Compound, method for detecting biomarker and amphiphilic polymer linker

Abstract

The invention relates to a compound, a method for detecting a biomarker and an amphiphilic polymer linker. The invention provides a compound which comprises a first connector with a first end connected with a carrier, a second connector with a first end connected with the carrier, a third connector with a first end connected with the carrier, a fourth connector connected with the carrier and a first fluorescent entity connected with the second end of the first connector, the second fluorescent entity is different from the first fluorescent entity and connected with the second end of the second connector, the third fluorescent entity is different from the first fluorescent entity and the second fluorescent entity and connected with the fourth connector, and the biomolecule is connected with the second end of the third connector. The biomolecule is configured to be linked to a biomarker. Wherein the three fluorescent entities each emit at approximately the same fluorescence intensity as one another. A method of detecting a biomarker and an amphiphilic polymer linker are also disclosed.

Description

[0001] This application is a divisional application of the invention patent application filed on September 30, 2020, entitled "A composite high-brightness fluorophore with a controllable spectral shape and a method for using a composite high-brightness fluorophore", with application number 2020800683755. Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial 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. The entire contents of U.S. Provisional Application Serial No. 62 / 908,023, International Application No. PCT / US2020 / 035568, and International Application No. PCT / US2020 / 035574 are incorporated herein by reference.

[0003] Government Support Statement The invention described herein was made with government support under Grant #1261910, Grant #1521057, and Grant #1738466 granted by the National Science Foundation. The government holds certain rights to this invention. Technical Field

[0004] This invention relates to fluorophore compounds and methods for detecting biomarkers using fluorophores. Background Technology

[0005] A fluorophore is a compound with fluorescent properties that has biomedical applications. For example, fluorophores can be used as tracers or dyes for staining certain molecules or structures. More specifically, fluorophores can be used to stain tissues, cells, or biological materials in various analytical methods such as fluorescence imaging and spectroscopy.

[0006] Flow cytometry (FCM) can count and sort thousands of cells per second and can be used for biomarker phenotype analysis. For such applications, each type of biomarker is specifically labeled with a fluorophore. By labeling specific biomarkers, the population of specific biomarkers can be quantified by quantifying the fluorescence signal from the specific fluorescent entity labeled on the biomarker. Conventional FCM uses a bandpass filter to sequentially separate and detect signals only near the peak of the fluorescence wavelength range for each fluorophore. Spectroscopic flow cytometry (spectral FCM) collects continuous fluorescence spectra from one or more fluorophores across the entire detection window using a series of numerous detectors. Therefore, spectral FCM can also distinguish signals by the shape of the fluorescence spectrum. Summary of the Invention

[0007] The compounds according to exemplary embodiments of this disclosure, among other possibilities, include: a first connector having a first end connected to a carrier; a second connector having a first end connected to the carrier; a third connector having a first end connected to the carrier; a first fluorescent entity connected to a second end of the first connector; a second fluorescent entity, different from the first fluorescent entity, connected to a second end of the second connector; and a biomolecule connected to a second end of the third connector. The biomolecule is configured to be linked to a biomarker.

[0008] In the other examples mentioned above, the carrier is boron nitride nanotubes (BNNTs) or carbon nanotubes (CNTs).

[0009] In another example of any of the foregoing, the carrier is a nanodot.

[0010] In another example of any of the foregoing, the first end of at least one of the first, second, and third connectors is covalently bonded to the carrier.

[0011] In another example of any of the foregoing, the first end of at least one of the first, second, and third connectors includes a functional group, and the functional group covalently binds the connector to the carrier.

[0012] In another example of any of the foregoing, the second end of at least one of the first, second, and third connectors is covalently bound to one of the first and second fluorescent entities or the biomolecule via a functional group.

[0013] In another example of any of the foregoing, the first end of at least one of the first, second, and third connectors is non-covalently bonded to the carrier.

[0014] In another example described above, at least one of the first, second, and third connectors is amphiphilic and includes both hydrophobic and hydrophilic regions. The hydrophobic region is non-covalently bonded to the carrier.

[0015] In another example of any of the foregoing, at least one of the first, second, and third connectors has a molecular weight between about 1,000 and 10,000 Da.

[0016] In another example of any of the foregoing, the carrier is boron nitride nanotubes.

[0017] In another example of any of the foregoing, at least one of the first, second and third connectors is DSPE-PEGn (1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[(polyethylene glycol)n]), where n is the number of polyethylene glycol (PEG) molecules in the PEG chain.

[0018] A method for detecting biomarkers according to exemplary embodiments of the present disclosure, among other possibilities, includes providing a plurality of fluorophores to a sample, each of the plurality of fluorophores comprising a biomolecule linked to a carrier, a first fluorescent entity, and a second fluorescent entity. The biomolecule is configured to interact with a plurality of biomarkers in the sample. The method further includes exciting the plurality of fluorophores in the sample with a laser and detecting at least one of the characteristics and amounts of the plurality of biomarkers in the sample based on the fluorescence spectra emitted by the excited plurality of fluorophores.

[0019] In another example of any of the foregoing embodiments, the first and second fluorescent entities and the biomolecule are connected to the carrier via first, second, and third connectors. At least one of the first, second, and third connectors is connected to the carrier via a covalent bond.

[0020] In another example of any of the foregoing embodiments, the first and second fluorescent entities and the biomolecule are respectively connected to the carrier via first, second, and third connectors. At least one of the first, second, and third connectors is connected to the carrier via a non-covalent bond.

[0021] In another example of any of the foregoing embodiments, at least one of the first, second, and third connectors is amphiphilic and includes both hydrophobic and hydrophilic regions. The hydrophobic region is non-covalently bonded to the carrier.

[0022] In another example of any of the foregoing, at least one of the first, second, and third connectors has a molecular weight between about 1,000 and 10,000 Da.

[0023] In another example of any of the foregoing, the carrier is boron nitride nanotubes.

[0024] In another example of any of the foregoing embodiments, the plurality of fluorophores are a first plurality of fluorophores, the biomarker is a first biomarker, and the biomolecule is a second biomolecule, and further includes providing a second plurality of fluorophores to the sample, each of the second plurality of fluorophores comprising a second biomolecule, a third fluorescent entity, and a fourth fluorescent entity linked to a carrier. The second biomolecule is configured to interact with the second plurality of biomarkers in the sample. The second fluorophore is linked to the second biomolecule.

[0025] In another example of any of the foregoing, the second plurality of fluorophores are excited by the laser, and further include detecting at least one of the characteristics and amounts of the second plurality of biomarkers in the sample based on the fluorescence spectrum emitted by the excited second plurality of fluorophores. Attached Figure Description

[0026] Figure 1 A in the diagram schematically shows the structure of the antibody-conjugated high-brightness fluorophore.

[0027] Figure 1 B in the diagram schematically illustrates the dye-connector structure.

[0028] Figure 1 The C in the diagram schematically illustrates the antibody-connector structure.

[0029] Figure 2 A in the figure shows simulated fluorescence signals from dyes 1 and 3, which emit with the same fluorescence intensity.

[0030] Figure 2 B in the diagram shows the simulated fluorescence signal from the complex fluorophore composed of dye 1 and dye 3.

[0031] Figure 2 C in the figure represents the simulated fluorescence signal from dyes 4 and 6, which emit with the same fluorescence intensity.

[0032] Figure 2 D in the figure represents the simulated fluorescence signal from the complex fluorophore composed of dye 4 and dye 6.

[0033] Figure 3 A in the diagram shows simulated fluorescence signals from dyes 1, 2, and 3 with the same fluorescence intensity.

[0034] Figure 3 B in the diagram represents the simulated fluorescence signal from the complex fluorophore composed of dyes 1, 2, and 3.

[0035] Figure 3 C in the figure represents the simulated fluorescence signal from dyes 4, 5, and 6 with the same fluorescence intensity.

[0036] Figure 3 D in the figure represents the simulated fluorescence signal from the complex fluorophore composed of dyes 4, 5, and 6.

[0037] Figure 4 A in the figure shows a simulated fluorescence signal from a complex fluorophore composed of dyes 1, 2, and 3 emitted at an intensity ratio of 1:1:1.5.

[0038] Figure 4B in the figure shows a simulated fluorescence signal from a complex fluorophore of dyes 1, 2, and 3 emitting at an intensity ratio of 1:1.5:1.

[0039] Figure 4 C in the figure represents the simulated fluorescence signal from a complex fluorophore of dyes 1, 2, and 3 emitting at an intensity ratio of 1.5:1:1.

[0040] Figure 5 Figure A shows the experimental absorption bands (solid lines) and fluorescence bands (dashed lines) for FITC, SRD, and Cy5 dyes. For illustrative purposes, all curves have been normalized to the same scale.

[0041] Figure 5 Figure B shows the fluorescence signal of a composite fluorophore composed of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization at a dye-linker concentration ratio of 1:1:1. The solid, dotted, and dashed curves represent the actual fluorescence signals of these composite fluorophores excited at 492 nm, 568 nm, and 647 nm.

[0042] Figure 5 C in the figure represents the fluorescence signal of a composite fluorophore composed of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization at a dye-linker concentration ratio of 1:1:2. The solid, dotted, and dashed curves represent the actual fluorescence signals of these composite fluorophores excited at 492 nm, 568 nm, and 647 nm.

[0043] Figure 5 D in the figure represents the fluorescence signal of a composite fluorophore composed of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization at a dye-linker concentration ratio of 1:4:1. The solid, dotted, and dashed curves represent the actual fluorescence signals of these composite fluorophores excited at 492 nm, 568 nm, and 647 nm.

[0044] Figure 6 Figure A compares the fluorescence signals of composite fluorophores composed of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals were excited at 492 nm.

[0045] Figure 6 Figure B compares the fluorescence signals of composite fluorophores composed of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals were excited at 568 nm.

[0046] Figure 6The C in the figure compares the fluorescence signals of composite fluorophores composed of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals were excited at 647 nm.

[0047] Figure 7 Figure A shows the fluorescence signal of a complex fluorophore composed 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 represent the actual fluorescence signals excited at 492 nm, 568 nm, and 647 nm.

[0048] Figure 7 Figure B shows the fluorescence signal of a complex fluorophore composed 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 represent the actual fluorescence signals excited at 492 nm, 568 nm, and 647 nm.

[0049] Figure 7 C in the figure represents the fluorescence signal of a complex fluorophore composed 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 represent the actual fluorescence signals excited at 492 nm, 568 nm, and 647 nm.

[0050] Figure 8 Figure A compares the fluorescence signals of composite fluorophores composed of FITC, SRD, and Cy5 dyes prepared by covalent functionalization using different dye concentration ratios. All signals were excited at 492 nm.

[0051] Figure 8 Figure B compares the fluorescence signals of composite fluorophores composed of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals were excited at 568 nm.

[0052] Figure 8 The C in the figure compares the fluorescence signals of composite fluorophores composed of FITC, SRD, and Cy5 dyes prepared by non-covalent functionalization using different dye concentration ratios. All signals were excited at 647 nm. Detailed Implementation

[0053] Very generally, a high-brightness fluorophore comprises a carrier element, a fluorescent element, and a connector that links the carrier element to the fluorescent element. For biomedical applications, each of the carrier element, connector, and fluorescent element must be biocompatible (although biocompatibility requirements may vary from application to application).

[0054] One example of a carrier element is nanomaterials such as carbon nanotubes (CNTs) and boron nitride nanotubes (BNNTs), both of which are considered biologically compatible nanomaterials for biomedical applications such as cell-based drug delivery and spectroscopic applications. However, fluorescent elements coupled with nanotubes have been shown to exhibit quenching or a reduction in fluorescence brightness.

[0055] It has been found that certain fluorophores with nanomaterial supports not only do not exhibit quenching effects, but also exhibit brightness several orders of magnitude higher than other known fluorophores, as described below in: U.S. Patent Application Serial No. 15 / 953,200, filed April 13, 2018, published as U.S. Patent Publication No. 2018 / 0296705; International Application No. PCT / US2020 / 035568, filed June 1, 2020; and International Application No. PCT / US2020 / 035574. The entire contents of U.S. Patent Application Serial No. 15 / 953,200, International Application Nos. PCT / US2020 / 035568 and PCT / US2020 / 035574 are incorporated herein by reference.

[0056] Now for reference Figure 1 In diagram A, fluorophore 20 is schematically shown. Fluorophore 20 typically 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 those on the cell wall, extracellular vesicles (such as extracellular bodies), etc. Example biomarkers include surface markers such as CD9 and CD127, as well as intracellular markers. This interaction links fluorophore 20 to the biomarker, allowing the biomarker to be detected (and counted, identified, etc.) by detecting the shape of the fluorescence spectrum of fluorophore 20, as discussed in more detail below.

[0057] 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.

[0058] In specific instances, the support 22 is a multi-walled BNNT or CNT support, wherein each BNNT or CNT has multiple coaxial shells of hexagonal boron nitride (h-BN for BNNTs) or graphene (for CNTs) with a typical outer diameter greater than about 1 nm but less than about 80 nm. The lengths of these BNNTs and CNTs are between about 1–5000 nm. In other instances, the support 22 can 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 considered. In one instance, as more fully described in International Application PCT / US2020 / 035574, the nanodots are mechanically stirred to promote the formation of defects in the nanostructure of the dots, which facilitate / enable bonding with the connectors 24. This, in turn, allows more connectors 24 and therefore more fluorescent entities 26 to bind to the nanodots and improve the fluorescence of the resulting fluorophore 20.

[0059] Now for reference Figure 1 In example B, connector 24 is an amphiphilic polymer connector. That is, connector 24 comprises a hydrophobic region 25 and a hydrophilic region 27. The hydrophobic region 25 is non-covalently bonded to the nanotube carrier 22, while the functional groups on the hydrophilic region 27 are covalently bonded to the phosphor 26 (or another entity, as will be discussed below). One example of connector 24 is DSPE-PEG. n -NH2(1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)]) n ]), where n is the number of polyethylene glycol (PEG) molecules in the PEG chain. Other connectors 24 may similarly include PEG chains of different lengths (or different chains).

[0060] Now for reference Figure 1 In the C region, the hydrophobic region 25 is non-covalently bound to the nanotube carrier 22, while the functional groups on the hydrophilic region 27 are covalently bound to the antibody 28 (or another biomolecule, such as nucleic acid). An example linker 24 is DSPE-PEG. n -Maleimide (1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[maleimide (polyethylene glycol)]) n ]), where n is the number of polyethylene glycol (PEG) molecules in the PEG chain. Other connectors 24 may similarly include PEG chains (or different chains) of varying lengths and with various functional groups.

[0061] In one example, as more fully described in patent application serial number 15 / 953,200, the connector 24 has a molecular weight greater than about 1000 Da (which, for a connector 24 with a PEG chain, corresponds to an extended connector length of about 5-10 nm) and less than about 10000 Da, which allows for improved fluorescence of the resulting fluorophore 20 compared to prior art fluorophores. In another example, the molecular weight of the connector 24 is greater than about 2400 Da and less than about 10000 Da.

[0062] Besides the DSPE-PEG linker 24 discussed above, many other potential linkers are known in the art. For example, 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 should be understood that linkers including more than one of the above groups will be selected such that linker 24 is stable; for example, linker 24 may not include two adjacent -O- groups, which would form an unstable peroxide bond. Linker 24 may be straight-chain, branched, or may include one or more ring systems. Non-limiting exemplary connectors include hydrophobic regions that can be fatty acids, phospholipids, sphingolipids, sphingomyelins [such as DSPE, 1-O-hexadecyl-2-O-(9Z-octadecenyl)-sn-glycerol-3-phosphate-(1'-rac-glycerol) (ammonium salt), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)5000, D-erythrosine-sphingosine ethanolamine, 1,2-diphydanyl-sn-glycerol-3-phosphate-L-serine, 3-sn-phosphatidyl-L-serine (PS), glycosylphosphatidylinositol, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, but not limited thereto]. The hydrophobic unit can be used to anchor to water-soluble polymer chains such as PEG (or PEO polyoxyethylene), PMO (polymethyloxazoline), PEI (polyethyleneimine), polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, peptides, and carbohydrates. The water-soluble polymer chain is attached to a linker at one end and to a fluorescent entity (or another part, as described below) at the second end. These hydrophobic and hydrophilic units must have the reactive groups mentioned above and such that these groups are concatenated together to form an amphiphilic linker.

[0063] In another example, as more fully described in International Application PCT / US2020 / 035574, fluorophore 20 can be created by covalently functionalizing linker 24 onto support 22. In this example, linker 24 comprises a functional group “R” that interacts with support 22 and a functional group “R’” that interacts with other parts of support 22 such as fluorescent dye molecule 26 and antibody 28. Such linker 24 in this example is not necessarily amphiphilic (and may or may not have hydrophobic / hydrophilic regions 25 / 27), although it may be. Additionally, in some examples, multiple linkers 24 may be connected in tandem with each other. The example functional group is an azide group, but any known functional group will be considered. In another example described in International Application PCT / US2020 / 035574, support 22 is treated, for example, by mechanical stirring in a polar liquid, to form defects in the nanostructure of support 22 that promote / cause binding to linker 24 via functional group R.

[0064] Fluorescent entity 26 is any known fluorescent dye, including but not limited to coumarins, benzoxadiazoles, acridinones, acridiniums, bisbenzimides, indole, benzisoquinoline, naphthalene, anthracene, xanthones, pyrene, porphyrin, fluorescein, rhodamine, boron-dipyrrole methylene (BODIPY), and anthocyanin derivatives. Many such fluorescent dyes are commercially available. Fluorescent entity 26 is bound to connector 24 by any suitable method.

[0065] Any high-brightness fluorophore 20 (“HFB”) described herein may include more than one type of fluorescent entity 26 on each carrier 22 to create a “composite fluorophore”. For example, Figure 2 Figure A shows the fluorescence spectra of two example fluorescent dyes, “Dye 1” and “Dye 3”. These dyes have emission spectra similar to those of fluorescein isothiocyanate (FITC) and cyanine-5 (Cy5), with peak emission at ~515 nm and 665 nm, respectively.

[0066] When "dye 1" and "dye 3" emit with the same fluorescence intensity (in a 1:1 ratio), the fluorescence spectrum of the composite fluorophore 20 containing these dyes will be as follows: Figure 2 As shown in B, it has two peaks.

[0067] In another example, the composite fluorophore 20 includes two other example dyes, "Dye 4" and "Dye 6," which have different fluorescence spectra from the examples above, and the spacing between the spectral peaks is relatively narrower compared to the examples above, such as... Figure 2As shown in C. In this example, "Dye 4" and "Dye 6" simulate the emission spectra of Alexa 555 and Alexa 568, with emission peaks at ~573 nm and 603 nm, respectively.

[0068] When dye 4 and dye 6 emit with the same fluorescence intensity (in a 1:1 ratio), the fluorescence spectrum of the composite fluorophore 20 composed of these dyes will be as follows: Figure 2 As shown in D, with Figure 2 Compared to those shown in B, these have two closer peaks. In this case, the second peak at 603 nm is more intense due to the overlap of the tails of the spectra with those of adjacent spectra (as shown in B). Figure 2 (as shown in C).

[0069] Therefore, it has been found that composite fluorophores 20 made from different types of dyes will produce fluorescence spectra with different, recognizable shapes, such as Figure 2 As shown in B and D.

[0070] As another example, the composite fluorophore 20 may include three types of dyes 1, 2, and 3 with different fluorescence spectra, such as Figure 3 As shown in A. In this example, dyes 1, 2, and 3 simulate the emission spectra of fluorescein isothiocyanate (FITC), sulfonylrhodamine B (SRD), and cyanine-5 (Cy5), with emission peaks at ~515 nm, 588 nm, and 665 nm, respectively.

[0071] When dyes 1, 2, and 3 emit at the same fluorescence intensity (in a 1:1:1 ratio), the fluorescence spectrum of the composite fluorophore 20 composed of these dyes will be as follows: Figure 3 As shown in B, it has three peaks. As illustrated, due to the overlap of the tails of adjacent spectra, the intensity of peak 588 nm is higher than that shown. Figure 3 The individual dye 2 shown in A ( Figure 3 A in the middle.

[0072] As another example, the composite fluorophore 20 may include dyes 4, 5, and 6, and... Figure 3 Compared to the aforementioned examples shown in Figure C, these dyes exhibit different fluorescence spectra and narrower spacing between spectral peaks. These dyes 4, 5, and 6 simulate the emission spectra of Alexa 555, SRD, and Alexa 568, with emission peaks at ~573 nm, 585 nm, and 603 nm, respectively. The spacing between these emission peaks ranges from approximately 12 nm to 18 nm.

[0073] When dyes 4, 5, and 6 emit at the same fluorescence intensity (in a 1:1:1 ratio), the fluorescence spectrum of the composite fluorophore 20 composed of these dyes will be as follows: Figure 3 As shown in D. As illustrated, due to the close spacing of the individual emission peaks, all three individual spectra merge into a broad spectrum with a unique shape.

[0074] like Figure 3 As shown in B and D, the choice of dye and the spacing between their emission peaks affect the fluorescence spectral shape of the complex fluorophore 20 of the dye.

[0075] In another example, the composite fluorophore 20 may include dyes that emit fluorescence intensities different from each other. Figure 4 A in the figure shows the combined spectrum of dyes 1, 2, and 3 with a fluorescence intensity ratio of 1:1:1.5. Figure 4 B in the figure shows the combined spectrum of dyes 1, 2, and 3 with a fluorescence intensity ratio of 1:1.5:1. Figure 4 Figure C shows the combined spectrum of dyes 1, 2, and 3 with a fluorescence intensity ratio of 1.5:1:1. As shown, although these composite fluorophores 20 include the same group of dyes, the shapes of all the resulting spectra are unique. The shape of the fluorescence spectrum of the composite fluorophore 20 depends on the fluorescence intensity ratio of the individual dyes at a specific excitation laser source / wavelength.

[0076] Figure 5 Figure A shows the normalized absorption band (solid line) and fluorescence band (dashed line) of the complex fluorophore 20 with three types of dyes 26 on each support 22. In this example, support 22 is a BNNT support. 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.

[0077] Figure 5 B in the figure shows the fluorescence signals of these composite fluorophores 20 excited at 492 nm (solid line), 568 nm (dotted line), and 647 nm (dashed line), respectively.

[0078] Since the laser at 492 nm is entirely within the absorption band of FITC ( Figure 5 Within spectrum 1) of A, strong fluorescence of FITC was detected at 520 nm, such as... Figure 5 The solid line spectrum in B shows that the long tail is contributed by the weak fluorescence of SRD (588 nm) and Cy5 (665 nm). A 492 nm laser should not cause emission of SRD and Cy5 because the excitation laser is far from their absorption bands (…). Figure 5In A, these are spectra 3 and 5, respectively. Therefore, the small fluorescence from the SRD at 588 nm is excited by fluorescence from FITC. Similarly, the small fluorescence from Cy5 at 665 nm is excited by fluorescence from the SRD.

[0079] The fluorescence signal of these composite fluorophores 20 excited at 568 nm ( Figure 5 The dotted-line spectrum in B is described below. A 568 nm laser excites the SRD and results in strong emission at 588 nm, with a weak fluorescence tail of Cy5 at 665 nm. This 568 nm laser should not excite FITC and Cy5 because the excitation laser is far from their absorption bands. Therefore, the small fluorescence of CY5 at 665 nm is excited by the fluorescence from the SRD.

[0080] The fluorescence signal excited by these composite fluorophores 20 at 647 nm ( Figure 5 The dashed spectrum in B is described below. The 647 nm laser excites only the CY5 dye and results in strong emission at 665 nm.

[0081] like Figure 5 As shown in B, the shape of the fluorescence spectrum of the composite fluorophore 20 is different when excited by different lasers.

[0082] Figure 5 C and D in the table summarize the effect of dye concentration used to manufacture composite fluorophore 20. Dye concentration is defined as the number of dye molecules per unit volume of synthesis solution.

[0083] Figure 5 The C in the figure shows the fluorescence signals of the composite HBF 20 excited at 492 nm (solid line), 568 nm (dotted line), and 647 nm (dashed line), respectively. These composite HBF 20 were prepared by mixing FITC, SRD, and Cy5 dye molecules in a 1:1:2 concentration ratio. The total dye concentration used is the same as that used above for... Figure 5 The same as those described in B in the text.

[0084] Figure 5 The figure shows the fluorescence signals of the composite HBF 20 excited at 492 nm (solid line), 568 nm (dotted line), and 647 nm (dashed line), respectively. These composite HBF 20s were prepared by mixing FITC, SRD, and Cy5 dye molecules in a 1:4:1 concentration ratio. The total dye concentration used is related to the concentration of the target dye. Figure 5 The ones described by B and C in the text are the same.

[0085] As shown, because these HBF 20s have different dye concentration ratios, even with the same total dye concentration, when excited by a laser at 492 nm, Figure 5The solid line spectra in C and D are different in shape. For comparison purposes, in Figure 6 The spectrum of all spectra excited at 492 nm is shown together in A. As shown, composite HBF 20 with different dye concentration ratios (1:1:1, 1:1:2, 1:4:1) but the same total dye concentration emits fluorescence with different spectral shapes even when excited by the same laser wavelength. When comparing all the dotted spectra ( Figure 6 B in the spectrum, excited at 568 nm) or all dashed line spectra ( Figure 6 The same applies when C in the sample is excited at 647 nm.

[0086] Any of the examples of composite fluorophores 20 discussed above may include BNNT as a carrier 22 and DSPE-PEG5000 as a connector 24, as described in U.S. Patent Application Serial No. 15 / 953,200, filed April 13, 2018 and published as U.S. Patent Publication No. 2018 / 0296705.

[0087] In another example, fluorophore 20 includes a connector 24 covalently bonded to the carrier 22 and the fluorescent entity 26, as described above. These example fluorophores 20 are thus prepared by covalent functionalization. For example, fluorophore 20 comprises a mixture of three different types of dyes: FITC, SRD, and Cy5 dye molecules, in three concentration ratios (1:1:1, 2:2:1, 2:1:1), as shown below. Figure 7 As shown in A, B, and C. The total dye concentration used for each of the three composite HBF 20s is the same.

[0088] like Figure 7 As shown in A, using different excitation lasers (492 nm, 568 nm, and 647 nm) will result in different spectral shapes, as illustrated by solid, dotted, and dashed spectra, respectively, in the case of a 1:1:1 dye concentration ratio.

[0089] like Figure 7 As shown in B, using different excitation lasers (492 nm, 568 nm, and 647 nm) will result in different spectral shapes, as illustrated by solid, dotted, and dashed spectra, respectively, when the dye concentration is in a 2:2:1 ratio.

[0090] like Figure 7 As shown in C, using different excitation lasers (492 nm, 568 nm, and 647 nm) will result in different spectral shapes, as illustrated by solid, dotted, and dashed spectra, respectively, when the dye concentration is in a 2:1:1 ratio.

[0091] Figure 8Figure A compares the spectral shapes of composite fluorophores 20 prepared at 492 nm with dye concentration ratios of 1:1:1, 2:2:1, and 2:1:1. As shown, the spectral shape of the composite fluorophore 20 with a dye-connector 24 / 26 concentration ratio of 1:1:1 is significantly different from those with dye-connector 24 / 26 concentration ratios of 2:2:1 and 2:1:1. The spectral shapes of the samples with 2:2:1 and 2:1:1 ratios are similar to those of SRD and Cy5 dyes, respectively, and they cannot be well excited at 492 nm.

[0092] Figure 8 B in the paper compares the spectral shapes of composite fluorophores 20 excited at 568 nm with dye-connector 24 / 26 concentration ratios of 1:1:1, 2:2:1, and 2:1:1. As shown, all of these spectra have different shapes.

[0093] Figure 8 C in the figure compares the spectral shapes of the composite fluorophore 20 excited at 647 nm with dye-connector 24 / 26 concentration ratios of 1:1:1, 2:2:1, and 2:1:1. As shown, all of these spectra have different shapes.

[0094] Therefore, the spectrum of the composite fluorophore 20 described herein varies depending on the selected excitation laser.

[0095] The composite fluorophore 20 described herein can be used in flow cytometry. For conventional flow cytometry (FCM), one or more biomarkers are labeled with a unique fluorophore 20 (such as the composite fluorophore 20 discussed above) via biomolecules 28 as described above. The fluorophore 20 is excited by a laser, causing it to fluoresce and emit detectable photons. Conventional FCM uses a bandpass filter to sequentially separate and detect photon-related signals only near the peak of the fluorescence wavelength range of each fluorophore. By labeling specific biomarkers, a population of those biomarkers can be quantified by quantifying the fluorescence signals from the unique fluorophores 20 labeled on one or more biomarkers.

[0096] In spectral flow cytometry (spectral FCM), the fluorescence spectrum of a sample is detected within a predetermined detection window (e.g., a wavelength range). Each fluorescent entity 20 in the sample possesses a unique fluorescence spectrum, which can be detected and identified as known in the art, for example by spectral separation, to identify and quantify biomarkers in the sample.

[0097] Typically, for spectral FCM, there is a trade-off between detection speed, sensitivity, and spectral resolution. For example, to collect many data points within a reasonably short detection duration, the detection time for each signal is very short. However, because the fluorophore 20 described herein has such high fluorescence intensity, less laser exposure (e.g., time) is required to obtain a recordable fluorescence signal. Therefore, the fluorophore 20 described herein allows spectral FCM to be used in high-throughput applications. Furthermore, because the composite fluorophore 20 can comprise two, three, or more fluorescent entities 26 selected to produce fluorophores 20 with unique fluorescence spectra when combined in various amounts, many different fluorophores 20, each with a unique fluorescence spectrum, can be prepared using readily available fluorescent dyes.

[0098] The foregoing description is exemplary in nature and not restrictive. Variations and modifications to the disclosed examples will become apparent to those skilled in the art without departing from the spirit of the invention. The legal scope of protection of this invention can only be determined by studying the following claims.

Claims

1. A compound 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 fourth linker 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 third fluorescent entity different from the first and second fluorescent entities connected to the fourth linker; and a biomolecule connected to a second end of the third linker, wherein the biomolecule is configured to connect to a biomarker, wherein the three fluorescent entities each emit at about the same fluorescent intensity as each other.

2. The compound of claim 1, wherein, The carrier is a boron nitride nanotube (BNNT) or a carbon nanotube (CNT).

3. The compound of claim 1, wherein, 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 bound to the carrier.

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

6. The compound of claim 4, wherein, The second end of at least one of the first, second, and third linkers is covalently bound to one of the first and second fluorescent entities or the 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 bound to the carrier.

8. The compound of claim 7, wherein, At least one of the first, second, and third linkers is amphiphilic and includes a hydrophobic region and a hydrophilic region, and wherein the hydrophobic region is non-covalently bound to the carrier.

9. The compound of claim 7, wherein, At least one of the first, second, and third linkers has a molecular weight between about 1000 and 10000 Da.

10. The compound of claim 7, wherein, The carrier is a boron nitride nanotube.

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

12. A method of detecting a biomarker comprising: providing a plurality of fluorophores to a sample, each of the plurality of fluorophores including a biomolecule connected to a carrier, a first fluorescent entity, a second fluorescent entity, and a third fluorescent entity different from the first and second fluorescent entities, wherein the three fluorescent entities each emit at about the same fluorescent intensity as each other, wherein the biomolecule is configured to interact with a plurality of biomarkers in the sample and whereby the fluorophore is connected to the biomolecule; exciting the plurality of fluorophores in the sample with a laser; and detecting at least one of a characteristic and an amount of the plurality of biomarkers in the sample based on a fluorescent spectrum emitted by the excited plurality of fluorophores.

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

14. The method of claim 12, wherein, The first and second fluorescent entities and the biomolecule are connected to the carrier by first, second, and third linkers, and wherein at least one of the first, second, and third linkers is connected to the carrier via a covalent bond.

15. The method of claim 12, wherein, The first and second fluorescent entities and the biomolecule are linked to the carrier by first, second, and third linkers, respectively, and wherein at least one of the first, second, and third linkers is linked to the carrier via a non-covalent bond.

16. The method of claim 15, wherein, At least one of the first, second, and third linkers is amphiphilic and includes a hydrophobic region and a hydrophilic region, and wherein the hydrophobic region is non-covalently bound to the carrier.

17. The method of claim 16, wherein, At least one of the first, second, and third linkers has a molecular weight of between about 1000 and 10000 Da.

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

19. The method of claim 12, wherein, The plurality of fluorophores is a first plurality of fluorophores, the biomarkers are first biomarkers, and the biomolecule is a second biomolecule, and further comprising providing a second plurality of fluorophores to the sample, each of the second plurality of fluorophores including a second biomolecule linked to a carrier, a third fluorescent entity, and a fourth fluorescent entity, wherein the second biomolecule is configured to interact with a second plurality of biomarkers in the sample, and whereby the second fluorophore is linked to the second biomolecule.

20. The method of claim 19, wherein, The second plurality of fluorophores is excited by the laser, and further comprising detecting at least one of a characteristic and an amount of the second plurality of biomarkers in the sample based on a fluorescence spectrum emitted by the excited second plurality of fluorophores.

21. An amphiphilic polymer linker comprising a hydrophobic region and a hydrophilic region, wherein, The molecular weight of the linker is greater than about 2400 Da and less than about 10000 Da; the linker includes 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; and the hydrophobic region of the linker is selected from a fatty acid, a phospholipid, a sphingolipid, and a sphingomyelin.

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