1:1 is very different from that with a dye-linker 24 / 26 concentration ratio of 2:2:1 and that with a dye-linker 24 / 26 concentration ratio of 2:1:1. The spectral shapes of the samples with 2:2:1 and 2:1:1 ratios are similar to SRD and Cy5 dyes, respectively, which are not sufficiently excited at 492 nm.

[0064] Figure 8B compares the spectral shapes of conjugated fluorescent dyes 20 with dye-to-linker 24 / 26 concentration ratios of 1:1:1, 2:2:1, and 2:1:1, excited at 568 nm. As shown, the spectra are all different in shape.

[0065] Figure 8C compares the spectral shapes of conjugated fluorescent dye 20 with dye-to-linker 24 / 26 concentration ratios of 1:1:1, 2:2:1, and 2:1:1, excited at 647 nm. As shown, the spectra are all different in shape.

[0066] Thus, the spectra of the conjugated fluorescent dyes 20 described herein vary depending on the excitation laser selected.

[0067] The conjugated fluorochromes 20 described herein can be used for flow cytometry. For conventional flow cytometry (FCM), one or more biomarkers are tagged with a unique fluorochrome 20, such as the conjugated fluorochrome 20 described above, via a biomolecule 28 described above. The fluorochrome 20 is excited with a laser, which causes the fluorochrome 20 to fluoresce and emit detectable photons. Conventional FCM uses bandpass filters to sequentially separate and detect signals associated with photons only near the peak of the fluorescence wavelength range of each fluorescent entity. By tagging specific biomarkers, a population of those biomarkers can be quantified by quantifying the fluorescent signal from the unique fluorescent entity 20 tagged to the biomarker(s).

[0068] In spectral flow cytometry (spectral FCM), the fluorescence spectrum of a sample is detected over a given detection window (e.g., wavelength range, etc.). Each fluorescent entity 20 in the sample has a unique fluorescence spectrum that can be detected and identified, for example, by spectral unmixing, as is well known in the art, to identify and quantify biomarkers in the sample.

[0069] In general, for spectral FCM, there is a trade-off between detection speed, sensitivity, and spectral resolution. For example, in order to collect many data points within a reasonably short detection period, the detection time for each signal becomes very short. However, because the fluorochromes 20 described herein have such high fluorescence intensity, they require less laser exposure (e.g., time) to obtain a registerable fluorescent signal. Thus, the fluorochromes 20 described herein enable the use of spectral FCM in high-throughput applications. Furthermore, because the composite fluorochromes 20 can include two, three, or more fluorescent entities 26 that are selected to generate a fluorochrome 20 with a unique fluorescence spectrum when combined in various amounts, many different fluorochromes 20, each with a unique fluorescence spectrum, can be made by using readily available fluorescent dyes.

[0070] The foregoing description is exemplary rather than limiting in nature. Variations and modifications of the disclosed examples will become apparent to those skilled in the art that do not necessarily depart from the essence of this invention. The scope of legal protection given to this invention can only be determined by studying the following claims.

Claims

1. A carrier; a first linker having a first end connected to the support; a second linker having a first end connected to the support; a third linker having a first end connected to the support; a first fluorescent entity attached to a second end of the first linker; a second fluorescent entity different from the first fluorescent entity attached to a second end of the second linker; a biomolecule connected to a second end of the third linker, the biomolecule being configured to connect to a biomarker; A compound comprising:

2. 2. The compound according to claim 1, characterized in that the support is a boron nitride nanotube (BNNT) or a carbon nanotube (CNT).

3. The compound according to claim 1, characterized in that the carrier is a nanodot.

4. The compound of claim 1, wherein the first end of at least one of the first, second, and third linkers is covalently attached to the carrier.

5. 5. The compound of claim 4, wherein the first end of at least one of the first, second, and third linkers comprises a functional group, the functional group covalently attaching the linker to the carrier.

6. 5. The compound of claim 4, wherein the second end of at least one of the first, second and third linkers is covalently attached to one of the first and second fluorescent entities or a biomolecule via a functional group.

7. The compound of claim 1, wherein the first end of at least one of the first, second, and third linkers is non-covalently attached to the carrier.

8. The compound of claim 7, wherein at least one of the first, second, and third linkers is amphipathic and comprises a hydrophobic region and a hydrophilic region, the hydrophobic region being non-covalently attached to the carrier.

9. The compound of claim 7, wherein at least one of the first, second and third linkers has a molecular weight of 1,000 to 10,000 Da.

10. 8. The compound according to claim 7, characterized in that the support is a boron nitride nanotube.

11. At least one of the first, second, and third linkers is DSPE-PEG ｎ (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol) ｎ 2. The compound of claim 1, wherein n is the number of polyethylene glycol (PEG) molecules in the PEG chain.

12. providing a plurality of fluorescent dyes to a sample, each of the fluorescent dyes comprising a carrier, a first linker having a first end connected to the carrier, a second linker having a first end connected to the carrier, a third linker having a first end connected to the carrier, a first fluorescent entity connected to a second end of the first linker, a second fluorescent entity different from the first fluorescent entity connected to a second end of the second linker, and a biomolecule connected to a second end of the third linker, the biomolecule being configured to connect to a biomarker, the biomolecule being configured to interact with a plurality of biomarkers in the sample, whereby the fluorescent dyes connect to the biomarkers; exciting the plurality of fluorescent dyes in the sample with a laser; detecting at least one of the identity and the amount of the plurality of biomarkers in the sample based on the fluorescence spectra emitted by the excited plurality of fluorescent dyes. A method for detecting a biomarker, comprising:

13. 13. The method of claim 12, wherein the support is a boron nitride nanotube (BNNT) support, a carbon nanotube (CNT) support, or a nanodot.

14. 13. The method of claim 12, wherein the first fluorescent entity, the second fluorescent entity, and the biological molecule are connected to the support by the first, second, and third linkers, respectively, and at least one of the first, second, and third linkers is linked to the support via a covalent bond.

15. 13. The method of claim 12, wherein the first fluorescent entity, the second fluorescent entity and the biological molecule are connected to the support by the first, second and third linkers, respectively, and at least one of the first, second and third linkers is linked to the support via a non-covalent bond.

16. 16. The method of claim 15, wherein at least one of the first, second, and third linkers is amphipathic and comprises a hydrophobic region and a hydrophilic region, the hydrophobic region non-covalently binding to the carrier.

17. 17. The method of claim 16, wherein at least one of the first, second and third linkers has a molecular weight of 1,000 to 10,000 Da.

18. 18. The method of claim 17, wherein the support is a boron nitride nanotube.

19. the plurality of fluorescent dyes is a first plurality of fluorescent dyes, the biomarker is a first biomarker, and the biomolecule is a first biomolecule; providing a second plurality of fluorescent dyes to the sample, the second plurality of fluorescent dyes including a second biomolecule, a third fluorescent entity, and a fourth fluorescent entity each attached to a carrier, the second biomolecule being configured to interact with a second plurality of biomarkers in the sample, whereby the second fluorescent dyes attach to the second biomarkers; 13. The method of claim 12, further comprising:

20. the second plurality of fluorescent dyes are excited by a laser; 20. The method of claim 19, further comprising detecting at least one of the identity and amount of the second plurality of biomarkers in the sample based on the fluorescence spectra emitted by the excited second plurality of fluorescent dyes.

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