UV-visible fluorophores

Abstract

In one example of the present application, a method of creating UV-visible fluorophores includes heating an optically transparent liquid under reflux such that solid fluorescent nanoparticles that emit fluorescence in the UV-visible spectral range between about 320nm to 600nm form in the solvent, and wherein the liquid is one of an organic solvent and water-soluble small molecules dissolved in water. Other example methods and example fluorophores are also disclosed.

Description

UV- VISIBLE FLUOROPHORESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application serial no. 63 / 673,823 filed on July 22, 2024, which is hereby incorporated herein in its entirety.BACKGROUND

[0002] Fluorophores are compounds that emit fluorescence or include fluorescenceemission moieties such as fluorescent dyes. Examples of such moieties include small organic dye molecules, protein dye molecules, and quantum dots (QDs). Fluorophores can be used for biological detection, such as fluorescent probes that specifically stain biomolecules which can then be detected by fluorescent imaging and spectroscopy. In addition, fluorophores are used for lightemitting devices (LEDs) and dye-sensitized or quantum-dot-sensitized solar cells.

[0003] Fluorophores can be conjugated with molecules such as antibodies, oligomers, aptamers, and streptavidins for specific biological detection. All known fluorophores (dye and QDs) emit light in visible (wavelength about 400-700nm) and near-infrared (about 800-1000nm) spectra. Finding fluorophores that emit beyond the visible and near-IR spectra is crucial to extend the detection spectra ranges, such as those in multicolor flow cytometry.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1A shows fluorescent signals at various excitation wavelengths from HPLC-grade ethanol without reflux treatment.

[0005] Figure IB shows fluorescent signals at various excitation wavelengths from HPLC-grade ethanol after an eight-hour reflux treatment.

[0006] Figure 1C shows fluorescent signals at various excitation wavelengths from HPLC-grade IPA without reflux treatment.

[0007] Figure ID shows fluorescent signals at various excitation wavelengths fromHPLC-grade IPA after an eight-hour reflux treatment.

[0008] Figure IE shows the ethanol and IPA samples from Figures IB and ID after an eight-hour reflux treatment.

[0009] Figure 2A shows fluorescent signals at various excitation wavelengths from hexane without reflux treatment.

[0010] Figure 2B shows fluorescent signals at various excitation wavelengths from hexane after a four-hour reflux treatment.

[0011] Figure 2C shows the hexane samples of Figures 2A-B with (left) and without (right) reflux treatment.

[0012] Figure 3A shows fluorescent signals at various excitation wavelengths from HPLC-grade DMF without reflux treatment.

[0013] Figure 3B shows fluorescent signals at various excitation wavelengths from HPLC-grade DMF after an reflux eight-hour treatment.

[0014] Figure 3C shows the HPLC-grade DMF samples of Figures 3A-B without reflux treatment (left), and after eight hours of reflux treatment (right).

[0015] Figure 4A shows fluorescent signals at various excitation wavelengths from reagent grade DMF after 3 -hour reflux treatment.

[0016] Figure 4B shows fluorescent signals at various excitation wavelengths from reagent grade DMF after 24-hour reflux treatment.

[0017] Figure 4C shows fluorescent signals at various excitation wavelengths from reagent grade DMF after 36-hour reflux treatment.

[0018] Figures 5A and 5B show TEM and AFM images, respectively, of nanoparticles from DMF refluxed for 15 minutes.

[0019] Figures 6A and 6B show high-resolution TEM images of isolated and aggregated nanoparticles, respectively, from DMF refluxed for 24 hours.

[0020] Figure 7A shows fluorescent signals at various excitation wavelengths from L- Ascorbic solution without reflux treatment.

[0021] Figure 7B shows fluorescent signals at various excitation wavelengths from L- Ascorbic solution after a four-hour reflux treatment.

[0022] Figure 8A shows fluorescent signals at various excitation wavelengths from HPLC-grade DMF after 4-hour reflux treatment in ambient air.

[0023] Figure 8B shows fluorescent signals at various excitation wavelengths from HPLC-grade DMF after 24-hour reflux treatment in Nitrogen ambient.

[0024] Figure 9A shows fluorescent signals at various excitation wavelengths from HPLC-grade IPA before 10-day sonication in the air.

[0025] Figure 9B shows fluorescent signals at various excitation wavelengths from HPLC-grade IPA after 10-day sonication in the air.

[0026] Figure 10A shows fluorescent signals at various excitation wavelengths from 50% HPLC-grade IPA-50% water mixture before 10-day sonication in air.

[0027] Figure 10B shows fluorescent signals at various excitation wavelengths from 50% HPLC-grade IPA-50% water mixture before after 10-day sonication in air.

[0028] Figure HA shows fluorescent signals at various excitation wavelengths from the supernatant of refluxed BNNT-DMF suspension.

[0029] Figure 11B shows fluorescent signals at various excitation wavelengths from pallet BNNTs.

[0030] Figure 12 schematically shows example fluorophores of the present description.SUMMARY

[0031] In one example of the present application, a method of creating UV-visible fluorophores includes heating an optically transparent liquid under reflux such that solid fluorescent nanoparticles that emit fluorescence in the UV-visible spectral range between about 320nm to 600nm form in the liquid. The liquid is one of an organic solvent and water-soluble small molecules dissolved in water.

[0032] In another example of the present application, a method of creating UV-visible fluorophores includes transferring mechanical energy to optically transparent liquid under reflux such that solid nanoparticles that emit fluorescence in the UV-visible spectral range between about 320nm to 600nm form in the liquid. The liquid is one of an organic solvent and water-soluble small molecules dissolved in water.

[0033] In another example of the present application, a fluorophore includes a boron nitride nanotube, and solid fluorescent nanoparticles inside the boron nitride nanotube, wherein the nanoparticles emit fluorescence in the UV-visible spectral range between about 320nm to 600nm.DETAILED DESCRIPTION

[0034] Known fluorescent moieties are small organic dye molecules, protein dye molecules, or QDs. Dye molecules and protein dye consist of aromatic groups. On the other hand, QDs are inorganic nanoparticles of semiconductors. QDs are much more expensive that organic dyes or protein dyes, and contain heavy metals or toxic elements. Examples of such QDs are Cadmium Selenide (CdSe), and Lead Sulfide (PbS).

[0035] The inventors have discovered new fluorophores that emit fluorescence in the UV- visible spectral range between about 320nm to 600nm. These fluorophores, unlike any others, are synthesized using small molecules without aromatic groups or organic optically transparent solvents. Imaging reveals that the present UV-visible fluorophores appear as solid nanoparticles with diameters between about 2nm to about lOnm, much like QDs, but without toxic elements, as will be discussed herein.

[0036] (A) Production of UV-visible fluorophores from common organic solvents.

[0037] In one example, UV-visible fluorophores can be synthesized by chemical reflux of optically transparent organic solvents near their boiling temperature (plus minus about 20 °C) in ambient air. In this example, the process uses a regular glass flask to heat the liquid while cooling the vapors back to the liquid by a standard water-cool reflux-condenser setup. “Reflux” is a well- known technique where a liquid is heated close to its boiling point in a vessel while continually condensing the resulting vapor and allowing the condensed vapor to return to the liquid in the vessel.

[0038] One example type of optically transparent organic solvent that can be treated to create fluorophores are alcohols with hydroxyl groups. Figures 1A-B show the fluorescent spectra of one such example alcohol, ethanol (C2H6O), and in this particular example, HPLC (High- Performance Liquid Chromatograph)-grade ethanol without treatment (A) and after an eight-hour reflux treatment at about 76 °C (B). The graphs in Figures 1A and B are at the same scale for comparison. As shown in Figure 1A, the fresh HPLC-grade ethanol (without reflux treatment) shows no fluorescence. In contrast, Figure IB shows that the HPLC grade ethanol refluxed for eight hours produces robust UV fluorescence at about 318nm and about 329nm as excited at 300nm and 320nm.

[0039] Figures 1C-D similarly compare the fluorescent spectra of a second example alcohol, isopropanol alcohol (IPA, CLHeO), and in this particular example, HLPC-gradeisopropanol alcohol, without treatment (C) and after an eight-hour reflux treatment at about 80 °C (D). The graphs of Figures 1C and D are at the same scale for comparison. As shown in Figure 1C, the fresh HPLC grade IPA (without reflux treatment) shows no fluorescence. In contrast, Figure ID shows that the HPLC-grade IPA refluxed for eight hours produced UV fluorescence at about 327nm, about 33 Inm, about 351nm, about 375nm, as excited at 280nm, 300nm, 320nm, and 340nm. The production of fluorophores in these refluxed samples is surprising as both refluxed ethanol and IPA remain optically transparent, as shown in Figure IE.

[0040] Another example type of optically transparent organic solvent that can be treated to create fluorophores is alkanes with single-bonded carbon and hydrogen atoms without any functional groups. Figures 2A-B show the fluorescent spectra of an example alkane with singlebonded carbon and hydrogen atoms without any functional groups, reagent-grade hexane (CeHu) without treatment (A) and after a four-hour reflux treatment at about 66 °C (B). The graphs in Figures 2A-B are at the same scale for comparison. As shown in Figure 2A, the untreated hexane shows no fluorescence. In contrast, Figure 2B shows that hexane refluxed for four hours produced robust UV fluorescence at about 313nm and about 329nm, as excited at 280 and 300nm. This result indicates the production of UV fluorophores in the refluxed hexane. Again, it was surprising that fluorophores were produced in the refluxed hexane while the liquid remains optically transparent, as shown in Figure 2C.

[0041] A third example type of optically transparent organic solvent that can be treated to create fluorophores is formamide or carboxylic acid amides. A particular example is N- Dimethylformamide (DMF, C3H7NO) and in a particular example, HPLC-grade DMF.

[0042] Figures 3 A-B shows the fluorescent spectra at the same scale of HPLC-grade DMF without treatment (A) and after an eight-hour reflux treatment at 150 °C (B). The graphs of Figures 3A-B are shown at the same scale for comparison. The untreated HPLC-grade DMF is not fluorescent, as shown in Figure 3 A. Figure 3B shows that the HPLC-grade DMF refluxed for eight hours produced UV fluorescence at about 319nm and about 376nm as excited at 300nm and 340nm. A minor fluorescence peaked at about 405nm, as excited at 380nm was also detected. Figure 3C is a photograph of HPLC grade DMF without reflux treatment (left) and after eight- hour treatment (left) at 150 °C. Both liquids are visibly transparent.

[0043] Reagent-grade solvents of ethanol, IPA, and DMF also produced fluorophores when treated as described herein. All the refluxed samples produced UV-visible fluorophores.

[0044] For example, Figure 4 A shows the fluorescent spectra of refluxed reagent-grade DMF at 150 °C for three hours, the product of which is transparent, as excited by various UV wavelengths. As shown, there are robust fluorescence peaks at about 320nm, about 331nm, and about 349nm when optically excited at 280nm, 300nm, and 320nm. The production of UV fluorophores using reagent-grade DMF required much shorter reflux treatment as compared to the examples discussed above, likely due to the presence of trace oxygen and water.

[0045] In another example, reagent-grade DMF was refluxed at 150 °C for twenty-four hours. The product became yellowish. Referring to Figure 4B, UV fluorescent peaks were detected at about 343nm, about 353nm, and about 381nm when excited at 280nm, 300nm, and 320nm, respectively. Surprisingly, the UV fluorescence is at longer wavelengths than those recorded in Figure 3 A for HPLC-grade DMF, even when excited by the same 280nm, 300nm, and 320nm light sources, respectively. This result suggests that the Stoke fluorescence shifts from the excitation source are larger in this case.

[0046] As shown in Figure 4B, visible fluorescence was further detected at about 390nm, about 43 Inm, about 453nm, about 469nm, about 494nm, about 508nm, about 518nm, when excited at 340nm, 360nm, 380nm, 400nm, 420nm, 440nm, and 460nm, respectively. These unexpected results suggest that UV and visible fluorophores are being produced simultaneously.

[0047] In another example, reagent-grade DMF was refluxed at 150 °C for thirty-six hours. The product became brownish. Referring to Figure 4C, visible fluorescence was detected at about 506nm, about 512nm, about 516nm, about 526nm, about 535nm, about 551nm, about 569nm, about 587nm and about 587nm when excited at 360nm, 380nm, 400nm, 420nm, 440nm, 460nm, 480nm, and 500nm, respectively. These results suggest that only visible fluorophores are being produced in this example.

[0048] The results mentioned above indicate that the duration of reflux treatment will affect the type of fluorophores produced. The longer reflux treatment duration will produce fluorophores that emit light at longer wavelengths. Short reflux treatment will explicitly produce UV fluorophores. UV fluorophores were detectable after 15 minutes of reflux of reagent-grade solvents.

[0049] The new UV-visible fluorophores are solid nanoparticles. For example, Figures 5A-B show images from (A) transmission electron microscopy (TEM) and (B) atomic force microscopy (AFM) of UV fluorophores produced after 15 minutes of reflux of reagent-grade DMFat 150 °C. This result shows that the UV-visible fluorophores discussed herein are solid nanoparticles.

[0050] Figure 6 shows high-resolution TEM images of the UV-visible fluorescent nanoparticles produced from refluxed DMF. As shown, nanosized crystals are detected as small as about 2-5nm (A). Aggregation of these crystals is also shown (B) larger than about 20nm. The TEM and AFM images confirm the formation of crystallized nanoparticles from the reflux DMF samples that produce UV-visible fluorescence.

[0051] (B) Production of UV-visible fluorophores from water-soluble compounds.

[0052] In another example, water-soluble small molecules dissolved in water were refluxed. Particular examples are L-ascorbic acid (C2H8O6, vitamin C) and niacinamide (CeFUNhO, vitamin B3). Reflux was performed near the evaporation temperature of Deionized (DI) water.

[0053] Figure 7A shows the fluorescent spectra of L-Ascorbic acid solution in DI water without treatment (A) and after a four-hour reflux treatment at about 98 °C (B). As shown in Figure 7A, the fresh L-Ascorbic acid solution shows no fluorescence. In contrast, Figure 7B shows that the L- Ascorbic acid solution refluxed for four hours produced visible fluorescence at about 409nm, about 415nm, about 435nm, about 453nm, about 474nm, about 496nm, and about 516nm, as excited at 320nm, 340nm, 360nm, 380nm. 400nm, 420nm, and 440nm. Based on the examples described above, it is expected that a shorter reflux duration of less than four hours may also produce UV fluorescence for water-soluble small molecules dissolved in water.

[0054] (C) Effect of the reflux ambients

[0055] It was discovered that the formation of the new UV-Visible fluorophores depends on the atmosphere inside the reflux flasks. Figure 8A shows the fluorescence signals of UV fluorophores after a 4-hour-long reflux of HPLC-grade DMF in air. Figure 8B shows the same intensity scale after a 24-hour-long reflux after the flask is fdled with dried nitrogen gas. No UV fluorescence was detected even after a 24-hour reflux in nitrogen ambient. These results suggest that air induced the formation of UV-Visible fluorophores. As shown in the inset, only very weak (noise-level) UV fluorescence was detected in Figure 8B.

[0056] (D) Formation of fluorophores by ultra-sound sonication

[0057] It has also been discovered that mechanical agitation can form UV fluorophores. Such agitation will transfer mechanical and heat energy into the precursor liquids to induce the formation of UV fluorophores.

[0058] Figures 9A-B shows the fluorescent signals at the same intensity scale at various excitation wavelengths from HPLC-grade IPA before sonication (A) and after 10-day sonication in the air (B). As shown, the UV fluorescence is barely detectable.

[0059] In another example, water was added to the HPLC-grade IPA (a mixture of 50 volume % HPLC-grade IPA and 50 volume % DI water). Figures 10A-B show the fluorescent signals at the same intensity scale at various excitation wavelengths from the IPA:DI Water mixture before (A) and after 10-day sonication in the air (B). As shown, the UV fluorescence becomes more obvious. These results show that mechanical agitation stimulates the formation of UV fluorophores from IPA (and other liquid discussed herein). Furthermore, water increases the efficiency of such UV fluorophores in formation.

[0060] (E) Size-selective Extraction by filling the new UV-visible nanoparticles into nanotubes.

[0061] It was also determined that UV fluorescent nanoparticles can be extracted by in- situ filling them inside nano capsules such as nanotubes during the reflux treatment. In a particular example, the nanotubes are boron nitride nanotubes (BNNTs), which are optically transparent and electrically insulating. BNNTs will prevent quenching of the filled nanoparticle fluorophores. Filling fluorescent compounds inside BNNTs enables the formation of high-brightness fluorophores with high molar extinction coefficients.

[0062] In a more particular example, the BNNTs have empty tubular channels of about 3 to 1 Onm in diameter. About 15mg of BNNTs were baked at 820 °C for an hour and then cooled to room temperature. These BNNTs were resuspended in DMF by bath sonication for about 5 minutes. The BNNT suspension in DMF was then subjected to reflux treatment at 150 °C for 24 hours to make UV-visible fluorophores, under the same conditions as that for data shown in Figure 4B.

[0063] The refluxed BNNT suspension was centrifuged at 10,000g for 30 minutes to condense the BNNTs into a pallet and separate them from the refluxed DMF (supernatant). Figure 11A shows the fluorescence spectra of the supernatant with strong visible fluorescence and some UV fluorescence, similar to Figure 4B. This data shows the reflux process produce UV-visible fluorophores within the DMF.

[0064] The BNNT pallet was then resuspended in acetone and centrifuged at 10,000g for 15 minutes 3-4 times. This rinsing and washing process removes free UV-visible fluorophoresfrom the surfaces of BNNTs. The final BNNT pallet is clean and resuspended in ethanol for fluorescence measurement. Figure 11B shows that all visible fluorescence signals are gone, leaving with the trace light from the excitation sources (see for comparison Figure 11A plotted at the same scale). The result indicates that the most visible fluorophores were removed from the BNNT surfaces during cleaning.

[0065] Figure 1 IB also shows some UV fluorescence from these clean BNNTs. The UV fluorescence comes from UV fluorophores, which were filled inside BNNTs, preventing them from being removed during cleaning.

[0066] Figure 12 schematically shows an example fluorophore 100 according to the present example. As shown, the fluorophore 100 includes a BNNT 102 with a tubular channel 103. UV fluorophores 104 are situated inside the tubular channel 103.

[0067] This application relates in some examples to optically transparent organic solvents, several examples of which are described above. It should be understood that mixtures of two or more optically transparent organic solvents can also be used. Likewise, this application contemplates the use of water-soluble small molecules dissolved in water. It should be understood that mixtures of two or more water-soluble small molecules dissolved in water could also be used.

[0068] The “grade” of solvents described herein is a well-understood term that refers to their purity / lack of certain impurities.

[0069] As used herein, the terms “approximately” and “about” have the typical meaning in the art, however in a particular example “about” or “approximately” can mean deviations of up to 10% of the values described herein.

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

Claims

CLAIMS1. A method of creating UV-visible fluorophores, comprising: heating an optically transparent liquid under reflux such that solid fluorescent nanoparticles that emit fluorescence in the UV-visible spectral range between about 320nm to 600nm form in the liquid, wherein the liquid is one of an organic solvent and water-soluble small molecules dissolved in water.

2. The method of claim 1, wherein the optically transparent liquid remains optically transparent after the heating.

3. The method of claim 1, wherein the optically transparent liquid changes color after the heating.

4. The method of claim 1, wherein the optically transparent liquid is an alcohol with a hydroxy group.

5. The method of claim 4, wherein the alcohol is ethanol.

6. The method of claim 4, wherein the alcohol is isopropanol alcohol.

7. The method of claim 1, wherein the optically transparent liquid is an alkane with single-bonded carbon and hydrogen atoms and without any functional groups.

8. The method of claim 7, wherein the alkane is hexane.

9. The method of claim 1, wherein the optically transparent liquid is formamide or a carboxylic acid amide.

10. The method of claim 9, wherein the optically transparent liquid is N- Dimethylformamide (DMF).

11. The method of claim 1, wherein the optically transparent liquid is water-soluble small molecules dissolved in water.

12. The method of claim 11, wherein the water-soluble small molecules are L-ascorbic acid molecules.

13. The method of claim 11, wherein the water-soluble small molecules are niacinamide molecules.

14. The method of claim 1, wherein the optically transparent liquid is an HPLC (High- Performance Liquid Chromatograph)-grade organic solvent.

15. The method of claim 1, wherein the optically transparent liquid is a reagent-grade organic solvent.

16. The method of claim 1, wherein the nanoparticles have diamenters between about 2 and 10 nanometers.

17. The method of claim 1, wherein the reflux is performed in an air environment.

18. The method of claim 1, further comprising detecting fluorescence of the solid nanoparticles after the heating.

19. The method of claim 1, further comprising adding boron nitride nanotubes to the solvent during the heating such that the nanoparticles fill inside the boron nitride nanotubes.

20. A method of creating UV-visible fluorophores, comprising: transferring mechanical energy to an optically transparent liquid under reflux such that solid nanoparticles that emit fluorescence in the UV-visible spectral range between about 320nm to 600nm form in the liquid, wherein the liquid is one of an organic solvent and water-soluble small molecules dissolved in water.

21. The method of claim 20, wherein the transferring is by ultra-sound sonication.

22. The method of claim 20, further comprising detecting fluorescence of the solid nanoparticles after the transferring step.

23. The method of claim 20, wherein the optically transparent liquid is an alcohol with a hydroxy group, an alkane with single-bonded carbon and hydrogen atoms and without any functional groups, formamide, a carboxylic acid amide, or water-soluble small molecules dissolved in water.

24. A fluorophore, comprising: a boron nitride nanotube; solid fluorescent nanoparticles inside the boron nitride nanotube, wherein the nanoparticles emit fluorescence in the UV-visible spectral range between about 320nm to 600nm.

25. The fluorophore of claim 24, wherein the boron nitride nanotube has a tubular channel of about 3 to about lOnm in diameter.

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