Highly efficient fluorescent thin films

Abstract

The invention relates to a fluorescent film comprising nanobeads emitters, e.g., the nanobeads emitters are fluorescent dye-conjugated copolymer nanobeads. A fluorescent device comprising a base substrate and the fluorescent film deposited thereon is also provided. The copolymer nanobeads are copolymers of a mixture of amphiphilic macromonomers, with fluorescent dyes covalently bonded to the chains the copolymer, e.g., through an amide bond.

Description

[0001] Highly efficient fluorescent thin films

[0002] FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of fluorescent thin films and is directed to highly efficient fluorescent thin films, which may be transparent and / or translucent and / or bio-compatible, and preparation and uses thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Fluorescent molecules and materials have been synthesized and studied for long, and across many areas and disciplines.1-6Compared to in-organic emitters like semiconductor quantum dots7(QDs), carbon dots8, perovskite QDs9, or rare earth based nanoparticles, the interest in small molecules fluorophores10has consistently persisted because of their light weight, high stability, solubility, bio-compatibility and emission tunability. An extensive number of fluorescent materials has thus been explored and optimized, providing a uniquely rich and ever-growing toolbox. Molecular assemblies, supramolecular structures11and hostguest materials4have been synthesized incorporating fluorescent molecules and studied with the aim of improving their photostability, intensity, sensing functionality, or photocataly sis.3,12’14

[0006] Fluorescent molecules fall into two distinct groups; the first comprises those molecules that fluoresce intensely in dilute solution but that lack emission in the solid-state due to aggregation-caused-quenching (TT-TT stacking), for example: pyrene, perylene derivatives and fluorescein.15The second group shows strong emission when forming aggregates or in crystalline (amorphous) form, making them suitable for solid-state devices and opto-electronic applications.16J-aggregates is a good examples for the latter. Yet, this is relatively a small group of molecules, excluding a large group of conventional dyes which could potentially be used in hybrid functional materials and smart surfaces.

[0007] In this context, it is not surprising that during the last years research groups have focused on designing organic fluorophores having high emission - both in solution and in solid state16, and thus can be used not only, e.g., as biological labels or probes, but also as emitters and components in solid-state devices.

[0008] Despite all these efforts, major challenges remain in fabrication of fluorescent thin films, among which are (i) insufficient brightness (ii) aggregation and (iii) isotropic emission resulting in signal loss within a given collection aperture. For at least of these reasons there is a need for new smooth and highly efficient fluorescent thin films, which may be transparent or transcluent and / or bio-compatible, and processes for preparation thereof.

[0009] SUMMARY

[0010] Experimental results reported below show that fluorescent films can be created by depositing nanobead emitters onto a base substrate, e.g., fluorescent dye-conjugated copolymer nanobeads. For example, the copolymer nanobeads are obtained by copolymerization of a mixture of monomers, including amphiphilic macromonomers. Fluorescent dyes are subsequently covalently attached to the ends of the chains the copolymer nanobeads, through a chemical reaction resulting in a formation of, e.g., an amide bond. Drop casting of a dispersion of fluorescent dye-conjugated copolymer nanobeads onto a range of various substrates (e.g., different types of glasses, aluminium, magnsium fluoride) resulted upon drying in formation of thin, storage-stable, smooth and uniform films. The films showed low surface roughness (unaffected by the conjugated dye molecule) and high homogenity indicated by moderate variation in fluorescence intensity across the film (i.e., the intensity of the maximal emission peak (at Xmax) measured at distant points over the film is very similar).

[0011] Accordingly, in one embodiment, the present disclosure provides fluorescent thin films comprising nanobeads emitters. Specifically, the invention relates to fluorescent thin films comprising fluorescent dye-conjugated copolymer nanobeads. A fluorescent device, comprising a base substrate and the fluorescent film deposited thereon, forms another aspect of the present invention.

[0012] In another embodiment, the present disclosure provides a process for preparing the fluorescent films of the present disclosure. The process comprises: a. preparing nanobeads emitters by a process comprising combining (hydrogel nano particles) HNPs with a dye molecule having an electrophilic conjugation domain, such as azides, maleimides or tetrazines conjugation domains, for example N- hydroxy succinimide ester conjugation domain; b depositing a layer of the NBEs on a substrate to obtain the fluorescence layer.

[0013] In yet another embodiment, the present disclosure provides the use of the fluorescent film of the invention for optoelectronic devices for solar technology or light emitting diodes or for functional hybrid materials when deposited onto plasmonic systems and metamaterials surfaces. Since those layers are transparent and can be deposited one on the other, another application can be related to cryptography.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1: Emission of NBEs. Normalized emission spectrum of all prepared NBEs on the same graph.

[0016] Figure 2: NBE thin films deposited onto fused silica surfaces characterization (A) Emission spectra of the different layers made of NBE of different conjugated molecules. All spectra were obtained using 405nm excitation and 460LP emission filter except NBE-R3 that was illuminated with 540nm and Em collected with 600LP. The emission spectra of the native dyes solution as well as absorption spectra of the NBE and the dyes is shown in Figure 7 for comparison. (B) photographs taken by smartphone camera, expressing the intense emission of those layers. The samples of NBE G, Y and R are illuminated with 488nm nm beam and NBE B, R3 and R2 are illuminated with 405nm beam (C) AFM images. The resulted surface roughness (Ra) is less than Inm and maximum height is less than 20nm for all the different deposited NBEs. The average layer height is 8nm as shown in Figure 8. That is, the same roughness / morphology is observed independent on the conjugated dye molecule. The scale bar is 2.5 m.

[0017] Figure 3. (A) A single-color fluorescent layer giving rise to intense green emission (NBE-G). (B) Two-color fluorescent layers (NBE-G & NBE-R) giving rise to white emission. Next to each fluorescent sample, one can see its transparency (samples are hold with tweezer, with no direct illumination).

[0018] Figure 4: NBE vs native dye film comparison. (A) Average (4-5 area of 30x30 pm) of emission spectrum of thin films deposited onto glass substrate from solutions with similar absorption intensity (shown in Figure 7). The spectrum was obtained for every pair at the same settings. (B) Fluorescent images of the layers. The dynamic range of all NBE film images was increased to accommodate their substantially higher brightness compared to the native dye layers. Scale bar is 1 Opm for all images.

[0019] Figure 5: Stability and homogeneity of NBE films. (A) aging test: emission spectrum of NBE layers on glass after they were deposited (black line) and after 6 months of storage in dark case at atmospheric conditions (dashed color line). The decrease in the intensity -7.4% for NBE-G and -20° / o for NBE-R The spectrum presented is the average intensity of several areas across the layer (3-6 areas) and the value was calculated from the average spectra. The spectrum for all measurements was performed at similar conditions: excitation provided by 488 5nm and collected using 500LP filter. (B) FL intensity cross -section: plot of the intensity of the maximal emission peak across NBE layer. NBE-G ’s plot shows intensity at 530nm along 2.5 mm distance with deviation of 5.6% with average intensity of 3425 180 counts. For NBE- R the peak is 590nm along 3mm distance with deviation of8.5%> and average6540 388 counts. Both spectra were acquired using 488 5nm. for NBE-G 150pm slit with exposure of 5sec parameters were used, while for NBE-R 250pm slit with lOsec exposure. More FL intensity cross -sections of different NBE films are shown in Figure 10. (C) Stability test over continuous illumination, upper row: confocal scanning (63X NA1.4, 488nm, 2% intensity 50 pW, 400Hz scan), bottom row: EPI-illumination (100X NA 1.46, 488nm laser, 600pW). The left: NBE-G, right: NBE-R. both deposited on glass without capping or protection layer. In confocal scanning conditions over 180sec the NBE-G layer showed 30% decrease, the NBE-R intensity decreased by 28%>. In EPI illumination over 550sec the NBE-G intensity decreased by 29%> decrease while the NBE-R intensity reduced by 36%>. (D) Confocal images of the layers, left: NBE-G, right: NBE-R. Scale bar 37 m.

[0020] Figure 6: NBE layers on different substrates. Characterizations of NBE-G layers on (from top): Aluminum with native oxide (Ra<2nm, oxide ~3nm), Silver with lOnm SiO2 (Ra<lnm), Silicon with native oxide (Ra<0.5), Magnesium Fluoride (160nm thick, Ra<Inm). (A) Fluorescent images of the layer. Scale bar 10pm. (B) AFM scans (10x10pm, 2.5pm special scale bar). (C) Height cross section of the AFM scans across the shown colored lines.

[0021] Figure 7: Absorbance and Fluorescence spectra in solution (A) Emission spectra of the native dyes (unconjugated) in aqueous solutions. (B) absorption spectra of NBE solutions (colored line) vs native dye solution (black line).

[0022] Figure 8: physical properties of NBE film on glass. (A) AFM height images (axial range 0- 6nm). First ro : 10x10pm scans, secondrow: 2x2pm scans. The average roughness (Ra) values were almost identical for all the different NBE layers: about 0.4nm in all presented scans. All scans had maximum Z value under 16nm (10.9 nm average with SD 3.4). (B) Height profile of NBE films. The cross-section line is 14pm across the sample (shown as dashed line on the images). The presented cross-section line color matching the corresponding curve.

[0023] Figure 9: Cryo-TEM characterization ofNBEs. NBE-G (ATTO488 labeled NBEs) size and shape were examined in two environments: (A) in solution (cryogenically frozen thin layer on grid), (B) Dry NBE sample (water was removed by low pressure dying). The average size of particles in the dry sample was llnm (7-24nm size range was detected) while in solution the average size was 36nm (24-105nm range). Figure 10: NBE Layers homogeneity. Line scan of NBE layers on fused silica substrate: Fluorescence (FL) intensity deviation over different points of the layer.

[0024] Figures 11, 12 and 13 show schemes 1, 2, and 3, respectively.

[0025] DETAILED DESCRIPTION

[0026] The present disclosure provides fluorescent thin films with different colors as well as a facile process for the preparation thereof. The fluorescent films of the present disclosure comprise nanobeads emitters (NBEs), which are deposited on surfaces.

[0027] The NBEs of the present disclosure comprise hydrogel nanoparticles (HNPs) to which different type of emitters (dye molecules) are attached. For example, the HNPs may be prepared by polymerization and cross-linking of N-isopropyl acrylamide (PNIPAAM) in a micelle template of PEO-PPO block copolymers and could be formed in varied sizes from 10- 400 nm17'18. Typically, the HNPs may be decorated with primary amine groups (low density) onto their surface to enable amide bond with dye molecules having ester terminal groups.

[0028] In more general terms, the HNPs are provided as copolymerization products of a mixture of amphiphilic macromonomers, with additional monomer(s) and crosslinker(s) also being present. The NBEs consist of copolymer nanobeads, with fluorescent dyes covalently bonded to the chains of the copolymer, usually at the end of the polymeric backbone.

[0029] For example, the copolymer nanobeads are copolymerization products of a mixture comprising a first amphiphilic macromonomer, a second amphiphilic macromonomer, a third monomer, all having acrylamide functionality as the polymerizable group, and optionally a crosslinker. The terms 'copolymer nanobeads' and 'HNPs' are used herein interchangeably.

[0030] Preferred HNPs suitable for use in the invention are the copolymerization products of a mixture comprising: a first macromonomer, which is CH2=CH-C(O)-NH-A-B-A-NH-C(O)-CH=CH2; a second macromonomer, which is CH2=CH-C(O)-NH-A-B-A-NH3+X‘ a third monomer, which is N-isopropyl acrylamide; a crosslinker, which may be selected from the group consisting of bis-aciylamide (BIS), 1,2- ethanediol diacrylate (EDDA) and 2, 2', 2" - nitrilotriethyl triacrylate (NTETA), an initiator, such as potassium persulfate or 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride; and a stabilizer, e.g., a non-ionic surfactant such as polyvinyl pyrrolidone (PVP); wherein A and B are preferably polypropylene oxide and polyethylene oxide blocks, respectively, for example a total of 4-6 polypropylene oxide and from 40-48 polyethylene oxide units. X is a counter anion, such as halide, e.g., chloride. Thus, preferred copolymer nanobeads of the invention have structural units corresponding to the monomers identified above.

[0031] The preparation method (i.e., polymerization in water) is described in the experimental section below and was also reported previously17, where suitably proportioned mixtures of the macromonomers were shown to adjust the size of the nanobeads. Suitable weight ratios of the components in the polymerizable mixture, i.e., the total of the first macromonomer / second macromonomer, N-isopropyl acrylamide, bis-acrylamide as the crosslinker, potassium persulfate as the initiator, and PVP as the surfactant are tabulated in Table 1 below. The copolymer nanobeads, with diameter usually ranging from 10 nm to 400 nm, e.g., from 10 to 100 nm, for example, from 10 to 50 nm, are obtained in the form of an aqueous suspension. They may possess a negative or a positive zeta potential, a factor influencing the method of deposition onto the base substrate as described below.

[0032] It is seen that one of the macromonomers introduces terminal amine groups to the chains of the copolymer, to enable chemical bonding of the fluorescent molecules. Because the concentration of the amine end groups in the chains of the copolymer determines the loading of fluorescent molecules, in some cases it may be beneficial to incorporate an additional amine- containing component into the nanobeads. For example, acrylonitrile may take part in the copolymerization reaction alongside N-isopropyl acrylamide (e.g., N-isopropyl acrylamide / acrylonitrile molar ratio 1 : 10 to 10: 1). Upon completion of the copolymerization reaction, the nitrile groups supplied by the acrylonitrile are reduced to give primary amines groups, available for reaction with the fluorescent molecules. Suitable reduction agents transforming -ON to -NH2 are described, for example, in WO 2024 / 075114 The resulting (post-reduction) nanobeads possess positive zeta potential, e.g. from +15 mV to +30 mV.

[0033] Fluorescent dyes bearing, or modified with, an amine-reactive group are combined with the copolymer nanobeads in a suitable solvent under appropriate conditions to achieve the conjugation, usually through formation of amide bonds. Amine-reactive groups that result in an amide bond include succinimide (e.g., N-hydroxy succinimide ester, abbreviated NHS), mal eimides and acyl chloride, to name a few. Some preferred fluorescent dyes are tabulated below, including NHS-dyes such as NHS-fluorescein. NHS-dyes useful for the coupling reaction are commercially available (NHS-fluorescein, 5-carboxyfluorescein N-succinimidyl ester, NHS-rhodamine) or can be prepared by known methods. Converting a dye to the NHS ester form is a useful approach for enabling bioconjugation, particularly for labeling proteins, peptides, or amines on surfaces. This process typically involves the activation of a carboxyl (-COOH) group on the dye molecule using an activating reagent like N,N'- di cyclohexylcarbodiimide (DCC) orN-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS). Another method of reacting N-hydroxy succinimide with -COOH-bearing compound is in the presence of diphenyl chlorophosphate, as described in US 5,734,064 to afford the succinimide ester

[0034] For example, to covalently bind the NHS-dye to the copolymer nanobeads, a solution of the NHS-dye in anhydrous DMSO or DMF is added to a reaction vessel that was previously charged with water and the HNPs under slightly alkaline pH (generated by sodium carbonate buffer). About 50-80 dye molecules may be linked to a nanobead. It should be noted that the invention is not limited to conjugation of NHS-dyes, e.g., azide-functionalized dyes are also useful.

[0035] The fluorescent dye-conjugated copolymer nanobeads are recovered from the coupling reaction in the form of an aqueous suspension, usually at concentration of a few mg per ml, e.g., 1-5 mg / ml, e.g., around 3 mg / ml, amenable to application on a base substrate.

[0036] Thus, additional aspects of the invention include a fluorescent device comprising a base substrate and a fluorescent film deposited on the base substrate, the fluorescent film comprising the fluorescent dye-conjugated copolymer nanobeads as previously described, and a process for fabricating such fluorescent device, comprising depositing a dispersion of fluorescent dye- conjugated copolymer nanobeads onto a base substrate and optionally drying to create a thin film. The fluorescent dye-conjugated copolymer nanobeads are prepared by a copolymerization reaction to produce the copolymer nanobeads followed by covalent binding of the fluorescent compounds as previously described.

[0037] The base substrate of the present disclosure may comprise glass (e.g., fused glass, borosilicate glass), magnesium fluoride, crystalline silicon, optionally with native oxide layer on its surface; silver, optionally with a thin silica layer on its surface; and aluminum, usually with a thin native oxide layer on its surface. Different thin film deposition methods can be applied, such as drop casting, spin coating, dip coating. Drop casting was found to be especially useful, requiring only few tens of microliters of dye solution to deposit the film on a specific area on the surface of the base substrate, with minimal back side staining and protect thin crisp layers to crack during the coloring processes, affording smooth and uniform films.

[0038] The drop casting of a suspension of fluorescent dye-conjugated copolymer nanobeads is done either directly onto the surface of the base substrate or preferably after surface activation / modification, e.g., following corona treatment or with the aid of an oppositely charged adhesion layer (a primer layer) that is first deposited onto the surface. This way, it is ensured that a subsequently deposited layer of fluorescent dye-conjugated copolymer nanobeads is fixed by a strong electrostatic attraction to the adhesion layer (as pointed out above, the copolymer nanobeads may possess negative zeta potential, e.g., from -10 mV to -40 mV)

[0039] This approach, which represents a preferred embodiment of the invention, is a Layer- by-Layer (LbL) deposition technique, comprising the steps of applying, e.g., by drop casting, a solution of a cationic polyelectrolyte onto the surface of the base substrate to create an adhesion layer and applying, e.g. by drop casting, a suspension of the fluorescent dye- conjugated copolymer nanobeads onto the adhesion layer.

[0040] Suitable cationic polyelectrolytes include quaternary nitrogen sites, such as nitrogencontaining rings, as in the case of poly (diallyl dimethylammonium chloride). The nitrogen bearing the positive charge may be part of a non-aromatic or aromatic ring. For example, in addition to poly (di allyl dimethylammonium chloride) (PDADMAC), the following polycations may be used: poly(allylamine hydrochloride) (PAH) , poly(ethyleneimine) (PEI) - branched or linear, chitosan - biocompatible, cationic at pH < 6.5; poly(vinylbenzyl trimethylammonium chloride) (PVBTA) ; poly(L-lysine) (PLL), Quatemized poly(vinylpyridine) (QPVP), poly(2- (dimethylamino)ethyl methacrylate) (PDMAEMA) , poly(4-vinylpyridine) (P4VP) and other polycations with quaternary ammonium groups.

[0041] Water evaporation usually follows each step of layer deposition, e.g., by blowing a stream of nitrogen over the layer, with dehydration occurring leading to reduction in the volume of nanobeads, and the formation of films with thickness of less than 1000 nm, <500 nm, e g., <100 nm, <50 nm, < 25 nm, and even thin or ultra-thin films with a thickness of less than 10 nanometers. It should be noted that the nanobeads deposited on the base substrate are not necessarily spherical particles, as deviation from sphericity may occur during the layer-by- layer assembly. The term nanobead, as used herein connection with the shape of the deposited emitter particle, is meant to include also particles showing low sphericity. AFM scanning analysis shows that the loading of the fluorescent dye-conjugated copolymer nanobeads on the base substrate is above 100 particles per 100 pm2, i.e., above 1,000,000 particles per square mm, when 3 mg / ml suspension is applied on the base substrate, e.g., ~ 1,200,000 particles per square mm.

[0042] A hydrophobic polymeric protective layer can be deposited on top of the fluorescent layer, to confer non-stickiness and good repellent properties to the fluorescent device of the invention. To this end, a liquid (i.e., the non-cured) form of the polymer is applied to the device, e.g., by drop casting, to create, upon curing, a clear, relatively thick (>lpm, >2pm) coating confining and protecting the fluorescent film. Protective coatings consisting of optical coating materials with refracting index similar to that of water (—1.3) that are useful are commercially available, such as the moisture cured MY-133-MC (MY Polymers Ltd.).

[0043] The films of the present disclosure may be prepared using a large group of fluorescent molecules (fluorescence emitters), resulting in nanometric thin homogenous layers having bright emission. The fluorescent films of the present disclosure have several advantages. For example, the films that are formed by the process of the present disclosure are homogeneous with no aggregation of the dye molecules, providing stable high quantum yield emitters. In addition, the films of the present disclosure may be transparent and / or translucent and / or biocompatible and depending on the substrate they may also be flexible or solid. Furthermore, the thickness of the films may be controlled, by the beads size or by the numbers of deposited layers.

[0044] In some embodiments the fluorescent films of the present disclosure are very homogenous, that is, the same morphology / and film thickness is observed for areas of about 0.1 cm2with no wrinkles and / or holes, and independent on the conjugated dye molecule, meaning that the morphology is determined by the HNPs.

[0045] Accordingly, the invention preferably provides a fluorescent device characterized by uniform distribution of the fluorescent dyes across the film, indicated by essentially constant fluorescent emission intensity measured at / .max at different points of the film over distance of 1 mm, or 2 mm, with maximal deviation of 10.0% from the mean observed. Experimental results reported below show that better uniformity can also be achieved, e.g., <5%. Preferably, a fluorescent device is provided, wherein the fluorescent film is characterized by average surface roughness measured by atomic force microscopy of less than 1 nm.

[0046] In some embodiments, the thickness of the films may be controlled. Typically, the thickness of the films of the present disclosure is from about 8 nm to about 1 micron. In other embodiments, the area of films of the present disclosure may be in the range from 1mm2to tens square centimeters.

[0047] In some embodiments two colors or more may be combined in order to obtain other colors. For example, when using 488 nm laser beam, 3 different emission layers can be excited (530 nm, 570 nm and 620 nm). Thus, green, yellow, orange and white emission may be obtained using the transparent films of the present disclosure When more than one color is deposited, the axial distance, i.e., the distance from the surface is controlled, with no cross talks between the different colors. The fluorescent films of the present disclosure may have white emission.

[0048] In another embodiment the present disclosure provides a process for preparing the fluorescent films of the present disclosure. The process comprises: a. preparing nanobeads emitters by a process comprising combining HNPs with a dye molecule having an electrophilic conjugation domain, such as azides, maleimides or tetrazines conjugation domains, for example N-Hydroxy succinimide ester conjugation domain; and b depositing a layer of the NBEs on a substrate to obtain the fluorescence layer.

[0049] The bright, ultra-thin, and photostable NBE layers provide a reliable means for focal verification and precise control of stage tip-tilt relative to the image plane. These capabilities are essential for ensuring optimal imaging conditions, making them valuable tools for both quality control (QC) and quality assessment (QA). By enabling users and service technicians to accurately evaluate and adjust the optical setup, our NBE layers contribute to maintaining high imaging performance, consistency, and reproducibility across various applications.

[0050] The present disclosure provides the use of the fluorescent films for optoelectronic devices for solar technology or light emitting diodes. In another embodiment, the fluorescent film of the present disclosure can be used as functional hybrid materials when deposited onto plasmonic systems and metamaterials surfaces, for example as 1 micron size RGB pixel, when deposited on plasmonic structures, described in US Patent 11,249,226.

[0051] In yet another embodiment, the present disclosure provides the use of the fluorescent film of the present disclosure for functional hybrid materials when deposited onto plasmonic systems and metamaterials surfaces.

[0052] Having described the invention with reference to certain preferred embodiments, other embodiments will become apparent to one skilled in the art from consideration of the specification. The invention is further defined by reference to the following examples describing in detail the preparation of the composition and methods of use of the invention. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention.

[0053] EXAMPLES

[0054] 1. Materials and methods

[0055] Jeffamine® ED-2003 (Mn = 1900), acryloyl chloride, and polyvinylpyrrolidone (PVP360000) were purchased from Sigma-Aldrich (USA), N-isopropylacrylamide (NIPAAM, 99.0%) was purchased from Acros organics CO., Ltd. (USA), and N,N-methylenebisacrylamide (BIS, 97.0%) was purchased from Alfa Aesar CO., Ltd. (UK. XTT). The N-Hydroxysuccinimide ester (NHS) dye derivatives of: Atto 488 NHS ester(#41698), 5-TAMRA NHS ester (#53048), Atto 490LS NHS ester(#78362) were purchased from Sigma-Aldrich Poly diallyl dimethylammonium chloride (PDDA) solution (35%wt in water, 100kDa>Mw) was bought from Sigma Aldrich (#522376).

[0056] 1.2 Atomic force microscope (AFM)

[0057] The average roughness (Ra) of the deposited layers as well as imaging of crystals and aggregates was performed by AFM (Bio FastScan AFM machine -Bruker AXS). The measurements were performed in soft tapping mode using silicon tip with silicon nitride cantilever (Brucker FASTSCAB-B model T:0.3micron, L30 micron, k=l,8N / m, f=450kHz). The resolution of the images was 512 samples / line. The microscope was covered with an acoustic hood to minimize vibrational noise. The measurements were performed under environmental conditions. Image analysis was performed using Gwydion with 2nd degree polynomial leveling.

[0058] 1.3. Fluorospectrometric measurements

[0059] Emission spectra were obtained with inverted microscope (1X83 Olympus) equipped with PIXIS 1024 eXcelonTM charge-coupled device (CDD) camera (TeleDyn Princeton Instruments) coupled to IsoPlane SCT320 spectrophotometer (600-nm blaze / 50 grooves / nm grating). The quantum yield of the CCD camera is above 90% for a wavelength range of 400- 900 nm. The setup was routinely calibrated using Princeton Instruments IntelliCal® calibration device. Excitation light was provided by Xe-arc lamp (110% power) with compatible excitation and emission filters sets (detailed below). Emission was captured employing a 250pm aperture slit and exposure time of 1-5 sec. The emission spectra showcased are the mean spectra acquired from 1023 pixel rows, analyzed using MATLAB. For the assessment of uniformity, line scans were conducted at 400pm intervals (using piezo based motorized stage) across various regions of the sample, with emission recorded at the peak wavelength plotted against distance. Filter sets:

[0060] - Ex: ET405 / 40x, De : AT455dc, Em: AT4651p

[0061] - Ex: ET470 / 40M, De: T4951pxr, Em: ET 5001p

[0062] - Ex: AT540 / 25x, De: AT 565 de, Em: AT6001p

[0063] 1.4 Fluorescent imaging

[0064] Epi -fluorescent images of the layers were captured using two setups: an inverted microscope fitted with a CCD camera (1X83 Olympus with PIXIS 1024 eXcel on TM from Princeton Instruments). The images were acquired using filter sets tailored to the specific emission and excitation wavelengths of different dyes (detailed in the fluorospectrometric measurements section above). For high-resolution imaging and axial scanning, a confocal microscope (Leica Stellaris) equipped with tunable excitation and emission bands and a 63X NA1.4 oil lens will be utilized.

[0065] 1.5 Absorption spectra

[0066] The spectra were acquired from solutions containing both NBE and unconjugated dye, with 50uL of each solution loaded into sub-micro-cuvettes. Measurements were conducted using a spectrophotometer-coupled microscope, utilizing the same setup as described in the fluorospectrometric section. Illumination was provided by a halogen lamp (Broadband 100 W from Olympus) at maximum intensity, equipped with an IR filter (750nm LP). Transmitted light was collected through a 150pm slit. The absorbance spectrum (A) was derived from the transmission spectrum (T) according to the Beer-Lambert law (eq 1), where P0 represents the initial light intensity and P denotes the light intensity measured after passing through the sample. eq 1: A = log101 / T= log10Po / P

[0067] 1.6 Cryo-TEM

[0068] Cryo TEM was used for Nano-Beads Emitters (NBE) size and shape characterization in a solution environment. For those measurement, 3 pl of NBE-G sample was loaded on a glow discharged (EmiTech KI 00 machine) lacey grids that were blotted and plunged into liquid ethane using a Gatan CP3 automated plunger and stored in liquid nitrogen until use Frozen specimen was transferred to the Gatan 914 cryo-holder and maintained at temperatures below -176 °C inside the microscope (to prevent ice crystals formation due to temperature rise). Samples were inspected with a Tecnai G2 microscope (FEI -Teramo fisher) with an acceleration voltage of 120 kV, which is equipped with a cryobox decontaminator. Images were taken using Digital Micrograph with a Mulitiscan Camera model 794 (Gatan) in different resolutions.

[0069] 2. Preparation of fluorescent thin films

[0070] 2.1 NBEs preparation:

[0071] NBEs synthesis is divided to two main parts: hydrogel nanoparticles (HNPs) preparation followed by attachment of fluorescence molecules to the HNPs as described below:

[0072] Step 1: Synthesis of hydrogel nanoparticles (HNPs)

[0073] HNPs were prepared according to the procedure described in Journal of Nanoparticle Research 2014, 16 (12)17wherein HNP is cross-linked PNIPAAM-co-PPO-PEO NP and PNIPAAM is Poly (JV-isopropylacrylamide. Briefly, mixture of 640 mg (Acr)Jeffaminei9oo and 188 mg NIPAAM (1.66 mmol) with 5mg BIS (0.03 mmol) and 5 mg of PVP360000 was dissolved in 8ml double distilled water (DDW). The reaction mixture was placed in water bath shaker and after adding KPS initiator solution (5 mg, 0.018 mmol in 2ml DDW) the polymerization process was allowed to continue at 73°C for 23 h in with nitrogen atmosphere. The resulting NP dispersion was dialyzed against 25 1 DDW for 2 weeks using 1,000 kDa cut-off molecular weight dialysis membrane (Spectrum Laboratories, Inc ).

[0074] The obtained HNP’s size (hydrodynamic diameter) and zeta potential were determined to be 25 3nm and -6 9m V. Those parameters were determined by DLS (Dynamic Light Scattering) at 25°C using Zetasizer Nano Series ZS (Nano-ZS, Malvern Instrument Ltd., UK) operating with a 4 mW HeNe laser (632.8nm), a detector positioned at a scattering angle of 173°, and a temperature-controlled jacket for the cuvette.

[0075] The Synthesis of partially acryloylated Jeffamine® ((Acr)Jeffamineigoo) was carried out as described in Auzanneau et al., J Pept Scil :31— 44, 1995 from Commercially available Jeffamine® (Mn=1900) and acryloyl chloride.

[0076] A preferred polymerization process for preparing the HNPs is shown in scheme 1 below:

[0077] Scheme 1: HNPs synthesis. The NPs can be synthesized in different sizes (10 nm - 400 nm) and are decorated with amine groups (for further bonding with dye molecules, as shown in Step

[0078] 2).

[0079] Copolymer nanobeadsaproduced by the method depicted in Scheme 1, and physico-chemical characterization thereof, are tabulated below: a] All the polymerizations performed in 10 ml H2O containing 188 mg NIPAAM, 5 mg BIS, 5 mg PVP and 5 mg K2S2O8 [b] “Block” refers to (Acr)i.i Jeffamine 1900. [c] The hydrodynamic diameter (Dh) represents the particle size of nanoparticles as measured by DLS at 25°C and 37°C. Each value is the average number-based values from 3-5 measurements by DLS. [d] Poly dispersity index (PDI) represents the relative variance in the particle size distribution, as further described in the Malvern Zetasizer instrument manual. In general, the more monodisperse the particles, the lower the PDI. Each value is the average number values calculated from Cumulant s analysis of the 3-5 DLS-measured intensities autocorrelation function. [e]The ((-potential is presented as mean value of five measurements ± standard deviation. Step 2: Attachment of fluorescence molecules to the HNPs for the preparation of NBEs

[0080] HNPs (lOnm) were labeled with several different dye molecules. The labeling process was conducted in a mild basic solution: lOmg sodium carbonate was added to 1ml of water containing 3mg hydrogel nanoparticles. The Img of the dye was dissolved in 50pL of dry DMSO and then added to the HNPs solution. The solution was stirred over night at room temperature. To remove non-bound excess dye, a dialysis period of three days was implemented using a Cellulose Ester Spectra / Pore membrane with a 1,000 kDa cut-off (from Spectrum Laboratories, Inc.) to achieve purified NBE solutions.

[0081] The process for attaching the dye molecules to the HNPs may be illustrated according to scheme 2 below:

[0082] Scheme 2: attachment of dye to HNPs. Dye molecules with / -Hydroxysuccinimide (NHS) ester conjugation domain are mixed with HNPs solution in mild basic conditions (NaCCh). A nucleophilic attack of the primary amines (NH2 groups on the NHP surface) on the NHS ester domain release the NHS leaving group and bonds the dye to the nanoparticle. After proton removal with basic catalysis, a stable amide bond is formed attaching the emitter molecule to the NHP’ s surface.

[0083] The molecule types, along with their respective excitation and emission wavelengths, are outlined in Table 2 below:

[0084]

[0085] Table2: Molecular Emitters used for NBE preparation. For ATTO dye info taken from Atto¬

[0086] Tec data base. For TAMRA ref

[0019] , for FAM ref.

[0020] , The chosen set includes a series of emitters, with small to large Stokes’ shift and their emission spans from 480 nm to 650 nm, covering quite abroad optical range (as checked experimentally and presented in Figure 1).

[0087] 2.2. Deposition of thin fluorescence films

[0088] Substrate cleaning

[0089] Borosilicate (BK-7) optical-grade round cover slips (170pm thick, 25mm diameter, from Menzel-Glaser) were used as substrates for sample preparation. The substrates were cleaned using Hellmanex®(III) solution and sonication. Afterwards the substrates were washed with ethanol and deionized water and dried under compressed air stream.

[0090] Thin film deposition

[0091] The NBEs are deposited onto different surfaces using a modified layer-by-layer (“drop- LBL ”21technique. First the sample is docked to a on thick microscope slide (2000pm) with stripes of Capton® tape (polyimide film). Then surface preparation treatments are performed to increase the connectivity of the negatively charged NBEs:

[0092] • For enhanced wetting of dielectric substrates, such as fused silica, silicon wafers, and polymer and MgF? spacers, the Corona surface activation treatment (CT or air plasma) is beneficial. This treatment, typically performed using the VETAPHONE iCorona TF- 415 machine, lasts for 45-60 seconds with 1 kW power for inorganic materials and 30 seconds with 0.3 kW for polymer surfaces.

[0093] • A Primer layer of PDDA is deposited onto the surface to improve the adhesion of negatively charged dyes. The PDDA solution, prepared by diluting a mixed molecular weight stock to 2% wt (Sigma Aldrich #522376), is applied using drop layer-by-layer (LBL) deposition prior to introducing the dye to the surface.

[0094] Optimal film deposition conditions were established for each substrate, and the process remains indifferent to the NBE type and it’s labeling. Accordingly, the NBE solution was drop-casted onto the samples surface using a micrometric pipettor. To remove the excess solution, it was blown away by nitrogen gas steam (pure N2, 5-6 atm) providing also brief drying of the surface.

[0095] The entire process for the preparation of thin fluorescence films may be illustrated according to scheme 3 below: A

[0096] Scheme 3: A process for fabrication of homogenous thin films of amine-reactive fluorescent molecules. (A) NBE synthesis: the dye and the Hydrogel nano particle (HNP) are chemically attached by reaction of amine groups on the surface of the particle and NHS ester domain of the dye. This step is done at room temperature in mild basic solution, to obtain NBEs liquid solution after dialysis process. (B) Layer deposition: the deposition onto surfaces take place after cleaning and surface activation done by plasma (CT) orPDDA base layer. The molecules are electrostatically attached to the surface, forming thin fluorescent homogenous film. (C) The Layer-by-Layer (LBL) deposition process involves several steps: initially, a positively charged PDDA polymer base layer is deposited onto the substrate from a solution, followed by the removal of excess material using a nitrogen blow. Subsequently, a negatively charged NBE solution is introduced to the surface and similarly removed after a brief exposure period of 20- 30 seconds. The electrostatic attachment of NBEs to the surface occurs during this phase, accompanied by a dehydration process under low-pressure conditions. This results in a substantial reduction in the volume of NBEs, leading to the formation of an ultra-thin film with a thickness of less than 10 nanometers.

[0097] Polymer protective layer deposition

[0098] MY-133-MC polymer (manufactured by MY Polymers LTD, Ness-Ziona, Israel) was applied by drop casting the undiluted solution onto the surface of the dry sample. This polymer undergoes humidity curing, requiring the samples to be stored under a wet beaker overnight. As a result, the cured polymer formed a thick capping layer (exceeding 2pm) over the sample, offering an optical environment akin to water (refractive index of 1.33) and provide physical protection and isolation from air to the thin layers.

[0099] 3. Characteristics of the fluorescent thin films

[0100] 3.1 Morphological and Optical characterizations of NBE thin films

[0101] Seven distinct NBEs were successfully synthesized with a set of the dye molecules, as summarized in Table 2. Utilizing the drop layer-by-layer (LBL) technique with plasma and PDDA surface treatment, thin films were deposited on fused silica substrates from these NBEs. Clearly, the attachment of the molecules to the beads prevents their aggregation, stabilizes them and retains their fluorescence.

[0102] The resulting transparent layers exhibited bright emission easily captured by a smartphone camera (see Figure. 3B). Furthermore, the emission spectra of the films, as depicted in Figure 5A, demonstrate a significant resemblance to the emission of the corresponding free dye solution (refer to Figure 7). This similarity suggests that the chemical attachment of molecules to the beads does not change their photophysical behavior in solution. It is assumed that such assembly prevents their aggregation, stabilizes them, and thus maintains their fluorescence properties in thin dry films. In this platform, the fluorescence molecules are spatially isolated from each other, a distance that could potentially be controlled by the number of functional groups on the hydrogel beads Additionally, their deposition onto fused silica resulted in homogeneous, smooth films over a considerable surface area. AFM scanning (Figure 2C) revealed that all NBE films exhibited the same morphological properties, independent of the dye attached to the beads. High-profile AFM scans of the films indicated low roughness and thickness of approximately 8 nm (Figure 8). This reduced thickness is attributed to the volume reduction of the 20nm beads (hydrodynamic radius) during the drying process, as confirmed by cryo TEM imaging (presented in Figure 9). As can be seen in Figure 9, the average diameter of the particles was 1 Inm (7-24nm size range was detected) while in solution the average size was 36nm (24-105nm range). Both values are different from the blank HNPs size measured by the DLS. The effect of the volume reduction during the drying is related to the HNPs and not to the fluorescent molecules which are attached to them.

[0103] The spread of the beads on the surface is homogenous without a tendency to aggregate, as demonstrated in the AFM scans (Zmax < 15nm) and highlighted by the low deviation in emission intensity across the millimeter area (presented in Figure 10). This high homogeneity can be explained by the negative charge of the hydrogel core of the NBE, inducing the spreading of the nanoparticles due to electrostatic repulsion between them and preventing agglomeration. Additionally, this charge provides a good affinity to the positively charged surface covered by PDDA chains.

[0104] The NBEs of the present disclosure were used not only to form single color fluorescent layers (Figure 3A), but also two-color fluorescent layers giving rise to white emission, for example, by using green and red NBEs as shown in Figure 3B.

[0105] 3.2 Comparison between native dye vs NBE dry films

[0106] To illustrate the advantages of using NBE in the fabrication of thin fluorescence films, native dyes (unconjugated) were also deposited onto the surface. The concentration of the solutions containing native dyes was adjusted to provide a similar absorption intensity as the corresponding NBE solutions (Absorption spectra presented in Figure 7B). Each pair of samples (native dye and NBE film) underwent examination using a fluorescent microscope coupled to a spectrograph under similar conditions. Spectra from several areas of each film (4- 5 area of 30x30pm) were averaged and presented in Figure 4A Fluorescent images were obtained from the different areas during the measurements.

[0107] The emission spectra, as depicted in Figure 4A, revealed that the NBE layers exhibited significantly higher brightness. Regardless of their labeling dye, all NBE films displayed emission intensities several times higher than those observed in films of native dyes. Additionally, during the measurements, the shape and intensity of the peaks from NBE layers remained stable and uniform, while in their native dye counterparts, both parameters varied noticeably. Fluorescent imaging (Figure 4B) of the layers provided insight into the reason behind these observations. In samples made from unconjugated dyes, crystals and agglomerations of molecules were commonly found. In contrast, NBE films of all colors formed fluorescent, smooth, and homogeneous films with a uniform texture. This property of the films demonstrates the platform's ability to eliminate the inherent tendency of dye molecules to interact with themselves in solventless conditions, enabling the formation of thin films.

[0108] 3.3 Stability and homogeneity of NBE films

[0109] The study focused on the aging and stability of the thin molecular films. In Figure 5, two case studies, NBE-G and NBE-R, are presented. Initially, it was demonstrated that even after several months (more than 6 months) without any protective layer, these films maintained relatively high fluorescence intensities, with a maximum reduction of approximately 20% for NBE-R and only 7.4% for NBE-G. Some minor modifications in the emission spectrum of NBE-R were observed (see Figure 5 A).

[0110] Introducing a protective polymer layer increases the stability of the thin NBE layer by offering physical shielding, isolating it from air to prevent oxidation, and creating the desired optical environment for the emitters. This layer should consist of a hydrophobic polymer to ensure that the deposition does not disrupt the NBE film. Protective layers composed ofMY133 (see 2.3 in M&M) have been shown to substantially decrease degradation in molecular thin layers.

[0111] Additionally, the homogeneity of these NBE thin layers was assessed by scanning and imaging fluorescence intensities over large distances / areas. The results showed moderate variations of less than 10% over distances larger than two microns.

[0112] The stability of NBE-G / R thin layers was also examined under continuous laser illumination. The stability results are depicted in Figure 5C and Figure 5D. In confocal microscopy, where the laser illumination is relatively strong (50uW in a confined spot), the reduction in fluorescence intensities after 180 seconds of continuous illumination was not more than 30%. Comparable findings were demonstrated in continuous EPI illumination over 550 seconds, confirming the robust photostability of the NBEs.

[0113] 3.4 NBE films on different substrates

[0114] As illustrated in Figure 6, thin and homogeneous films were deposited on substrates of varying nature, including silver with a silica iSiOr) thin passivation layer, smooth aluminum with native oxide only on the surface, atomically flat silicon wafer, and magnesium fluoride surface.

[0115] While the results in Figure 6 specifically pertain to NBE-G, it is reasonable to infer, based on previous outcomes with different NBEs (as shown in Figure 5), that the film properties remain independent of the labeling molecules. The deposition procedure in all cases was drop layer-by-layer (LBL) based, with slight modifications optimizing the results. For instance, fine films on silicon wafers were achieved after a 60-second long CT treatment before introducing PDDA It's noteworthy that the oxide layer, post-treatment, didn't undergo noticeable changes (confirmed with ellipsometer), but the film quality improved.

[0116] On the other hand, substrates such as MgF2, Ag+SiO2, and Al demonstrated optimal results with no CT treatment at all. Across all substrates, the layers exhibited millimeter-sized homogeneous areas, bright emission (with no change in the spectrum), and nanometric thickness. Variations in thickness and surface patterns were attributed to hydrophilicity: charged PDDA and NBE adhered better to more hydrophilic surfaces, such as fused silica or MgFz When hydrophilicity was lower, NBE tended to concentrate in tiny water droplets on the surface until complete evaporation occurred, resulting in a dotted pattern and increased layer thickness (as observed in Si substrate, for example).

[0117] RFERENCES

[0118] (1) Ha, J. M.; Hur, S. H ; Pathak, A.; Jeong, J. E.; Woo, H. Y. Recent Advances in Organic Luminescent Materials with Narrowband Emission. NPG Asia Mater 2021, 13 (1). https: / / doi org / 10 1038 / s41427-021-00318-8

[0119] (2) Grimm, J. B.; Lavis, L. D. Caveat Fluorophore: An Insiders’ Guide to Small-Molecule Fluorescent Labels. Nature Methods. 2022, pp 149-158. https: / / doi.org / 10.1038 / s41592- 021-01338-6.

[0120] (3) Li, S.; Mu, B.; Wang, X.; Wang, A. Recent Researches on Natural Pigments Stabilized by Clay Minerals: A Review. Dyes and Pigments 2021, 190 (February), 109322. https: / / doi.org / 10.1016 / j dyepig.2021.109322.

[0121] (4) Aharon, E.; Kalina, M ; Frey, G. L. Inhibition of Energy Transfer between Conjugated Polymer Chains in Host / Guest Nanocomposites Generates White Photo- and Electroluminescence. J Am Chem Soc 2006, 128 (50), 15968-15969. https: / / doi . org / 10. 1021 / j a066290m.

[0122] (5) Amirav, L ; Alivisatos, a. P. Luminescence Studies of Individual Quantum Dot Photocatalysts. J Am Chem Soc 2013, 135 (35), 13049-13053. https : / / doi . org / 10. 1021 / j a404918z.

[0123] (6) Baldo, M. A.; O’Brien, D. F.; You, Y.; Shoustikov, A.; Sibley, S.; Thompson, M. E.; Forrest, S. R. Highly Efficient Phosphorescent Emission from Organic Electroluminescent Devices. Nature 1998, 395 (6698), 151-154. https: / / doi.org / 10.1038 / 25954.

[0124] (7) Panfil, Y. E.; Oded, M.; Banin, U. Colloidal Quantum Nanostructures: Emerging Materials for Display Applications. Angewandte Chemie International Edition 2018, 57 (16), 4274-4295. https: / / doi.Org / https: / / doi.org / 10.1002 / anie.201708510.

[0125] (8) Xia, C ; Zhu, S.; Feng, T.; Yang, M.; Yang, B. Evolution and Synthesis of Carbon Dots: From Carbon Dots to Carbonized Polymer Dots. Advanced Science 2019, 6 (23), 1 01316. http s : / / doi . org / http s : / / doi . org / 10.1002 / advs .201901316.

[0126] (9) Liu, L ; Najar, A.; Wang, K.; Du, M.; Liu, S. (Frank). Perovskite Quantum Dots in Solar Cells. Advanced Science 2022, 9 (7), 2104577. https: / / doi.Org / https: / / doi.org / 10.1002 / advs.202104577.

[0127] (10) Prasad, P. K.; Motiei, L.; Margulies, D. Steps toward Enhancing the Fluorescence of Small-Molecule-Based Protein Labels Using Supramolecular Hosts. Results Chem 2021, 3, 100134. https: / / doi.Org / https: / / doi.org / 10.1016 / j.rechem.2021.100134.

[0128] (11) Aida, T.; Meijer, E. W.; Stupp, S. I. Functional Supramolecular Polymers. Science (1979) 2012, 335 (6070), 813-817. https: / / doi.org / 10.1126 / science.1205962.

[0129] (12) Perego, J.; Bezuidenhout, C. X.; Villa, I.; Cova, F.; Crapanzano, R.; Frank, I.; Pagano, F.; Kratochwill, N.; Auffray, E.; Bracco, S.; Vedda, A.; Dujardin, C.; Sozzani, P. E.; Meinardi, F.; Comotti, A.; Monguzzi, A Highly Luminescent Scintillating Hetero- Ligand MOF Nanocrystals with Engineered Stokes Shift for Photonic Applications. Nat Commun 2022, 13 (1). https: / / doi.org / 10.1038 / s41467-022-31163-0.

[0130] (13) Wang, L.; Li, W.; Yin, L.; Liu, Y.; Guo, H.; Lai, J.; Han, Y.; Li, G.; Li, M.; Zhang, J.; Vajtai, R , Ajayan, P M ; Wu, M. Full-Color Fluorescent Carbon Quantum Dots Sci Adv 2020, 6 (40), 1-9. https: / / doi.org / 10.1126 / sciadv.abb6772.

[0131] (14) Miao, Y.; Yin, M. Recent Progress on Organic Light-Emitting Diodes with Phosphorescent Ultrathin (<lnm) Light-Emitting Layers. iScience 2022, 25 (2), 103804. https: / / doi.org / 10. 1016 / j isci.2022.103804.

[0132] (15) Li, Q , Li, Z. The Strong Light-Emission Materials in the Aggregated State: What Happens from a Single Molecule to the Collective Group. Advanced Science 2017, 4 (7), 1-15. https: / / doi.org / 10.1002 / advs.201600484.

[0133] (16) Benson, C. R.; Kacenauskaite, L.; VanDenburgh, K. L., Zhao, W.; Qiao, B.; Sadhukhan, T.; Pink, M.; Chen, J.; Borgi, S.; Chen, C. H.; Davis, B. J.; Simon, Y. C.; Raghavachari, K.; Laursen, B. W.; Flood, A. H. Plug-and-Play Optical Materials from Fluorescent Dyes and Macrocycles. Chem 2020, 6 (8), 1978-1997. https : / / doi . org / 10. 1016 / j chempr.2020.06 029.

[0134] (17) Khandadash, R , Machtey, V., Shainer, I. et al. Novel biocompatible hydrogel nanoparticles: generation and size-tuning of nanoparticles by the formation of micelle templates obtained from thermo-responsive monomers mixtures. J Nanopart Res 16, 2796 (2014).

[0135] (18) Palakkal, S., Logviniuk, D. & Byk, G. Tuning the size and hydrophobicity of nanohydrogels exploiting a self-assembly assisted polymerization mechanism for controlled drug delivery. J Nanopart Res 22, 354 (2020).

[0136] (19) N. Archipowa et al., “Characterization of Fluorescent Dyes Frequently Used for Bioimaging: Photophysics and Photocatalytical Reactions with Proteins,” J Phys Chem B, vol. 127, no. 44, pp. 9532-9542, 2023.

[0137] (20) A. L. F. Baptista, P. J. G. Coutinho, M. R. Oliveira, and J. I N R. Gomes, “Effect of pH on the control release of microencapsulated dye in lecithin liposomes. II,” J Liposome Res, vol. 13, no. 2, pp. 123-130, 2003.

[0138] (21) L.-R. Cai, Organic-inorganic layer-by-layer self-assembled multilayer films: Preparation, characterization and applications. Hong Kong University of Science and Technology (Hong Kong), 2004.

Claims

CLAIMS1. A fluorescent film comprising nanobeads emitters.

2. A fluorescent film according to claim 1, wherein the nanobeads emitters are fluorescent dye-conjugated copolymer nanobeads3. A fluorescent device comprising a base substrate and a fluorescent film of claim 1 or 2 deposited thereon.

4. A fluorescent device according to claim 3, wherein the base substrate comprises fused glass, borosilicate glass; magnesium fluoride; crystalline silicon, optionally with native oxide layer on its surface; silver, optionally with a thin silica layer on its surface; or aluminum, optionally with a thin native oxide layer on its surface.

5. A fluorescent device according to claim 3 or 4, wherein the copolymer nanobeads are copolymers of a mixture of amphiphilic macromonomers, with fluorescent dyes covalently bonded to the chains the copolymer.

6. A fluorescent device according to claim 5, wherein the fluorescent dyes are covalently bonded to the ends of the copolymer's chains through an amide bond.

7. A fluorescent device according to claim 5 or 6, wherein the copolymer nanobeads are copolymers of a mixture comprising a first amphiphilic macromonomer, a second amphiphilic macromonomer, a third monomer, and crosslinker, all having acrylamide functionality as the polymerizable site.

8. A fluorescent device according to claim 7, wherein: the first macromonomer is CH2=CH-C(O)-NH-A-B-A-NH-C(O)-CH=CH2; the second macromonomer CH2=CH-C(O)-NH-A-B-A-NH3+X‘; the third monomer is N-isopropylacrylamide; and the crosslinker is bis-acrylamide; wherein A and B polypropylene oxide and polyethylene oxide blocks, respectively, and X is a counter anion.

9. A fluorescent device according to claim 8, wherein the nanobead copolymer is the copolymerization product of a mixture comprising: a first macromonomer, which is CH2=CH-C(O)-NH-A-B-A-NH-C(O)-CH=CH2; a second macromonomer, which is CH =CH-C(O)-NH-A-B-A-NH3+X"; a third monomer, which is N-isopropyl acrylamide, a crosslinker, which is bis-acrylamide; a free radical initiator, which is potassium persulfate; anda stabilizer, which is a non-ionic surfactant.

10. A fluorescent device according to any one of claim 3 to 9, wherein the fluorescent film is 5 to 500 nm thick transparent or translucent film.11 . A fluorescent device according to any one of claims 3 to 10, comprising an adhesion layer applied on the surface of the base substrate, interposed between the base substrate and the fluorescent film.

12. A fluorescent device according to claim 11, wherein the adhesion layer consists of a cationic polyelectrolyte.

13. A fluorescent device according to any one of claims 3 to 12, further comprising a protective layer applied atop of the fluorescent film.

14. A fluorescent device according to any one of claims 3 to 13, characterized by uniform distribution of the fluorescent dyes across the film, indicated by essentially constant fluorescent emission intensity measured at '. ax at different points of the film over distance of 1 mm, with maximal deviation of 10.0% from the mean observed15. A fluorescent device according to any one of claims 3 to 14, wherein the film is characterized by average surface roughness measured by atomic force microscopy of less than 1 nm.

16. A fluorescent device according to any one of claims 3 to 15, comprising multiple color film.

17. A process for fabricating a fluorescent device, comprising depositing a dispersion of fluorescent dye-conjugated copolymer nanobeads onto a base substrate and optionally drying to create a thin film.

18. The process according to claim 17, wherein the deposition of the dispersion is done by drop-casting.

19. The process according to any one of claims 17-18, wherein the base substrate undergoes a pretreatment step, such as cleaning and corona surface activation treatment, prior to the deposition of the dispersion onto the surface of the base substrate.

20. The process according to any one of claims 17-19, wherein the deposition is a layer-by layer deposition, comprising applying a solution of a cationic poly electrolyte onto the surface of the base substrate to create an adhesion layer and applying a suspension of the fluorescent dye-conjugated copolymer nanobeads onto the adhesion layer.

21. The process according to any one of claims 17-20, further comprising applying a polymeric protective layer on top of the fluorescent film.

Images