The present invention relates to processes for producing gold nanoclusters. More specifically the processes enable very precise and discrete patterning of fluorescent features in polymer films and products thereof. Especially interesting is the process for production of fluorescent gold nanoclusters and applications thereof, such as anti-counterfeiting.

BACKGROUND OF THE INVENTION

Fluorescence is a widely used technique to tackle counterfeiting. For example, a genuine banknote reveals fluorescent patterns under UV light and, in turn, prevents tampering and counterfeiting. Ink-based fluorescent marks can be printed using rubber stamps or inkjet printers. For some applications, where higher level of security or identification of a single product is required, it would be beneficial to print information as unique, or more specifically serialized, fluorescent security codes and labels, such as barcodes or QR codes. To this end, rubber stamps are not useful as making unique rubber stamps for each item is not practical. On the other hand, inkjet printers perform serial point-by-point scanning and printing which is nevertheless time-consuming, hindering the potential of fluorescent patterning for applications where speed and scalability are crucial.

As such, metal nanoclusters present a specific subgroup within nanoparticles (NPs) and provide interesting chemical and optical features, including strong absorption and intense luminescence. A variety of applications have been proposed for nanoclusters from biological labeling, to sensing and imaging. Synthesis and stabilization of metal nanoclusters are major issues.

A variety of noble metal nanoparticles or nanoclusters have been prepared either as a“top- down” or as a“bottom-up” approach. In the former, large particles are etched to form smaller nanoclusters. The bottom-up approach comprises various synthetic methods, including thermal reduction, microwave heating, photoreduction, ligand-induced etching, sonochemistry and template-assisted synthesis. Fluorescent noble metal nanoclusters are usually formed in the presence of the correct stabilizing templates or ligands and have a ligand shell-metal core structure. In addition to the metal core, the ligand shell typically consists of proteins, DNA, polymers, dendrimers, thiolates or other small molecules. In addition to the ultra-small size, the ligand shell of noble metal nanoclusters and the surface of the core could significantly contribute to their chemical and physical properties and influence further applications of fluorescent noble metal nanoclusters.

Among all types of metal nanoclusters, gold is of greatest importance since it is less toxic, more biocompatible, and has relatively bio-inert surfaces and extraordinary stability. In addition, the modification of the chemistry of gold surface is more straightforward and there is more control over the size and shape of gold nanoclusters. Consequently, gold nanoclusters with broad spectrum provide a strong platform for many applications.

As an example of chemical method, nanocrystalline superlattices in gold colloidal solutions have been prepared by ligand-induction using AuC reduced with sodium borohydride. However, these structures are larger than nanoclusters and unable to provide desired characteristics.

Gold nanomaterials have also been synthesized using microorganisms, such as by bio reduction of chloroauric acid (HAuCU) using the fungal culture filtrate of Alternaria alternate with a protein shell outside the nanoparticles for stabilization. Another study evaluated the accumulation of gold across taxonomically diverse plant species (alfalfa, cucumber, red clover, ryegrass, sunflower, and oregano). Plants accumulated gold in their roots and plants forming gold nanoparticles within 6 h of treatment. Even though the geometries of in planta synthesized gold nanoparticles was interesting, their size range fell outside present interest, thus nanoclusters.

Direct laser writing has been used to efficiently form and stabilize silver nanoclusters in solid- state matrices, such as glass, zeolite, and polymer, enabling compelling applications such as optical data storage and micro-labeling. Fluorescent silver nanoclusters have been reported in polymer matrices. As a drawback, formation of silver nanoclusters requires high intensity, typically pulsed laser. As a side effect, such intensities generate heat and stress to the polymer matrix. Even though the silver nanoclusters can successfully be produced, visible detrimental effect on the polymer film limits the use of such material.

In an attempt to study the photochemical reactivity of metal clusters Sakamoto et al. have described a process to prepare gold nanoclusters within PVAc film where a mixture of a radical precursor and gold chloride with ratio of 5:1 mole/mole have been dissolved in a PVAc solution. Activation of said radical precursor by UV-light is needed for reduction of Au-ions to produce the gold nanoclusters. Next, the prepared film was exposed to UV irradiation for 30 min and then stored in dark for two weeks for aging. The authors found the aging period in the dark to be essential for obtaining the nanoclusters they desired, namely Au _m . Relatively high radical precursor content remains in the final product. (Langmuir 2009, 25 (24), 13888- 13893).

Madhuri and Radhakrishnan also reported on generation of gold nanoclusters in poly (methyl methacrylate) films through thermal annealing, in practice, heating in a convection oven at 150 °C for 15 min. Thermal annealing does not provide means for exposure only of a predetermined part of the solid polymer matrix, but applies equally to all parts of said film. Patterns of fluorescent and non-fluorescent areas cannot be produced. ( Dalton Trans. 2017, 46 (46), 16236-16243).

There is a need for a process of producing localized fluorescent gold nanoclusters within solid matrices in a cost-effective and facile technique. In addition, there is a need for solid matrices containing localized fluorescent gold nanoclusters.

BRIEF DESCRIPTION OF THE INVENTION

An object of the present invention is to provide a process and means to alleviate the disadvantages discussed above. The present invention relates to a process of producing fluorescent gold nanoclusters according to claim 1 , composition according to claim 13 and polymer matrix according to claim 16.

The invention is based on studies on formation of localized fluorescent gold nanoclusters in polymer thin films by light exposure. The present process provides advantages since dramatically lower intensity is required to form and activate fluorescent features compared to that needed in case of silver nanoclusters in polymer films. Therefore, low-cost light sources, such as LEDs, can be used for this purpose.

Further, parallel patterning is plausible by projecting light through digital micromirror devices (DMDs) or spatial light modulators (SLMs). Projecting light through digital micromirrors provides considerable advantages in comparison to e.g. use of masks in patterning. Masks are expensive and very rigid to produce. The pattern cannot be altered, but remains constant when a mask is used. Conversely, projecting light through digital micromirrors offers practically unlimited possibilities for varying the pattern without set-up times.

The present inventors have surprisingly found a synergistic effect through the low light intensity needed for gold nanoparticle formation and the non-detrimental effect thereof on the polymer matrix. When combined to speed gained by projecting the light through digital micromirrors, new applications for producing polymer products are opened. To distinguish from methods based on gold nanoparticle or nanocluster formation through chemical reactions, the present process requires no chemical reducing agent, specific radical source, or specific anion source to be coupled with gold cations. This provides advantages through simpler chemical compositions. Even more significantly, contrary to chemical nanoparticle formation which takes place throughout the mixed matrix, the present nanocluster formation can be steered with high precision forming nanoclusters only to cites exposed to light energy. Further, the prior art processes based on chemical nanocluster formation often require an isolation step for recovering formed nanoparticles from the matrix where they are produced, which according to the present invention is not needed. Compared to chemical or biological nanocluster formation, which typically requires certain incubation time for reaction between the precursors, present process provides further advantages with very rapid nanocluster formation.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described in greater detail by means of preferred embodiments with reference to the attached accompanying drawings, in which

Figure 1 shows a schematic illustration of laser patterning process on a polymer matrix comprising gold nanoclusters. A desired ratio of gold chloride and PVA were dissolved in water and the resulting aqueous solution was spun cast on a glass coverslip to film thickness of 50 nm. Light source was focused into the film to activate localized fluorescent patterns of gold nanoclusters.

Figure 2 presents as schematic outlines the formation (Figure 2 a) and excitation (Figure 2b) of gold nanoclusters when implemented in industrial scale.

Figure 3 describes studies conducted at different wavelengths (430, 473, 520 nm) giving normalized fluorescence intensity of a 1 .28 pm x 1 .28 pm square region of gold nanoclusters embedded in PVA film with 20 % Au/OH ratio, exposed to light with 30 W/cm ² intensity. The relatively long time-frame reveals how gold nanoclusters are formed and how they, after the peak fluorescence intensity, turn to photobleaching phase.

Figure 4 shows spectral (a) growth, and (b) decay of gold nanoclusters embedded in PVA film with 20 % Au/OH ratio. The emission spectra were recorded at different stages when the film was irradiated by a laser beam with 60 pm diameter and maximum intensity of 700 W/cm ² . Figure 5 illustrates the experimental emission spectrum corresponding to a film with 30 % Au/OH ratio, where two constituent Gaussian components are shown, with peaks at 560 nm and 660 nm, and FWHMs of 100 nm and 1 15 nm, respectively.

Figure 6 shows fluorescence intensity evolution of a 1 .28 pm x 1 .28 pm square region of a polymer matrix film comprising gold precursors exposed to different laser intensities with wavelength of 473 nm. Inset represents the dependence of rise time (circles) and photobleaching time constants (triangles) with respect to the laser intensity.

Figure 7 shows false-color emission images of patterns generated by scanning a polymer matrix films comprising gold nanoclusters against a focused laser beam with 473 nm wavelength. The zigzag pattern (a) was written within a film of 10 % Au/OH ratio, with a laser power of 2 pW and the scanning speed was 5 pm/s. Tampere University logo (b) was written in a film of 20 % Au/OH ratio, with a laser power of 0.5 pW and the scanning speed was 2 pm/s. Patterns were excited by a collimated beam of the same laser used for writing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process for producing gold nanoclusters comprising a. providing a polymer and a gold cation source, and admixing to obtain an intermediate, b. solidifying said intermediate comprising the polymer and gold cations therein, to obtain a solid polymer matrix, c. exposing a predetermined part of the solid polymer matrix for an exposure time from 0.0001 to 1 s, to light having intensity from 1 to 1000 W/cm ² , preferably from about 10 to 500 W/cm ² to produce fluorescent gold nanoclusters.

The light induces simultaneous photo-reduction and agglomeration of reduced neutral-state gold, leading to the formation and stabilization of fluorescent gold nanoclusters. Solidifying the intermediate contributes to localizing the gold nanoclusters. The polymer ensures the further gold nanoclusters growth. As a result, fluorescent gold nanoclusters at a solid polymer matrix are produced. For exposure, light having wavelength from 200 to 700 nm, preferably from 300 to 500 nm and most preferably from 350 to 490 nm is used in step c.

When the as-formed structures are excited they have broadband emission over the visible wavelengths. Here the gold nanocluster formation is also referred to as gold nanocluster generation. It is understood, that during gold nanocluster formation, the exposure to light also excites the fluorescent gold nanoclusters formed. Hence, immediately after formation of nanoclusters in the polymer matrix, said nanoclusters emit light. However, they can be excited later on under desired conditions for reading the fluorescent pattern coded to the gold nanocluster containing polymer matrix.

Patterning to solid polymer matrix is formed by exposing a predetermined part of the solid polymer matrix to light. Preferably the exposure in step c) follows a predetermined pattern, wherein at least part of the solid polymer matrix remains non-exposed and hence non- fluorescent as in figure 7.

Hence, according to a specific embodiment of the present process, the process produces fluorescent gold nanoclusters and comprises a further step, namely d. exciting said gold nanoclusters for an exposure time from 0.0001 to 1 s, with light intensity from 1 to 1000 W/cm ² , preferably from 10 to 500 W/cm ² to obtain fluorescence.

Fluorescence is hence produced. Depending on the application, said step of exciting may be performed at any time after producing said nanoclusters. Light having wavelength from 400 to 700 nm, preferably from 500 to 660 nm, preferably from 520 to 560 nm, hence green light, is used in step d. Such selection of wavelength provides better control for avoidance of photobleaching or overgrowth of gold nanoclusters at the solid polymer matrix (Au@solid polymer).

After production of fluorescent gold nanoclusters at a solid polymer matrix, the process may further comprise a step of detection of said polymer matrix by collecting fluorescent light from said polymer matrix. In embodiments, where fluorescent gold nanoclusters at a solid polymer matrix, such as labels, are made in micro-scale, then microscope objectives with high numerical aperture are required to collect fluorescent light and detect labeling.

As used herein, micro-scale refers to objects, such as polymer matrix objects having two dimensions, or at least the largest dimension measurable in micrometers, such as largest dimension from 10 to 990 pm, preferably from 40 to 500 pm. An extreme example of micro scale patterns is given in figure 7, patterns (a) and (b), wherein the pattern dimensions are about 20 pm x 20 pm and about 10 pm x 10 pm respectively. Macroscopic labels made using said process could be good enablers for anti-counterfeiting, where invisible authentication codes made from gold nanoclusters become visible only upon proper excitation. According to embodiments in macro-scale the fluorescent gold nanoclusters at a solid polymer matrix provide objects having two dimensions, or at least the largest dimension from 1 mm to 50 mm, preferably from 2 mm to 26 mm. In macro-scale, fluorescent gold nanoclusters at a solid polymer matrix provide a fluorescent QR-code detected upon excitation and read by a reading device. Accordinly, one can make fluorescent QR code and read it by a common consumer device containing a camera, such as a smartphone. In the simplest case, if the flash light of the phone is filtered to produce green light, the smartphone could be the sole device for reading, and thereby the labels are excited by proper light.

According to a preferred embodiment, the step a. further comprises providing a solvent, and step b. further comprises removing at least part of the solvent from said intermediate to initiate solidification. The process for producing fluorescent gold nanoclusters can then be described comprising a. providing a polymer, a gold cation source, and a solvent, and admixing to provide an intermediate, b. removing at least part of the solvent from said intermediate to solidify the polymer and gold cations therein, to obtain solid polymer matrix, c. exposing a predetermined part of the solid polymer matrix, for an exposure time from 0.0001 to 1 s, to light having intensity from 1 to 1000 W/cm ² , preferably from 10 to 500 W/cm ² to produce fluorescent gold nanoclusters.

The present inventors have surprisingly found that gold nanoclusters can be synthesized within polymer matrix using considerably lower light intensity than expected, leading to a remarkably higher speed and less expensive light patterning process.

The term“fluorescent gold nanoclusters” refers to agglomerates formed from gold cations reduced and clustered together. Nanocluster is an established definition used in the field of nanotechnology and used in order to differentiate from nanoparticles, which usually fall between sizes from 10 to 100 nm. The nuclearities of nanoclusters generally vary from 2 atoms to roughly a few hundreds of atoms, typically tens of atoms. Hence, gold nanoclusters consist of only from a few Au atoms to several hundreds Au atoms. Nanoclusters typically have distinct optical and electrochemical properties, caused by their discrete energy levels due to size quantization. The nanoclusters are highly organized forming typically symmetrical assemblies. As to size, the nanoclusters are defined to have all dimensions < 2 nm. Hence, none of the dimensions of said nanoclusters exceeds 2 nm or in other words, the largest diameter is below 2 nm. Here,“gold” and“Au” are used interchangeably referring to clusters comprising Au atoms, not to elemental, metallic gold.

Fluorescence is a form of luminescence and one of special characteristics of gold nanoclusters. Consequently, “fluorescent” refers to an entity which emits light upon appropriate excitation. Fluorescent gold nanoclusters emit detectable light when exposed to excitation light. Further growth, that is growth beyond the largest diameter of 2 nm is undesirable. With“bleaching” is here referred to decrease of the fluorescence, caused by excessive growth of the gold nanoclusters and loss of fluorescent characteristics through said growth.

With term“fluorescent gold nanoclusters” is herein referred to gold nanoclusters as defined above having broadband emission over the visible wavelengths. The typical wavelengths are between 400 and 800 nm.

As used herein,“direct light patterning” refers to exposing the predetermined part or sites of a solid polymer matrix to light having intensity from about 1 to 1000 W/cm ² . According to an embodiment, the exposure is provided as direct light patterning. Direct laser writing is a term used in literature correspondingly to refer to such light exposure when laser is used as the light source. Said light exposure may be provided by different light sources. Collimated light of a laser can be focused onto the polymer film and the polymer film is scanned against the beam to make desired pattern. Collimated laser beam can be also reflected off a spatial light modulator (SLM) or a digital micromirror device, in such a way that patterned light strikes desired parts of the film and generates patterned fluorescent gold nanoclusters. Likewise, any other light source, such as LED, or filtered white light source, can be patterned by a digital micromirror device to create patterned fluorescent gold nanoclusters. Irradiation of “predetermined sites” of the solid polymer matrix is referred to here as exposure to light selectively, enabling at least a part of the solid polymer matrix remaining non-exposed and hence non-fluorescent.

According to an embodiment, the light source comprises continuous wave laser irradiation. Continuous wave laser was used in the experimental part, since it provides precise means for light exposure, as so-called laser writing. Intensity may vary from 1 to 1000, preferably from 10 to 500 W/cm ² to produce said gold nanoclusters. The wavelength used for light patterning can vary between 200 and 700 nm, preferably between 300 and 500 nm, and more preferably between 350 - 490 nm. Continuous wave lasers operating within this wavelength are common and readily available commercially. Use of such common laser enables applications where exciting takes place distinctly from nanocluster formation process.

According to another embodiment, light-emitting diodes, (LEDs) are used as a light source. Typical wavelengths provided by LEDs may be between 200 and 700 nm, preferably between 300 and 500 nm and more preferably between 350 - 490 nm. Intensity may vary from 1 to 1000, more preferably from 10 to 500 W/cm ² to produce said gold nanoclusters.

The formation of gold nanoclusters by photoreduction from Au ³⁺ in polymer matrix requires set amount of energy. The codependence of intensity, exposure time and wavelength can be calculated based on common general knowledge. If the energy is too low, no nanocluster formation occurs or said formation is too slow for industrial applications. However, exposure to the light at an intensity too high or for an excessive time, leads to photobleaching. This is known to relate to growth of the nanoclusters to reach dimensions in the range of nanoparticles, where characteristics typical for nanoclusters, such as fluorescence, are gradually lost. The intensity required for gold nanocluster formation was surprisingly low in the experiments herein conducted.

One of the benefits of the present invention is that the energy needed for gold nanocluster formation is surprisingly low. Contrarily to the prior art silver nanocluster formation, which had to be performed with a pulsed laser with intensity of 8 - 80 MW/cm ² , and with continuous wave laser with intensity of 6-12 MW/cm ² , the present process has now been successful using intensities from about 1 to 1000 W/cm ² .

The time needed for nanocluster formation is dependent on the other parameters selected for the light (wavelength and intensity). However, the present inventors have now found that due to low energy requirement, the time for light exposure can be reduced from that needed for prior art applications. The exposure time in step c. of the process of the present invention may be from 0.0001 , from 0.001 s, from 0.1 to 100 s, to 10 s, to 1 s or to 0.1 s. Time is especially relevant when macroscale patterning is performed.

In embodiments, where the speed of gold nanocluster generation is of essence, shorter wavelengths are more appropriate, such as wavelengths from 400 to 440 nm, preferably 430 nm. The experiments conducted here showed how nanoclusters were generated much faster at wavelength of 430 nm. This is demonstrated in Fig. 3. According to embodiments, wherein high precision is desired and delicate patterns need to be formed to the solid polymer matrix, a focused light beam is used to enable forming spots. The solid polymer matrix, mounted on a computer-controlled 3D translation stage, is then scanned against the laser beam to generate desired fluorescent patterns. Even with relatively simple experimental setting, surprisingly sophisticated fluorescent patterns were produced on the solid polymer matrix in the experiments conducted.

A specific embodiment for creating complex patterns involves patterned light (using digital micromirrors). Parallel light exposure using‘digital micromirrors’ is very useful for‘micro’- scale labels. The present invention enables the use of parallel light exposure to‘macro’-scale parallel patterning as well. Since the intensity level required for generation of nanoclusters is very low in the present process, the power distributed on a larger region of the film making the intensity lower, is still sufficient. Thereby, parallel writing of macro-scale labels is feasible too. Patterning and labels thereof can be provided even faster than scanning with a focused laser beam. Where macroscale patterns are desired, the process is also referred to as “parallel writing”. Such embodiment may be understood with reference to figure 2a.

The gold cations are provided to the intermediate as gold salts, typically as Au ³⁺ or gold(lll) derivatives, or as Au ⁺ or gold(l) derivatives. Terms“gold cations” and“Au ⁺ or Au ³⁺ “ are used herein interchangeably. More specifically, these comprise gold(lll) bromide, gold(lll) hydroxide, gold(l) iodide and gold chlorides. Gold chlorides commercially available comprise AuCI, AuC , HAuCU and hydrates HAuCU ^* xhhO thereof, such as Gold(lll) chloride trihydrate HAuCU ^* 3H2O. Corresponding to gold(l) chloride, also gold(l) iodide, Aul, is available. There, the gold cation is Au ⁺ . Reagents comprising further elements to gold and chlorine are also known, and for similar purposes as herein described, for example «(AuCU) has been recommended. Commercial products comprise both powders and solutions, such as 30 wt. % HAuCU in dilute HCI. Gold provides advantages over other metals through its stability and biocompatibility. Furthermore, gold has now been found to require lower intensity for nanocluster formation.

The amount of gold cations as gold precursors can be optimized and are dependent on the polymer used. As used herein, the content of gold-precursor in intermediates of step a. and b. are expressed in relation to the amount of hydroxyl groups in the polymer. For example, 1 .5 wt% PVA solutions with different Au/OH ratios (i.e., number of gold atoms to number of hydroxyls), from 0 % to 50 % were studied in experiments herein conducted. Preferred Au/OH molar ratios were between 1 and 100 %, preferably between 5 and 70 %, and the most preferred ratios between 10 and 30 %. As used here, the molar Au/OH ratio in said intermediate is calculated from the proportions of component gold cation source and hydroxyl-groups in the polymer.

In general, the use of macromolecules to stabilize nanoclusters is known in the prior art. Many of the prior art applications relate to nanoclusters in liquid matrices, such as polymer- scaffolded metal nanoclusters in solution, but here the focus is on solid matrices, preferably polymer films. However, the high intensity laser light required to produce polymer-scaffolded silver nanoclusters in solid matrices has been reported having a detrimental impact on the polymer films, and leaving visible grooves on the laser written regions. In addition, high laser intensity requirement of silver nanoclusters hinders the possibility of making macro-scale fluorescent marks through parallel writing.

According to the present method, the gold cation sources and the polymer should be in a form allowing admixing. This enables substantially even distribution of gold cation sources to the continuous matrix of polymer. According to an embodiment, the polymer and gold cation source are soluble to a solvent, and admixing takes place in a solution. Depending on polymer, solvents may vary. However, since traces of the solvent could remain in the solid matrix formed, preferred solvents are those of low toxicity, such as ethanol, water or mixtures thereof. Exemplary polymers comprise synthetic water-soluble polymers, such as PVA. Polymers can alternatively be molded as melts, where gold cations may be mixed in. Means for doping polymer matrix and processing it to typical products are known in the art. Typically monomers are mixed and doped before polymerization and formation of continuous solid matrix. Alternatively, doping could be done as solution, consequently solidifying the mixed intermediate. Preferred embodiments comprise polymer films, which may be produced by conventional methods for polymer film formation, such as casting, blow molding, extrusion, coating, electrospraying, eletrospinning, etc. methods. According to an embodiment, a polymer film is formed on a substrate. When referring to“solid polymer matrix” the present disclosure encompasses polymer matrices which are solid at ambient temperature. For the success of distinct and specific patterning, solid matrix is needed to prevent free movement of formed gold nanoclusters, as would be possible in liquid solutions. Preferably a solid polymer matrix is solid at 20 °C, more preferably at 70 °C and most preferably at 100 °C.

Because of the lower intensity in the light exposure step of the present process, the choice of polymer matrix can be broadened. In general, polymers suitable here comprise high molecular weight acrylate and vinyl type polymers. In order to create distinguishable patterns to the matrix, it is necessary to use a polymer which does not have fluorescent properties overlapping with those of gold nanoclusters, preferably no fluorescent properties at all. Even though the number of variants within these groups are high, the characteristics of different materials are commonly known as well. Depending on the end use, for example specific requirements for pharmaceuticals should be met, a man skilled in the art can select a suitable matrix material. The polymers usable herein comprise poly(methacrylic acid)(PMAA), poly(vinyl alcohol)(PVA), poly(methyl methacrylate) (PTMP-PMMA), poly(n-butyl methacrylate) (PTMP-PBMA), poly(tert-butyl methacrylate) (PTMP-PtBMA), poly (N-vinyl-2- pyrrolidone), Vinyl Ether Star Polymers, polycarbonate, polystyrene, SU-8. So far, best results have now been reported with varieties of poly(methacrylic acid)(PMAA) and poly(vinyl alcohol)(PVA). Preferably the polymer matrix comprises poly(vinyl alcohol), more preferably poly(vinyl alcohol) having molecular weight, Mw from 25 000 to 125 000, specifically 89 000- 98 000.

As used herein, admixing the polymer and gold cations, provides an intermediate. According to the present process said intermediate comprising the polymer and gold cations therein is solidified to obtain a solid polymer matrix. Solidification methods are typically known and recommended by the polymer provider. A solid polymer matrix before formation of gold nanoclusters provides benefits over liquid form matrices.

According to another aspect, herein is provided a solid polymer matrix, preferably a poly (vinyl alcohol) (PVA) polymer matrix, comprising fluorescent gold nanoclusters produced by a process according to the process described herein. Such matrix allows provision of fluorescent patterns, labels and markings on said matrix. Preferably said solid polymer matrix is in the form of a polymer film. The film thickness may vary according to the application thereof. Said polymer matrix may be used as a fluorescent label.

According to an embodiment, the solid polymer provides the matrix for a micro-scale label. Said micro-scale labels are very small tags having two dimensions, or at least the largest dimension from 10 to 990 pm, preferably from 40 to 500 pm.

According to another embodiment, the solid polymer provides the matrix for a macro-scale label. A macro-scale label is visible to the naked eye or readable by hand-held devices, such as a smart phone, said macro-scale label is characterized by having two dimensions, or at least the largest dimension from 1 mm to 50 mm, preferably from 2 mm to 26 mm.

Gold nanoclusters embedded within such polymeric matrices provide surprisingly simple and hence controllable matrix compared to prior art nanodots in liquid matrices requiring complex systems with reducing agents, specific anions or colloid-forming agents. Increasing complexity of nanoclusters as such increases the system complexity respectively limiting options with regard to i.e. solvent, stabilizer and matrix choices leading to further restriction when end uses are concerned. To distinguish from methods based on gold nanoparticle or nanocluster formation through chemical reactions, the present process requires no specific anion source to be coupled with gold cations. Suitability to very different applications is increased through avoidance of several components.

As an important part of the nanomaterial synthesis, is also detection, verification, monitoring and characterization of material gained. Analysis methods and techniques are known in the art. The synthesis of the nanoparticles and nanoclusters has been monitored by UV-visible spectroscopy. The particles thereby obtained may be characterized by UV, dynamic light scattering (DLS), X-ray diffraction (XRD), energy dispersive X-ray (EDX) analysis, Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). Energy-dispersive X-ray has been used to verify the presence of gold in the nanoparticles.

According to another aspect of the present invention, herein is provided a composition comprising a poly(vinyl alcohol) polymer, a gold cation source, and fluorescent gold nanoclusters. Preferably, said composition is produced according to an embodiment of the process described herein. Said composition is preferably in the form of a poly(vinyl alcohol) film. Preferably the gold cation source is a gold salt selected from gold(lll) bromide, gold(l) iodide and gold chlorides, most preferably HAuCU. The fluorescent gold nanoclusters are preferably not spread throughout the film evenly, but form text, figures, patterns, or other forms of information. The composition was studied as a thin film in the present examples. Very thin polymer film with fluorescent nanocluster embedded therein provided the desired effects and furthermore was convenient for experimental purposes. However, increasing the film thickness may vary according to the application where it is used and other requirements therein. For example, if the polymer matrix is used for providing surface properties, protection of some kind, rigidity, barrier, coating etc. the polymer matrix layer may be adjusted accordingly.

According to an embodiment, the molar Au/OH ratio in the PVA composition is between 1 and 100 %, preferably between 5 and 70 % and most preferably between 10 and 30 %. A lower limit for said molar Au/OH ratio in the PVA composition is set by the distribution of the gold cations in the matrix; too low percentage results in a matrix, wherein the gold cations are far from another not being able to form nanoclusters, on the other hand, too high a molar Au/OH ratio can interfere with nanoparticle formation and cause increased tendency for photobleaching. A composition described herein may be used as a fluorescent label. Molar Au/OH ratio is calculated from the proportions of component gold cation source and hydroxyl-groups in the polymer. Molar Au/OH ratio in the composition comprising a poly(vinyl alcohol) polymer, a gold cation source, and fluorescent gold nanoclusters is calculated from the proportions of component gold cation source and fluorescent gold nanoclusters against the hydroxyl-groups in the polymer.

The appended figures provide schematic outlines of the process and results from the measurements made. Figure 1 discloses a schematic outline of the experimental setting used in the laboratory. It is understood that such setting in merely provided as an example and a man skilled in the art is able to vary it to larger scale production. To produce the solid matrix on the substrate of figure 1 , a PVA polymer, water as a solvent, and a gold cation source are admixed to obtain an aqueous intermediate, which then is coated on the substrate. The intermediate solidified by forming by spin coating a very thin film, from which solvent evaporates. A predetermined part of the solid polymer matrix is exposed to light obtained from light source (writing LS) having varying intensities. The light beam is guided by dichroic mirrors (DM) and microscope objective (OB), to the desired site of the solid polymer matrix. Examples of exposure of a predetermined part of the solid polymer matrix can be seen in figure 7.

The same arrangement for dichroic mirrors (DM) and microscope objective (OB) is used to direct the light source from excitation (excitation LS) to the matrix. However, the intensity needed for excitation is lower and light source may be selected accordingly.

Both the writing and excitation can be monitored and verified by camera and spectrometer recording fluorescence as shown in figure 1 , with help of the system of mirrors (M, DM), lenses (L), and filters (F).

The simultaneous production and excitation were unavoidable when studying evolution of gold nanoclusters in solid polymer matrix in laboratory. However, in general, gold nanoclusters in solid polymer matrix are produced by light irradiation for a short time, as shown in figure 2a. Then, no matter what the application is, they need to be excited (figure 2b) with proper light (at longer wavelength - like green light) to avoid further growth and photobleaching.

Hence, figure 2a discloses a schematic arrangement for industrial scale gold nanocluster formation. Using state of the art digital micromirror (DMD), spatial light modulator (SLM) or photomasking systems, the light exposure may be directed with high specificity to multiple points or areas of the polymer matrix containing gold cations, here label. According to an embodiment of the present process, projecting the light through a spatial light modulator (SLM) or a digital micromirror device provides the exposure needed for gold nanocluster formation. By such arrangement, a predetermined pattern of exposed and non-exposed matrix is produced in very short time.

Studying of the effect of laser irradiation on Au-ion containing polymer films and the evolution of the fluorescent gold nanoclusters

A collimated laser beam with 473 nm and maximum intensity of 1 W/cm ² was launched to strike the polymer film while the growth and decay of the emission was traced by in-situ imaging of the sample using an EMCCD camera (see Fig. 1 ). The laser writing process is shown in Fig. 1 and described in detail in the Methods section. A series of fluorescent images was captured at different times, during which the polymer film was irradiated by a laser beam with a Gaussian profile and peak intensity of 1 W/cm ² . The fluorescence intensity evolved with radial gradient, with smaller rise time (t _diw ) and photobleaching time constant (t) at the center, where the peak intensity resided. Thanks to the Gaussian profile of the laser beam, NCs evolution for different excitation intensities were calculated by extracting the fluorescence intensity of 1 .28 pm x 1.28 pm squares at different parts of the images (not shown). The resulting evolution curves are selectively shown in Fig. 2(b). The emission intensities exhibit a logistic growth in gold nanoclusters population where / _max is the maximum intensity, k is steepness of the curve, and to is the time to reach to the midpoint of the maximum fluorescence intensity.

The higher the laser writing intensity, the faster the rate at which gold nanoclusters are generated. Likewise, the higher the excitation intensity, the faster the rate at which the written fluorescent gold nanoclusters bleach. The inset in Fig. 2(b) represents the exponential dependence of the aforementioned time constants to the applied intensities, in which t is about four times larger than t _diw assuming identical laser intensity for both writing and excitation process. Therefore, by stopping the irradiation at the peak and exciting the as- formed gold nanoclusters with lower intensities, photobleaching occurs at far longer times. Interestingly, the curve corresponding to 29 W/cm ² intersects the one related to 100 W/cm ² at t = 1 10 sec and shows higher emission intensity, despite being excited with three times higher intensities. The effect of laser wavelength on the writing process

A supercontinuum laser beam was coupled into a computer-controlled monochromator whose output light impinging a sample with 20 % Au/OH ratio, with maximum intensity around 30 W/cm ² . The evolution of the fluorescence intensity corresponding to various wavelength was traced by in-situ imaging of the sample. Figure 3 demonstrates normalized fluorescence intensities corresponding to 1 .28 pm x 1 .28 pm square region on the sample for violet, blue, and green excitation light. While shorter wavelengths cause faster writing and photobleaching, longer wavelengths result in significantly slower formation of nanoclusters, and in turn, longer bleaching time constants. Therefore, violet light would be the most efficient and fastest for writing, while green light enables efficient reading as gold nanoclusters display superior photostability.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations be included within the scope of the invention.

Experiments were set up for preparing matrices with gold cation sources therein, exposing said matrices to laser beam and characterizing the obtained material. In addition to optical results discussed in detail below, the polymer matrix was observed by bright field microscope. Given the microwatt power level of the laser, no detrimental impact on the polymer film was observed.

Sample preparation

To provide an aqueous polymeric solution, 240 mg (5 mmol) of poly(vinyl alcohol) (PVA) powder (Sigma-Aldrich, Mw = 89 000 - 98 000, 99 + % hydrolyzed) was dissolved in 8 ml of water to obtain 3 wt% PVA solution. Next, aqueous gold precursor solutions, thus the gold cation sources, were prepared by dissolving different quantities (0 to 640 mg, 0 to 1 .6 mmol) of gold(lll) chloride trihydrate (Sigma-Aldrich, > 99.9%) in 8 ml of water. The polymeric and gold-precursor solutions were subsequently mixed to obtain intermediates, resulting in 1.5 wt% PVA solutions with different Au/OH ratio (i.e., number of gold atoms to number of hydroxyl groups, from 0 % to 30 %). The blends were subsequently spun cast on borosilicate coverslips (22 mm x 22 mm x 0.17 mm), with 1500 RPM and for 2 minutes, leading to thin PVA films of 50 nm thickness containing gold cation sources as gold precursors.

Optical setup

The laser writing and the fluorescence spectroscopy on gold nanoclusters were conducted at a 90-degree angle with respect to each other to prevent the transmitted or reflected incident light reaching the detectors. The polymer-coated substrate was flipped over such that the polymer film was sandwiched in between the coverslip substrate and a glass microscope slide, and was fixed on a three-axis motorized scanning stage (Thorlabs, MAX303/M). Continuous wave (CW) laser beam from a single-mode laser diode (Cobolt 06-01 Series) with wavelength of 473 nm was cleaned and expanded to obtain collimated beam of 6 mm diameter using a spatial filter system.

This laser was employed for the both laser writing and excitation. A polarizing beamsplitter cube housed in a rotation mount was utilized to adjust the laser output power. The resulting beam reflected off a dichroic mirror (Semrock, FF484-FDi01 -25x36), redirected via a 45- degree-mounted mirror, and delivered to the sample through an oil-immersion objective lens (Leica HCX PL APO 100X). This lens was responsible to deliver the excitation light and to collect the emission from the specimen. The laser beam was usually focused onto the back aperture of the microscope objective by a convex lens of 200 mm focal point, resulting in a collimated beam on the film. In this way, we could produce and excite a greater population of nanoclusters. For scanning purposes, the aforementioned convex lens was removed, and a collimated beam was delivered to the microscope objective, thus making a focused beam spot on the polymer film.

A shutter (Thorlabs SH05) along with a controller (Thorlabs SC10) was utilized in the beam path to control exposure times. Collected fluorescence from gold nanoclusters were passed through the dichroic mirror and was redirected to a bandpass filter (Semroch, FF01 -612/69- 25) and was focused onto an EMCCD camera (Andor, iXon3 897). The fluorescence was redirected to spectrometer (Avantes, Avac-pec 2048) by a flipping mirror or a beamsplitter in order to measure the spectra. To investigate the effect of wavelength on the writing process, a supercontinuum white light source (Fianium Ltd.) was coupled to a computer-controlled monochromator (HORIBA, iHR550), with slit width set to 2 mm leading to spectral resolution of 12 nm. Optical characterization

The evolution of the gold nanoclusters emission spectra for Au@PVA films was studied under the laser irradiation. Emission spectra was measured for samples with different Au/PVA concentration (from 2.5 % to 30 % Au/OH ratio) by irradiating the collimated laser beam of 60 pm diameter to non-exposed sites of Au@PVA films, and spectra was recorded on 100 ms time steps. Figure 4 (a) shows the growth of the spectrum corresponding to an Au@PVA film with 20 % Au/OH ratio, exposed to a beam of 700 W/cm ² . As can be clearly seen, the fluorescence exists even immediately after the laser exposure, and it logistically evolves (as described above), reaching to a maximum after 3 seconds elapse time. Subsequently, the emission enters to a photobleaching phase, as irradiation continues (see Fig. 4 (b)). Interestingly, there is no visible change in the profile of the spectra from the beginning of the growth and after the photobleaching, suggesting that the sources of fluorescence remain unchanged. The broad spectrum from 500 nm to 800 nm is attributed to the fluorescence obtained from gold nanoclusters. The sharp cutoff around 500 nm is associated with the dichroic mirror used for this measurement.

Given the broad and asymmetric experimental line shapes, one could predict that the emission spectra of Au@PVA nanoclusters consist of more than one component. Accordingly, to deconvolute photoluminescence curves, we employed a two-Gaussian fitting model. Figure 5 illustrates the experimental emission spectrum corresponding to a film with 30 % Au/OH ratio, where two constituent Gaussian components are shown, with peaks at 560 nm and 660 nm, and FWHMs of 100 nm and 1 15 nm, respectively. 473 nm laser was used for excitation with 60 pm diameter and maximum intensity of 1.7 kW/cm ² .. The emission profile was fit using a two-Gaussian model, with dashed and dash-dotted lines being the constituent elements and the red line being the total fit. Inset depicts the relation between fluorescence intensity of AuNCs against Au/OH ratio. The emission spectra are well replicated (solid line) by summing up the two constituent components (dashed and dash- dotted lines), with coefficient of determination being R ² ~0.998. The two-element emission spectra suggest that the generation of gold nanoclusters follows a size-focusing growth route, which could be related to the stability of certain size of gold nanoclusters.

Scanning transmission electron microscopy (STEM)

Advances in transmission electron microscopy techniques allows researchers to directly probe the geometry of fluorescent nanoclusters with resolutions down to the atomic levels. Having said that, application of TEM to examine nanoclusters encapsulated in solid templates is fairly scarce, partly due to the difficulties in TEM-grids preparation. To accomplish such a task, thin film needs to be deposited on fragile grids and be exposed to the laser writing process, which is not trivial. Nonetheless, owing to the ability in casting the polymer as a thin film, we could prepare the grids (described below), and exploit STEM to obtain detailed nanoscale information about the changes in Au@PVA film sites after being exposed to laser irradiation. Fig. 6 (a) shows a photograph corresponding to a PVA film containing Au ions with no exposure to laser light. As can be clearly seen, numerous ultra-small gold dots of size < 0.5 nm already reside in the film, which could be formed during the sample preparation and were kinetically trapped in the film. In contrast, Fig. 6 (b) represents related sites of Au@PVA film which was subject to laser exposure for sufficient time. The laser exposure was performed by in-situ imaging of its fluorescence, and was stopped once the emission became maximum. As can be clearly seen, all of < 0.5 nm gold dots were washed off, and instead, particles of size 1 -3 nm were found ubiquitous in the film. The gold composition of particles was confirmed by energy-dispersive X-ray spectroscopy (not shown).

STEM imaging

Thin Au@PVA film with 10 % Au/OH ratio was spun cast on TEM grids (Agar Scientific, AGS147-3H) with 2500 RPM for 2 minutes to guarantee film thickness of < 50 nm, which is crucial for TEM measurements. A second step spinning, set at 4000 RPM for 30 s, was immediately run to cast off the edge beads, thus preventing film quality deterioration. Several grid cells were selectively exposed to laser irradiation for sufficient time to reach maximum population of nanoclusters. The STEM photography was performed at the Nanomicroscopy Center in Aalto University, using JEM-2200FS double Cs-corrected electron microscope (JEOL Ltd) operated at 200kV.

The results confirm that photostable, fluorescent gold nanoclusters can be produced within PVA films using a low intensity laser light. Study on the evolution of gold nanoclusters through fluorescence microscopy and spectroscopy confirmed that the required intensity level for exciting gold nanoclusters lies within a few tens of watts per square centimeter. Furthermore, the evolution exhibits two regimes, with first being the formation of gold nanoclusters and second being the photobleaching where large nonfluorescent NPs form. High direct laser writing intensity causes abrupt formation of Gold nanoclusters and fast photo-bleaching. Therefore, for effective use of Au@PVA films, one need to select higher powers for fast gold nanoclusters generation, while exciting the as-formed structures with lower powers. Related time constants for laser writing were shown to be about four times smaller than that of photo- bleaching for a certain wavelength and intensity level, suggesting the photo-stability of PVA- scaffolded gold nanoclusters. We also confirmed that the formation of Gold nanoclusters and photo-bleaching occur with higher pace at shorter wavelengths. Therefore, the excitation light sources should be at different colors to result in effective and fast writing while resistant to photo-bleaching during excitation. In addition, evolution study on fluorescence spectra displayed no visible change in the lineshapes, with two peaks at 560 nm and 660 nm, suggesting that the source of fluorescence remains identical during the laser writing process. We also find in this new material system that the required light intensity to generate nanoclusters is far below the threshold at which polymer film undergoes topological changes, with no grooves leaving behind, contrary to that reported for silver nanoclusters. This is particularly advantageous for applications in anti-counterfeiting where the secret information -in the form of bar codes or QR codes- is fluorescent under special light source while invisible under ambient lights. It is worth noting that, whereas the required intensity for activating AuNCs in the polymer film is very low, the Au@PVA film is not sensitive to the ambient light. Regarding to photo-bleaching, time constant exceeds a few minutes provided that the written features are excited by intensities less than 20 W/cm ² . This intensity is significantly higher compared to the intensity of solar irradiance, which is about 0.1 W/cm ² . We also find that, although the fluorescent images made in Au@PVA display descent contrasts, the brightness is weak as compared to quantum dots. However, estimating the brightness and quantum yield of laser written Au@PVA requires rigorous pertinent experiments which is left for future work. Having said that, there is possibilities to augment the QY by employing other polymers, such as PTMP- PtBMA, which has shown high QY in liquid form. Thanks to the biocompatibility of both gold and PVA, this facile and cost-effective method along with the new material system could be used to create fluorescent microtaggants for in-dose drug and food authentication and other anti-counterfeiting applications.

Experiments with LED as a light source

As the light activation of gold nanoclusters is reliant on wavelength and intensity, any light source, including light emitting diode (LED), with correct said parameters can be used for light activation. Since any source other than lasers provide incoherent light, it should be firstly collimated via a single convex lens. Then the collimated light is delivered to the sample through the identical setup mentioned in the previous example for continuous wave laser. If the collimated light reflects off a DMD, then arbitrarily patterned light strikes the sample, leading to the activation of patterned fluorescent gold nanoclusters. In this way, the activation occurs in parallel by the use of a cost-effective light source, thus providing a fast and low- cost method for light patterning of fluorescent markers. A set of examples corresponding to those conducted above with laser, were conducted using LED as a light source. The placement of the equipment was the same. Collected fluorescence from gold was detected with camera and spectrometer like described earlier.

Here, LED with 470 nm wavelength, was employed for both the exposure and excitation. Given the incoherent light of the LED, it was firstly collimated via a single lens. Then the collimated light reflected off a dichroic mirror (Semrock, FF484-FDi01 -25x36), redirected via a 45-degree-mounted mirror, and delivered to the sample through an oil-immersion objective lens (Leica HCX PL APO 100X). This lens was responsible to deliver the excitation light and to collect the emission from the specimen. For patterning purposes, the collimated light could reflect off a DMD, then arbitrarily patterned light strikes the sample, leading to the activation of patterned fluorescent gold nanoclusters. In this way, the activation occurs in parallel by the use of a cost-effective light source, thus providing a fast and low-cost method for light patterning of fluorescent markers and labels.