Production of Fluorescent Colloidal Nanoparticles

Abstract

comprising: providing a solid substrate; depositing a combination of vaporizable gold atoms and one or more organosulfur ligands on the surface of said substrate by physical vapour deposition (PVD) un

Description

Field of the Invention The invention concerns a hybrid physical-chemical method for producing fluorescent colloidal nanoparticles. The disclosed processes represent a simple, environmentally friendly method to produce size-controlled colloidal nanoparticles that absorb light in the ultraviolet region of the electromagnetic spectrum, and emit light in the visible region. Background of the Invention Any nanoscale material which is small enough to show discrete energy levels as a result of its size is conventionally referred to as a Quantum Dot. Quantum Dots may show fluorescence which can be exploited in a number of applications including biomedicine, photovoltaics, catalysis, light emitting diodes and photoconductors and photodetectors. A notable use of Quantum Dots is their use in LEDs, whose optical properties are exploited in screens and displays. [M. A. Cotta, ACS Appl. Nano Mater., 2020, 3, 4920-4924.] Quantum Dots have been produced from a variety of different materials including group IV and III—V compounds (e.g. InAs and GaAs), ll-VI and l-lll-VI compounds (e.g. CulnSa), and transition-metal dichalcogenides (e.g. CdSe and ZnS), perovskites, and carbon (based on graphene). [S. Kubendhiran, Z. Bao, K. Dave and R.-S. Liu, ACS Appl. Nano Mater., 2019, 2, 1773-1790.] Nanoparticles, also known as atomic clusters, are assemblies of atoms (or molecules) with a size of upto about 100 nm. The ability to exploit, and in some cases tailor, the unique chemical or physical properties of such nanoparticles has now enabled them to be used in a wide spectrum of applications, such as catalysis, medicine (both diagnostics and therapy) and photonics. Metal, in particular gold, nanoparticles, represent one particular subset of nanomaterials that has shown distinctive behaviour on the nanoscale, and this has contributed to progress in nanomedicine and nanotechnology. Specifically, 1 colloidal gold suspensions (in which a gold core is capped by an organic molecular, e.g. polymer, layer) are now commercially available, with the largest application area being biomedicine, with an estimated annual worldwide value of ~ $5 billion in 2022, which is expected to grow to ~$10 billion by 2027 with a compound annual growth rate in excess of 10%. Fluorescent gold nanoclusters have abundant potential uses in varying types of biological imaging due to their unique optical properties coupled with gold’s biocompatibility. [C. Zhang, X. Gao, W. Chen, M. He, Y. Yu, G. Gao and T. Sun, iScience, 2022, 25(10), 105022], Further, gold’s high atomic number and chemical reactivity on the nanoscale plus its biocompatibility, stability and efficient renal clearance means gold nanoclusters have also shown promise in other applications in biomedicine, including but not limited to antimicrobial applications, contrast agents and in radiation therapy. [M. F. Matus and H. Hakkinen, Small, 2021, 17, 2005499.] It is also known that metal, in particular gold, nanoclusters form discrete ordered sizes described by the number of metal atoms in the cluster. For gold, clusters with ‘magic’ numbers of atoms exist associated with closed electronic shell structures (structural or electronic), which possess a higher thermodynamic stability. Examples include bare Aun, where n could be 8, 18, 20, 34, 40, 58, 92, 138. [J. Wang, C. F. Mbah, T. Przybilla, B. Apeleo Zubiri, E. Spiecker, M. Engel and N. Vogel, Nat Commun, 2018, 9, 5259. T. Mori and T. Hegmann, J Nanopart Res, 2016, 18, 295.] Similarly (but for more complex reasons including effects of both electronic structure and geometrical / chemical factors), in the case of thiol-protected nanoclusters, arrangements of Au atoms are also observed with discrete known ratios of thiol ligands, denoted as Aux(SR)y. where SR is a thiol ligand and x and y denote the number of gold atoms and thiol molecules in the clusters. For thiolated gold nanoclusters varying x and y have been determined such as the most common and well-studied AU25SR18. It should be noted that the number of gold atoms in a more stable cluster is generally different for bare and protected gold nanoparticles. [X. Kang, H. Chong and M. Zhu, Nanoscale, 2018,10,10758-10834. P. Maity, S. Xie, M. Yamauchi and T. Tsukuda, Nanoscale, 2012, 4, 4027-4037.] The main conventional methods for the synthesis of colloidal gold nanoparticles, which enhances the commercial value of gold per gram by a factor of greater than 100 compared with gold bullion, can be divided into two types. The first type is based on the top-down processing of bulk gold under high-voltage electrical current or powerful irradiation (e.g., laser ablation) in a liquid, wherein the metal atoms are separated and then condense into nanoparticles in the liquid dispersion medium. However, such bulk gold processing methods tend to produce gold nanoparticles with rather non-uniform sizes, which can make the processes inefficient and / or the colloidal nanoparticles unsuitable for use in key market areas such as biomedicine as a result of, e.g., non-uniform optical properties. The second, more popular, type is based on the wet-chemical reduction of gold salts (e.g. halides), with subsequent centrifugation, and has been widely used for colloidal gold synthesis for a number of decades. Such wet-chemical reduction processes do offer a good degree of size control and, by extension, optical properties; greater power and concentration of reducing agent generally equates to smaller gold nanoparticles. However, the processes utilise harsh chemical reducing agents such as sodium borohydride and, typically, strong acids such as tetrachloroauric acid (HAuCk) in aqueous solution, which are reduced to produce approximately spherical, or non-spherical, gold nanoparticles. Such wet-chemical reduction processes, therefore, have the potential to result in the formation of impure nanoparticles that include some degree of contaminants (e.g. chlorine and / or boron from the residual salt and reducing agent, respectively), and may render the colloidal nanoparticles unsuitable for ideal use in key market areas such as biomedicine and cosmetics. There is, therefore, a hitherto unmet need for an efficient, environmentally friendly, route to producing high purity size-controlled fluorescent nanoparticle colloids. This unmet need is addressed by the unique hybrid physical-chemical process disclosed herein, which provides a simple, cost-effective route to prepare size controlled protected nanoparticles (wherein a vaporizable gold core is capped by a molecular shell-layer) in a contaminant free suspension that reduces solvent and reagent usage and thus environmental and health risks. Statements of Invention The present invention is as set out in the accompanying claims. The invention provides a method for preparing fluorescent colloidal nanoparticles, said method comprising: (i) providing a solid substrate; (ii) depositing a combination of vaporizable gold atoms and one or more organosulfur ligands on the surface of said substrate by physical vapour deposition under vacuum conditions to form a nanocomposite, film-coated substrate; (iii) removing said coated substrate from vacuum conditions; and (iv) washing said coated substrate in a liquid solvent under non vacuum conditions to dissolve the nanocomposite coating and form a suspension of fluorescent colloidal nanoparticles in said solvent. It has been surprisingly found that such a hybrid physical-chemical method represents a cost-effective route to prepare highly pure size-controlled nanoparticles which minimises / reduces, and preferably eliminates, usage of expensive and / or toxic solvents and reagents compared with conventional nanoparticle synthetic processes, thereby reducing environmental and health risks. Without being bound to any particular theory, it is believed that the organosulfur ligands bind to the gold atoms via strong Au-S interactions, providing a strong covalent interaction that results in small, size controlled stable nanoclusters. Moreover, it has been found that the method results in the formation of small (i.e. sub 5 nm), stable nanoclusters that fluoresce. As used herein the terms ‘fluorescent’ refers to particles that emit electromagnetic radiation in the visible region of the electromagnetic spectrum as a result of absorbing shorter wavelength electromagnetic radiation such as light in the X-ray or ultraviolet region of the electromagnetic spectrum. As used herein the term ‘organosulfur’ refers to organic compounds that contain at least one sulfur atom and a saturated or unsaturated and substituted or unsubstituted hydrocarbon. As used herein, the term ‘nanoparticles’ refers to particles that are between about 1 to about 100 nm in diameter, and comprise clusters or assemblies of vaporizable atoms or molecules. In preferred embodiments, such nanoparticles comprise or consist of clusters or assemblies of one or more metal atoms or metal containing molecules. In alternative embodiments, such nanoparticles may comprise or consist of a clusters or assemblies of one or more atoms or molecules which show non-metallic semiconductor behaviour (e.g. silicon) or insulating behaviour (as in glass) in the corresponding bulk materials. Similarly, the term ‘colloidal nanoparticles’ refers, in the context of the present specification, to a stabilized solution of core-shell nanoparticles, i.e. a vaporizable gold particle core disposed within a capping layer formed from an organosulfur organic or inorganic ligand structure. As used herein, the term ‘vaporizable atoms’ refers to any atom or particle that can be vaporized and deposited onto a surface by physical vapor deposition under vacuum conditions. In the present invention, the vaporizable atoms comprise or consist of gold atoms. As would be readily appreciated by the skilled reader, the term ‘vacuum conditions’ refer in the context of the present specification to a closed environment having gas pressure of about 10-1 Pa or below, preferably about 10-3 Pa or below, and more preferably about 10-4 Pa or below. Conversely, in the context of the present specification, ‘non-vacuum conditions’ refers to an open or closed environment having a gas pressure of greater than about 102 Pa, preferably greater than about 103 Pa, and more preferably greater than about than about 104 Pa. In preferred embodiments, ‘non-vacuum conditions’ refer to a standard atmospheric pressure environment (at sea level, this equates to a pressure of about 101 kPa). As the skilled person will readily appreciate, capping agents or ligands, which in the methods of the present invention are organosulfur compounds, are 5 utilised in colloidal nanoparticle syntheses to stabilize the interface where nanoparticles interact with the preparation medium and control aggregation / prevent coagulation during colloidal synthesis and subsequent storage / use. Further, these stabilizing agents can play a key role in altering the physicochemical, biological and / or optical characteristics of the formed nanoparticles, with the steric and chemical bonding (e.g. charge exchange, atomic restructuring) effects of such capping ligands being responsible for modifying said characteristics. In particular, as noted above and without being bound to any particular theory, it is believed that organosulfur ligands, in particular thiol containing organosulfur compounds, bind to gold atoms in the methods of the present invention via strong Au-S interactions, providing a strong covalent interaction that results in small, i.e. sub 5 nm, size-controlled stable nanoclusters that are free from contamination and have been shown to fluoresce. Therefore, in its broadest aspect, the method of the invention is not limited to the deposition of any particular organosulfur capping ligand, and may be selected by the skilled reader based on need and / or availability without difficulty. However, in preferred embodiments, the deposition process of step (ii) comprises depositing one or more organosulfur ligands selected from thiols (i.e. compounds of formula R-S-H), thiosulfinates (i.e. compounds of formula R-S-(O)-S-R), thioethers (i.e. compounds of formula R-S-R), disulfides (i.e. compounds of formula R-S-S-R), sulfonic acids (i.e. compounds of formula R-S(O)(O)-OH), and any combination thereof, where R denotes a saturated or unsaturated hydrocarbon. In particularly preferred embodiments, the one or more organosulfur ligands comprise or consist of one or more thiol containing compounds. As will also be appreciated, the size of the organosulfur ligand can affect the final size, and so the optical properties, of the resultant nanocluster. Therefore, in preferred embodiments, the one or more organosulfur ligands have a molecular mass of at least about 45 Da, more preferably at least about 75 Da, and still more preferably at least about 100 Da. Similarly, in preferred embodiments, the one or more organosulfur ligands have a molecular mass of no more than about 300 Da, more preferably no more than about 200 Da, and still more preferably no more than about 150 Da. In certain preferred embodiments, the one or more organosulfur ligands is an amino acid, and more preferably is a L-amino acid such as L-cysteine or L-methionine. In particularly preferred embodiments, the organosulfur ligand is cysteine, and most preferably is L-cysteine. The use of such biological ligands results in the formation of biospecific fluorescent nanoclusters that are free from contamination. The solid substrate on which the nanocomposite is formed is also not particularly limited and includes, but is not limited to, silicon or carbon-based materials (such as graphene, carbon nanotubes, fullerenes and amorphous-Carbon), an oxide, a nitride, a glass, or a MXene. However, in some embodiment, the substrate is a silicon material, which may optionally be doped with one or more heteroatoms (e.g., boron or phosphorus) which regulate the electrical and thermal properties. The presence of dopants or defects at the surface of the substrate may be advantageous in controlling surface diffusion (and so, by extension, nanoparticle size) of the deposited atoms on the substrate. The extent of film thickness of the deposited nanocomposite on the surface of the substrate can be measured or calculated by a variety of methods. For example, film thickness is typically calculated by layer deposition rate and deposition time, and / or measured with spectroscopic ellipsometry. Preferably, said nanocomposite film thickness varies from a single monolayer to about 100 pm, more preferably from about 5 nm to about 50 pm, and most preferably from about 50 nm to 10 pm, on the substrate. Additionally or alternatively, the weight ratio of deposited vaporizable gold atoms to organosulfur capping ligand within the nanocomposite layer, which can be controlled and calculated by the values from quartz crystal microbalance (QCM) measurements, preferably ranges from about 10:90 to about 99:1, and more preferably from about 20:80 to about 95:5. As already noted, the method of the invention comprises depositing a combination of vaporizable gold atoms and organosulfur capping ligands on the surface of the substrate by a physical vapour deposition (PVD) process such as evaporation, sputtering, pulsed laser deposition or pulsed injection 7 deposition under vacuum conditions. As would be readily apparent to a person of ordinary skill in the art, the vaporizable gold atoms and organosulfur ligands may be deposited via any conventional PVD method. However, such processes exclude cluster deposition processes, wherein atom clusters are formed (e.g., via condensation in the gas phase) prior to deposition onto the surface of the substrate. Instead, said PVD techniques comprise an atom deposition process, wherein individual atoms or molecules are deposited, and then diffuse and aggregate into nanoparticles, on the substrate surface or within, or following dissolution of, the nanocomposite hybrid film. As will be readily appreciated, the term ‘pulsed injection deposition’ refers to a process in which a solution comprising molecules to be deposited are transferred into a vacuum deposition chamber, where the solvent evaporates and the molecules are subsequently deposited in the vapour phase onto a substrate surface. The use of such a deposition method thus avoids the need to couple thermal energy to sublimate the material prior to deposition, and so is particularly suitable for the deposition of fragile molecules, such as the organosulfur capping ligands used in the present invention, without damage resulting from excitation of rotation / vibration modes. In preferred embodiments, the gold atoms and / or organosulfur ligands are independently deposited on the surface of the substrate by evaporation deposition or by pulsed injection deposition. In particularly preferred embodiments, the gold atoms are deposited on the surface of the substrate by evaporation deposition, and the organosulfur ligands are deposited on the surface of the substrate by pulsed injection deposition. Advantageously, by utilising pulse injection deposition, the deposition rate can be tightly controlled, and high deposition rates achieved. For example, by utilising pulse injection deposition, the deposition rate of organosulfur ligand can be increased by a factor of about 40 or more compared with the use of evaporation deposition. The PVD deposition of vaporizable gold atoms and organo-sulfur capping ligands in step (ii) of the method of the invention can be carried out in separate independent deposition steps, or both the gold atoms and capping ligands may be co-deposited in a single deposition step. In either of said processes, the substrate is coated with one or more layers, wherein each of said layers preferably comprises or consists of said vaporizable gold atoms and / or said organosulfur ligands. Therefore, in said embodiments, the substrate preferably does not comprise any solvent (e.g. water) soluble layer(s) such as an intermediate salt layer, and preferably does not contain any other layers in addition to the PVD deposited vaporizable gold atom and / or organosulfur capping ligand layer(s). Therefore, in some embodiments, deposition step (ii) comprises or consists of the simultaneous co-deposition of vaporizable gold atoms and organosulfur capping ligands on the surface of the substrate by physical vapour deposition under vacuum conditions to form a nanocomposite film of nanoparticles embedded in a continuous phase of ligand material. This co-deposition step may be repeated one or more times to build film thickness as required. In alternative embodiments, the deposition step (ii) comprises or consists of the sequential deposition of an organosulfur ligand layer followed by a vaporizable gold atom layer onto the surface of the substrate by physical vapour deposition under vacuum conditions to form a nanocomposite laminate film comprising a layer of gold nanoparticles captured on a layer, or between two layers, of organosulfur ligand material. However, said gold atoms may also penetrate into the ligand layer(s) and form nanoparticles therein. As will be readily appreciated, these sequential deposition steps may be repeated one or more times to form two or more layers of organosulfur ligand material and, optionally, two or more layers of gold nanoparticles. Further in such sequential deposition embodiments, the nanocomposite laminate film preferably forms a sandwich structure in which n layers of gold nanoparticles are deposited between n+1 layers of organosulfur ligand material. As already noted, following deposition of the gold atoms and organosulfur ligands onto the solid substrate, individual gold atoms diffuse and aggregate into nanoparticles, thereby forming the nanocomposite coated substrate on the substrate surface. The nanocomposite coated substrate is then removed from vacuum conditions and, in step (iv), washed in a liquid solvent under non- vacuum conditions to dissolve the nanocomposite coating, thereby forming a suspension of colloidal gold nanoparticles in said solvent. As will be readily appreciated, the choice of solvent used in wash / dissolution step (iv) is not particularly limited, provided the chosen solvent is compatible with the organosulfur ligand component of the nanocomposite coating. For example, in preferred embodiments the solvent is water (for environmental and biocompatibility reasons). Wash step (iv) may immediately follow (e.g. within 1 day) the removal of the nanocomposite from vacuum conditions. However, it will also be readily appreciated that said wash step may be delayed for a period of time (e.g. from 2 days to 6 months or more), thereby permitting storage and / or transport of the nanocomposite coated substrate in a preferred solid form, before forming the colloidal nanoparticles at a later time point and / or location as desired. The specific method by which the coated substrate is washed in dissolution step (iv) is not limited, provided that the substrate is brought into contact with the solvent to dissolve the nanocomposite coating, thereby forming a suspension of colloidal gold nanoparticles in said solvent. Therefore, in some embodiments, the coated substrate is washed by exposure to a flow of solvent, e.g., by spraying. However, in preferred embodiments, the coated substrate is washed by immersion of the coated substrate in a fixed volume of solvent, thereby forming a high concentration of colloidal nanoparticles in said solvent. In particularly preferred embodiments the coated substrate is washed by immersion in a fixed volume of solvent and subjected to sonication (i.e. the application of ultrasonic (> 20 kHz) sound energy) to agitate the substrate and enhance the release of the fluorescent nanoparticles into the solvent. In an alternative, also preferred, embodiment dissolution step (iv) comprises contacting the surface of said coated substrate with an absorbent pad or swab to at least partially, and preferably fully, transfer the nanocomposite film to said absorbent pad or swab, and then submerging said pad or swab in a fixed volume of said solvent thereby forming a suspension of said nanoparticles. Such a dissolution process enables the production of a highly concentrated suspension of fluorescent colloidal nanoparticles in said solvent. In preferred 10 embodiments the suspension of nanoparticles is subjected to sonication to ensure full dissolution of the nanoparticles in the solvent. The skilled reader will also appreciate that the method of the invention may advantageously comprise one or more additional process steps. However, in preferred embodiments, if any additional processing step(s) are included, said steps do not include the deposition of one or more additional solvent (e.g. water) soluble layer(s), and more preferably does not include the deposition of any other layer(s) to the nanocomposite film coated substrate formed in step (ii). In one such preferred example, the nanocomposite coated substrate is subjected to a processing step (ii-a), after step (ii) but prior to step (iii), to modify particle size and density on the substrate surface. Suitable processing methods include, but are not limited to, heating, ion-beam irradiation, laser irradiation, and electron beam irradiation. Where ion-beam or electron beam irradiation is employed, the substrate upon which the nanocomposite is formed is preferably doped with one or more dopant materials to provide a charge conducting substrate or enhance the conducting property of an inherently conducting substrate. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises” mean “including but not limited to” and do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Throughout the description and claims of this specification, the term “about” includes a variation of ± 5%, preferably ± 4%, more preferably ± 3%, still more preferably ± 2% and most preferably ± 1% unless the context otherwise requires. Any references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art. Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose. The Invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein: Figure 1. Schematic illustration of the apparatus and method for preparing fluorescent colloidal gold nanoparticles; Figure 2. Image of the vacuum system used for the method described here. (A) Main Chamber; (B) Load Lock; (C); Sample Stage; (D) Manipulator arm; (E) Gate Valve; (F) QCM; (G) High Temperature (gold) evaporator; (H) HTC Evaporator Controller; (I) OLED (ligand) Evaporator; (J) OLED Evaporator Controller; (K) Pulse Valve; (L) Pulse Valve Controller; Figure 3.Schematic representation of the pulse valve system used for vacuum spray deposition of L-cysteine; Figure 4. Colloidal gold nanoparticles formed by Co-evaporation of gold and L-cysteine. (A) Representative STEM image showing Au clusters; (B) size distribution determined from STEM image; (C) UV-Visible and photoluminescence spectra; (D) photoluminescence emission was measured at an excitation wavelength of 350 nm. The pink emission observed in PL when excited at 350 nm (D), the image shows the cuvette filled with sample within the PL instrument with the 350 nm light directed at the circular window on the left, and the coloured emission from this excitation was observed; Figure 5. Colloidal gold nanoparticles formed by Co-deposition of gold (Evaporation) and L-cysteine (Vacuum Spray). (A) UV-Visible and photoluminescence spectra; (B) photoluminescence emission was measured at an excitation wavelength of 360 nm. The visible red emission observed in PL when excited at 360 nm, the image shows the cuvette filled with the sample within the PL instrument with the 360 nm light directed at the circular window and the coloured emission at this excitation was observed. MATERIALS AND METHODS The production of fluorescent colloidal gold, i.e. a suspension of fluorescent gold nanoparticles capped with an organosulfur stabilising ligand such as L-cysteine in a solvent, was explored by a two-step process as illustrated in Figure 1. In particular, in a first (deposition) step, a nanocomposite matrix has been produced by physical vapour deposition of both gold atoms and organosulfur molecules onto a solid support substrate under vacuum conditions. In a specific example of this approach, a silicon wafer, used as the support, was mounted onto a stage at the end of a manipulator arm inside the load lock of the vacuum system shown in Figure 2. Once the load lock chamber had been evacuated to a vacuum in the order of 10-5 to 10-8 mbar, a gate valve was opened, and the wafer transferred into the main chamber for deposition. To produce the nanocomposite film the capping ligand (L-cysteine) was first deposited either by thermal evaporation of solid L-cysteine or by pulsed injection spraying into the vacuum chamber of an aqueous solution of L-cysteine (via a pulsed valve). This step was subsequently followed by the simultaneous deposition of both gold atoms and L-cysteine as a layer onto the support, either by metal and ligand evaporation (co-evaporation) or by metal evaporation and ligand pulse valve spray (co-deposition), or by sequential deposition of layers of the two materials. Deposition rates of ligand and gold were determined inside the vacuum chamber using a Quartz Crystal Monitor 13 (QCM) fitted with a 6 MHz gold coated crystal, the deposition rates were measured in A / s. For accurate deposition rates the sample stage was removed from the main chamber and the QCM inserted into the centre of the chamber to ensure rates were comparable to those deposited on the centre of the sample. Detailed descriptions of each method step are given below. Thermal evaporation of L-cysteine was conducted using an OLED Effusion Cell, from Createc, and the evaporator was fitted with a quartz crucible which was controlled by a PID control box. For evaporation experiments the crucible was filled with around 6 g of L-cysteine and placed into the evaporator when the system was at atmospheric conditions. The system was then pumped down to between 10-5 to 10-8 mbar and, once under sufficient vacuum, L-cysteine was heated slowly (0.1 °C / s) to 110 °C and left to de-gas for approximately 24 hours. De-gassing of the ligand was only required when the main chamber had been vented and opened to atmosphere. For thermal evaporation of L-cysteine the evaporator was set to 125 °C, once the evaporator had reached temperature the deposition rate was first measured using QCM. For vacuum spray deposition of L-cysteine, a solution of 1 wt.% L-cysteine in deionised water was produced and placed into a Swagelok 125 cm3 cylinder which was attached, via a two-way ball valve, to a pulsed valve. The pulsed valve (Parker Series 9 High-Speed Pulsed Solenoid Valve, 0.031 in. Orifice, 28 V, Stainless Steel Body) was attached to the main chamber via a custom DN40 flange containing a 3 mm hole for the liquid to enter the vacuum chamber. The valve was controlled via a custom control box fitted with an Arduino controller, the open and closed times of the pulse valve could be changed to control the vacuum spray deposition rate. Prior to vacuum spray deposition the pulsed valve set up (shown schematically in Figure 3) was purged with Argon, the two-way valve connecting the sample reservoir to the valve was then opened, and the control box used to commence deposition. QCM measurements were also used to determine the deposition rate of L-cysteine; however due to the water also landing on the crystal accurate rate measurements were challenging to determine. Once the deposition rate of L-cysteine, either by evaporation or vacuum spray had been determined, the sample stage loaded with the silicon wafer was moved in and orientated to face the thermal evaporators and pulsed valve. L-cysteine deposition commenced when either the evaporator shutter lid was opened, or the pulsed valve electrical control turned on. Gold evaporation was conducted at varying evaporator temperatures, with deposition rate being exponentially related to the temperature. Gold was placed into a tungsten crucible which was placed inside the high temperature evaporator (High Temperature Cell, HTC, Createc). The temperature was set and controlled via a PID control box. Once the gold evaporator was at temperature, with the sample stage removed, the deposition rate was measured using the QCM by opening the shutter lid of the evaporator. Gold deposition along with ligand deposition was then conducted once the sample stage was in the centre of the main chamber with the stage, and silicon wafer, facing the bottom of the chamber. If necessary, the processes of metal and ligand evaporation and deposition can be repeated multiple times (in any sequence) until a desired thickness and amount of the hybrid film of gold and organosulfur ligand molecules is obtained. For a layer-by-layer structure, a L-cysteine layer of ~ 5 nm thickness can be used in the subsequent layers. In these proof-of-concept experiments, the nanoparticle matrix architecture consisted of “one layer of L-cysteine alone + one layer of gold and L-cysteine deposited simultaneously.” Upon completion of the deposition step, the sample stage was rotated and removed from the main chamber and into the load lock. The gate valve could then be closed, and the load lock vented which enabled the vacuum in the main chamber to be preserved. Once the load lock was vented to atmospheric pressure the sample stage could be removed. The wafer supporting the nanocomposite film was then transferred into deionised water (3 mL), this is then sonicated in a water bath, for typically 60 minutes, to form a suspension of colloidal capped gold nanoclusters in water. Alternatively, to ensure complete removal of the nanocomposite film, the surface of the wafer could be swabbed using an absorbent pad or swab such as, e.g., a cotton bud. The absorbent pad or swab can then be placed into water to remove the sample and produce a concentrated colloidal suspension. The suspension can then be sonicated to ensure full dissolution of the sample in the solvent. Sonication was carried out with a Bransonic CPX2800-E Ultrasonic bath, 110W, 40kHz, operating in high power Sonics mode. For TEM imaging of the colloidal particles, solutions were drop cast onto TEM grids. 3 pL of colloid was placed onto a Carbon Film supported on a Cu TEM support grid and left to dry at room temperature. STEM images were taken using an FEI TALOS F200X TEM using a STEM High Angle Annular Dark Field (HAADF) detector at 200 kV acceleration voltage. UV-Visible spectra were measured on a Jenway 7135 Spectrophotometer fitted with a Xenon lamp light source. Spectra were collected between wavelengths of 300 to 800 nm at 1 nm intervals. Baseline spectra were determined prior to analysis using deionised water. UV-Visible measurements were taken of the colloids as produced after sonication, unless stated otherwise. The optical characteristics of the colloids were further assessed by measuring Photoluminescence (PL) using an FS5 Spectrofluorometer (Edinburgh Instruments) fitted with a 150 W CW Ozone-free Xenon arc lamp. Emission scan spectra were collected from 420 to 800 nm with a step of 2 nm and a dwell time of 0.1s. All PL spectra were run at both 350 and 360 nm excitation wavelengths. All PL measurements were taken of the colloids as produced after sonication, unless stated otherwise. Example 1: Formation of Colloidal Gold Nanoparticles by Co-Evaporation of Gold and L-Cysteine A structure comprised of “one layer of L-Cysteine (6 nm) plus one layer of gold and also L-Cysteine (60 min deposition)” was built on the surface of the silicon wafer by two phases of direct evaporation; L-cysteine was evaporated at 125 °C for 60 minutes (0.016 A / s) to give an initial layer of approximately 6 nm; this was followed by co-evaporation of Au and L-cysteine at 1100°C (0.018 A / s) and 125 °C (0.016 A / s) respectively for 60 minutes. Figure 4 displays the results of such a deposition sequence which resulted in colloidal gold formation. Notably, Figure 4 (A) shows a Scanning Transmission Electron Microscope (STEM) image of colloidal gold nanoclusters which were generated by dissolution by sonication in water of the coated wafer, and the colloid was subsequently deposited and dried onto a TEM grid for imaging and size analysis. The STEM image shows an array of clusters in a cloud like film, with average particle diameter of 3.07 ± 0.53 nm. Figure 4 also shows the UV-Visible absorbance spectrum and photoluminescence emission under UV excitation (350 nm). This colloid shows a peak absorption wavelength of 359 nm in the UV-Visible spectrum and a peak emission wavelength of 650 nm when excited at 350nm. Notably, PL emission spectra for this sample were identical when excited at both 350 and 360 nm, hence only one spectrum is shown in Figure 4. Example 2: Formation of Colloidal Gold Nanoparticles by Co-Deposition of Gold (Evaporation) and L-cysteine (Vacuum Spray) In an alternative method to that disclosed in Example 1, colloidal gold nanoclusters were prepared using a combination of evaporation and vacuum spray methods to deposit a layer of gold atoms and ligands respectively, upon the surface of the solid support (the silicon wafer) mounted on the stage of the manipulator arm. Here a solution of L-Cysteine in deionised water was gravity fed into a high-speed pulsed valve mounted directly onto a flange of the main vacuum chamber. The solution of L-cysteine was pulsed into the main chamber at various frequencies. A structure comprised of “one layer of L-Cysteine (20 minutes pulsed deposition) plus one layer of gold co-deposited with L-Cysteine (300 minutes)” was produced. Figure 5 displays the results of such a deposition and the effect on colloidal gold formation. L-cysteine was first vacuum spray deposited for 20 minutes (pulsed valve open time: 5ms, closed time: 10 s) followed by co-deposition of L-cysteine spray (pulsed valve open time: 5ms, closed time: 10 s) and gold evaporation (1100°C, 0.018 A / s) for 300 minutes. The pulsed valve spray deposition has increased the deposition rate of ligand by a factor of about 40 compared with evaporation. With the use of the pulsed valve the deposition rate of the ligand can be increased further by changing the open and closed frequencies and by altering the concentration of the liquid solution used, thus providing the possibility of increasing the ligand:Au ratio compared to that achievable by a coevaporation process as disclosed in Example 1. Figure 5 displays the results of such a deposition sequence which resulted in colloidal gold formation. Notably, Figure 5 (A) shows the UV-Visible absorbance spectrum and photoluminescence emission under UV excitation (360 nm). Figure 5 also shows a photo of the red visible emission from the sample when excited at 360 nm. This colloid has a peak absorption wavelength of 362 nm in the UV-Visible spectrum and a peak emission wavelength of 638 nm when using an excitation wavelength of 360 nm. PL emissions at excitation wavelengths of 350 and 360 nm showed identical spectra hence only one is shown in Figure 5. The clusters produced had a sub 5 nm average particle diameter. Using the pulsed valve to increase the ligand deposition rates has resulted in longer possible Au deposition times. Notably, Example 2 increased the gold loading 5 times compared to Example 1. These examples show that vacuum deposition methods can be used to produce gold nanoclusters of less than 5 nm which emit visible light when excited in the UV range. Further, it is envisaged that many experimental parameters can be controlled further to produce more uniform cluster size distributions and therefore produce sharper optical spectra. SUMMARY Proof of principle experiments have successfully demonstrated a novel, simple, environmentally friendly and cost-effective route to produce fluorescent colloidal gold nanoclusters (quantum dots) capped with sulfur-containing ligands. The method employs evaporation of metal atoms plus deposition of organosulfur, such as in particular thiol containing ligands including L-Cysteine, by either evaporation (co-evaporation) or by pulsed injection deposition, i.e. vacuum spray via a pulsed valve (co-deposition), onto a supporting wafer under vacuum. 5 In future, the fluorescent properties of these colloids might be tuned (colour selected) and enhanced by filtering the size of the colloidal particles into different fractions (fractionation) and also by further studies of the organosulfur ligand types and / or the ligand to gold ratio and deposition rates. Conventionally, fluorescent gold nanoclusters have found application I uses as 10 biosensors and biomarkers in Oncology and have multiple other potential technologies, e.g. in displays, and are typically produced by chemical methods using metal salts such as chloroauric acid and reducing agents such as sodium borohyride. However, the present invention provides for the first time a process to produce fluorescent, organosulfur ligand capped gold nanoclusters without 15 salts and reducing agents and thus with much reduced effluent issues and enhanced purity during manufacture.

Claims

1) A method for preparing fluorescent colloidal nanoparticles, said method comprising:i. providing a solid substrate;ii. depositing a combination of vaporizable gold atoms and one or more organosulfur ligands on the surface of said substrate by physical vapour deposition under vacuum conditions to form a nanocomposite film coated substrate;iii. removing said coated substrate from vacuum conditions; andiv. washing said coated substrate in a liquid solvent under non vacuum conditions to dissolve the nanocomposite coating and form a suspension of fluorescent colloidal nanoparticles in said solvent.2) The method of claim 1, wherein step (ii) comprises depositing one or more organosulfur ligands selected from thiols, thiosulfinates, thioethers, disulfides, sulfonic acids, and any combination thereof.3) The method of claim 2, wherein said one or more organosulfur ligands comprise or consist of one or more thiol containing compounds.4) The method according to any of the preceding claims, wherein said one or more organosulfur ligands have a molecular mass of at least about 45 Da and / or no more than about 300 Da.5) The method according to any of the preceding claims, wherein said one or more organosulfur ligands is an amino acid, and preferably is a L-amino acid.6) The method according to claim 5, wherein said amino acid is cysteine.7) The method according to any one of the preceding claims, wherein said substrate is selected from silicon; a carbon-based material such as graphene, carbon nanotubes, fullerenes or amorphous-carbon; an oxide, a nitride, a glass, or a MXene.8) The method according to claim 7, wherein said substrate is a silicon material, optionally doped with one or more heteroatoms (e.g. boron or phosphorus).9) The method according to any one of the preceding claims, wherein said nanocomposite film has a thickness of from about 5 nm to about 50 pm.10)The method according to any one of the preceding claims, wherein the weight ratio of said vaporizable gold atoms to organosulfur ligand within the nanocomposite layer ranges from about 10:90 to about 99:1.11 )The method according to any one of the preceding claims, wherein said vaporizable gold atoms and / or said organosulfur ligands are independently deposited on the surface of the substrate by evaporation deposition or by pulsed injection deposition.12)The method according to claim 11, wherein said vaporizable gold atoms are deposited on the surface of the substrate by evaporation deposition, and said organosulfur ligands are deposited on the surface of the substrate by pulsed injection deposition.13)The method according to any of the preceding claims, wherein said substrate is coated with one or more layers, wherein each of said layers comprises or consists of said vaporizable gold atoms and / or said organosulfur ligands.14)The method according to any one of the preceding claims, wherein step (ii) comprises or consists of the simultaneous co-deposition of said vaporizable gold atoms and said organosulfur ligands on the surface of the substrate by physical vapour deposition under vacuum conditions to form a nanocomposite film of nanoparticles embedded in a continuous phase of ligand material.15)The method according to any one of claims 1 to 14, wherein step (ii) comprises or consists of the sequential deposition of an organosulfur ligand layer followed by a vaporizable gold atom layer onto the surface of the substrate by physical vapour deposition under vacuum conditions to form a nanocomposite laminate film comprising a layer of gold nanoparticles captured on a layer, or between two layers, of organosulfur ligand material.16)The method according to claim 15, wherein said nanocomposite laminate film forms a sandwich structure in which n layers of gold nanoparticles are deposited between n+1 layers of organosulfur ligand material.17)The method according any one of the preceding claims, wherein said liquid solvent is water.18)The method according to any one of the preceding claims, wherein step (iv) comprises immersion of the coated substrate in a fixed volume of said liquid solvent.19)The method according to claim 18, wherein the coated substrate is washed by immersion in a fixed volume of solvent and subjected to sonication.20)The method according to any one of claims 1 to 17, wherein step (iv) comprises contacting the surface of said coated substrate with an absorbent pad or swab to at least partially transfer the nanocomposite film, and then submerging said absorbent pad or swab in a fixed volume of said solvent to form the suspension of said nanoparticles.21)The method according to claim 20, wherein the suspension of nanoparticles is subjected to sonication.22)The method according to any one of the preceding claims, wherein said nanocomposite coated substrate is subjected to a processing step (ii-a), after step (ii) but prior to step (iii).23)The method according to claim 20, wherein said processing step does not include the deposition of one or more additional solvent soluble layer(s), to the nanocomposite film formed in step (ii).24)The method according to claim 20 or claim 21, wherein step (ii-a) comprises heating, ion-beam irradiation, laser irradiation, and / or electron beam irradiation.

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