Method for manufacturing carbon quantum dots with a shifted fluorescence emission spectrum

Abstract

The method of obtaining modified carbon quantum dots by N-substitution of compounds from the coumarin group, consists, according to the invention, in the fact that the carbon quantum dots, obtained by carbonisation of biomass constituting, inter alia, 1 biologically active substance having free carboxyl groups, are modified by the formation of an amide bond in an aqueous medium, under normal conditions, and then purified using dialysis membranes. During grafting, the ratio of dye to EDC is 1 to 1.1. The invention also relates to the carbon quantum dots with a fluorescence emission spectrum in the range 380- 800 nm, obtained by this method.

Description

[0001] METHOD FOR MANUFACTURING CARBON QUANTUM DOTS WITH A SHIFTED FLUORESCENCE EMISSION SPECTRUM

[0002] TECHNICAL FIELD

[0003] The object of the invention is a method for manufacturing carbon quantum dots and their modification with organic compounds from the coumarin group in an aqueous medium and carbon quantum dots with a shifted fluorescence emission spectrum, obtained by this method, suitable for use as agents for biodetection of cancer cells, elements of theranostic systems, detection of cations and compounds of high and low molar mass, bioimaging of cell organelles and whole eukaryotic and prokaryotic cells, component of biosensors and sensors, optical fibres, controlled drug delivery and release systems, inks and bio-inks for 3D printing, as well as for online and offline monitoring of chemical and biochemical processes.

[0004] BACKGROUND

[0005] Zero-dimensional carbon nanomaterials are an important nanomaterial group due to their unique physicochemical, including optoelectronic and biological properties. They are characterised by a size below 10 nm, so they can penetrate the cell membrane and bioaccumulate in selected cell organelles. Importantly, they lack a metallic core and therefore have much lower cytotoxicity and can be successfully used in medicine and pharmacy as components of theranostic systems enabling simultaneous diagnostics and therapy, as well as controlled drug delivery and release systems or fluorescent probes.

[0006] Due to the presence of hydrophilic functional groups, such as hydroxyl, amine and carboxyl groups, are water-soluble, significantly increasing their applicability in both in vitro and in vivo applications. A unique feature of carbon nanodots is the dependence of fluorescence emission on the excitation wavelength. Nanodots, unlike organic molecular dyes, show resistance to photobleaching and photo-aging.

[0007] One of the most important parameters of carbon nanomaterials is the fluorescence emission range, which determines their applicability. Fluorescence properties result from both perturbations in the crystal lattice, deformation of the surface structure and molecular properties due to the presence of the C=C conjugated bond system and N, S, O heteroatoms.

[0008] The best-known two-step surface modification methods for this group of carbon-core nanomaterials include:

[0009] 1. surface functionalisation of carbon nanodots obtained by oxidation of carbon black with nitric acid, using amine derivatives;

[0010] 2. surface oxidation of carbon nanodots obtained by graphite electrode electrolysis leading to their auto-passivation;

[0011] 3. surface passivation of nanodots obtained by laser ablation of graphite, using poly(ethylene glycol).

[0012] The best-known methods for one-step surface modification of carbon nanodots include:

[0013] 1. self-passivation of the nascent carbon nanodot core with heteroatoms such as S, N, O derived from the biomass constituting the substrate; and

[0014] 2. modification of the nascent carbon nanodot core by the formation of chemical bonds present on its surface, mainly ester, amide, with chemical compounds containing N, O or S heteroatoms.

[0015] The described strategies for modifying carbon quantum dots by functionalisation, require the use of toxic solvents or modifying compounds, and are unique in terms of end-product characteristics, which significantly hampers their scalability and commercial use in diagnostics or pharmacy.

[0016] Methods to modify CQDs by grafting or substitution are currently known and in use, however, these only result in an increase in fluorescence quantum yield in a range that does not allow use in combination with dyes found in human tissues. This has led to a search for new methods of modifying carbon quantum dots, which will have the effect of altering the fluorescence emission range so that it does not overlap with that of luminescent compounds, naturally found in the human body, such as oxy- and deoxyhaemoglobin and melanin. Of the known methods of modifying carbon quantum dots, the most similar to the solution according to the present invention is the method presented in the Polish patent PL 243214 B1, which discloses a method for synthesising carbon nanodots using a microwave pressure reactor. According to that description, carbon nanomaterials are obtained by carbonisation of chitosan, glucose or sucrose at elevated pressure in a microwave radiation field, purified and then subjected to a surface modification reaction with an organic dye in a microwave radiation field at an elevated temperature of 140-200°C and purified again.

[0017] The article entitled “A coumarin-modified graphene quantum dot-based luminogen for the detection of cysteine in aqueous media”, Deepa Sebastian, Kala Ramakrishnan, Photochemistry and Photobiology, 2023, 1-12, presents a method for modifying the surface of graphene quantum dots by a sonication-assisted amidation reaction with graphene oxide as a carbon source, leading to obtaining nanomaterials characterised by cyan fluorescence. Briefly, approximately 50 mg of GQDs was dissolved in 20 mL of deionised water (Dl- Water) and subjected to reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 76.8 mg), followed by the addition of 0.01 M HCI solution until pH 5 and stirred for 30 min. N-hydroxysuccinimide (NHS, 23.0 mg) was then added and the pH was adjusted to 9 by adding 0.01 N NaOH solution and stirring for 30 minutes. Finally, 69.20 mg of 3- aminocoumarin was added and subjected to ultrasound for 30 minutes.

[0018] A similar example is the article entitled “Coumarin-Modified Graphene Quantum Dots as a Sensing Platform for Multicomponent Detection and Its Applications in Fruits and Living Cells”, by Zhaochuan Yu, Wenhui Ma, Tao Wu, Jing Wen, Yong Zhang, Liyan Wang, Yuqian He, Hongtao Chu, and Minggang Hu ACS Omega 2020 5 (13), 7369-7378, where the synthesis of C-GQDs exhibiting cyanine fluorescence is also described. Briefly, 50 mg GQDs were mixed with 20 mL DMF (dimethylformamide) and were subjected to ultrasound treatment for 1 h to form a homogeneous dispersion, after which EDC (76.8 mg, 0.4 mmol) was added to the system. Subsequently, 0.01 M HCI was dropped into the above solution until the pH of the system reached 5. After vigorous stirring for 30 minutes at 25°C, NHS (23.0 mg, 0.2 mmol) and 0.01 M NaOH were added to the dispersion to raise the pH of the system to 9, and subjected to activation for 3 hours. Then, coumarin (69.2 mg, 0.2 mmol) was added to the mixture and stirred continuously for 48 h at 25°C. Finally, the reaction solution was dispersed in 100 mL of water and extracted repeatedly with CH2CI2. The extracted aqueous phase was further purified with a dialysis membrane (1000 Da) for 48 h and then lyophilised to obtain C-GQD (46 mg) as a powder.

[0019] Another example of quantum dots surface-modified with a coumarin group dye is the one described in the publication entitled "Fluorescence spectrum-based dual-parameter detection method for pH and DO in cancer cell metabolic fluid", by Yanli Hu, Chao Zheng, Wei Tao, Hui Zhao, Journal of Nanoparticle Research, 2020, 22, 186, 1-8. In brief, the obtained CdSe quantum dots, containing a CdSe core and a shell made of ZnS, were surface- modified by a coupling reaction using EDC. The finished product exhibited cyan fluorescence and potential use in early cancer diagnosis by demonstrating the dependence of fluorescence on pH and concentration of oxygen dissolved in the analysed medium (phosphate buffer).

[0020] As reported in the specialist and patent literature, there is currently no known method for obtaining modified carbon quantum dots with a shifted fluorescence emission spectrum by coupling reactions of CQDs with an organic dye under normal conditions using EDC in an aqueous medium.

[0021] PURPOSE OF THE INVENTION

[0022] The purpose of the invention was to develop a method of modifying carbon quantum dots, obtained by hydrothermal reaction, through N-substitution of coumarin compounds in an aqueous medium, to change the fluorescence emission spectrum to one that does not overlap with biological dyes found in vivo, so that they can be used as agents for cancer cell biodetection, components of theranostic systems, detection of cations and high and low molar mass chemical compounds, bioimaging of cell organelles and whole eukaryotic and prokaryotic cells, element of biosensors and sensors, optical fibres, controlled drug delivery and release systems, inks and bio-inks for 3D printing, and online and offline monitoring of chemical and biochemical processes, and furthermore to obtain surface-modified carbon quantum dots substituted with selected coumarins so as to alter their fluorescence range, and so that they may be suitable for the above-mentioned applications.

[0023] Carrying out the chemical modification reaction of CQDs with amine derivatives of coumarins, using the coupling agent EDC exclusively in an aqueous solution, at room temperature under atmospheric pressure, in any reaction vessel (open glass / Teflon vessels), thus eliminating heating processes and carrying out chemical processes in pressurised apparatus. In addition, it is possible to arbitrarily scale down / up the process of chemical modification of CQDs with an amine derivative of coumarin and the coupling agent 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0024] SUMMARY OF THE INVENTION

[0025] The object of the present invention is a method for manufacturing carbon quantum dots modified with organic compounds of the coumarin group, with a shifted fluorescence emission spectrum, comprising the steps in which: a) carbon quantum dots containing free carboxyl groups are manufactured, using at least one biologically active substance as a carbon source, b) the carbon quantum dots containing free carboxyl groups obtained in step a) are modified with coumarin derivatives containing a free amino group in the aromatic ring, with the formation of an amide bond between the carbon quantum dots and the amine coumarin derivative, characterised in that the carbon quantum dots containing free carboxyl groups are obtained by a carbonisation reaction, preferably hydrothermal or thermal, the above-mentioned carbon quantum dots containing free carboxyl groups are subjected to a chemical coupling reaction with an amine coumarin derivative using the chemical agent, 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide, in an aqueous medium, at room temperature, under normal pressure, and then purified using dialysis membranes, preferably with MWCO = 1000 Da.

[0026] Preferably, biomass containing at least one substrate with biological activity is used as a raw material for the manufacture of the carbon quantum dots.

[0027] Preferably, a solution of unmodified carbon quantum dots containing free carboxyl groups on their surface is used as an aqueous solution at a concentration of 2-5 mg / mL for the modification reaction of carbon quantum dots with an amine derivative of coumarin and 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0028] Preferably, 3 to 3.7 pmol of the fluorescent dye from the coumarin group containing a free amino group in the aromatic ring, preferably 7-amino-4-methylcoumarin and 7-amino-4- (trifluoromethyl)coumarin), are used per 1 mg of carbon quantum dots, using a dye concentration in the range of 0.2-1.0 mg / mL, preferably 1 mg / mL.

[0029] Preferably, 1-1.2 moles of EDC coupling agent, preferably 1.1 moles of EDC agent in the form of an aqueous solution, is used per 1 mole of the fluorescent dye of the coumarin group containing one amino group in the aromatic ring.

[0030] Preferably, a non-toxic amine coumarin derivative with the fluorescence emission range of 380-800 nm is used for the chemical modification of carbon quantum dots, to visualise biological structures in the visible range using fluorescence microscopy.

[0031] Preferably, the carbonisation reaction is carried out in a closed reaction vessel in a 200 W microwave radiation field for 60 minutes at a pressure of 9-10 atm.

[0032] Preferably, the carbonisation reaction is carried out in a closed reaction vessel in a microwave radiation field of 800 W, for 4 minutes.

[0033] The essence of the invention is that carbon quantum dots obtained by carbonisation under hydrothermal and thermal conditions, are subjected to modification by N-substitution of organic compounds of the coumarin group containing free amino groups in an aqueous medium, using the coupling agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and then are subjected to purification on dialysis membranes.

[0034] According to the invention, in the first step, the starting raw material, being a solution of carbon quantum dots obtained from biomass, at least one component of which is a low or high molecular weight compound with bioactive properties, is placed in a glass or Teflon reaction vessel and subjected to stirring on a magnetic stirrer, then a freshly prepared solution of EDC coupling agent is added and, after its activation, an aqueous coumarin solution is added, and the grafting reaction is carried out for a period of 24 hours.

[0035] The reaction products obtained are purified from unreacted urea substrates and other byproducts using a dialysis process with dialysis membranes (MWCO 1000 Da), using distilled water as a purification agent, obtaining the final product as a solution of carbon quantum dots with fluorescence emission in the range 380 to 800 nm.

[0036] Preferably, the surface modification reaction uses an aqueous solution of carbon quantum dots obtained by biomass carbonisation at a concentration of 5 mg / mL.

[0037] Preferably, the carbon quantum dots obtained by hydrothermal carbonisation using at least one biologically active substance as a carbon source are used as the starting material.

[0038] Preferably, the carbon source is a compound selected from the group of amino acids, proteins, polysaccharides, cannabinoids, vitamins and / or neurotransmitters.

[0039] Preferably, compounds from the coumarin group, containing free reactive amine groups in their chemical structure, are used to modify the surface of the carbon quantum dots.

[0040] Preferably, compounds from the coumarin group, with fluorescence emission in the range 380-800 nm, are used for the modification.

[0041] Preferably, a CQDs solution of 1 to 5 mg / mL is used for the modification.

[0042] Preferably, 5 mg / mL EDC is used as the coupling agent.

[0043] Preferably, an amount of 1.02 to 2.27 mL of EDC solution is used.

[0044] Preferably, the ratio of EDC to dye is 1:1 -1.2 moles.

[0045] Preferably, the modification reaction is carried out for 24 hours.

[0046] Preferably, the reaction is carried out at room temperature.

[0047] Preferably, the reaction is carried out under normal pressure.

[0048] Preferably, the reaction is carried out with continuous stirring of 300 rpm.

[0049] Preferably, the finished reaction products are purified using dialysis membranes MWCQ=1000.

[0050] Modified carbon quantum dots are obtained in an environmentally safe manner under normal conditions by N-substitution with organic compounds from the coumarin group, making them non-toxic to eukaryotic and prokaryotic cells and displaying fluorescence in the 380-800 nm range. The solution according to the invention is illustrated in the following examples, not limiting the scope of its protection.

[0051] The object of the present invention is illustrated in the drawings, where:

[0052] Fig. 1 shows a reaction scheme for the modification of CQDs with 7-amino-4- methylcoumarin. Step 1 - activation of carboxyl groups from the surface of CQDs by EDC. Step 2 - coupling reaction of the CQDs with 7-amino-4-methylcoumarin, resulting in the formation of an amide bond between the CQDs and the free amine group of the aromatic ring;

[0053] Fig. 2 shows the fluorescence spectra of unmodified CQDs used for the modification reactions obtained according to Example 1. Excitation of fluorescence emission with radiation in the range of 300-500 nm. Fluorescence spectra of modified CQDs used for the modification reactions obtained according to Example 1. Excitation of fluorescence emission with radiation in the range 330-380 nm;

[0054] Fig. 3 shows the FTIR spectra of the CQDs obtained according to Example 1. The broad absorption band in the range 2400-3600 cm’1confirms the presence of free carboxyl groups on the surface of the CQDs. In addition, the band visible in the spectrum at 1693 cm’1confirms the presence of free grafted carboxyl groups on the surface of the nanomaterial;

[0055] Fig. 4 shows a reaction scheme for the modification of CQDs with 7-amino-4- (trifluoromethyl)coumarin. Step 1 - activation of carboxyl groups from the surface of CQDs with EDC, Step 2 - coupling reaction of CQDs with 7-amino-4-(trifluoromethyl)coumarin resulting in the formation of an amide bond between CQDs and the free amine group of the aromatic ring;

[0056] Fig. 5 shows the fluorescence spectra of unmodified CQDs used for the modification reactions obtained according to Example 2. Excitation of fluorescence emission with radiation in the range of 300-500 nm. Fluorescence spectra of modified CQDs used in the modification reactions obtained according to Example 2;

[0057] Fig. 6 shows FTIR spectra of CQDs obtained according to Example 2. The broad absorption band in the range 2400-3600 cm’1confirms the presence of free carboxyl groups on the surface of the CQDs; additionally, the band visible in the spectrum at 1693 cm-1confirms the presence of free grafted carboxyl groups on the surface of the nanomaterial;

[0058] Fig. 7 shows the fluorescence spectra of unmodified CQDs used for the modification reaction obtained according to Example 3. Excitation of fluorescence emission with radiation in the range 300-450 nm. Fluorescence spectra of modified CQDs used in the modification reactions obtained according to Example 3;

[0059] Fig. 8 shows the FTIR spectra of the CQDs obtained according to Example 3. The broad absorption band in the range 2400-3600 cm’1confirms the presence of free carboxyl groups on the surface of the CQDs;

[0060] Fig. 9 shows the fluorescence spectra of unmodified CQDs used for the modification reaction obtained according to Example 4. Excitation of fluorescence emission with radiation in the range of 300-450 nm. Fluorescence spectra of modified CQDs used for the modification reactions obtained according to Example 4. Excitation of fluorescence emission with radiation in the range 300-450 nm. Fluorescence spectra of the CQDs modified with 7- amino-4-(trifluoromethyl)coumarin according to Example 4;

[0061] Fig. 10 shows the FTIR spectra of the CQDs obtained according to Example 4. The broad absorption band in the range 2400-3500 cm’1confirms the presence of free carboxyl groups on the surface of the CQDs, which is further confirmed by the strong signal at 1708 cm’1coming from the vibration of the carbonyl group of the carboxyl moiety.

[0062] The attached diagram (Fig. 1) presents the modification pathway of the carbon nanomaterials.

[0063] EXAMPLES

[0064] EXAMPLE 1

[0065] Modification of carbon quantum dots obtained by a hydrothermal method with 7-amino-4- methylcoumarin

[0066] For the chemical modification reaction of CQDs, nanomaterials obtained by a hydrothermal method from a mixture of 1 g glucose, 0.25 g glycine, 0.1 g cannabidiol (CBD), 0.5 mL of 35% hydrochloric acid solution and 20 mL of water were used in a reaction vessel that was sealed and exposed to a 200 W microwave radiation field for 45 minutes. The pressure in the reaction vessel was maintained at 9-10 atm. After completion of the carbonisation reaction, the contents of the reaction vessel were placed in a 50 mL beaker, which was immersed in an ultrasonic bath for 15 minutes. After the extraction of the CQDs, a 10% NaOH solution was added to the solution until a pH value of 7 was reached, which was monitored using a pH meter equipped with a hydrogen ion-sensitive combined glass electrode. After the neutralisation of the sample, the mixture was filtered through filter paper and then placed in a dialysis membrane (MWCO = 1000 Da). The CQDs solution was purified for 4 days, until the nanomaterial was completely cleared of low molecular weight by-products of the carbonisation process. The chemical modification reaction of the CQDs was carried out by adding 5 mL of a 3 mg / mL CQDs solution to a 25 mL glass beaker. The solution was stirred at room temperature with a magnetic stirrer, and then an aqueous solution of the coupling agent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)) prepared just before the reaction was added, at a concentration of 5.0 mg / mL in an amount of 1.71 mL. Immediately after mixing the solutions, an aqueous dye solution of 7-amino-4- methylcoumarin was added at a concentration of 1 mg / mL and in an amount of 8.8 mL. The reaction mixture was left at room temperature for 24 hours to react, maintaining continuous stirring of the solution at 300 rpm. The resulting aqueous solution of modified CQDs was then purified from by-products and unreacted dye by dialysis using dialysis membranes (MWCO = 1000 Da). Deionised water was used as the solvent for the purification of the modified CQDs. The reaction product was purified for 4 days, yielding a pure CQDs solution containing no low molecular weight reaction by-products.

[0067] EXAMPLE 2

[0068] Modification of carbon quantum dots obtained by a hydrothermal method with 7-amino-4- (trifluoromethyl)coumarin

[0069] The chemical modification reaction of CQDs used nanomaterials obtained by a hydrothermal method from a mixture of 22 cm3deionised water followed by 1 g glucose, 0.1 g aspartic acid, 0.2 g glycine, 0.3 g arginine, 0.5 g CBD, 0.25 g ascorbic acid and 0.6 mL 35% hydrochloric acid solution. The reaction vessel was sealed and exposed to microwave radiation at 200 W for 60 minutes. The pressure in the reaction vessel was maintained at 9- 10 atm. After completion of the carbonisation reaction, the contents of the reaction vessel were placed in a 50 mL beaker, which was immersed in an ultrasonic bath and exposed to ultrasound for 15 minutes. After completing the CQDs extraction process, a 5% NaOH solution was added to the CQDs solution until a pH value of 7 was reached, which was monitored using a pH meter equipped with a hydrogen ion-sensitive combined glass electrode. After the neutralisation of the sample, the mixture was filtered through filter paper and then placed in a dialysis membrane (MWCO = 1000 Da). The CQDs solution was purified for 4 days, until the nanomaterial was completely cleared of low molecular weight by-products of the carbonisation process. To carry out the chemical modification reaction of CQDs by coupling to 7-amino-4-(trifluoromethyl)coumarin via an amide bond, 5 mL of a 2 mg / mL solution of CQDs was added to a 25 mL glass beaker. The solution was stirred with a magnetic stirrer, and then an aqueous solution of the coupling agent, prepared just before the reaction was carried out, was added at a concentration of 5.0 mg / mL in an amount of 1.14 mL. Immediately after mixing the solutions, an aqueous solution of 7-amino- 4-(trifluoromethyl)coumarin dye was added at a concentration of 1 mg / mL in an amount of 7.6 mL. The reaction mixture was left at room temperature for 24 hours to react, maintaining continuous stirring of the solution at 250 rpm. The resulting aqueous solution of the modified CQDs was then purified from by-products and unreacted dye by dialysis using dialysis membranes (MWCO = 1000 Da). Deionised water was used as the solvent for the purification of the modified CQDs. The reaction product was purified for 4 days, yielding a pure CQDs solution containing no low molecular weight reaction by-products.

[0070] EXAMPLE 3

[0071] Modification of carbon quantum dots obtained by non-pressurised carbonisation in a microwave radiation field with 7-amino-4-(trifluoromethyl)coumarin

[0072] To obtain CQDs for chemical modification, 15 cm3of water, 1 g of glucosamine sulphate and 0.1 g of dopamine were added to a 50 cm3porcelain crucible-shaped reaction vessel. 1 cm3of 35% hydrochloric acid solution was added to the mixture. The mixture was heated at 40°C until the reactants were completely dissolved. The crucible was exposed to an 800 W microwave radiation field for 4 minutes. To the cooled reaction mixture, 20 cm3of water was added, the sample was placed in an ultrasonic bath for 5 minutes and then a 10% NaOH solution was added until a pH of 7 was reached. The pH of the solution was controlled with a pH meter. The solutions were filtered through filter paper and then transferred to dialysis membranes made of regenerated cellulose (MWCO = 1000 Da). The membrane was placed in a beaker containing 500 cm3of distilled water and dialysed for 4 days. For chemical modification by coupling using the coupling agent EDC with 7-amino-4- (trifluoromethyl)coumarin, 5 mL of a 4 mg / mL solution of CQDs was added to a 50 mL glass beaker. The solution was stirred with a magnetic stirrer, and then an aqueous solution of the coupling agent, prepared just before the reaction was carried out, was added at a concentration of 5.0 mg / mL in an amount of 2.27 mL. Immediately after mixing the solutions, an aqueous solution of 7-amino-4-(trifluoromethyl)coumarin dye at a concentration of 1 mg / mL was added in an amount of 15.3 mL. The reaction mixture was left at room temperature for 24 hours to react, maintaining continuous stirring of the solution at 300 rpm. The resulting aqueous solution of modified CQDs was then purified of by-products and unreacted dye by dialysis using dialysis tubing (MWCO = 1000 Da). Deionised water was used as the solvent for the purification of the modified CQDs. The reaction product was purified for 4 days, yielding a pure CQDs solution containing no low molecular weight reaction by-products.

[0073] EXAMPLE 4

[0074] Modification of carbon quantum dots obtained by non-pressurised carbonisation in a microwave radiation field with 7-amino-4-(trifluoromethyl)coumarin

[0075] 15 cm3of water, 1 g of glucosamine sulphate and 0.1 g of dopamine were added to a 50 cm3crucible-shaped reaction vessel made of Teflon. 1 cm3of 35% hydrochloric acid solution was added to the mixture. The mixture was heated at 40°C until the reactants were completely dissolved. The crucible was exposed to a 800 W microwave radiation field for 4 minutes. To the cooled reaction mixture, 20 cm3of water was added, the sample was placed in an ultrasonic bath for 5 minutes, and then a 5% NaOH solution was added until a pH value of 7 was obtained. The pH of the solution was controlled using a pH meter. The solutions were filtered through filter paper and then transferred to dialysis membranes made of regenerated cellulose (MWCO = 1000 Da). The membrane was placed in a beaker containing 500 cm3of distilled water and dialysed for 4 days. To carry out the chemical modification of CQDs with 7-amino-4-(trifluoromethyl)coumarin by coupling with EDC agent in aqueous solution, 3 mL of CQDs solution at a concentration of 3 mg / mL was added to a 50 mL glass beaker.

[0076] The solution was stirred with a magnetic stirrer and then an aqueous solution of the coupling agent prepared just before the reaction was added, at a concentration of 5.0 mg / mL in an amount of 1.02 mL. Immediately after mixing the solutions, an aqueous solution of 7- amino-4-(trifluoromethyl)coumarin dye was added at a concentration of 1 mg / mL in an amount of 6.9 mL. The reaction mixture was left at room temperature for 24 hours to react, maintaining continuous stirring of the solution at 100 rpm. The resulting aqueous solution of modified CQDs was then purified of by-products and unreacted dye by dialysis using dialysis membranes (MWCO = 1000 Da). Deionised water was used as the solvent for the purification of the modified CQDs. The reaction product was purified for 4 days, yielding a pure CQDs solution containing no low molecular weight reaction by-products.

[0077] BENEFICIAL EFFECTS OF THE INVENTION

[0078] An advantageous aspect of the invention is the method for chemically modifying the surface of carbon quantum dots with dimensions of 2-10 nm, containing carboxyl groups on their surface, carried out in an aqueous solution, at room temperature under atmospheric pressure, using amine derivatives of coumarins with high fluorescence quantum yield, carried out using a water-soluble EDC coupling agent, resulting in the formation of chemical-amide bonds between the dye molecules and the CQDs. Carbon quantum dots with low fluorescence quantum yields, containing carboxyl groups on their surface, obtained with high yield in a microwave radiation field using pressure or non-pressure method, are chemically modified with amine derivatives of coumarins, which have high fluorescence quantum yields, thereby imparting improved fluorescence properties to the CQDs. In addition, the use of mild coupling reaction conditions (room temperature, no elevated pressure, no presence of concentrated acids or other aggressive reactants) contributes to the preservation of the chemical structure and morphology of the CQDs, resulting only in a chemical modification of the surface carboxyl groups of the CQDs. Reducing the amount of free carboxyl groups does not contribute to a reduction in the water solubility of the modified nanomaterials, since the high content of free hydroxyl groups on the surface of the CQDs, which strongly interact with water allows the grafting of amine derivatives of coumarins exhibiting low solubility in water. The chemical modification of CQDs that do not exhibit high fluorescence quantum yields makes it possible to obtain nanomaterials exhibiting high fluorescence quantum yields capable of visualising cellular structures by fluorescence microscopy. The method according to the present invention allows any chemical modification of the surface of CQDs with coumarin group dyes, containing a free amino group in the aromatic ring, thereby imparting fluorescent properties to the CQDs with similar fluorescence emission characteristics as those exhibited by pure amino coumarin derivatives in aqueous solution. The by-products formed during the reaction of the chemical modification of CQDs with a fluorescent dye using the coupling agent EDC in aqueous solution are easily separated using regenerated cellulose dialysis membranes (MWCO = 1000 Da), resulting in a pure aqueous solution of the chemically modified CQDs. At no stage of the synthesis of CQDs and their chemical modification is it necessary to use anhydrous chemical reaction conditions, as is the case, for example, with other coupling agents (a solution of dicyclocarbodiimide (DCC) in tetrahydrofuran, so it is possible to use a lyophilisation process or classical drying to remove water from the CQDs and obtain the final product in a solid form.

Claims

AMENDED CLAIMS received by the International Bureau on 24 September 2025 (24.09.2025)1. A method for manufacturing carbon quantum dots modified with coumarin derivatives, with a shifted fluorescence emission spectrum, comprising the steps in which: a) carbon quantum dots containing free carboxyl groups are manufactured using biomass comprising at least one biologically active compound selected from amino acids, proteins, polysaccharides, cannabinoids, vitamins and neurotransmitters as a carbon source, b) the carbon quantum dots obtained in step (a) are modified with an amino coumarin derivative to form an amide bond using 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), characterised in that the above-mentioned carbon quantum dots are subjected to a coupling reaction in aqueous medium, at room temperature and atmospheric pressure, without addition of N-hydroxysuccinimide, and the reaction products are purified by dialysis using membranes with MWCO = 1000 Da, yielding carbon quantum dots with a fluorescence emission spectrum in the range 380-800 nm.

2. The method according to claim 1, wherein the biomass comprises cannabinoids, preferably cannabidiol (CBD).

3. The method according to claim 1 or 2, wherein the biomass further comprises compounds selected from dopamine and ascorbic acid.

4. The method according to any of the preceding claims, wherein the amino coumarin derivative is selected from 7-amino-4-methylcoumarin and 7-amino-4-(trifluoromethyl)coumarin.

5. The method according to any one of the preceding claims, wherein the molar ratio of coumarin derivative to EDC is 1 :1 -1.2.

6. The method according to any one of the preceding claims, wherein the CQDs are used as an aqueous solution at a concentration of 2-5 mg / mL, and the coupling reaction is carried out for 24 hours under continuous stirring.

7. The method according to any one of the preceding claims, wherein the carbonization reaction is carried out in a closed reaction vessel in a 200 W microwave radiation field for 60 minutes at a pressure of 9-10 atm.

8. The method according to any one of the preceding claims, wherein the carbonization reaction is carried out in a closed reaction vessel in a microwave radiation field of 800 W for 4 minutes.

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