NZ570093A - Method of making luminescent nanoparticles from carbohydrates - Google Patents

Abstract

Disclosed is a method of preparing luminescent carbon nanoparticles by reacting one or more carbohydrates with sulphuric acid and subsequently oxidising a product of the reaction.

Description

28/0? 2009 TUB Hi 01 FAX +61 3 8618 4199 F.B.Rice & Co. 0006/067 III *10057489715* 1 "700^ METHOD OF MAKING LUMINESCENT NANOPARTICLES FROM CARBOHYDRATES FIELD OF THE INVENTION The present invention relates to a method of preparing luminescent carbon nanoparticles and to luminescent carbon nanoparticles prepared by that method.

BACKGROUND OF THE INVENTION Luminescent quantum dots (QDs) have become an important photonic tool in the past two decades due to their unique properties, such as high chemical stability, resistance to photodegradation and readily tunable optical properties. They have a wide variety of promising applications in biology, medicine and so on. However, the high price, known toxicity and potential environmental hazard associated with many of these materials 10 may greatly limit their applications.

More recently, luminescent carbon dots have been discovered. These carbon dots have less toxicity than quantum dots but may have similar applications. The carbon dots are carbon nanoparticles which have been functionalized by oxidation with, for example, nitric acid. In certain cases the carbon dots are then passivated with organic or other 15 molecules. There is variation in the fluorescence and luminescence properties of these carbon dots and this may be due to differences in size, composition, surface charge or type of passivation of different dots.

One method for producing carbon dots is the laser ablation of a carbon target, eg [1], [2], For instance, in Sun et ai [1], a carbon target was ablated by using a Q-switched 20 Nd: YAG laser (1064 rnn, 10 Hz) in a flow of argon gas carrying water vapor at 900 °C and 75 kPa. The obtained sample was treated in an aqueous nitric acid for 12 h (no detectable fluorescence at this step), followed by surface passivation by attaching organic species to the acid-treated carbon particles. The passivated particles were luminescent The particles were approximately 5 nm in diameter.

INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 8 JUL 2009 RECEIVED 28/07 2009 TUE ll! 01 FAX +61 3 8618 4199 F.B.Rice & Co. 0007/067 2 The other method is to use the combustion soot of candles as the starting material [3], In Liu et al [3], the collected soot was treated with oxidative acid. Then the luminescent carbon nanoparticles were separated by using polyacrylamide gel electrophoresis.

The two methods described above are time consuming and have a high cost.

The present inventors have found a simpler and cheaper alternative method of preparing carbon dots from carbohydrates.

SUMMARY OF THE INVENTION The present invention provides a method of preparing luminescent carbon nanoparticles by reacting one or more carbohydrates with sulphuric acid and subsequently oxidising a 10 product of the reaction.

In another embodiment, the present invention provides method of preparing luminescent carbon nanoparticles by reacting one or more carbohydrates with sulphuric acid to form aggregates of luminescent carbon nanoparticles and subsequently breaking up the aggregates so that the luminescent carbon nanoparticles readily disperse.

The present invention also provides luminescent carbon nanoparticle prepared by the methods of the present invention.

Further provided is a luminescent carbon nanoparticle prepared from one or more carbohydrates.

Also provided is a carbon nanoparticle comprising sulphur.

The present invention further provides a luminescent carbon nanoparticle wherein the emission intensity of the carbon nanoparticle does not decrease by more than 20% after 20 hrs of continuous excitation at 360 nm.

There is also provided a plurality of luminescent carbon nanoparticles wherein the full width at half maximum of the peak at the maximum emission wavelength of the 25 plurality of carbon nanoparticles is less than 125 nm. 28/07 2009 TUE lis 01 FAX +61 3 8618 4199 F.B.Rice «■ Co. @008/067 3 The present invention provides for the use of the luminescent carbon nanoparticles of the present invention in diagnostic imaging, in biological markers or drug delivery systems In addition, the present invention further provides for the use of luminescent carbon 5 nanoparticles according to the present invention in the preparation of a medicament for use in diagnostic imaging of a patient, in biological markers or drug delivery systems.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Temporal evolution of absorption (A) and corresponding emission (B) spectra of luminescent carbon nanoparticles prepared according to the present invention 10 during treatment in 5 M of nitric acid solution. The reaction time is indicated in the figure. The excitation wavelength for photoluminescence measurement was 380 nm.

Figure 2. Normalized photoluminescent spectra of luminescent carbon nanoparticles prepared from glucose (a) and sugar (b).

Figure 3. Normalized photoluminescent spectra of luminescent carbon nanoparticles 15 prepared from glucose (a) and candle soot (b).

Figure 4. shows emission spectra of luminescent carbon nanoparticles prepared according to the present invention from glucose before (a) and after (b) passivation Figure 5. shows an image of luminescent carbon nanoparticles prepared according to the present invention under ambient light and UV lamp (365nm) Figure 6, shows an FT-IR spectrum of products obtained by H2SO4 treatment of sucrose.

Figure 7. shows a Raman spectrum of products obtained by H2SO4 treatment of sucrose.

Figure 8. shows UV and emission spectra of luminescent carbon nanoparticles 25 prepared according to the present invention after reaction with TTDA 28/07 2009 TUE 11:01 FAX +61 3 8618 4199 F.B.Rice & Co. 0009/067 4 Figure 9. shows a UV and an emission spectra of luminescent carbon nanoparticles prepared according to the present invention after reaction with poly(ethylene glycol) bis(3-aminopropyl) terminated (PEG 1 soon).

Figure 10. shows a UV and an emission spectra of luminescent carbon nanoparticles 5 prepared according to the present invention after reaction with ethylenediamine Figure 11. shows a FT-IR spectrum of products obtained by H2SO4 treatment of starch Figure 12. shows a Raman spectrum of products obtained by H2SO4 treatment of starch Figure 13. shows a UV and an emission spectra of luminescent carbon nanoparticles prepared according to the present invention after reaction with TTDA Figure 14. FT-IR (A) and Raman (B) spectra of active carbon powder (a) and carbon nanoparticles prepared from glucose (b), sucrose (c) and starch (d).

Figure 15. High resolution XPS spectra for CNPs. A: full scan. B: C Is; C: Ols; D: S2p Figure 16. Time evolution of emission spectra of aggregated CNPs prepared from 15 glucose measured during different time of nitric acid (2.0 M) treatment The treatment time is indicated on the figure.

Figure 17. Absorption (A) and emission (B) spectra of carbon dots prepared from glucose, before (a) and after (b) TTDA passivation. (Emission spectra were recorded using different emission slits) Inset: passivated CNPs under ambient light (left) and (B) 20 UV light (365 nm) (right).

Figure 18. TEM images of TTDA passivated CNPs prepared from glucose.

Figure 19. XRD patterns of glucose-derived CNPs before (a) and after (b) 4 h of nitric acid (2.0 M) treatment and active carbon powder (c).

Figure 20. Emission spectra of TTDA passivated CNP at different excitation 25 wavelength which was progressively increased from 360 rnn on the left with a 10 nm 28/07 2009 TUE 11:01 FAX +61 3 8618 4199 F. B. Rice & Co. @010/067 increment. CNP were obtained by 12 h of nitric acid (2.0 M) treatment. Inset: normalized emission spectra Figure 21. Emission spectra of TTDA passivated CNP at different excitation wavelength which was progressively increased from 360 nm on the left with a 10 nm 5 increment. CNPs were obtained by 4 h of nitric acid (2.0 M) treatment.

Figure 22. Emission spectra of PEGuoon passivated carbon dots at different excitation wavelength which progressively increased from 350 nm, in 20 nm increments. CNPs which were obtained by using glucose as starting materials and 12 h of nitric acid (2.0 M) treatment.

Figure 23. Normalized emission intensity of TTDA passivated carbon dots during continuous excitation at 360 nm.

Figure 24. Effect of pH on the photoluminescence of carbon dots passivated with TTDA Figure 25. Emission spectra of TTDA passivated carbon dots at different excitation 15 wavelength which progressively increased from 350 nm, in 10 nm increments. CNPs were obtained by using starch as starting material and 12 h of nitric acid (2.0 M) treatment.

Figure 26. Emission spectra of carbon dots passivated with different ligands. A: ethylenediamine; B: PEG1500; C: TTDA; D: oleylamine. CNPs were obtained by 20 using glucose as starting material and 12 h of nitric acid (2.0 M, 50 mL) treatment.

Figure 27. Quantum yields of carbon dots passivated with different ligands. A: ethylenediamine; B: PEG1500; C: TTDA and D: oleylamine.

Figure 28. Confocal microscopy images of mice heart cell labeled with the carbon dots after incubation for 1 h at 37 °C. Top left: image of mice heart cell without carbon dots 25 labeling. 28/07 2009 TUE 11:02 FAX +61 3 8618 4199 F.B.Rice & Co. @011/067 6 Figure 29. Confocal microscopy images of mice heart cell labeled with the carbon dots after incubation for 2 h at room temperature. Top left: image of mice heart cell without carbon dots labeling.

Figure 30. SEM image of CNPs prepared form sugar.

Figure 31. Absorbance (A) and emission (B) spectra of TTDA passivated carbon dots at different excitation wavelength which progressively increased from 360 nm, in 10 nm increments. CNPs were obtained by using sugar as starting material and 12 h of nitric acid (2.0 M, 50 mL) treatment.

Figure 32. Absorbance (A) and emission (B) spectra of TTDA passivated carbon dots 10 at different excitation wavelength which progressively increased from 360 nm, in 10 nm increments. CNPs were obtained by using sugar as starting material and 12 h of nitric acid (2.0 M, 25 mL) treatment.

Figure 33. Absorbance (A) and emission (B) spectra of TTDA passivated carbon dots at different excitation wavelength which progressively increased from 360 nm, in 10 15 nm increments. CNPs were obtained by using sugar as starting material and 12 h of nitric acid (2.0 M, 15 mL) treatment.

Figure 34. Absorbance (A) and emission (B) spectra of TTDA passivated carbon dots at different excitation wavelength which progressively increased from 360 nm, in 10 nm increments. CNPs were obtained by using sugar as starting material and 4 h of 20 nitric acid (2.0 M, 15 mL) treatment.

Figure 35. Quantum yield of carbon dots prepared using A: 50 mL; B: 25 mL; C: 15 mL of 2.0 M nitric acid and 12 hrs of the treatment; and D: using 15 mL of 2.0 M nitric acid and 4 hrs of the treatment.

Figure 36. Absorbance (A) and emission (B) spectra of ATBA passivated carbon dots 25 at different excitation wavelength which progressively increased from 360 nm, in 10 nm increments. CNPs were obtained by using sugar as starting material and 12 h of nitric acid (2.0 M, 50 mL) treatment. Quantum yield measured: 0.045. 28/07 2009 TUE 11:02 FAX +61 3 8618 4199 F.B.Rice & Co. @012/067 7 Figure 37. Absorbance (A) and emission (B) spectra of ATBA passivated carbon dots at different excitation wavelength which progressively increased from 360 nm, in 10 nm increments. CNPs were obtained by using sugar as starting material and 12 h of nitric acid (2,0 M, 25 mL) treatment Quantum yield: 0.0279 DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides a method of preparing luminescent carbon nanoparticles by reacting one or more carbohydrates with sulphuric acid and subsequently oxidising a product of the reaction.

Preferably, the sulphuric acid is concentrated sulphuric acid.

The luminescent carbon nanoparticles can also be termed "carbon dots". As used herein, the terms "carbon nanoparticles" and "CNPs" indicate carbon nanoparticles which may or may not be luminescent Without being bound by theory, the present inventors believe that the reaction of carbohydrates with sulphuric acid produces aggregates of carbon nanoparticles. The 15 step of oxidising the nanoparticles serves to introduce hydroxyl and carboxylic acid groups to the surface of the nanoparticles. This breaks up the aggregates and allows the nanoparticles to disperse well in solution. The introduced hydroxyl and carboxylic acid groups allow for attachment of other components to the surface of the groups.

The surfaces of the luminescent carbon nanoparticles prepared by the methods of the 20 present invention appear to have relatively few defects. This is supported by the fact that carbon nanoparticles prepared according to the present invention show luminescence after nitric acid treatment whereas carbon nanoparticles prepared by other methods often show luminescence only after a passivation step.

The aggregates of nanoparticles formed after reaction of a carbohydrate with sulphuric 25 acid may display luminescence even before nitric acid treatment. Accordingly, in an alternative embodiment, the aggregates of nanoparticles formed after the reaction of the carbohydrates with sulphuric acid may be broken up by means other than oxidation 28/07 2009 TUE 11:02 FAX +61 3 8618 4199 F.B.Rice & Co. @013/067 8 such as by sonication and the like. In that alternative embodiment, the present invention provides a method of preparing luminescent carbon nanoparticles by reacting one or more carbohydrates with sulphuric acid to form aggregates of luminescent carbon nanoparticles and subsequently breaking up the aggregates so that the 5 luminescent carbon nanoparticles readily disperse.

The luminescent carbon nanoparticles may have a diameter of between 1 to 100 nm, preferably 2-50 nanometeres, more preferably 2-10 nanometres. In a preferred from, the carbon nanoparticles are about 5 nm in diameter.

The carbohydrate can be any carbohydrate ranging from simple sugars to more 10 complex carbohydrates such as the starches and cyclodextrins. The present inventors have found that different carbohydrates provide luminescent carbon nanoparticles having different properties. For instance, under the same reaction conditions, glucose and sucrose produce luminescent carbon dots having maximum emission wavelengths of442 nm and 460 nm respectively (see Example 1). This allows the property of the 15 luminescent carbon nanoparticles to be controlled according to the desired application. Further control can be achieved, for example, by varying the length of time that the carbohydrate is exposed to the sulphuric acid, by varying the concentration of the acid and the carbohydrate, and by controlling the temperature of the reaction.

In one embodiment, the product of the reaction is oxidised by an oxidising agent such 20 as an oxidising acid, KMnO^ H2O2, KCrCU, or K2C1SO7. Preferably, the oxidising agent is an oxidising acid. More preferably, the oxidising acid is nitric acid.

If nitric acid is used, preferably the concentration of the nitric acid is from 1M to 5M, preferably from 2M to 3 M. even more preferably about 2M.

The product of the reaction may be oxidised with nitric acid from 1 to 24 hours, 25 preferably from 4 to 12 hours.

Alternatively, other suitable oxidation methods, such as treatment with plasma, can be used. 28/07 2009 TUE lis 02 FAX +61 3 8618 4199 F.B.Rice & Co. @014/067 9 In a preferred from, the carbohydrate is selected from the group consisting of glucose, sugar (sucrose), and starch. Preferably, the carbohydrate is glucose.

Preferably, the reaction step and the subsequent oxidation step are each carried out in solution. More preferably, the reaction step and the subsequent oxidation step are each 5 carried out in aqueous solution.

In a preferred form, following the reaction of the one or more carbohydrates with the sulphuric acid, the reaction mixture is neutralised with the addition of base.

In a more preferred form, the oxidising agent is added directly to the neutralised reaction mixture.

The luminescent carbon nanoparticles can be isolated by common methods such as precipitation, filtration, extraction and evaporation of solvent They can be purified by any suitable means, such as by electrophoresis or dialysis.

Although the present inventors have found that the luminescent carbon nanoparticles prepared by the method of the present invention have luminescent properties, these 15 properties may be improved by passivating the surface of the luminescent carbon dots with a passivating agent. Other properties of the luminescent carbon dote such as stability and ease of handling may also be improved.

Accordingly, in a preferred form, the method further comprises coupling the luminescent carbon nanoparticles to a passivation agent Suitable coupling techniques 20 include covalent bonding and physical adsorption. Suitable passivating agents are described, for example, in US 2008/0113448, the disclosure of which is hereby incorporated by reference.

A passivation agent can be any material that can bind to a carbon nanoparticle surface and encourage or stabilise the radiative recombination of excitons, which it is theorised 25 in US 2008/0113448 comes about through stabilisation of tibe excitation energy 'traps' existing at the surface as a result of quantum confinement effects and the large surface area to volume ratio of a nanoparticle. Moreover, a passivation agent can be polymeric, s 28/07 2009 TUE 11:03 FAX +61 3 8618 4199 F.B.Rice & Co. @015/067 molecular, biomolecular, or any other material that can passivate a nanoparticle surface. For instance, the passivation agent can be synthetic polymer such as poly(lactic acid) (PLA), poly(ethylene glycol) (PEG), poly(propionylethyleneimine-co-ethyleneimine) (PPEI-EI), and poiy(vinyl alcohol) (PVA). In one embodiment, the 5 passivation agent can be a biopolymer, for instance a protein or a peptide.

The passivation agent and/or additional materials graft to the core nanoparticle via the passivation agent can provide the luminescent particles with additional desirable characteristics. For example, a hydrophilic passivation agent can be bound to the core carbon nanoparticle to improve the solubility/dispersability of the nanoparticle in water, 10 In another embodiment, a passivation agent can be selected so as to improve the solubility of the carbon nanoparticle in an organic solvent.

In particular, the passivating agent can be an amino-functionalised compound. Examples of passivating agents include 4,7,10-triox-1,13-tridecanediamine (TTDA), PEG1500, polyethylene glycol) bis(3-aminopropyl) terminated (eg PEGisoon), 15 oleylamine, poly(lactic acid(, poly(propionylethyleneimine-co-ethyleneimine) (PPEI-EI), and poly(vinyl alcohol) (PVA), and ethylenediamine.

In a preferred form, the passivation agent is 4,7,10-triox-l,13-tridecanediamine (TTDA), Unfortunately the photoluminescence mechanism of carbon dots is not very clear at the 20 present time. In Sun's paper (4), it is suggested that the photoluminescence may be attributed to the presence of surface energy traps that become emissive upon stabilization as a result of the surface passivation. In Papadimitrakopoulos's paper, the authors were of the view that oxygen, particularly in the presence of an acid or neutral environment, can quench photoluminescence through hole doping and subsequent 25 nonradiative Auger recombination. Accordingly, they used an aliphatic (dodecyl) analog of flavin mononucleotide to passivate the surface of single-wall carbon nanotube. The environment around the carbon nanotube was sufficiently tight to exclude oxygen from the SWNT surface, resulting in a quantum yield of 20%. 28/07 2009 TUE lis 03 FAX +61 3 8618 4199 F.B.Rice & Co. 0016/067 11 Without being bound by theory, it is the view of the present inventors that the photoluminescence of the carbon nanoparticles is also due to the electron transfer between C and N.

Accordingly, in one embodiment, the luminescent carbon nanoparticles comprise 5 nitrogen. This may be by the use of a nitrogen-rich passivating agent. In one embodiment, therefore, the passivating agent is a nitrogen-rich compound such as a flavin.

In certain embodiments, the passivating agents are capping ligands with biotin, such as: The luminescent carbon nanoparticles which are coupled to capping ligands with biotin may be in turn coupled to cell-targeting antibodies for use in targeted cell-imaging.

As can be seen from the examples, the passivation step greatly improves the luminescence and quantum yield of the luminescent carbon nanoparticles.

In a second aspect, the present invention provides a luminescent carbon nanoparticle 15 prepared by the methods of the present invention.

In one embodiment, the luminescent carbon nanoparticle is coupled to a passivation agent.

The quantum yields of the luminescent carbon nanoparticles prepared according to the present invention can vary substantially depending on the reaction conditions used to 20 prepare them and whether they are coupled to passivating agents. o o 28/07 2009 TUE 11:03 FAX +61 3 8618 4199 F.E.Rice & Co. @017/067 12 In one embodiment, if the luminescent carbon nanoparticles are not coupled to a passivating agent, then the quantum yield ranges from 1 to 4%.

In another embodiment, when the luminescent carbon nanoparticles are coupled to a passivating agent, then the quantum yield ranges from 5% to 20%.

In a preferred form, the product of the reaction (ie the aggregate of carbon nanoparticles) comprises sulphur prior to the oxidation step.

In one embodiment, the carbon nanoparticle comprises greater than 0.1% sulphur. Preferably, greater than 0.2% sulphur. More preferably, greater than 0.5% sulphur.

In a further preferred form, the product of the reaction (ie the aggregate of carbon 10 nanoparticles) comprises from 40%-95% carbon and from 5%-50% oxygen prior to the oxidation step. Mote preferably, from 56%-66% carbon and from 28%-36% oxygen.

In a third aspect, the present invention provides a luminescent carbon nanoparticle prepared from one or more carbohydrates.

Preferably, the luminescent carbon nanoparticle is coupled to a passivation agent.

In a preferred form, the process of preparing the luminescent carbon nanoparticle includes the step of reacting one or more carbohydrates with sulphuric acid.

The carbon nanoparticles prepared according to the methods of the present invention may comprise trace amounts of sulphur.

Accordingly, in a fourth aspect, the present invention provides a carbon nanoparticle 20 comprising sulphur.

In one embodiment, the carbon nanoparticle comprises greater than 0.1% sulphur. Preferably, greater than 0.2% sulphur. More preferably, greater than 0.5% sulphur.

The presence of sulphur in the nanoparticles can be measured by any technique known in the art including elemental analysis and XPS. 28/07 2009 TUE Hi 03 FAX +61 3 8618 4199 F.B.Rice 5> Co. @018/067 13 In a preferred form, the carbon nanoparticle further comprises from 56%-66% carbon and from 28%-36% oxygen.

In one embodiment, the carbon nanoparticle is formed by reacting one or more carbohydrates with sulphuric acid.

In one embodiment, the carbon nanoparticle comprised sulphur before the addition of a passivating agent or is not coupled to a passivating agent.

In a preferred form, the carbon nanoparticle is luminescent Preferably, the analysis for sulphur is conducted on the carbon nanoparticle before coupling the carbon nanoparticle to a passivation agent In a fifth aspect, the present invention provides a luminescent carbon nanoparticle wherein the emission intensity of the carbon nanoparticle does not decrease by more than 20% after 20 hrs of continuous excitation at 360 nm.

In a sixth aspect the present invention provides a plurality of luminescent carbon nanoparticles wherein the full width at half maximum of the peak at the maximum 15 emission wavelength of the carbon nanoparticles is less than 125 nm.

The maximum emission peak of carbon dots prepared according to the methods of the present invention is much narrower than that of carbon dots prepared using other techniques such as the oxidation of candle soot (Figure 3) which implies that the size distribution of carbon dots is narrower for the carbon dots prepared in accordance with 20 the present invention. For instance, the full width at half maximum of the emission peak of carbon dots prepared at optimized conditions is 76 nm, which is much narrower than those prepared from candle soot where the narrowest emission peak has a full width at half maximum of 125 nm (see table 1 of the supporting data of reference 2).

Preferably, the full width at half maximum of the peak at the maximum emission 25 wavelength is less than 100 nm, more preferably less than 80 nm, even more preferably less than 70 nm. 28/07 2009 TUE Hi 03 FAX +61 3 8618 4199 F.B.Rice & Co. @019/067 14 Preferably, the luminescent carbon nanoparticle of the fifth aspect is prepared by the method of the first aspect.

In one embodiment, the luminescent carbon nanoparticles of the present invention display luminescence prior to, or without, coupling to a passivating agent.

It would be understood by those skilled in the art that the carbon nanoparticles of the present invention can be used in any application in which luminescent nanoparticles are known to be useful. Suitable applications are described for instance in US 2008/0113448, the disclosure of which is hereby incorporated by reference.

In one embodiment, a carbon nanoparticle can be formed to include a reactive 10 functional chemistry suitable for use in a desired application, eg a tagging or analyte recognition protocol. For instance, a passivating agent can include a reactive functionality that can be used directly in a protocol, for example to tag a particular analyte or class of materials that may be found in a sample. Exemplary materials can include, for example, carbohydrate molecules that may conjugate with carbohydrates 15 on an analyte or biological species.

In another embodiment, a functional chemistry of a passivation agent can be further derivatised with a particular chemistry suitable for a particular application. For example, in one embodiment, a reactive functionality of a passivating agent can be further derivatised via a secondary surface chemistry functionalisation to serve as a 20 binding site for a substance. For example, a member of a specific binding pair, ie two different molecules where one of the molecules chemically and/or physically binds to the second molecule, such as an antigen or antibody can be bound to a nanoiparticle either directly or indirectly via a functional chemistry of the passivation agent that is retained on the nanoparticle following the passivation of the core carbon nanoparticle. 25 The passivation mid further derivatisation of the core carbon nanoparticle need not be carried out in separate reaction steps, however, abd in one embodiment, the passivation and derivatisation of the carbon nanoparticle can be carried out in a single process step. 28/07 2009 TUE 11:04 FAX +61 3 8618 4199 F.B.Rice & Co. 0020/067 Accordingly, a luminescent carbon nanoparticle can be advantageously utilized to tag, stain or mark materials, including biologically active materials, e.g., drugs, poisons, viruses, antibodies, antigens, proteins, and the like; biological materials themselves, e.g., cells, bacteria, fungi, parasites, etc; as well as environmental materials such as 5 gaseous, liquid, or solid (e.g., particulates) pollutants that may be found in a sample to be analysed. For example, the passivating material can include or can be derivatized to include functionality specific for surface receptors of bacteria, such as E.coli and L. monocytogenes, for instance. Upon recognition and binding, the bacteria can be clearly discernable due to the photoluminescent tag bound to the surface. 1Q Suitable reactive functionality particular for targeted materials are generally known to those of skill in the art. For example, when considering development of a protocol designed for recognition or tagging of a particular antibody in a fluid sample, suitable ligands for that antibody such as haptens particular to that antibody, complete antigens, epitopes of antigens, and the like can be bound to the polymeric material via the 15 reactive functionality of the passivating material. For instance, via a biotinylated functionality.

In another embodiment, a nanoparticle can be utilized to tag or mark the presence of a particular substance through the development of the photoluminescent characteristic on the nanomaterials only when the nanoparticle is in the presence of the targeted 20 substance. For example, a carbon nanoparticle can be formed and not subjected to a passivation reaction or optionally only partially passivated, such that the nanoparticle exhibits little or no photoluminescence. Upon contact with a passivating material (eg, a targeted substance) under reaction conditions, the nanoparticle can be passivated by the targeted substance in the sample and the nanoparticle can then exhibit increased 25 photoluminescence, and the presence of the targeted substance can be confirmed via the increased luminescence of the nanoparticle.

In another embodiment, the luminescence from a passivated, highly luminescent carbon nanoparticle can be quenched in the presence of a particular targeted substance. For example, the visible luminescence can be quenched in the presence of a potentially 30 harmful environmental substance such as a nitro-derivatized benzene, TNT, or a key 28/07 2009 TUE 11:04 FAX +61 3 8618 4199 F.B.Rice & Co. 0021/067 16 ingredient in explosives. For example, upon contact of the passivated, luminescent nanoparticle with the targeted substance, the luminescent properties of the nanoparticle can be quenched via collision or contact of the quencher molecules (i.e., the detectable substance) with the luminescent carbon nanoparticles that result in electron transfers or 5 other quenching mechanisms as are generally known to those in the art.

A photoluminescent nanoparticle can obviously be utilized in many other applications as well, in addition to tagging and recognition protocols such as those described above. For example, the disclosed luminescent nanoparticles can generally be utilized in applications previously described as suitable for photoluminescent silicon 10 nanoparticles. In some embodiments, luminescent nanoparticles as herein described can be utilized in applications suitable for luminescent nanoparticles. For instance, disclosed luminescent nanoparticles can be utilized in applications such as are common for luminescent quantum dots.

Luminescent carbon nanoparticles can be environmentally and biologically compatible. 15 For instance, a luminescent carbon nanoparticle can be formed so as to pose little or no environmental or health hazards during use. As such, a luminescent carbon nanoparticle prepared by the methods described herein can be utilized in light emission applications, data storage applications such as optical storage mediums, photo-detection applications, luminescent inks, and optical gratings, filters, switches, and the like, just 20 to name a few possible applications that are generally known to those of skill in the art..

Moreover, as the luminescent carbon nanoparticles can emit different colours at different excitation wavelengths, they can be used economically in practical, real-world applications. For instance, in using the nanoparticles of the present invention in labelling applications, detection and/or analysis (for instance through utilization of 25 confocal fluorescence microscopy) can be performed at multiple colours without the need for multiple sets of different luminescent materials.

The carbon nanoparticles of the present invention may also find applications in LEDs (light emitting diodes). 28/07 2009 TUE 11:04 FAX +61 3 8618 4199 F. E. Rice & Co. 0022/067 17 In one aspect, the present invention provides for the use of the luminescent carbon nanoparticles of the present invention in diagnostic imaging.

In another aspect, the present invention provides for the use of luminescent carbon nanoparticles according to the present invention in Hie preparation of a medicament for S use in diagnostic imaging of a patient The imaging can be of any cells or tissues, such as tumours, organs, and the like. In a preferred form, the imaging is imaging of heart tissue.

In a further aspect, the present invention provides for the use of the carbon nanoparticles of the present invention as markers for cell tagging or in drug delivery 10 systems.

EXAMPLES Materials D-(+)-glucose, sucrose, starch (potato starch, soluble), 4,7,10-trioxa-l,13-tridecanediamine, and poly(ethylene glycol) bis(3-aminopropyl) terminated (PEGisoon) 15 were purchased from Sigma-Aldrich. Spectra/Por DispoDialyzers (cellulose ester membranes, MW cut off: 1000) were also obtained from Sigma-Aldrich, The other chemicals used were obtained from local suppliers.

Instruments AH UV-vis and emission spectra were recorded by using a Shimadzu UV-1700 20 Spectrophotometer and a PerkinElmer LS55 luminescence spectrometer, respectively. Raman spectra were obtained with a Renishaw Raman spectrometer (System 1000) with 785 nm (red) laser excitation. FT-IR spectra were recorded by using FT-IR/FT-FIR spectrometer (Spectrum 400, PerkinElmer).

Quantum Yield Measurements Quantum yield was measured according to established procedure (J, R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., 1999, Kluwer Academic/Plenum 28/07 2009 TUE 11:04 FAX +61 3 8618 4199 F.B.Rice & Co. 0023/067 18 Publishers, New York) by using quinine sulfate in 0.10 M H2SO4 solution as the standard. The absorbance was measured on a Shimadzu UV-1700 Spectrophotometer. Absolute values are calculated according to the following equation: IB A n I Aan2 R - (1) where Q is the quantum yield, I is the measured integrated emission intensity, A is the optical density and n is the refractive index (taken here as the refractive index of the respective solvents). The subscript R refers to the reference fluorophore, quinine sulfate. In order to minimize re-absoiption effects, absorbencies in the 10 mm fluorescence cuvette were kept under 0.05 at the excitation wavelength of 360 nm.

Example 1 2 g of glucose (or saccharide) was dissolved in 5 mL of distilled water. Under violent stirring, 8 ml of concentrated sulphuric acid was added. The reaction was kept for 40 min, and the mixture was neutralized by using NaOH (12 g NaOH in 50 ml water). 50 mL of 5 M nitric acid solution was added and the mixture was refluxed for 12 h.

Figure 1 shows the absorption and emission spectra of carbon dots prepared from glucose during the treatment in 5 M of nitric acid. The photoluminescent (PL) intensity increased with the increase in reaction time up to 4 hrs, then followed by a decrease in the PL intensity between 4 -5 hours of treatment and then become stable. This result shows the possibility of preparing luminescent carbon dots from glucose.

The size of carbon dots which corresponds to the different emission wavelength can be controlled by using different starting materials. Figure 2 shows the normalized photoluminescent spectra of carbon dots prepared from glucose and sugar. The maximum emission wavelengths are 442 and 460 nm for carbon dots prepared from glucose and sugar, respectively.

Figure 3 shows the PL spectra of carbon dots prepared from candle soot without electrophoresis purification according to the reference^]. The emission peak of carbon 28/07 2009 TUE 11:05 FAX +61 3 8618 4199 F.B.Rice & Co. 0024/067 19 dots prepared from glucose is much narrower than that of carbon dots prepared from candle soot, which implies that the size distribution of carbon dots is narrower for the carbon dots prepared in accordance with the present invention.

After the treatment by nitric acid, the surfaces of the carbon dots were functionalized 5 with -OH and -COOH groups, which allows other chemicals to be grafted to, and thus passivate the surface and improve the quantum yield- Example 2 2 g of glucose was dissolved in 5 mL of distilled water. Under violent stirring, 8 ml of concentrated sulphuric acid was added. The reaction was kept for 40 min, and the 10 mixture was neutralized by using NaOH (12 g of NaOH in 50 ml water). 50 mL of nitric acid solution (2 M) was added and the mixture was refluxed for 12 h. After cooling to room temperature, the solution was adjusted to pH 7 by using NaOH solution. Then the solution was extracted by using ethyl acetate for three times. Ethyl acetate was removed under vacuum to give crude products. In order to passivate the 15 nanoparticle surface, 1 g of 4,7,10-trioxa-1,13-tridecanediamine (TTDA) was added and the mixture was heated to 120 °C for 72 h under N2. Figure 4 gives the emission spectra before and after passivation. The quantum yield changed from 0.01 before to 0.12 after passivation. Figure 5 shows a photograph of nanoparticles in water after passivation under ambient light and UV lamp (365 nm).

Example 3 2 g of sucrose was dissolved in 5 mL of distilled water. Under violent stirring, 8 ml of concentrated sulphuric acid was added. The reaction was kept for 40 min, and 50 mL of MiliQ water was added, then the mixture was filtered and solid portion obtained was washed with MiliQ water. Figure 6 and Figure 7 give the IR and Raman spectrum of 25 obtained product. a) 0.45 g solid product obtained was reacted with TTDA at 120 °C for 72 h under N2. 28/07 2009 TUE lis 05 FAX +61 3 8618 4199 F.B.Rice & Co. 0025/067 Figure 8 gives the UV and emission spectra after passivation. The quantum yield was 0.092 after passivation. b) 50 mg solid product obtained was reacted with 290 mg poly(ethylene glycol) bis(3-aminopropyl) terminated at 120 °C for 72 h under N2.

Figure 9 gives the UV and emission spectra after passivation. The quantum yield was 0.05 after passivation with poly(ethylene glycol) bis(3-aminopropyI) terminated (PEGuoon). c) 0.45 g solid product obtained was reacted with ethylenediamine at 120 °C for 72 h under Nj.

Figure 10 gives the UV and emission spectra after passivation. The quantum yield was 0.032 after passivation.

Example 4 2 g of starch was added to 2 mL of distilled water. Under violent stirring, 12 ml of concentrated sulphuric acid was added. The reaction was kept for 40 min, and 50 mL of 15 MiliQ water was added, then the mixture was filtered and solid portion obtained was washed with MiliQ water. Figure 11 and Figure 12 give the ER. and Raman spectrum of obtained product. 0.914 g of product obtained was added to 50 mL of nitric acid solution (2 M) and the mixture was refluxed for 12 h. After cooling to room temperature, the solution was 20 adjusted to pH 7 by using NaOH solution. Then the solution was extracted by using ethyl acetate for three times. Ethyl acetate was removed under vacuum to give crude products. In order to passivate the nanoparticle surface, 2 g of 4,7,10-trioxa-l,13-tridecanediamine (TTDA) was added and the mixture was heated to 120 °C for 72 h under N2. Figure 13 gives the UV and emission spectra after reaction. The quantum 25 yield was 0.055 after passivation. 28/07 2009 TOE 11:05 FAX +61 3 8618 4199 F.B.Rice & Co. 0026/067 21 Example 5 Synthesis of carbon dots 2 g of carbohydrate (glucose, sucrose or starch depending on the experiment) was added to 5 mL of distilled water. Under vigorous stirring, 8 ml of concentrated 5 sulphuric acid was added. The reaction was allowed to proceed for 40 min, followed by the addition of 40 ml of water. The black carbon powder produced was collected by centrifuging, and was washed with water for three times. The resulting carbon powder was dispersed in 50 mL of nitric acid solution (2,0 M) and sonicated for 30 min. The mixture was then refluxed for 12 h. After cooling to room temperature, the solution was 10 neutralized by Na2C(>3 solution and most of the water was removed under vacuum. The CNPs obtained were dialyzed for one day using the Spectra/Por DispoDialyzer (a membrane with molecular weight cutoff of approximately 1000) to remove all salts.

In order to passivate the nanoparticle surface, 1 g of 4,7,10-trioxa-l,13-tridecanediamine (TTDA) was added to the CNPs and the mixture was heated to 120 °C 15 for 72 h under N2. Then the CNPs were again purified via dialysis (a membrane with molecular weight cutoff of approximately 1000) for two days.

Characterization by IR, Raman, XPS and XKD spectroscopies FT-IR and Raman spectroscopy were used to characterize the aggregated carbon particle obtained from the treatment of glucose, sucrose and starch carbohydrates with 20 concentrated sulfuric acid (see Figure 14). The FT-IR spectra clearly identify the carboxyl group, both through the very broad 3300 cm"10-H stretching absorption and the 1705 cm"1 C=0 stretching vibration. The Raman spectra of these samples feature the characteristic graphite bands - a D-band at 1301 cm"1 and G-band at 1598 cm'1 -but against a broader fluorescence background. XPS results (Figure 15) show the CNPs 25 contain carbon, oxygen and sulfur. Elemental analysis found the ratio of carbon, hydrogen and oxygen to be 2.8 :2,3 :1 (Table 1). 28/07 2009 TUE 11:05 FAX +61 3 8618 4199 F.B.Rice & Co. 0027/067 22 Table 1 The results of elemental analysis.

C(%) H (%) S(%) O (calculated, %) CNPs from glucose 59.88 4.77 0.67 34.68 CNPs from sucrose 65.60 4.54 <0.2 29.86 CNPs from starch 64.39 4.35 0.57 .69 Example 6 Carbon dots prepared from glucose After refluxing the CNPs prepared from glucose in a nitric acid solution for 12 h according to the technique set out in Example 5, the resulting solution exhibited a weak photoluminescence. In addition to the emergence of photoluminescence, the nitric acid treatment also blue shifted the maximum emission wavelength (see Figure 16). Bright-photoluminescence was observed after the CNP surface was further passivated by 10 treatment with 4,7,10-trioxa-l,13-tridecanediamine (TTDA) at 120 °C for 72 h under nitrogen (Figure 17).

Transmission electron microscopy (TEM) showed the carbon dots have a crystalline structure consisting of parallel crystal planes with a lattice spacing of 3.2 A (Figure 18) that is very close to the graphite 002 lattice spacing. This value agrees well with XRD 15 pattern of CNPs before passivation, which shows a diffraction peak centred at door* 3.4 A (see Figure 19). The diameter of these carbon dots, as estimated from the TEM images, is approximately 5 nm. The calculated quantum yield was 12 %. 28/07 2009 TUE 11:05 FAX +61 3 8618 4199 F.B.Rice & Co. 0028/067 23 The optical properties of these carbon dots synthesized via the aqueous solution pathway are shown in Figure 20 and 21. Their emission is highly dependent on excitation energy: as the excitation wavelength is increased, the emission peak position shifts to longer wavelengths and the intensity decreases. Figure 22 shows the emission 5 spectra of carbon dots passivated with PEG 1500N. These carbon dots show considerable photostability, with the emission intensity decreasing by only 17 % after 19 h of continuous excitation at 360 nm (see Figure 23). The pH has small effect on the PL intensity of carbon dots (Figure 24).

Example 7 Optical properties of CNP obtainedfrom starch Figure 25 shows the emission spectra of TTDA passivated carbon dots at different excitation wavelength which progressively increased from 3 50 nm, in 10 nm increments. CNPs were obtained using the technique set out in Example 5 by using starch as starting material and 12 h of nitric acid (2.0 M) treatment.

Example 8 Effect of passivating ligands on quantum yields of carbon dots Different passivating ligands (ethylenediamine, PEG1500, TTDA and oleylamine) were used to passivate the carbon dots which were prepared from glucose under the protocol described in Example 5. The emission spectra are show in Figure 26. Among 20 the ligands investigated, carbon dots passivated with TTDA shows the highest quantum yield (Figure 27).

Example 9 Cell imaging Carbon dots were prepared according to procedure the described in Example 1 with the 25 difference that the purification was carried out by means of dialysis as explained in the Experimental part The carbon dots were dissolved in PBS buffer solution. 50 juL of 28/07 2009 TUE 11:06 FAX +61 3 8618 4199 F.B.Rice & Co. 0029/067 24 the solution was added to an eppendorf tube containing mice heart ceils. The mixture was then incubated for 2 h at either the room temperature or for 1 h at 37 C°. The cells were then imaged with confocal microscopy. The results are shown in Figure 28 and 29.

Example 10 Carbon dots prepared from sugar 2 g of sugar was added to 5 mL of distilled water. Under vigorous stirring, 8 mL of concentrated sulphuric acid was added. The reaction was allowed to proceed for 40 min, followed by the addition of 40 mL water. The black carbon powder was collected 10 by centrifuging and was washed with water three times. In order to optimize the conditions, the resulting carbon powder was dispersed in different volumes of 2 M nitric acid solution and sonicated for 30 min. The mixture was then refluxed for 4 or 12 h. After cooling to the room temperature, the CNPs obtained were dialysed for one day to remove all salts.

In order to passivate the nanoparticle surface, TTDA was added to the CNPs and the mixture was heated to 120 °C for 72 h under Na, followed by removal of water. The CNPs were then again purified via dialysis for two days.

Figure 30 shows the SEM image of CNPs obtained by using sugar as the starting material and before the nitric acid treatment. Figure 31-33 show the absorbance and 20 emission spectra of carbon dots prepared from CNPs obtained by using 50 mL, 25 mL and 15 mL of nitric acid (2.0 M) treatment for 12 hrs and Figure 34 by using 15 mL of nitric acid (2.0 M) treatment for 4 hrs. Figure 35 provides a summary of quantum yield of carbon dots prepared using 50 mL, 25 mL and 15 mL of 2.0 M nitric acid and 12 hrs of the treatment; and using 15 mL of 2.0 M nitric acid and 4 hrs of the treatment.

As can be seen from the Figures, the conditions for CNP synthesis from sugar have been optimized in terms of the nitric acid volume needed in the reaction and the time of the treatment. For CNPs prepared from 2 g sugar a 15 ml of 2 M nitric acid and 12 hrs of treatment were found as the optimum (or alternatively 25 ml of 2 M nitric acid and 4 28/07 2009 TUE 11:06 FAX +61 3 8618 4199 F.B.Rice & Co. 0030/067 hrs of the treatment). The maximum quantum yield for CNP obtained from sugar is 7 ■%.

The optimization of the time for nitric acid treatment during the synthesis of CNP from glucose showed that the lower wavelength CNP emission peak (and therefore the 5 smaller particle size) can be obtained by the longer treatment time.

Example 11 Synthesis of biotinated capping ligand Scheme 1. Synthesis procedure for N-(13-Amino-4,7,10-trioxatridecanyl) biotinamide Biotin N-hydroxysuccinimide ester (1) 1,3-Dicyclohexylcarbodiimide (5.49 g, 48.9 mmol) was added to a solution of biotin, 10 g, 40.9 mmol) and N-hydroxysuccinimide (4.72 g, 40.9 mmol) in anhydrous DMF (250 mL) under stirring. The reaction was kept at room temperature for 48 h. The 15 formed dicyclohexylurea was filtered off and the solvent was evaporated under reduced pressure to dryness. 500 mL of diethyl ether were added to the residue and the solution was stirred for 2 h, giving a white precipitate. After filtration, the solid was recrystallized in isopropanol, yielding the pure product as a white powder (11.2 g). 28/07 2009 TUE 11:06 FAX +61 3 8618 4199 F.B.Rice & Co. @031/067 26 NMR (300 MHz, CDC13): 4.32 (1H, m), 4.16 (1H, m), 3.11 (1H, m), 2.90 (1H, m), 2.81 (5H, 5), 2.68 (2H, m), 1.80-1.35 (6H, m).

N-(l3-AmiriO"4,7,10-trioxatridecanyl) biotinamide (2, ATBA) Compound 1 (8 g, 23.4 mmol) was dissolved in 150 mL of dry DMF. Under inert 5 atmosphere, this solution was added dropwise to a solution of 4.7,10-trioxa-l,13-tridecanediamine (20.7 g, 99.6 mmol) in 8 mL of triethylamine within lh. After stirring the reaction mixture at room temperature for 72 h, the solid formed was filtered off and DMF was evaporated under vaccum. The resulting oil is added dropwise to 1 L of hexane. A white precipitate is formed after a few minutes and then was recrystallized in 10 isopropanol (2 h at reflux, 12 h without heating and stirring), yielding 5.2 g of the pure product as white crystals.

*H NMR (300 MHz, DMSO): 7.72 (lH,m), 6.40 (lH,s), 6.34 (1H), 4.32 (1H, m,), 4.14 (1H, m), 3.52-3.35 (12H, m), 3.13-3.03 (3H, m), 2.85-2.79 (2H, m, H2), 2.59 (2H, m), 2.04 (2H, m), 1.65-1.55 (8H, m), 1.35 (2H, m).

Preparation N-(l3-Amino-4,7,10-trioxatridecanyl) biotinamide (A TBA) passivated Carbon dots TTDA was added to the CNPs obtained from sugar and the mixture was heated to 120 °C for 72 h under N2, after removal of all water. The CNPs were then purified via dialysis for two days.

Absorbance (A) and emission (B) spectra of ATBA passivated carbon dots at different excitation wavelength which progressively increased from 360 nm, in 10 nm increments are shown in Figure 36. CNPs were obtained by using 50 mL of nitric acid (2.0 M,) for 12 h. Quantum yield measured: 0.045.

Figure 37 present the absorbance (A) and emission (B) spectra of ATBA passivated 25 carbon dots at different excitation wavelength which progressively increased from 360 nm, in 10 nm increments. CNPs were obtained by using 25 mL of nitric acid 2.0 M for 12 h treatment. Quantum yield: 0.0279 28/07 2009 TUE 11:06 FAX +61 3 8618 4199 F. B. Rice & Co. 0032/067 27 It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodimnts without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not 5 restrictive.

REFERENCES [1] Sun, Y.-P., Zhou, B.s Lin, Y., Wang, W., et al., Quantum-Sized Carbon Dots for Bright and Colorful Photoluminescence. J. Am. Chem. Soc. 2006,128,7756-7757. [2] US 2008/0113348 [3] Liu, H„ Ye, T., Mao, C., Fluorescent carbon nanoparticles derived from candle soot. Angew. Chem., Int. Ed. 2007,46, 6473-6475. [4] JACS VOL. 128, NO. 24,2006 7757 [5] SCIENCE, VOL 323,6,2009,1319

Claims (16)

Received at IPONZ on 12 Oct 2010 28 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of preparing luminescent carbon nanoparticles by reacting one or more carbohydrates with sulphuric acid and subsequently oxidising a product of the reaction. 5

2. A method according to claim 1 wherein the product of the reaction is oxidised by an oxidising agent such as an oxidising acid, KMn04, H2O2, KCr04, or K^C^O?.

3. A method according to claim 2 wherein the oxidising agent is an oxidising acid.

4. A method according to claim 3 wherein the oxidising acid is nitric acid.

5. A method according to any one of claims 1 to 4 wherein the carbohydrate is 10 selected from the group consisting of glucose, sugar (sucrose), and starch.

6. A method according to claim 5 wherein the carbohydrate is glucose.

7. A method according to any one of claims 1 to 6 further comprising the step of coupling the luminescent carbon nanoparticles to a passivation agent.

8. A luminescent carbon nanoparticle prepared by the method of any one of claims 15 1 to 7.

9. A luminescent carbon nanoparticle prepared from one or more carbohydrates according to the method of any one of claims 1 to 7.

10. A luminescent carbon nanoparticle prepared by the method of any one of claims 1 to 7 comprising sulphur. 20

11. A luminescent carbon nanoparticle prepared by the method of any one of claims 1 to 7 wherein the emission intensity of the carbon nanoparticle does not decrease by more than 20% after 20 hrs of continuous excitation at 360 nm.

12. A plurality of luminescent carbon nanoparticles prepared by the method of any one of claims 1 to 7 wherein the full width at half maximum of the peak at the 25 maximum emission wavelength of the carbon nanoparticles is less than 125 nm. 395501 1 PCT/NZ2009/0! Received 29

13. The use of the luminescent carbon nanoparticles of any one of claims 8 to 12 in diagnostic imaging, in biological markers or drug delivery systems

14. The use of luminescent carbon nanoparticles according to any one of claims 8 to 12 in the preparation of a medicament for use in diagnostic imaging of a patient, in 5 biological markers or drug delivery systems.

15. A method of preparing luminescent carbon nanoparticles by reacting one or more carbohydrates with sulphuric acid to form aggregates of luminescent carbon nanoparticles and subsequently breaking up the aggregates so that they disperse well in solution. 10

16. A luminescent carbon nanoparticle prepared by the method of claim 15. 15 Amended Sheet 350347 1 IPEA/AU