Fluorescence probe and method of fabricating a fluorescent probe

Abstract

The present invention discloses a fluorescent probe, comprising a carbon dot surface functionalised with glucosamine. The present invention also provides a method of fabricating a fluorescent probe and a method of staining a nucleolus of a cell by contacting the fluorescent probe as disclosed herein with the cell.

Description

[0001]

[0002] Fluorescence probe and Method of Fabricating a Fluorescent Probe

[0003] Technical Field

[0004] The present invention relates, in general terms, to a fluorescent probe and method of fabricating a fluorescent probe. The present invention also relates to a method of staining a nucleolus of a cell.

[0005] Background

[0006] The nucleolus is the largest nuclear structure in the nucleus. The primary functions of the nucleolus are rRNA transcription and ribosomal subunit assembly. The rate of cell growth is strongly correlated to protein biogenesis by ribosomes. Deregulation of ribosome biogenesis can lead to cell transformation. Nucleolus also have significant roles in health and disease, response to cellular stresses, maintenance of genome stability and genome damage repair etc. Nucleolar changes associated with neurodegenerative diseases, cardiovascular diseases, and different forms of cancer are reported. Nucleolar functions are also directly related to life expectancy. Recent studies show that nucleolus functions have a significant role in treating cancer. Most of the cancer cells show intense ribosome biosynthesis. Therefore, control over the functions of the nucleus (or nucleolus) may be advantageous in cancer therapy as well. Hence, there is an urgent need for developing novel nucleolus targeting probes to understand nucleolar functions and advance the therapeutic efforts for controlling the nucleolus.

[0007] Fluorescence imaging technique has been widely used in biomedical research because of its high sensitivity, specificity, and excellent versatility. There are various types of fluorescent compounds that are routinely used in laboratory research. Among these, fluorescent organic small molecules, fluorescent proteins, and fluorescent nanomaterials are extensively applied both in in vitro and in vivo studies. Compared to the fluorescent proteins, the fluorescent nanomaterials are easy to handle as complex genetic modification is required to introduce the fluorescent protein within the cells which is highly time consuming and tedious. The nanoparticle-based probes also have several advantages over organic fluorescent dyes, such as high photostability, better chemical stability, and improved brightness etc. Besides, nanoparticles show higher retention time in the body and can be utilised for long-term imaging. In addition, nanoparticles can combine both diagnostic and therapeutic properties in a single platform. Hence, a nanoparticle-based fluorescent probe for nucleolus could help the theragnostic studies focusing on the nucleolus.

[0008] A variety of fluorescent nanoparticles are reported in the literature for probe and sensor development including nano particles based on silica, organic polymers, quantum dots, carbon dots, up-conversion materials, metal particles and metal oxides. Among all these categories, fluorescent carbon dots are continuously gaining tremendous interest because of its good solubility, cell permeability and excellent biocompatibility.

[0009] Compared to other organelles, fluorescent probes targeting nucleolus are currently limited due to their low selectivity and specificity towards the structures inside the nucleolus. Besides, the poor permeability of the probes also hinders the development of highly selective nucleolus-targeting probes. Even though more than a dozen fluorescent probes for targeting nucleolus have been developed, to the best of our knowledge, only one probe is commercially available, and its chemical structure is proprietary. SYTO RNASelect is the commercially available green, fluorescent dye used to stain rRNA. The practical application of the SYTO RNASelect is limited due to its relatively poor selectivity and specificity and low photostability. A rRNA selective fluorescent probe based on phthalimide derivative for imaging the nucleolus in live cells has been reported. There are also several reports on the RNA selective probes developed from styryl compounds. Two-photon fluorescent probes are also attempted for cell RNA imaging. A deep red light up compound probe for imaging nucleolar RNA in live cells has been reported. A PH sensitive chemical probe to target rRNA in the nucleolus has been reported. Even though these probes are partially successful in imaging RNA in cells, they are limited by specificity and selectivity toward RNA from DNA or suffer from lower detection limits. Most studies focus on improving these probes' cell permeability and biocompatibility.

[0010] It would be desirable to overcome or ameliorate at least one of the above-described problems.

[0011] Summary

[0012] The present invention concerns a fluorescent probe, comprising a carbon dot; wherein the carbon dot is nitrogen-doped; wherein the carbon dot is surface-functionalised with glucosamine; wherein the carbon dot is characterised by an average particle size of about 2 nm to about 5 nm; and wherein the fluorescent probe is characterised by a specificity and / or selectivity for a nucleolus of a cell.

[0013] In some embodiments, glucosamine is conjugated to the carbon dot via an amide bond.

[0014] In some embodiments, the fluorescent probe is characterised by 1 glucosamine per carbon dot to 20 glucosamine per carbon dot.

[0015] In some embodiments, the carbon dot further comprises carboxylic acid moieties on its surface thereof.

[0016] In some embodiments, the carbon dot is characterised by an average particle size of about 3.4 nm.

[0017] In some embodiments, the fluorescent probe is characterised by excitation dependant emission with an excitation wavelength of about 400 nm to about 600 nm.

[0018] In some embodiments, the fluorescent probe is characterised by an emission wavelength of about 420 nm to about 650 nm.

[0019] In some embodiments, the fluorescent probe is characterised by a surface state absorption of about 550 nm.

[0020] In some embodiments, the fluorescent probe is characterised by an edge state absorption of about 350 nm.

[0021] In some embodiments, the fluorescent probe is characterised by a core state absorption below about 300 nm.

[0022] In some embodiments, the fluorescent probe selectively binds to RNA.

[0023] In some embodiments, the fluorescent probe is characterised by a fluorescent intensity which is positively correlated to a RNA concentration.

[0024] In some embodiments, the fluorescent probe is characterised by a cytotoxicity of less than about 10% at a concentration of about 100 pg / mL.

[0025] The present invention also provides a method of fabricating a fluorescent probe, comprising : a) solvothermally synthesising a carbon dot in the presence of a nitrogen dopant at predetermined conditions; and b) surface functionalising the carbon dot with glucosamine.

[0026] In some embodiments, step a) comprises solvothermally synthesising the carbon dot in the presence of citric acid and formamide.

[0027] In some embodiments, the predetermined conditions is a temperature of about 130 °C for about 6 hours.

[0028] In some embodiments, a mole ratio of citric acid to formamide is about 1: 1 to about 1 : 100. In some embodiments, the mole ratio is about 1:60.

[0029] In some embodiments, the method further comprises a step after a) of purifying the carbon dot.

[0030] In some embodiments, step b) comprises surface functionalising the carbon dot with glucosamine in the presence of a carbodiimide compound and N-hydroxysuccinimide (NHS).

[0031] In some embodiments, the carbodiimide compound is l-ethyl-3-(-3- dimethylaminopropyl) carbodiimide hydrochloride (EDC).

[0032] In some embodiments, the weight ratio of the carbodiimide compound to NHS is about 1 : 1.

[0033] The present invention also provides a method of staining a nucleolus of a cell, comprising : a) contacting a fluorescent probe as described herein with the cell.

[0034] Brief description of the drawings

[0035] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which :

[0036] Figure 1 shows the transmission electron microscope image of the fluorescent probe, comprising carbon dot surface-functionalised with glucosamine (CD-red), (a) Transmission electron microscope image of CD-red. (b) Particle size histogram measure from TEM images. The average particle size measured is 3.4 ± 0.6 nm.

[0037] Figure 2 shows the absorption spectra of CD-red.

[0038] Figure 3 shows the photoluminescence spectra of CD-red excited at 405 nm.

[0039] Figure 4 shows FT-IR spectra of the original nitrogen-doped nanoparticle and CD-red. The black curve represents FTIR spectra of nitrogen-doped nanoparticle (CD) and red curve represents the FTIR spectra of CD-red.

[0040] Figure 5 shows confocal microscope images of HeLa cells labelled with CD-red and DAPI dye. (A) Confocal image of the HeLa cells showing nucleolus. The bright regions are the nucleolus. The excitation wavelength was 560 nm, and emission was collected from 580 nm to 650 nm. (B) Confocal image of the HeLa cells showing the nucleus labelled with DAPI dye. The dark regions in the nucleus are the nucleolus. The excitation wavelength is 405 nm, and emission was collected from 420 nm to 480 nm. (C) Composite image of (A) and (B) together. (D) The bright field image of the HeLa cells.

[0041] Figure 6 shows confocal microscope images of single HeLa cell labelled with CD-red and DAPI dye. (A) Confocal image of a single HeLa cell showing nucleolus. The bright regions are the nucleolus. The excitation wavelength was 560 nm, and emission was collected from 580 nm to 650 nm. (B) Confocal image of a single HeLa cell showing the nucleus labeled with DAPI dye. The dark regions in the nucleus are the nucleolus. The excitation wavelength was 405 nm, and emission was collected from 420 nm to 480 nm. (C) Composite image of (A) and (B) together. (D) The bright field image of the HeLa cell.

[0042] Figure 7 shows confocal microscope images of HeLa cells labelled with carbon dots and DAPI. The nanoparticle shows a slightly higher intensity in the nucleolus, but the labelling is not specific as it lights up the entire cell.

[0043] Figure 8 shows confocal microscope images of HeLa cells labelled with CD-red and

[0044] DAPI. The nanoparticle shows a higher intensity in the nucleolus.

[0045] Figure 9 shows methanol-fixed MRC-5 cells stained with SYTO® RNASelect™ green fluorescent cell stain [Thermofisher (Cat. no. S32703)].

[0046] Figure 10 shows a flow chart of CD-red synthesis.

[0047] Figure 11 shows the fluorescence emission of CD-red when different RNA concentrations are added to the CD-red. (a) Fluorescence spectra of CD-red with different concentrations of RNA. (b) The plot of normalized fluorescence intensity at 620 nm vs. Concentration of RNA.

[0048] Figure 12 shows confocal images of HeLa cells treated with CD-red, DAPI dye and composite images, (a) and (d) Confocal images of HeLa cells treated with CD-red and DAPI dye. (b) and (e) Confocal images of HeLa cells after the RNase digestion test, (c) and (f) Confocal images of the HeLa cells after the DNase digestion test, (g) Composite image of (a) and (d). (h) Composite image of (b) and (e). (i) Composite image of (c) and (f).

[0049] Figure 13 shows images of mouse red blood cells treated with CD-red. (a) Bright-field image of mouse red blood cells treated with CD-red. (b) Confocal image of mouse red blood cells treated with CD-red at 568 nm excitation.

[0050] Figure 14 shows the MTT reduction percentages of HeLa cells with different concentrations of CD-red.

[0051] Detailed description

[0052] The present invention is predicated on the understanding that a nucleolus targeting probe has to cross both cell and nuclear membranes and then distinguish the nucleolus from the widespread chromatin and from the nucleoplasm within the cells. The probe also needs to achieve selectivity and specificity towards the structures inside the nucleolus.

[0053] The present invention concerns a fluorescent probe for staining of the nucleolus with bright red fluorescence. The fluorescent probe comprising a carbon dot surface- functionalised with glucosamine (CD-red) allows for selective imaging of the nucleolus of live cells by binding to RNA in the nucleolus. The fluorescent probe is biocompatible, cell and nucleus permeable and may have high specificity and selectivity towards the nucleolus. This may allow the fluorescent probe to be used in the study of nucleolar functions and in other therapeutic applications. In contrast, carbon dot without functionalisation with glucosamine (CD) does not demonstrate this nucleolus specificity and selectivity as carbon dot without functionalisation with glucosamine lights up the entire cell when labelling the cell.

[0054] Accordingly, the present invention discloses a fluorescent probe, comprising a carbon dot surface-functionalised with glucosamine.

[0055] Carbon dots are carbon nanoparticles that are less than 10 nm in size. They exhibit different size-dependent optical properties such as fluorescence, photoluminescence, chemiluminescence and electrochemical luminescence. The fluorescence properties may be enhanced by surface functionalisation, such as with amine and hydroxamic acid, organic dyes, or glucosamine.

[0056] Surface functionalisation involves modifying a surface to give the material physical, chemical or biological characteristics different from the ones originally possessed by the material. It introduces chemical functional groups to a surface via physical mixing or chemical bonding. A functional group may be conjugated to the surface of a material via chemical bonding where a stable covalent bond is formed between two molecules, such as between glucosamine and carbon dots.

[0057] Carbon dots surface-functionalised with glucosamine (CD-red) may have higher specificity and selectivity towards the nucleolus compared to carbon dots without surface-functionalised glucosamine (CD). Figure 8 shows a higher intensity in the nucleolus when cells are labelled with CD-red and DAPI as compared to the cells labelled with carbon dots and DAPI in Figure 7, where the labelling is not specific to the nucleolus as the carbon dots light up the entire cell. CD-red may also have better solubility and cell permeability.

[0058] Specificity refers to the ability of the entity to accurately identify and / or measure a single target without interference from other components. Selectivity involves favouring one entity over others, especially when dealing with multiple potential targets or stimuli. Specificity focuses on precise measurement of a single entity, while selectivity involves prioritization among different entities.

[0059] In some embodiments, the carbon dot surface-functionalised with glucosamine is doped to modify the properties of the carbon dots. Dopants may comprise nitrogen, sulphur, and / or phosphorus. In some embodiments, the carbon dot surface-functionalised with glucosamine is nitrogen doped. The doping with nitrogen may improve the photoluminescence emissions of the carbon dot surface-functionalised with glucosamine.

[0060] In some embodiments, the carbon dot surface-functionalised with glucosamine comprises glucosamine conjugated to the carbon dot surface-functionalised with glucosamine via a linker. In this regard, glucosamine is covalently bonded to the carbon dot. The linker may be optionally substituted, and may be a straight chain linker or a branched linker. In some embodiments, the linker is characterised by a chain length of 1 to 50 atoms.

[0061] The term "linker" is used herein to refer to a bond or an atom or a collection of atoms optionally used to link interconnecting moieties such as hydroxyl and / or amine moieties on glucosamine and a surface of a carbon dot, or a ligand associated with a carbon dot to form the carbon dot surface-functionalised with glucosamine. The linker may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage. The linker may also impart additional properties such as watersolubility and cell membrane permeability so that it can permeate into cells and / or crosslink hydrophobic proteins within membranes. These properties may be determined by the composition of the spacer arm and / or reactive group.

[0062] The glucosamine may be conjugated to carbon dot using crosslinker chemistry. Towards this end, functional moieties such as amine, carboxyl, sulfhydryl, carbonyl may be used in combination to provide different types of crosslinks.

[0063] In some embodiments, glucosamine is directly conjugated to a surface of the carbon dot surface-functionalised with glucosamine. For example, the carbon dot surface- functionalised with glucosamine may comprise ligands such as citric acid and / or formamide, to which glucosamine may covalently bond to. Accordingly, glucosamine is conjugated to the carbon dot surface-functionalised with glucosamine via a ligand.

[0064] In some embodiments, glucosamine is conjugated to the carbon dot surface- functionalised with glucosamine via an amide bond. This may be effected using carbodiimides (such as l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N,N'-Dicyclohexylcarbodiimide (DCC)), and N-hydroxysuccinimide esters. For example, the carbon dot may be synthesised having carboxylic acid moieties on its surface, which may be used to conjugate with an amine moiety on the glucosamine.

[0065] EDC and other carbodiimides are zero-length crosslinkers; they cause direct conjugation of carboxylates (-COOH) to primary amines (-NH2) without becoming part of the final crosslink (amide bond) between target molecules. EDC crosslinking reactions may be performed in conditions devoid of extraneous carboxyls and amines. Acidic (pH 4.5 to 5.5) buffer such as MES buffer (4-morpholino-ethane-sulfonic acid) may be used, but phosphate buffers at pH < 7.2 are also compatible with the reaction chemistry. N- hydroxysuccinimide (NHS) or its water-soluble analog (Sulfo-NHS) is often included in EDC coupling protocols to improve efficiency or to create a more stable, amine-reactive intermediate.

[0066] NHS esters are reactive groups formed by EDC-activation of carboxylate molecules. NHS ester-activated crosslinkers and labeling compounds react with primary amines in slightly alkaline conditions (pH 7.2-8.5) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (MW 115 kDa), which can be removed easily by dialysis or desalting.

[0067] NHS-ester crosslinking reactions are usually performed in phosphate buffer at pH 7-9.0 for 0.5 to 4 hours at room temperature or 4°C. Primary amine buffers such as Tris (TBS) may be used at the end of a conjugation procedure to quench (stop) the reaction.

[0068] Sulfo-NHS esters are identical to NHS esters except that they contain a sulfonate (-SOs) group on the N-hydroxysuccinimide ring. This charged group has no effect on the reaction chemistry, but it does tend to increase the water-solubility of crosslinkers containing them. In addition, the charged group prevents Sulfo-NHS crosslinkers from permeating cell membranes, enabling them to be used for cell surface crosslinking methods.

[0069] Other crosslinking chemistry may also be used.

[0070] Imidoester crosslinkers react with primary amines to form amidine bonds. To ensure specificity for primary amines, imidoester reactions are best done in amine-free, alkaline conditions (pH 10), such as with borate buffer. Because the resulting amidine bond is protonated, the crosslink has a positive charge at physiological pH, much like the primary amine which it replaced. The amidine bonds formed are reversible at high pH.

[0071] Maleimide-activated crosslinkers and labelling reagents react specifically with sulfhydryl groups (-SH) at near neutral conditions (pH 6.5-7.5) to form stable thioether linkages. Short homobifunctional maleimide crosslinkers enable disulfide bridges in protein structures to be converted to permanent, irreducible linkages between cysteines.

[0072] Haloacetyl crosslinkers may contain an iodoacetyl or a bromoacetyl group. Haloacetyls react with sulfhydryl groups at physiologic to alkaline conditions (pH 7.2 to 9), resulting in stable thioether linkages.

[0073] Pyridyl disulfides react with sulfhydryl groups over a broad pH range to form disulfide bonds. As such, conjugates prepared using these crosslinkers are cleavable with typical disulfide reducing agents, such as dithiothreitol (DTT). During the reaction, a disulfide exchange occurs between the -SH group of the target molecule and the 2-pyridyldithiol group of the crosslinker. Pyridine-2-thione (MW = 111 kDa; Amax = 343 nm) is released as a byproduct that can be monitored spectrophotometrically and removed by dialysis or desalting.

[0074] Carbonyls (aldehydes and ketones) can be produced in glycoproteins and other polysaccharide-containing molecules by mild oxidation of certain sugar glycols using sodium meta-periodate. Hydrazide-activated crosslinkers and labelling compounds will then conjugate with these carbonyls at pH 5 to 7, resulting in formation of hydrazone bonds.

[0075] Alkoxyamine compounds conjugate to carbonyls (aldehydes and ketones) in much the same manner as hydrazides.

[0076] Photoreactive reagents are chemically inert compounds that become reactive when exposed to ultraviolet or visible light. Aryl azides (also called phenylazides) is a photoreactive chemical group used in crosslinking and labelling reagents. When an aryl azide compound is exposed to UV light, it forms a nitrene group that can initiate addition reactions with double bonds or insertion into C-H and N-H sites or can undergo ring expansion to react with a nucleophile (e.g., primary amine).

[0077] Diazirines are a class of photo-activatable chemical groups that may have better photostability than phenyl azide groups, and it is more easily and efficiently activated with long-wave UV light (330-370 nm). Photo-activation of diazirine creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths of the particular reagent.

[0078] The reaction between an azide and an alkyne either using a copper catalyst or a copper free strained alkyne results in the formation of a stable triazole linkage between the coupling partners. In the classic click reaction, an azide is coupled to an alkyne using Cu(I) to bring two coupling partners together and form a stable triazole linkage. One drawback of this approach is that copper ions— both Cu(II) as well as Cu(I), which is produced in the presence of ascorbate or TCEP— can harm cells, reduce the fluorescence of fluorophores, and impair protein function. To overcome this challenge, strained cyclic alkynes (DIBO / DBCO) have been developed to efficiently react with an azide to form the triazole linkage in the absence of copper under biological conditions. The strain in this eight-membered ring allows the reaction with azide-modified molecules to occur in the absence of catalysts or extreme temperatures, enabling the study of the surface of live cells, and preventing copper-induced damage of fluorescent proteins such as GFP in fixed and permeabilized cells.

[0079] The Staudinger reaction occurs between a methyl ester phosphine (PH3) and an azide (Ns-) to produce an aza-ylide intermediate that is trapped to form a stable covalent bond. Similar to click chemistry, the ligation reaction is highly specific and can be performed in aqueous environments at physiological pH. Staudinger ligations do not require copper to be reactive.

[0080] In some embodiments, the fluorescent probe is characterised by 1 glucosamine per carbon dot surface-functionalised with glucosamine to 20 glucosamine per carbon dot surface-functionalised with glucosamine. In other embodiments, ratio is 1 glucosamine per carbon dot surface-functionalised with glucosamine to 18 glucosamine per carbon dot surface-functionalised with glucosamine, 1 glucosamine per carbon dot surface- functionalised with glucosamine to 16 glucosamine per carbon dot surface- functionalised with glucosamine, 1 glucosamine per carbon dot surface-functionalised with glucosamine to 14 glucosamine per carbon dot surface-functionalised with glucosamine, 1 glucosamine per carbon dot surface-functionalised with glucosamine to 12 glucosamine per carbon dot surface-functionalised with glucosamine, or 1 glucosamine per carbon dot surface-functionalised with glucosamine to 10 glucosamine per carbon dot surface-functionalised with glucosamine.

[0081] In some embodiments, the carbon dot further comprises carboxylic acid moieties on its surface thereof. In some embodiments, the carbon dot is characterised by a mole ratio of carboxylic acid moieties to glucosamine of about 10: 1 to about 1000: 1. In other embodiments, the mole ratio is about 100:1 to about 1000: 1, about 100: 1 to about 900: 1, about 100:1 to about 800: 1, about 100:1 to about 700:1, about 100:1 to about 600: 1, about 100:1 to about 500: 1, about 100:1 to about 400:1, about 100:1 to about 300: 1, or about 100: 1 to about 200:1.

[0082] In some embodiments, the carbon dot surface-functionalised with glucosamine is further passivated with a passivating agent. Passivation refers to coating a surface of a nanomaterial so that its surface becomes "passive", that is, less readily affected by the environment. This however does not prevent a ligand from surface functionalising the nanomaterial. The passivating agent may be an ionisable molecule. In some embodiments, the carbon dot surface-functionalised with glucosamine is further passivated with citric acid.

[0083] The term "about" is used herein as a relative term, approximating various quantities such as measurements of size and wavelengths. The use of this term is appropriate depending on the specific context and does not render the scope indeterminate. This may in turn depend on the accuracy of the instrumentation used to determine the parameter. Where "about" is used, the degree of variance from the defined value will depend on what the skilled person would understand it to mean. For example, in the case of a temperature of about 50 degrees, this might mean 51 or 52 degrees. For example, in the case of pH of about 5, this might mean 4.6 or 5.4.

[0084] In some embodiments, the carbon dot surface-functionalised with glucosamine is characterised by an average particle size of about 2 nm to about 5 nm (Figure 1). Smaller sized particles may enter the nucleus through passive nuclear transport, where the size limit of the transport is about 5 nm. In other embodiments, the average particle size is about 2 nm to about 4.5 nm, about 2 nm to about 4 nm, about 2 nm to about

[0085] 3.5 nm, about 2 nm to about 3 nm, about 2.5 nm to about 5 nm, about 2.5 nm to about

[0086] 4.5 nm, about 2.5 nm to about 4 nm, about 2.5 nm to about 3.5 nm, about 2.5 nm to about 3 nm, about 3 nm to about 5 nm, about 3 nm to about 4.5 nm, about 3 nm to about 4 nm, or about 3 nm to about 3.5 nm. In some embodiments, the carbon dot surface-functionalised with glucosamine is characterised by an average particle size of about 3.4 nm.

[0087] In some embodiments, the fluorescent probe is characterised by excitation dependant emission with an excitation wavelength of about 400 nm to about 600 nm. In other embodiments, the excitation wavelength is about 400 nm to about 550 nm, about 400 nm to about 500 nm, about 425 nm to about 600 nm, about 425 nm to about 550 nm, about 425 nm to about 500 nm, about 450 nm to about 600 nm, about 450 nm to about 550 nm, about 450 nm to about 500 nm, or about 500 nm to about 600 nm.

[0088] In some embodiments, the fluorescent probe is characterised by an emission wavelength of about 420 nm to about 650 nm. In other embodiments, the emission wavelength is about 420 nm to about 600 nm, about 420 nm to about 550 nm, about 420 nm to about 500 nm, about 420 nm to about 450 nm, about 450 nm to about 650 nm, about 450 nm to about 600 nm, about 450 nm to about 550 nm, about 450 nm to about 500 nm, about 500 nm to about 650 nm, about 500 nm to about 600 nm, or about 500 nm to about 550 nm.

[0089] In some embodiments, the fluorescent probe is characterised by a surface state absorption of about 450 nm to about 650 nm. Surface states are electronic states found at the surface of materials and are formed due to the sharp transition from solid material that ends with a surface. In other embodiments, the surface state absorption is about 450 nm to about 600 nm, about 450 nm to about 550 nm, about 500 nm to about 650 nm, about 500 nm to about 600 nm, about 500 nm to about 550 nm, about 550 nm to about 650 nm, or about 550 nm to about 600 nm. In some embodiments, the fluorescent probe is characterised by a surface state absorption of about 550 nm.

[0090] In some embodiments, the fluorescent probe is characterised by an edge state absorption of about 300 nm to about 400 nm. In other embodiments, the edge state absorption is about 300 nm to about 375 nm, about 300 nm to about 350 nm, about 325 nm to about 400 nm, about 325 nm to about 375 m, about 325 nm to about 350 nm, about 350 nm to about 400 nm, or 350 nm to about 375 nm. In some embodiments, the fluorescent probe is characterised by an edge state absorption of about 350 nm.

[0091] In some embodiments, the fluorescent probe is characterised by a core state absorption below about 300 nm.

[0092] In some embodiments, the fluorescent probe is specific and selective to a nucleolus of a cell. As shown in Figures 5 and 6, the fluorescent probe labelled the nucleolus in the cells with bright fluorescence without lighting up the entire cell. As shown in Figure 13, the fluorescent probe is unable to stain the red blood cells of the mouse as the red blood cells lack a nucleolus, resulting in a missing fluorescence signal when the cells are irradiated with an excitation wavelength of about 560 nm.

[0093] In some embodiments, the fluorescent probe selectively binds to RNA. The nucleolus contains RNA and a low or non-existent concentration of DNA. The fluorescent probe is specific and selective by binding to RNA present within the nucleolus of a cell, resulting in the bright fluorescence when an excitation wavelength is applied onto the cell, as shown in Figure 12. Figure 12a shows the nucleolus being labelled by the fluorescent probe with a bright fluorescence while Figure 12b shows a diminished fluorescence in the cells treated with RNase as the hydrolysis of RNA substrates by RNase prevents the carbon dots in the fluorescent probe from binding with RNA. When the cells, stained with the carbon dots in the fluorescent probe, are treated with DNase, there was no change in the fluorescent intensity of the nucleolus in the cells (Figure 12c) as the carbon dots does not bind to DNA.

[0094] In some embodiments, the fluorescent probe is characterised by a fluorescent intensity which is positively correlated to a RNA concentration. In some embodiments, the fluorescent probe is characterised by a fluorescent intensity which is linearly correlated to a RNA concentration from more than 0 pg / mL to about 20 pg / mL. The fluorescent probe may show a turn-on fluorescence behaviour upon binding with RNA and an increase in fluorescent intensity with the concentration of RNA. As shown in Figure lib, the fluorescence of the fluorescent probe is increased to about 18 times when added with 150 pg / mL of RNA.

[0095] In some embodiments, the fluorescent probe is characterised by a cytotoxicity of less than about 10% at a concentration of 100 pg / mL (Figure 14). In other embodiments, the cytotoxicity is less than about 8%, less than about 6% or less than about 5%.

[0096] Accordingly, the present invention discloses a fluorescent probe, comprising a carbon dot; wherein the carbon dot is nitrogen doped; wherein the carbon dot is surface-functionalised with glucosamine; wherein the carbon dot is characterised by an average particle size of about 2 nm to about 5 nm; and wherein the fluorescent probe is characterised by a specificity and / or selectivity for a nucleolus of a cell.

[0097] The present invention also provides a method of fabricating a fluorescent probe, comprising : a) solvothermally synthesising a carbon dot in the presence of a nitrogen dopant at predetermined conditions; and b) surface functionalising the carbon dot with glucosamine.

[0098] Figure 10 shows a flow chart of the fabrication of the fluorescent probe.

[0099] The carbon dot may be solvothermally synthesised in the presence of a carbon source. A nitrogen source may also be present for the doping of the carbon dot. Carbon sources may include citric acid, polyurethane, biomass waste, or a combination thereof. Nitrogen sources may include formamide, ethylenediamine, dimethylformamide, or a combination thereof. In some embodiments, step a) comprises solvothermally synthesising the carbon dot in the presence of citric acid and formamide.

[0100] In some embodiments, the carbon dot is synthesised at a temperature of about 110 °C to about 180 °C. In other embodiments, the temperature is about 110 °C to about 170 °C, about 110 °C to about 160 °C, about 110 °C to about 150 °C, about 110 °C to about 140 °C, or about 110 °C to about 130 °C. In other embodiments, the temperature is about 130 °C.

[0101] In some embodiments, the carbon dot is synthesised for a duration of about 3 hours to about 8 hours. In other embodiments, the duration is about 4 hours to about 8 hours, about 5 hours to about 8 hours, or about 6 hours to about 8 hours. In other embodiments, the duration is about 6 hours.

[0102] In some embodiments, the predetermined conditions is a temperature of about 130 °C for about 6 hours.

[0103] In some embodiments, the synthesis is conducted in a polar protic solvent. In some embodiments, the solvent is an alcohol, such as methanol, ethanol, propanol, or butanol.

[0104] In some embodiments, the mole ratio of citric acid to formamide is about 1: 1 to about 1 : 100. In other embodiments, the mole ratio is about 1:1 to about 1:90, about 1 : 1 to about 1:80, about 1: 1 to about 1 :70, about 1 :1 to about 1:60, about 1:20 to about 1 :90, about 1:20 to about 1 :80, about 1:20 to about 1:70, about 1 :20 to about 1:60, about 1 :40 to about 1 : 100, about 1 :40 to about 1 :90, about 1 :40 to about 1 :80, about 1 :40 to about 1 :70, about 1 :40 to about 1:60, about 1 :60 to about 1 : 100, about 1 :60 to about 1 :90, about 1 :60 to about 1 :80, or about 1 :60 to about 1 :70. In some embodiments, the mole ratio is about 1 :60.

[0105] In some embodiments, the method further comprises a step after a) of purifying the carbon dot. The carbon dot may be purified using centrifugation, and / or dialysis.

[0106] In some embodiments, glucosamine is conjugated to the carbon dot via an amide coupling reaction.

[0107] In some embodiments, step b) comprises surface functionalising the carbon dot with glucosamine in the presence of a carbodiimide compound and N-hydroxysuccinimide (NHS). The carbodiimide compound couples NHS to carboxyls, forming an NHS ester for conjugation to amines, such as glucosamine.

[0108] In some embodiments, the carbodiimide compound is l-ethyl-3-(-3- dimethylaminopropyl) carbodiimide hydrochloride (EDC).

[0109] In some embodiments, the weight ratio of the carbodiimide compound to NHS is about 1 : 1.

[0110] The present invention also provides a method of staining a nucleolus of a cell, comprising : a) contacting a fluorescent probe as described herein with the cell.

[0111] The contacting step may be performed for at least 1 h, 2 h, 4 h, 8 h, 12 h, or 24 h.

[0112] The cell may then be washed to remove the excess fluorescent probe. The excess fluorescent probe may not be internalised in the cell, and may be removed via a media exchange.

[0113] Examples

[0114] Synthesis of the probe (CD-red)

[0115] Initially, nitrogen-doped carbon dots were synthesized from citric acid and formamide using a solvothermal method as per previously published literature. The reaction temperature and reaction times were slightly modified based on the TEM analysis of the carbon dots prepared. The formation of carbon dots and optical properties are sensitive to even minor changes in the reaction time and temperature. An optimized reaction temperature of 130°C and reaction time of 6 hours is used to synthesize carbon dots. Carbon dots are synthesized by a solvothermal method using citric acid, ethanol, and formamide. First, a mixture of 25 mL of ethanol and 25 mL of formamide was prepared. Then 2g of citric acid was added to the mixture and sonicated for 20 minutes. After that the solution was transferred into a lOOmL Teflon-lined stainless-steel autoclave and heat treated at 130°C in a hot air oven for 6 hours. The solution was then collected after cooling to room temperature and centrifuged at 1000 RPM for 30 minutes. The supernatant solution was discarded, and the precipitate was washed with ethanol multiple times. The final precipitate was dried and ground to make a fine powder. The powder was then dissolved in ethanol and purified using a 3KDa dialysis membrane for 24 hours.

[0116] The nanoparticles were then conjugated with glucosamine to improve their solubility and cell permeability using a simple but powerful EDC / NHS chemistry. 10 mg of EDC in 1 mL of Millipore water and 10 mg of NHS in 1 mL of Millipore water were prepared separately, added to the nanoparticle solution, and kept for mixing in a linear shaker for 2 hours. After two hours, 25 mg of glucosamine prepared in 1 mL of Millipore water was added to the solution and kept for mixing for another 24 hours. Then the solution was dialyzed with a 2KDa membrane for 24 hours. The final solution is collected and used for further experiments. The size and shape of the particles were characterized by their Transmission electron microscope (TEM) image, which indicates that the average size of the particle is around 3.4 ± 0.6 nm (Figure 1). The carbon dots surface- functionalised with glucosamine prepared are smaller in size compared to the literature followed for synthesis. Smaller size particles can enter the nucleus through passive nuclear transport. The passive nuclear transport limit is - 5 nm. Figure 4 shows the characteristic FTIR peak of glucosamine was observed in our probe, indicating the successful conjugation of glucosamine to nitrogen-doped carbon dots. The Zeta potential was -4.9 mV for original nitrogen-doped carbon dots and -1.1 mV for CD-red.

[0117] General procedure for cell imaging study

[0118] To examine its potential bioimaging applications, the glucosamine-conjugated carbon dots samples were incubated with live HeLa cells and imaged under a confocal microscope. Surprisingly, this probe selectively stains the nucleolus with bright red fluorescence (Figure 5, 6). The biomolecular imaging of the probe was performed with the HeLa cell lines. HeLa cells were grown on a coverslip in a 12-well plate. Approximately 50000 cells were seeded on the coverslip and incubated for 24 hours. After 24 hours, the media was removed, and washed the cells with PBS. 5 pg / ml solution of the probe prepared in DMEM was added to the cells and incubated for 2 hours. Then the probe-DMEM solution was aspirated and washed twice with PBS. The cells were treated with 300 nM DAPI dye solution in PBS for 10 minutes. The cells were then fixed using a 3.7% paraformaldehyde solution and imaged using a confocal microscope.

[0119] Discussions

[0120] Figure 1 shows the transmission electron microscope image of the CD-red. It indicates that CD-red is spherical and has an average particle size of 3 nm. From Figure 2, it is clearer that CD-red shows a surface state absorption of around 550 nm, an edge state absorption around 350 nm, and a core state absorption below 300 nm. Figure 3 shows the photoluminescence spectra of the CD-red. It shows an emission peak at 450 nm corresponding to a 405 nm excitation wavelength. Figure 4 shows the FTIR spectra of CD-red nanoparticles and glucosamine-conjugated probe nanoparticles. The FTIR peaks at 1032 nm corresponding to glucosamine conjugation. Figure 5 displays the confocal image of the HeLa cells labelled with the CD-red and DAPI dye. DAPI targets the DNA concentration inside cells, and it marks the nuclei. It is known that the DNA concentration is extremely low or non-existent in the nucleolus. So, the nucleolus regions will not get stained by the DAPI; consequently, it appears dark in the images. In Figure 5(B), the dark spots inside the nucleus are nucleolus, and in Figure 5(A), these regions are bright. The labelling ability of CD-red on the nucleolus can be confirmed from these images. Further, in Figure 6, the labelling of nucleolus by CD-red in a single isolated HeLa cell is shown.

[0121] CD-red titration against RNA

[0122] A titration experiment is conducted using CD-red and RNA to explore how the binding of CD-red to RNA affects the fluorescence emission. Different RNA concentrations are added to the CD-red, and the fluorescence emission is recorded. Figure 11(a) shows the fluorescence spectra of CD-red with different concentrations of RNA. The normalized peak fluorescence intensity at 620 nm as a function of RNA concentration is plotted in Figure 11(b). CD-red shows a turn-on fluorescence behaviour upon binding with RNA and a linear increase in the fluorescence intensity with the concentration of RNA. The CD-red fluorescence is increased to 18 times when added with 150 pg / mL of the RNA. To the best of the inventors' knowledge, CD-red shows much higher RNA sensitivity than previously reported probes.

[0123] RNase and DNase digestion experiments

[0124] The selectivity and specificity of the CD-red towards RNA are further analysed by performing Deoxyribonuclease (DNase) and ribonuclease (RNase) digestion experiments. The experiments are performed in HeLa cells. HeLa cells were seeded on a coverslip, kept in a 12-well plate, and incubated for 24 hours. After 24 hours, the cells are fixed with 3.7% paraformaldehyde and then treated with 1% triton X -100 in a phosphate-buffered saline (PBS) solution (pH 7.2). CD-red (5 pg / ml) is added to the cells and incubated for 30 minutes. The cells are then washed twice with PBS and treated with DNase and RNase. The control cells are treated with PBS. Figures 12(a), (d), and (g) show the confocal images of control HeLa cells treated with CD-red and DAPI dye and composite, respectively. Figure 12(b) shows the confocal image of HeLa cells incubated with CD-red after the RNase digestion test. The fluorescence from the nucleolus is completely diminished in the cells treated with RNase. This is because of the hydrolysis of RNA substrates by the RNase, and the nanoparticles are not able to bind with RNA. No changes were observed in the DAPI-stained image of HeLa cells (Figure 12(e)) in the RNase digestion test, as RNase does not influence DNA. Figure 12(h) shows the composite image of both Figure 12(b) and (e). The fluorescence intensity of the nucleus stained by DAPI (Figure 12(f)) showed an apparent diminishing in the DNase digestion test. While the DNase digestion test reveals no disruption in the fluorescence intensity of the CD-red labelled nucleolus (Figure 12(c)). This shows the superiority of the probe CD-red for labelling nucleolus by RNA binding mechanism without interference with DAPI.

[0125] Control experiments with mouse red blood cells

[0126] A control experiment was conducted to test the activity of CD-red in mouse red blood cells that lack a nucleolus. Figure 13(a) shows the bright field image of mouse red blood cells. Figure 13(b) shows the corresponding confocal image excited with a laser having an excitation wavelength of 560 nm. In Figure 13(b), the fluorescence signal is missing, which indicates that the CD-red are not stained on the red blood cells. This proves the selective staining activity of the CD-red on the nucleolus of the cells.

[0127] Cvtotoxicitv of CD-red

[0128] The cytotoxicity of the CD-red was examined via MTT reduction. Figure 14 shows the MTT reduction percentages of the HeLa cells incubated with different concentrations of CD-red. The result confirms no significant difference in cell viability, even for a 100 ug / mL concentration. It should be noted that only 5 ug / mL concentration of the probe is required for labelling the nucleolus.

[0129] Conclusion

[0130] In conclusion, the inventors have successfully developed CD-red, a highly selective and sensitive carbon dot based novel fluorescent probe for nucleolus. CD-red shows excellent photophysical and photochemical properties when applied to cell imaging. Its good solubility and low cytotoxicity could make it an attractive candidate for studying nucleolus dynamics and monitoring disease related to nucleolus, in future. The inventors believe that CD-red will open a new avenue which will help the scientist to discover several fundamental findings within the nucleolus.

[0131] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0132] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0133] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0134] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

Claims

Claims1. A fluorescent probe, comprising a carbon dot; wherein the carbon dot is nitrogen doped; wherein the carbon dot is surface-functionalised with glucosamine; wherein the carbon dot is characterised by an average particle size of about 2 nm to about 5 nm; and wherein the fluorescent probe is characterised by a specificity and / or selectivity for a nucleolus of a cell.

2. The fluorescent probe according to claim 1, wherein glucosamine is conjugated to the carbon dot via an amide bond.

3. The fluorescent probe according to claims 1 or 2, wherein the fluorescent probe is characterised by 1 glucosamine per carbon dot to 20 glucosamine per carbon dot.

4. The fluorescent probe according to any one of claims 1 to 3, wherein the carbon dot further comprises carboxylic acid moieties on its surface thereof.

5. The fluorescent probe according to any one of claims 1 to 4, wherein the carbon dot is characterised by an average particle size of about 3.4 nm.

6. The fluorescent probe according to any one of claims 1 to 5, wherein the fluorescent probe is characterised by excitation dependant emission with an excitation wavelength of about 400 nm to about 600 nm.

7. The fluorescent probe according to any one of claims 1 to 6, wherein the fluorescent probe is characterised by an emission wavelength of about 420 nm to about 650 nm.

8. The fluorescent probe according to any one of claims 1 to 7, wherein the fluorescent probe is characterised by a surface state absorption of about 550 nm.

9. The fluorescent probe according to any one of claims 1 to 8, wherein the fluorescent probe is characterised by an edge state absorption of about 350 nm.

10. The fluorescent probe according to any one of claims 1 to 9, wherein the fluorescent probe is characterised by a core state absorption below 300 nm.

11. The fluorescent probe according to any one of claims 1 to 10, wherein the fluorescent probe selectively binds to RNA.

12. The fluorescent probe according to any one of claims 1 to 11, wherein the fluorescent probe is characterised by a fluorescent intensity which is positively correlated to a RNA concentration.

13. The fluorescent probe according to any one of claims 1 to 12, wherein the fluorescent probe is characterised by a cytotoxicity of less than about 10% at a concentration of about 100 pg / mL.

14. A method of fabricating a fluorescent probe, comprising: a) solvothermally synthesising a carbon dot in the presence of a nitrogen dopant at predetermined conditions; and b) surface functionalising the carbon dot with glucosamine.

15. The method according to claim 14, wherein step a) comprises solvothermally synthesising the carbon dot in the presence of citric acid and formamide.

16. The method according to claims 14 or 15, wherein the predetermined conditions is a temperature of about 130 °C for about 6 hours.

17. The method according to any one of claims 14 to 16, wherein the mole ratio of citric acid to formamide is about 1:1 to about 1 : 100.

18. The method according to any one of claims 14 to 17, further comprising a step after a) of purifying the carbon dot.

19. The method according to any one of claims 14 to 18, wherein step b) comprises surface functionalising the carbon dot with glucosamine in the presence of a carbodiimide compound and N-hydroxysuccinimide (NHS).

20. The method according to claim 19, wherein the carbodiimide compound is 1- ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).

21. The method according to any one of claims 14 to 20, wherein the weight ratio of the carbodiimide compound to NHS is about 1: 1.

22. A method of staining a nucleolus of a cell, comprising: a) contacting a fluorescent probe according to any one of claims 1 to 13 with the cell.

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