Quantum dots

Abstract

The invention relates to a plurality of tin sulfide quantum dots having a mean atomic ratio of tin to sulfur of from 0.5:1 to 10.0:1, a mean diameter of 0.50 nm to 7.00 nm, and which are photoluminesc

Description

FIELD OF THE INVENTION

[0001] The invention relates to quantum dots, particularly tin sulfide quantum dots, and to a method for their manufacture. The invention further relates to uses of the quantum dots in bioimaging and for the manufacture of biofilms. BACKGROUND

[0002] Quantum dots (QDs) are nanoparticles of a semiconductor material with diameters that are typically 10.0 nm or less. QDs display quantum confinement effects. The semiconductor band gap of QDs is determined by their particle size. The band gap increases as the diameter of the QD decreases.

[0003] Since the band gap is determined by the diameter of the QD, when QDs are photoirradiated they produce photoluminescence at a wavelength (colour) determined by the diameter of the QD. One way of controlling the wavelength of the photoluminescence is by controlling the diameter of the QDs.

[0004] The imaging of cells and tissues through the use of detectable labels is a technique that is commonly used in bioanalysis. Such techniques are advantageous because they often have high sensitivity, and both fast acquisition times and responses, as a result of the fluorescence lifetimes of the labelling materials that are used.

[0005] Various materials have been used as labels in bioimaging, such as complex organic dyes, fluorescent proteins and fluorescent nanoparticles. The most common labelling materials are organic dyes and fluorescent proteins. However, there are serious concerns associated with the use of these materials because of their potential toxicity, particularly their carcinogenicity, to living organisms. These labelling materials may also suffer from poor photochemical stability and can have photobleaching characteristics, relatively short lifetimes and a narrow excitation range, which can significantly hinder their performance in life science applications.

[0006] To avoid these drawbacks, fluorescent quantum dots have been considered for use as labelling materials. There have been problems providing biocompatible quantum dots that can be used as labels in bioimaging. Despite the development of convenient synthetic methods for the manufacture of certain types of quantum dot, the materials used in these quantum dots are problematic for use in bioimaging. For example, cadmium- or lead-containing quantum dots, such as CdTe, CdSe, CdS, PbS and PbSe, are generally toxic to biological systems. Moreover, indium and tellurium are relatively rare materials, 1 which raises concerns about the production and the eventual use of indium- and telluriumcontaining quantum dots (e.g. InAs, InP and CdTe) in bioimaging applications. There have also been problems with solubility.

[0007] Quantum dots that have been described in the prior art, particularly tin sulfide quantum dots, have photoluminescence properties that only cover some subset of the visible spectrum or the near-infrared (NIR) spectrum, not the full spectrum. There thus remains the need for the development of new tin sulfide quantum dots which possess photoluminescence over a broader range of wavelengths.

[0008] The quantum dots of the invention are biocompatible and are non-toxic to living cells. The quantum dots are stable, particularly photostable, and can have photoluminescence properties that are suitable for bioimaging and / or multiplex bioimaging. The quantum dots are highly fluorescent and have size-dependent and compositiondependent, multi-colour ultraviolet, visible and near-infrared emission properties. The quantum dots may also be used to manufacture biofilms. SUMMARY OF THE INVENTION

[0009] In a first aspect, the invention provides a plurality of tin sulfide quantum dots having a mean atomic ratio of tin to sulfur of from 1.1:1 to 10.0:1, a mean diameter of 0.50 nm to 5.00 nm, and which are photoluminescent at wavelengths of (a) 360 to 470 nm, (b) 475 to 545 nm and (c) 550 to 740 nm.

[0010] In a second aspect, the invention provides a hot-injection synthesis method for manufacturing a plurality of tin sulfide (SnS) quantum dots as herein defined, wherein the method comprises: (i) dispersing a metallic precursor comprising tin (Sn) in an organic solvent at a first temperature of 50°C to 150°C to produce a reactant liquid; and (ii) mixing a precursor comprising sulfur (S) with the reactant liquid at a second temperature of 25°C to 55°C.

[0011] In a third aspect, the invention provides a monodisperse plurality of tin sulfide quantum dots having a mean atomic ratio of tin to sulfur of from 0.5:1 to 1.5:1, a mean diameter in the range of 2.00 nm to 7.00 nm, and which are photoluminescent at a wavelength from 450 nm to 550 nm, wherein said quantum dots comprise a surface modifying agent which comprises an organic polymer.

[0012] In a fourth aspect, the invention provides a hot-injection synthesis method of manufacturing a monodisperse plurality of tin sulfide (SnS) quantum dots as herein defined, wherein the method comprises: (i) dispersing an organic polymer in an organic solvent at a first temperature of 50°C to 150°C to produce a reactant liquid; and (ii) mixing a precursor comprising tin (Sn) and a precursor comprising sulfur (S) with the reactant liquid at a second temperature of 125°C to 160°C.

[0013] In a further aspect, the invention provides a method of imaging a biological target comprising: administering the plurality of tin sulfide quantum dots as herein defined to the biological target; and imaging the biological target

[0014] In another aspect, the invention provides a plurality of quantum dots as herein defined for use in a diagnostic method practised on the human or animal body.

[0015] In yet another aspect, the invention provides a method for manufacturing a biofilm comprising contacting the quantum dots as herein defined with a plurality of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention is further described hereinafter with reference to the accompanying drawings.

[0017] Figure 1 shows in (A) UV-Vis absorption spectra of some SnS quantum dots (QDs) of the invention, and (B) calculated band-gap energy values of the QDs.

[0018] Figure 2 shows TEM micrographs of SnS QDs produced from elemental sulfur (“SnS-S”; see (A) and (C)) and SnS QDs produced from (TMS)2S (“SnS-TMS”; see (B) and (D)). The images in (A) and (B) are low magnification images (the scale bar in the bottom right hand corner is 50 nm). Images (C) and (D) are HRTEM micrographs (the scale bar in the bottom right hand corner of these images is 2 nm).

[0019] Figure 3 shows normalized photoluminescence (PL) spectra of (A) SnS-TMS and (B) SnS-S QDs. The samples were illuminated with excitation wavelengths from 300 nm to 550 nm.

[0020] Figure 4 shows low resolution (in (A) and (B)) and high-resolution (in (C) and (D)) TEM images with FFT micrographs) of SnS QDs.

[0021] Figure 5 shows room temperature (A) 3D photoluminescence (PL) and (B) 3D photoluminescence excitation (PLE) spectra of SnS QDs of Example 3 showing multiple populations of QDs as a function of excitation wavelength. Figure 5 shows (C) an image of the emission properties of SnS QDs excited at 405 nm, 532 nm, and 650 nm laser wavelength.

[0022] Figure 6 shows low and high-resolution TEM (HRTEM) images of SnS QDs with a mean QD size of 4.8 nm.

[0023] Figure 7 shows (A) time-resolved photoluminescence (TRPL) decay spectra of SnS QDs of Example 3. (B) Biexponential fittings of the PL decay curves in (A). Data collected at 300 K.

[0024] Figure 8 shows (A) UV-Vis absorption spectra of some SnS / PVP QDs and (B) calculated optical band energy value of the QDs at reaction times ranging between 30 and 120 minutes

[0025] Figure 9 shows (A) PL spectrum and (B) PLE spectra of SnS / PVP QDs synthesized at different reaction times. Figure 9 shows (C) an image of the emission properties of SnS / PVP QDs collected at reaction times between 30 and 120 minutes. The samples were excited at 365 nm.

[0026] Figure 10 shows a summary graph of the PL emission characteristics of SnS / PVP QDs of Example 5 comparing the PL emission intensity measured at growing intervals between 20 and 120 minutes. Samples were excited at 470 nm reaching a maximum PL emission intensity.

[0027] Figure 11 shows a summary plot of the PL emission changes in SnS / PVP QDs of Example 6. The PL emission intensity of the highest intensity PL peaks at each concentration and reaction time were compared.

[0028] Figure 12 (A) shows (g) low and (a, d, h) high-resolution TEM micrographs of single QDs with clear lattice fringes, (c, f, j) FFT patterns and (b, e, i) HRTEM reconstruction images generated from the FFT patterns of SnS / PVP QDs.

[0029] Figure 13 shows (A) time-resolved photoluminescence (TRPL) decay spectra of SnS / PVP QDs of Example 6. The sample was excited at 430 nm measuring the TRPL lifetime at 490 nm. (B) Changes in the TRPL lifetime of QDs as a function of growing time obtained from the fitted TRPL decay curves in (A). Data collected at 300 K.

[0030] Figure 14 is a photograph of samples of a bacterial suspension of P. fluorescens that has been incubated for 20 hours with each of (a) a reference sample with no quantum dots, (b) 0.8% v / v of SnS / PVP quantum dots, (c) 0.8 % v / v SnS / OA-OLA quantum dots, (d) closer look of 0.8 % v / v SnS / OA-OLA quantum dots, which resulted in biofilm formation, and (f) a zoomed-in image showing a closer view of the biofilm region on the walls of the centrifuge tube.

[0031] Figure 15 shows (a) a high-resolution fluorescence image of a sample of P. fluorescens cells that were incubated with 0.8 % v / v SnS / OA-OLA quantum dots for 20 hours, (b) the sample imaged under fluorescence (FL), (c) a bright-field (BF) image of the sample, and (d) a merged mode image of the sample with an optical or confocal microscope.

[0032] Figure 16 shows growth curves for P. fluorescens bacterial cells incubated with different dosages of SnS / OA-OLA quantum dots for 5 hours.

[0033] Figure 17 shows lambda scan spectra of the fluorescence intensity of SnS / OA-OLA quantum dots, which were measured by collecting light in 10 nm bands from 420 nm to 600 nm, 490 nm to 690 nm and 570 nm to 760 nm at 405 nm, 488 nm and 561 nm excitation wavelengths, respectively.

[0034] Figure 18 shows a series of high-resolution fluorescence images of P. fluorescens biofilms formed in the presence of SnS / OA-OLA quantum dots after 20 hours of incubation imaged under (A) fluorescence (FL) excited at 405 nm (blue) laser wavelength, (B) fluorescence (FL) excited at 488 nm (green) laser wavelength, and (C) fluorescence (FL) excited at 561 nm (red) laser wavelength, (D) bright field mode with a confocal microscope, and (E) a merged mode image of the sample with an optical or confocal microscope. DEFINITIONS

[0035] The term “quantum dot” as used herein refers to a crystal, preferably of a semiconductor, having a particle size, particularly a diameter, of 10.0 nm or less. The crystal is typically a nanocrystal. For a crystal, the diameter may refer to the mean (e.g. arithmetic mean) of the largest lattice constant and the smallest lattice constant.

[0036] Any reference to, for example, “SnS quantum dots” as used herein refers to quantum dots that comprise a tin sulfide as a binary compound without any restriction to the stoichiometry of tin and sulfur or the phase of the compound, unless the context indicates otherwise. The quantum dots of the invention comprise, or consist essentially of, a tin sulfide. Thus, the invention relates to plurality of quantum dots comprising, or consisting essentially of, a tin sulfide.

[0037] The term “biocompatible” as used herein refers to the ability of the quantum dots to perform with an appropriate host response in a specific application (e.g. as set out in ISO 10993-1 (2018)). The definition of “biocompatibility”, its associated terms and tests for its evaluation in ISO 10993-1 (2018) are incorporated herein by reference. In general terms, biocompatibility refers to the ability of, for example, the quantum dots to perform their desired function, such as bioimaging, without eliciting any undesirable local or systemic effects in a biological recipient.

[0038] The term “alkyl” as used herein refers to a straight, branched or cyclic hydrocarbon radical consisting of carbon and hydrogen atoms, and containing no unsaturation. A “C1-6- alkyl” group contains one to six carbon atoms. In general, it is preferred that the alkyl group is acyclic (e.g. straight or branched). Unless stated otherwise specifically in the specification, an alkyl group is unsubstituted or may be substituted by one or more substituents. When the alkyl group is substituted, then each substituent may be selected from hydroxy and C1-6 alkoxy. Each substituent is preferably unsubstituted. It is preferred that the alkyl group is unsubstituted.

[0039] The term “alkoxy” as used herein refers to a radical bonded through an oxygen atom of the formula -O-alkyl, where the alkyl group is defined above and may, for example, be a Ci-6-alkyl group (i.e. Ci-6-alkoxy group). An alkoxy group is unsubstituted, unless state otherwise.

[0040] The term "arylalkyl" as used herein refers to a radical bonded to an alkyl group, such as defined above, which alkyl group is further substituted by (or bonded to) an aryl group. The term "aryl" as used herein refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen atoms and carbon atoms, where at least one of the rings in the ring system is fully unsaturated (i.e. it contains a cyclic, delocalized [4n+2] TT-electron system in accordance with the Huckel theory). The aryl group may have 6 to 10 carbon atoms (e.g. Cs-io-aryl), preferably 6 carbon atoms (e.g. phenyl). Examples of arylalkyl groups include benzyl (PhCH2-) and phenylethyl.

[0041] The term “polydispersity” as used herein (also referred to in the art as “dispersity”) is a parameter relating to a particle size distribution (e.g. a diameter distribution of the quantum dots). It is a measure of the heterogeneity of a particle size distribution. From the distribution, the polydispersity is calculated as the standard deviation divided by the arithmetic mean of the particle size (e.g. diameter), expressed as a percentage. The particle size distribution may be unimodal and / or non-uniform. A Gaussian curve typically can be fitted to the distribution. The Gaussian curve can be used to calculate the polydispersity.

[0042] For the avoidance of doubt, the expression “consists essentially of’ as used herein limits the scope of a feature to include the specified materials, and any other materials or steps that do not materially affect the basic and novel characteristics of that feature, such as, for example, minor impurities. The expression “consists essentially of’ embraces the expression “consisting of’. DETAILED DESCRIPTION

[0043] The invention provides a plurality of tin sulfide quantum dots. These quantum dots comprise, or consist essentially of, a tin sulfide. Each quantum dot of the plurality of quantum dots comprises, or consists essentially of, a particle of the tin sulfide.

[0044] The invention provides a plurality of tin sulfide quantum dots which are photoluminescent at wavelengths of (a) 360 to 470 nm, (b) 475 to 545 nm and (c) 550 to 740 nm (referred to herein as “the first aspect”) and a plurality of tin sulfide quantum dots which are photoluminescent at a wavelength of 450 to 550 nm (referred to herein as “the second aspect”).

[0045] The quantum dots (also referred to herein as a plurality of quantum dots) may be dispersed in a liquid (e.g. they are a liquid dispersion). The liquid may be a solvent, such as described below in the context of the method of the invention.

[0046] The dispersion may be a suspension of the quantum dots, a colloid of the quantum dots or a solution of the quantum dots. Typically, the dispersion is a colloid of the quantum dots.

[0047] Each quantum dot may be substantially spherical, substantially spheroidal or substantially semi-spherical. Thus, most (>50 % by number) of the quantum dots are substantially spherical, substantially spheroidal or substantially semi-spherical.

[0048] When the quantum dots are substantially spheroidal, then the aspect ratio of the quantum dots may be from 1.1 to 3.5, preferably from 1.5 to 3.0, more preferably from 2.0 to 2.5. The aspect ratio is the ratio of the length of the minor axis (e.g. the minimum diameter) to the length of the major axis (e.g. the maximum diameter).

[0049] The diameter of a quantum dot can be measured using transmission electron microscopy (TEM). Any diameter referred to herein refers to the primary diameter of the quantum dot (i.e. it does not refer to the diameter of an aggregate of quantum dots). When a quantum dot is non-spherical, then the diameter refers to the mean (e.g. arithmetic mean) of the maximum diameter and the minimum diameter that is measured for the quantum dot.

[0050] In the first aspect, the quantum dots may have diameters in the range of 0.76 nm to 7.00 nm, preferably diameters in the range of 0.78 nm to 6.00 nm, such as 1.12 nm to 5.00 nm. For example, the quantum dots may have diameters in the range of 0.76 nm to 4.50 nm, or in the range of 1.12 nm to 3.50 nm.

[0051] The quantum dots of the first aspect have a mean diameter of 0.50 nm to 5.00 nm, preferably 0.70 nm to 3.50 nm, more preferably 1.00 nm to 2.50 nm. The mean diameter may be from 0.50 nm to 2.00 nm, such as when the quantum dots have diameters in the range of 0.10 nm to 4.50 nm. The mean diameter may be from 1.40 nm to 2.50 nm, such as when the quantum dots have a diameter of 1.20 nm to 3.50 nm. As the quantum dots become smaller, the band gap becomes wider, thereby shifting the photoluminescence to shorter wavelengths.

[0052] The quantum dots may have a mean diameter of 0.80 nm to 5.00 nm, preferably 1.00 nm to 4.50 nm, more preferably 1.20 nm to 3.50 nm. The mean diameter may be from 1.00 nm to 2.00 nm, such as when the quantum dots have diameters in the range of 0.76 nm to 4.50 nm. The mean diameter may be from 1.40 nm to 2.50 nm, such as when the quantum dots have a diameter of 1.12 nm to 3.50 nm.

[0053] In the first aspect of the invention, the mean diameter of the quantum dots is most preferably in the range 1.00 nm to 2.50 nm.

[0054] In the second aspect of the invention, the tin sulfide quantum dots have diameters in the range of 2.00 nm to 7.00 nm, preferably in the range of 3.00 nm to 7.00 nm, more preferably about 5 nm.

[0055] In the first aspect of the invention, the quantum dots are typically polydisperse (also known in the art as heterodisperse). Thus, the quantum dots have a distribution of diameters. The numerical ranges for the diameter described herein, unless the context indicates otherwise, may refer to a distribution of quantum dots where at least 95% (e.g. by number), preferably at least 99%, more preferably at least 99.5%, of the quantum dots have a diameter falling within the numerical range.

[0056] The quantum dots may have a polydispersity of >30%, preferably >35%, such as >40%, and more preferably >50%.

[0057] Generally, the quantum dots have a polydispersity of <90%, preferably <80%.

[0058] The polydispersity of the quantum dots may be >30% and <90%, preferably >35% and <90%, such as >40% and <90%, more preferably >50% and <90%, and even more preferably >50% and <80%.

[0059] The diameter, the mean diameter and the polydispersity described above typically relate to the diameter, the mean diameter and the polydispersity of the particles of the tin sulfide (e.g. the tin sulfide core of the quantum dot), unless the context indicates otherwise.

[0060] In the second aspect of the invention, the quantum dots are typically monodisperse. Thus, the quantum dots are similar in size with, for example, a mean diameter of 5.00 nm and are typically well separated.

[0061] The samples of quantum dots that have been described previously in the art are predominantly monodisperse samples. The polydispersity of such samples is below 30% and is typically less than 20%.

[0062] It has been found that the quantum dots of the invention are highly stable, particularly when compared to many commercially available photostable organic dyes and other types of quantum dots. The quantum dots are also biocompatible and are non-toxic. The quantum dots do not show degradation upon prolonged exposure to live cells or biological media. They do not show photobleaching characteristics when performing longterm imaging observations.

[0063] The composition of each quantum dot in the plurality of quantum dots may vary. There may be a distribution of compositions.

[0064] The atomic ratio, such as of a sample of quantum dots, can be measured by energy-dispersive X-ray spectroscopy (EDS), such as by EDS in transmission electron microscopy. The mean atomic ratio (e.g. the arithmetic mean) can be calculated from the individual atomic ratios of a sample of the quantum dots.

[0065] In the first aspect, the mean atomic ratio of tin to sulfur, is from 1.1:1 to 10.0:1, such as 1.2:1 to 9.0:1, particularly 1.3:1 to 9.0:1. It is preferred that the mean atomic ratio of the metal to sulfur is from 1.1:1 to 5:1, more preferably 1.2:1 to 3.0:1, and even more preferably 1.5:1 to 2.5:1.

[0066] In the second aspect, the mean atomic ratio of tin to sulfur is from 0.5:1 to 1.5:1, preferably about 1.0:1.

[0067] The quantum dots may have a distribution of atomic ratios of the tin to sulfur, which is distributed about the mean atomic ratio by up to ± 99% of the mean atomic ratio, such as up to ± 95% or up to ± 90%. Thus, for example, when the mean atomic ratio of tin to sulfur is 5.0:1 and the atomic ratio of tin to sulfur is distributed about the mean atomic ratio by up to ± 99% of the mean atomic ratio, then the quantum dots may have an atomic ratio of tin to sulfur that ranges from 0.05:1 to 9.95:1.

[0068] In another aspect of the invention, the composition of each quantum dot in the plurality of quantum dots may be similar (e.g. all of the quantum dots have a stoichiometric atomic ratio of the metal to sulfur atoms).

[0069] In the first aspect of the invention, the quantum dots of the invention are photoluminescent at several distinct wavelengths and can be used to perform multiplechannel imaging, such as from the UV to the near-infrared regions of the electromagnetic spectrum, at the same time. This allows for the parallel detection of multiple biomolecular interactions in complex biological systems. The multi-colour photoluminescent properties of the quantum dots is believed to be due to their different sizes (e.g. range of diameters) and the variation in the composition of the quantum dots, as described above.

[0070] In this aspect the quantum dots are photoluminescent at wavelengths of (a) 360 nm to 470 nm, (b) 475 nm to 545 nm, and (c) 550 nm to 740 nm. The quantum dots produce photoluminescence upon photoirradiation.

[0071] The wavelengths in (a), (b) and / or (c) may refer to the full width at half maximum (FWHM), such as in an emission spectrum of the quantum dots. Thus, the quantum dots of the invention have at least three distinct wavelength regions at which they are photoluminescent.

[0072] The quantum dots of the first aspect are photoluminescent at the wavelengths described above when the quantum dots have (a) a mean atomic ratio as described above and (b) a mean diameter as described above. Preferably, the quantum dots are luminescent at the wavelengths described above when the quantum dots have (a) a mean atomic ratio as described above, and (b) a mean diameter as described above.

[0073] In the second aspect of the invention, the tin sulfide quantum dots are luminescent at a wavelength from 450 nm to 550 nm, preferably a wavelength of 475 nm to 525 nm, more preferably about 490 nm.

[0074] The quantum dots, including the first and second aspects of the invention, may have a band gap of from 2.0 to 3.5 eV, preferably from 2.1 eV to 3.2 eV, more preferably from 2.2 to 2.9 eV, even more preferably from 2.2 to 2.8 eV.

[0075] In the second aspect of the invention, it is preferred that the quantum dots have a band gap of about 2.8 eV.

[0076] The photoluminescent properties of the quantum dots of the invention render them potentially useful in bioimaging, such as in the ultraviolet, visible and near-infrared regions of the electromagnetic spectrum.

[0077] Quantum dots having the desired photoelectronic properties for bioimaging and / or multiplex bioimaging are often composed of toxic elements. The invention provides nontoxic quantum dots that can be used in bioimaging and / or multiplex bioimaging, such as when studying prokaryotes or live cells. The excellent biocompatibility of quantum dots does not result in toxicity-related changes in the cell community. In general, the quantum dots are non-toxic because they are composed of non-toxic constituent elements.

[0078] In one example, the quantum dots do not comprise a surface-modifying agent, such as a ligand or a coating. In some applications it may not be necessary to modify the surface of the quantum dots, such as to provide stability or for biocompatibility.

[0079] The invention also relates to quantum dots that further comprise a surfacemodifying agent. Thus, each quantum dot may comprise, or consist essentially of, a particle of a tin sulfide and a surface-modifying agent. It is possible to modify the surface of the quantum dots for several purposes, such as to enhance stability or biocompatibility, or to assist with the targeting and labelling of a biological sample. The surface-modifying agent that is used may depend on the intended application of the quantum dots.

[0080] The optical properties of the metal sulfide core material can be preserved when certain surface-modifying agents are used. The surface-modifying agent, such as ligands and / or coatings (polymers), can insulate the core material, which can stabilise and maximise fluorescence. The surface-modifying agent may provide better surface passivation and may enhance the optoelectronic properties of the core material.

[0081] When a surface-modifying agent is present, it may form a layer disposed on a surface (e.g. an outer surface) on a particle of the metal sulfide. This layer can concentrate the charge carriers in the metal sulfide core, instead of being lost at the surface.

[0082] A surface-modifying agent may provide or be the outermost surface of the quantum dots.

[0083] Typically, the surface-modifying agent is biocompatible. It is preferred that the surface-modifying agent is biocompatible for in vivo use.

[0084] When the quantum dots comprise a surface-modifying agent, then the quantum dots may be hydrophobic or hydrophilic (e.g. an outer surface of the quantum dots is hydrophobic or hydrophilic). The quantum dots may be described as hydrophobic or hydrophilic when the surface-modifying agent (e.g. the outermost surface-modifying agent if there is more than one surface-modifying agent) is hydrophobic or hydrophilic, respectively.

[0085] In one embodiment, the quantum dots may, preferably, be hydrophobic. It has been found that quantum dots comprising a hydrophobic surface-modifying agent can be used to manufacture a biofilm. Without wishing to be bound by theory, biofilm formation may be a surface-induced process dependent on the surface chemistry of the quantum dots.

[0086] The surface-modifying agent may be a ligand, wherein said ligand may coordinate (e.g. by a covalent bond) to the metal of a quantum dot. The ligand may coordinate to the metal by donating one or two electrons to form a covalent bond with the metal.

[0087] The ligand may be an X-type ligand or a L-type ligand, such as defined in the covalent bond classification (CBC) method. A ligand can be categorised as an X-type ligand or an L-type ligand based on the way in which it is covalently bonded to the metal.

[0088] An X-type ligand is a ligand that either (a) donates one electron to the metal and accepts one electron from the metal when using the neutral ligand method of electron counting, or (b) donates two electrons to the metal when using the donor pair method of electron counting. Examples of X-type ligands include a halogen (e.g. Cl, Br, F etc.), OH or CN.

[0089] An L-type ligand is a neutral ligand that donates two electrons to the metal. This is regardless of the electron counting method used. These electrons can, for example, come from a lone pair. The bonds formed between an L-type ligand and the metal may be referred to as a dative covalent bond or a coordinate bond. Examples of L-type ligands include a phosphine (e.g. PR3), an amine (e.g. NR3) or a thiol (e.g. RSH).

[0090] The ligand is, for example, a compound, which may comprise a coordinating group and a side chain. The side chain is bonded (e.g. covalently bonded) to the coordinating group. The coordinating group is for coordinating to the metal of the quantum dots.

[0091] The coordinating group may be a carboxylate group (-COO~) or a conjugate acid thereof, a phosphate group (-O-PO32') or a conjugate acid thereof, an amine group, or a phosphine group.

[0092] When the coordinating group is an amine group, then preferably the amine group is represented by the formula (A1): S-NR1R2 (A1) wherein: S is the side chain; and each of R1 and R2 may be the same or different and is independently selected from H, Ci-6-alkyl and Ce-io-aryl-Ci-6-alkyl.

[0093] It is preferred that R1 is H (e.g. hydrogen). More preferably, R1 and R2 is hydrogen.

[0094] When the coordinating group is a phosphine group, then preferably the phosphine group is represented by the formula (P1): S-PRARB (P1) wherein: S is the side chain; and each of RA and RB may be the same or different and is independently selected from H, Ci-6-alkyl and Ce-io-aryl-Ci-6-alkyl.

[0095] It is preferred that RA is H (e.g. hydrogen). More preferably, RA and RB is hydrogen.

[0096] In principle, the side chain of the ligand (as represented by S in formula (A1) or (P1) above) can be any moiety that will modify the surface of the quantum dots.

[0097] The side chain may, for example, be attached to a functional group or a targeting moiety. The functional group may modify the surface properties of the quantum dots. The targeting moiety may selectively bind to a biological target.

[0098] It is preferred that the side chain is a hydrophobic group, which is a saturated or an unsaturated hydrocarbon chain having from 3 to 25 carbon atoms. It is preferred that the hydrocarbon chain is a linear hydrocarbon chain.

[0099] When the coordinating group of the ligand is a carboxylate group (-COO~) or a conjugate acid thereof, then the ligand may be a C4-26 fatty acid ora conjugate base thereof. The C4-26 fatty acid may, for example, be selected from butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid and linoelaidic acid. It is preferred that the ligand is oleic acid or a conjugate base thereof (e.g. oleate anion).

[0100] When the coordinating group of the ligand is an amine, then the ligand may have a fatty acid side chain. The fatty acid side chain may, for example, be selected from n-butyl, n-pentyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-icosyl, n-docosyl, n-tetracosyl and n-hexacosyl. Additionally, or alternatively, the ligand may be a fatty amine, such as cocos amine, tallow amine, stearyl amine or oleyl amine. When the coordinating group of the ligand is an amine, it is preferred that the ligand is oleyl amine.

[0101] The surface-modifying agent may be two or more ligands (i.e. two or more different ligands). Each ligand may be independently selected from a ligand as defined above.

[0102] When the surface-modifying agent is two or more ligands, then it is preferred that a first ligand comprises an amine group as a coordinating group and a second ligand comprises a carboxylate group or a conjugate acid thereof as a coordinating group. The second ligand is preferably a fatty acid. More preferably, the first ligand is oleic acid or the conjugate base thereof (e.g. oleate anion) and the second ligand is oleyl amine or a conjugate acid thereof.

[0103] The molar ratio of oleic acid to oleyl amine may be from 2.5:1 to 1:2.5, preferably from 2:1 to 1:2, such as 1:1 to 1:2 or 2:1 to 1:1.

[0104] The surface-modifying agent may alternatively be a polymer. In principle, a variety of polymers could be used to provide a surface-modifying agent and to apply a coating to the metal sulfide metal core.

[0105] In the second aspect of the invention, the surface modifying agent is present and is an organic polymer. It will be understood that, by “organic polymer” we typically mean a polymer in which the molecular chain forming the backbone of the polymer comprises carbon. The polymer may be an amphiphilic polymer.

[0106] In all embodiments of the invention, the organic polymer may, for example, be a polyglycolic acid, a polylactic acid, a polycaprolactone, a poly(lactic-co-glycolic acid), a poly(N-isopropylacrylamide), a polysiloxane, polyethylene glycol, polyvinyl alcohol, or a polyvinylpyrrolidine.

[0107] It is preferred that the polymer is a polyvinylpyrrolidine (PVP). PVP is water soluble and is a hydrophilic surface-modifying agent.

[0108] The polymer may, for example, be composed of different molecular weights in the range of 10,000 to 1,300,000 g / mol, more preferably molecular weights in the range of 10,000 to 360,000 g / mol, even more preferably in the range of 40,000 to 55,000 g / mol, such as 55,000 g / mol.

[0109] The increase of the molecular weight corresponds to the length of the polymer chain. The higher the molecular weight, the longer the chain length of the polymer.

[0110] The molar ratio of polyvinylpyrrolidine may be from 2 to 50, preferably from 5 to 25, such as 10 to 20.

[0111] When the quantum dots comprise a surface-modifying agent, then the quantum dots may have diameters in the range of 1.00 nm to 15.00 nm, more preferably diameters in the range of 1.50 nm to 10.00 nm, such as 2.00 nm to 6.50 nm.

[0112] A further aspect of the invention relates to a composition for bioimaging. The composition comprises a liquid carrier and the quantum dots of the invention.

[0113] The liquid carrier is typically biocompatible.

[0114] The liquid carrier may be a biological medium (e.g. blood) or a transport medium, such as an aqueous solution.

[0115] It is preferred that the liquid carrier is an aqueous solution. More preferably, the liquid carrier is phosphate-buffered saline.

[0116] The invention also provides a method for manufacturing tin sulfide quantum dots as hereinbefore defined. The quantum dots of the invention, whether comprising a surfacemodifying agent or otherwise, are obtained or obtainable from the methods of the invention.

[0117] The methods of the invention are simple to perform and have a short production cycle. It allows quantum dots to be manufactured at low cost with good reproducibility and control over the optoelectronic and physical properties of the resulting product. The method can also be readily scaled-up.

[0118] The methods are hot-injection synthesis methods. Unlike other types of preparative method, the method of the invention avoids the use of harsh conditions and multiple reaction steps and permits good control over the sizes, composition and emission properties of the quantum dots that are obtained.

[0119] In the first aspect of the invention, the method comprises a first step (i) dispersing a metallic precursor comprising tin (Sn) in an organic solvent at a first temperature of 50°C to 150°C to produce a reactant liquid; and (ii) mixing a precursor comprising sulfur (S) with the reactant liquid at a second temperature of 25°C to 55°C.

[0120] The step of dispersing the metallic precursor in a solvent to produce a reactant liquid may be a step of dissolving or suspending the metallic precursor in the solvent to produce a reactant liquid.

[0121] The reactant liquid is typically a reactant dispersion, when the metallic precursor is dispersed in the solvent. The reactant liquid or the reactant dispersion may be a reactant solution or a reactant suspension. The form of the reactant liquid will depend on the properties of the metallic precursor and the solvent.

[0122] The step of dispersing the metallic precursor in a solvent is performed at a temperature of 50°C to 150°C, more preferably a temperature of 75°C to 125°C, such as 85°C to 110°C (e.g. about 105°C). The term “first” in the expression “first temperature” is used as a label to distinguish it from temperatures relating to other method steps and does not require that a “second temperature” should be used in this method step.

[0123] In a preferred embodiment of the first aspect, between steps (i) and (ii), the method comprises an additional step (i)(a) increasing the temperature of the reactant liquid to a temperature in the range 125 C to 150°C under an inert gas, such as nitrogen (N2) or argon (Ar). Step (i)(a) ideally further comprises the addition of a ligand comprising an amine group, preferably oleyl amine.

[0124] Before dispersing the metallic precursor in the solvent, the solvent may be at the first temperature (e.g. the solvent has been pre-heated to the first temperature). The addition of the metallic precursor to the solvent at the first temperature may produce the reactant liquid.

[0125] Alternatively, when the metallic precursor is dispersed in the solvent, then the solvent and the metallic precursor may be heated to the first temperature to produce the reactant liquid.

[0126] The step of dispersing the metallic precursor in the solvent may include decomposing the metallic precursor in the solvent, such as to produce the reactant liquid. Thus, the method may comprise a step of dispersing and decomposing the metallic precursor in the solvent to produce a reactant liquid.

[0127] In the second aspect, the method comprises (i) dispersing an organic polymer in an organic solvent at a first temperature of 50°C to 150°C to produce a reactant liquid; and (ii) mixing a precursor comprising tin (Sn) and a precursor comprising sulfur (S) in organic solvent with the reactant liquid at a second temperature of 125°C to 160°C.

[0128] The organic polymer may, for example, be a polyglycolic acid, a polylactic acid, a polycaprolactone, a poly(lactic-co-glycolic acid), a poly(N-isopropylacrylamide), a polysiloxane, polyethylene glycol, polyvinyl alcohol, or a polyvinylpyrrolidine (PVP), preferably PVP.

[0129] The description which follows is generally applicable to both the first and second aspects, unless otherwise indicated.

[0130] The step of producing a reactant liquid may be performed under an inert gas. 15

[0131] The solvent and / or the dispersion of the metallic precursor in the solvent may be heated to the first temperature under an inert gas.

[0132] The inert gas is substantially free of oxygen, preferably oxygen and water. The term “substantially free” in this context refers to the purity of the inert gas, which is typically at least 99.9% (e.g. total impurities <1000 ppm), preferably at least 99.99% (e.g. total impurities <100 vpm), more preferably at least 99.999% (e.g. total impurities <10 vpm), and even more preferably at least 99.999% (e.g. total impurities <1 vpm).

[0133] The inert gas may be nitrogen (N2) or argon (Ar).

[0134] The solvent may be a degassed solvent, preferably degassed for the removal or reduction of oxygen and / or water in the solvent. The solvent may have been degassed in the presence of an inert gas, such as the inert gas described above.

[0135] The step of dispersing the metallic precursor in the solvent may include stirring the metallic precursor in the solvent at the first temperature.

[0136] The stirring may be performed to dissolve the metallic precursor in the solvent, such as to produce a reactant solution.

[0137] In general, the metallic precursor is stirred in the solvent between 1 to 2 hours, preferably for at least 1 hour.

[0138] As part of the step of dispersing the metallic precursor in the solvent, the metallic precursor may be added to the solvent, preferably to the solvent at the first temperature. The metallic precursor may then be stirred in the first solvent, such as to produce the reactant solution.

[0139] The metallic precursor may be added to the solvent using any conventional technique. It is preferred that the metallic precursor is added to the solvent under an inert gas, such as an inert gas as described above.

[0140] The metallic precursor comprises a tin compound. The tin compound may be a tin salt.

[0141] The metal compound may be a tin halide, a tin oxide, a tin amide or a tin amine. The tin amide may be a tin silylamide. The tin amine may be a tin silylamine. It is preferred that the tin compound is a tin halide.

[0142] The metallic precursor may comprise, or consist essentially of, SnCh (Tin (II) chloride), SnCh (Tin (II) chloride) anhydrous, SnChx H2O (Stannous chloride, dihydrate), SnCU (Tin (IV) chloride), SnBr2 (Tin (II) bromide), SnF2 (Tin (II) fluoride), Snh (Tin (II) iodide), Sn(N(SiMe3)2)2 or a hydrate thereof, such as SnChxFW (Tin (II) chloride dihydrate). It is preferred that the metallic precursor comprises, or consists essentially of, SnCt (Tin (II) chloride) anhydrous.

[0143] In one example, the organic solvent typically has a high boiling point (e.g. >100°C, preferably >150°C). It may also be polar and / or weakly coordinating.

[0144] In general, and including the first aspect of the invention, the solvent may be selected from trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and 1-octadecene (ODE). It is preferred that the solvent is 1-octadecene (ODE).

[0145] In another example, including the second aspect of the invention, the solvent has a hydroxy group, such as in glycerol or ethylene glycol, preferably ethylene glycol.

[0146] The inert gas in this embodiment is substantially free of oxygen, preferably oxygen and water. The term “substantially free” in this context refers to the purity of the inert gas, which is typically at least 99.9% (e.g. total impurities <1000 ppm), preferably at least 99.99% (e.g. total impurities <100 vpm), more preferably at least 99.999% (e.g. total impurities <10 vpm), and even more preferably at least 99.999% (e.g. total impurities <1 vpm).

[0147] For convenience, the expression “precursor comprising sulfur” is referred to herein using the abbreviated term “sulfur precursor”. The temperature in this method step is referred to herein as the “second temperature”, where the term “second” is used as a label to distinguish the temperature from other temperatures that may be used as part of the method.

[0148] The precursor comprising sulfur is mixed with the reactant liquid to bring about a reaction with the metal from the metallic precursor.

[0149] The step of mixing the sulfur precursor with the reactant liquid may be a step of reacting the sulfur precursor with the reactant liquid. More preferably, the step of mixing the sulfur precursor with the reactant liquid is a step of reacting the sulfur precursor with a metal from the metallic precursor in the reactant liquid.

[0150] The second temperature can be used to control the size and the band-gap of the resulting quantum dots. Higher temperatures generally produce larger quantum dots having a smaller band gap.

[0151] In the first aspect of the invention, the second temperature is 25°C to 55°C. In comparison to prior art methods for the preparation of quantum dots, the method of the invention uses relatively moderate reaction conditions, such as low reaction temperatures, and precursors having low reactivity. The relatively moderate conditions allow better control over the composition and photoluminescent properties of the final product.

[0152] The step of mixing the sulfur precursor with the reactant solution produces a product liquid (e.g. a liquid for forming, or that contains, the product). The product liquid may be a product dispersion, such as a product solution or a product suspension. The form of the product liquid will depend on the solvent, the identity of the precursors and the reaction conditions that are used.

[0153] The sulfur precursor is typically admixed with the reactant liquid. The sulfur precursor may be mixed or admixed with the reactant liquid using any conventional technique. It is preferred that the sulfur precursor is mixed or admixed with the reactant solution under an inert gas, such as the inert gas described above.

[0154] The sulfur precursor may be mixed or admixed with the reactant liquid by being injected into the reactant liquid, preferably under the inert gas.

[0155] The sulfur precursor may be elemental sulfur or a compound comprising sulfur. The identity of the sulfur precursor, specifically its reactivity, can be used to control the properties, such as the diameter and / or the composition of the quantum dots. When a sulfur precursor has relatively low reactivity, such as elemental sulfur, then the diameter of the quantum dots may be smaller and the atomic ratio of metal to sulfur in the resulting metal sulfide may be higher. These properties may influence the luminescent properties of the quantum dots.

[0156] The compound comprising sulfur may be a metallic compound, such as a metal salt, or an organic compound.

[0157] The metallic compound may be a binary metallic compound, such as an alkali metal sulfide or an alkaline earth metal sulfide.

[0158] Examples of alkali metal sulfides include lithium sulfide (U2S), sodium sulfide (Na2S), sodium dithionite (Na2S2O4), and potassium sulfide (K2S). Sodium sulfide or potassium sulfide are preferred.

[0159] Examples of alkaline earth metal sulfides include calcium sulfide (CaS) and magnesium sulfide (MgS).

[0160] When the sulfur-containing compound is an organic compound, then the organic compound may be a thiol compound (e.g. a compound comprising an -SH group), a thioether compound (e.g. a compound comprising an -S- moiety, which is not a thiol compound), a disulfide compound (e.g. a compound comprising an -S-S- moiety) or a thiocarbonyl compound (e.g. a compound comprising a -C=S moiety). It is preferred that the organic compound is a thioether compound or a thiocarbonyl compound. More preferably, it is preferred that the organic compound is a thioether compound.

[0161] The thiocarbonyl compound may be thioacetamide (C2H5NS), thioacetic acid (CH3COSH), or thiourea (SC(NH2)2).

[0162] The thioether compound may be bis-(trimethylsilyl)sulfide (S[Si(CH3)s]2).

[0163] For the avoidance of doubt, the composition of the metallic precursor is preferably different to the composition of the sulfur precursor. In general, the metallic precursor is a different compound to the sulfur precursor.

[0164] The reactivity of the sulfur precursor can influence the nucleation and growth rate. It is believed that the higher the reactivity of the sulfur precursor, the higher the concentration of reactant sulfur species and the faster the nucleation rate, which can result in the formation of a larger concentration of small nuclei. A high concentration of reactant sulfur species could accelerate the growth rate forming quantum dots having larger sizes compared to when lower concentrations of reactant sulfur species are present.

[0165] In the first aspect of the invention, it is preferred that the sulfur precursor is elemental sulfur.

[0166] In the second aspect of the invention, it is preferred that the sulfur precursor is thiourea.

[0167] After the steps described above, the product liquid may be maintained at the second temperature, preferably with stirring, for up to 1 hour, such as from 5 to 60 minutes. This step can facilitate the nucleation and growth of the quantum dots.

[0168] The product liquid may be cooled to room temperature (e.g. 20°C - 25°C), preferably after maintaining the product liquid at the second temperature, such as described above. The product liquid may be allowed to cool (e.g. cool naturally) to room temperature or by immersing a reaction vessel containing the product liquid in a cold-water bath or an ice bath.

[0169] In the method of the invention, the molar ratio of the metal (e.g. from the metallic precursor) to sulfur (e.g. from the sulfur precursor) is typically 5:1 to 1:2, preferably 3:1 to 1:1, and more preferably 2.5:1 to 1.1:1. The molar ratios can affect the composition and the size distribution of the quantum dots.

[0170] The invention also provides a method of manufacturing quantum dots comprising a surface-modifying agent as described above.

[0171] The method of the invention may further comprise mixing, preferably admixing, a surface-modifying agent, in the solvent, in the reactant liquid or the product liquid. The product liquid in this context refers to a liquid comprising the quantum dots without a surface-modifying agent.

[0172] The mixing or admixing the surface-modifying agent, in the solvent, the reactant liquid or the product liquid may be a step of dispersing the surface-modifying agent in the solvent, the reactant liquid or the product liquid. The dispersing may be dissolving or suspending the surface-modifying agent.

[0173] The surface-modifying agent may be mixed or admixed with the metallic precursor before, or after the metallic precursor is mixed or admixed with the sulfur precursor.

[0174] The surface-modifying agent may be mixed or admixed with the solvent either before, after or simultaneously with the mixing or admixing of the metallic precursor and sulfur precursor with the solvent.

[0175] In the second aspect, the surface modifying agent is an organic polymer and is mixed or admixed with the solvent before the metallic precursor and precursor comprising sulfur are mixed or admixed with the solvent.

[0176] It is preferred to mix or admix the surface-modifying agent before the solvent or the reactant liquid is at (e.g. heated to) the first temperature.

[0177] The surface-modifying agent may be mixed or admixed with the solvent, the reaction liquid or the product liquid using any conventional technique.

[0178] Typically, the surface-modifying agent is mixed or admixed with the solvent, the reactant liquid or the product liquid under an inert gas, such as described above.

[0179] The step of mixing, admixing or dispersing the surface-modifying agent with the solvent, the reactant liquid or the product liquid may include stirring the surface-modifying agent in the solvent, the reactant liquid or the product liquid. It is preferred that the stirring is when the solvent or the reactant liquid is at the first temperature.

[0180] The stirring may, for example, be performed to disperse the surface-modifying agent and / or the metallic precursor in the solvent. It may be preferable to stir to disperse the surface-modifying agent and to decompose the metallic precursor in the solvent.

[0181] Typically, the surface-modifying agent (and the metallic precursor, if present) is stirred in the solvent or the reactant liquid between 1 and 2 hours, preferably for at least 1 hour.

[0182] In one aspect of the invention, the molar ratio of surface-modifying agent to the metal (e.g. from the metallic precursor) may be >2:1. It is preferred that the molar ratio is from 2:1 to 30:1, more preferably 5:1 to 25:1, such as 10:1 to 20:1. The molar ratio refers to the total number of moles of the surface-modifying agent, such as when the surfacemodifying agent comprises two or more ligands.

[0183] The surface-modifying agent may be two or more ligands, such as described above. When the surface-modifying agent is two or more ligands, then the ligands may be added separately, simultaneously or concurrently to the solvent, the reactant liquid or the product liquid.

[0184] When the surface-modifying agent is two or more ligands, particularly a first ligand and a second ligand as described above, then the molar ratio of the first ligand to the second ligand is from 5:1 to 1:10, preferably 2:1 to 1:5, such as 1:1 to 1:5, more preferably 1:1 to 1:3.

[0185] In another aspect of the invention, the surface-modifying agent may be a coating, such as a polymer. The molar ratio of the surface-modifying agent to the metal may be from 2:1 to 50:1, more preferably 5:1 to 25:1, such as 10:1 to 20:1.

[0186] The sulfur-precursor may be mixed with the reactant liquid as described above.

[0187] Like the sulfur-precursor, the surface-modifying agent, especially when the surface-modifying agent is a ligand or a coating, can influence the concentration of nuclei, such as by interacting with the sulfur-precursor. At high concentrations, the surfacemodifying agent may reduce the concentration of nuclei that are formed. The surfacemodifying agent, particularly when it is a ligand or a coating, may interact with the surface of the metal sulfide, which can aid the production of monodispersed quantum dots and can prevent the particles of the metal sulfide from aggregating.

[0188] It is also possible to control the number of layers of the surface-modifying agent (e.g. thickness of the coating) by varying the concentration of the surface-modifying agent.

[0189] In general, the method of manufacturing quantum dots may comprise a step of separating the quantum dots, such as from the product liquid.

[0190] The step of separating the quantum dots, such as from the product liquid, may be a step of precipitating the quantum dots (e.g. from the product liquid) using a solvent. The solvent used to perform the precipitation step will depend on the surface properties of the quantum dots (e.g. whether a hydrophobic or a hydrophilic surface-modifying agent is present).

[0191] The solvent for precipitating the quantum dots may be a polar solvent, preferably a polar aprotic solvent. Examples of polar solvents include acetone, methanol, tetrahydrofuran, DMSO and DMF. It is preferred that the polar aprotic solvent is acetone.

[0192] Alternatively, the step of separating the quantum dots, such as from the product liquid, may be a step of washing the product liquid with a first solvent, and then dispersing the quantum dots from the product liquid in a second solvent.

[0193] The first solvent for washing the product liquid may be a polar solvent as described above. The second solvent may be a non-polar solvent, such as a hydrocarbon solvent. It is preferred that the non-polar solvent is pentane or hexane.

[0194] The invention also provides a method of imaging a biological target. Typically, the method is a multiplex method of imaging a biological target.

[0195] In principle, the biological target can be a virus, a bacterium, a cell, a tissue or an organ. It is preferred that the biological target is a cell or a tissue.

[0196] The method involves administering the quantum dots or the composition for bioimaging of the invention to the biological target.

[0197] The method may be a diagnostic method, particularly an in vitro and / or ex vivo diagnostic method.

[0198] The quantum dots or the composition of the invention may be administered to a pre-obtained biological target, such as a pre-obtained cell, tissue or organ.

[0199] When the method is used as a diagnostic method, then the diagnostic method may not be carried out on the human or animal body or the diagnostic method may be carried out on a non-living (e.g. dead) human or animal body.

[0200] The method may be an in vitro method of imaging the biological target. The biological target is preferably a cell or a plurality of cells, a tissue or an organ. More preferably, the biological target is a cell or a plurality of cells.

[0201] Alternatively, the method may be an ex vivo method of imaging the biological target. The biological target is preferably a cell or a plurality of cells, a tissue or an organ. More preferably, the biological target is a tissue.

[0202] In a further alternative, the method may be an in vivo method of imaging the biological target. The biological target is preferably a tissue, a cell or a plurality of cells, or an organ.

[0203] In general, the method also includes a step of imaging the biological target. The biological target is imaged after the administration of the quantum dots or the composition. The quantum dots may label the biological target or act as a contrast agent.

[0204] A further aspect of the invention relates to the use of the quantum dots or the composition in cellular imaging and / or biological tissue imaging.

[0205] The quantum dots of the invention can accumulate in certain tissues, such as tumours, due to abnormalities or defects in the tissue vasculature. In the case of cancer, the quantum dots may be rapidly absorbed by the cancer cells, which tend to grow and divide more quickly than healthy cells.

[0206] The invention further provides a method for manufacturing a biofilm. The method comprises contacting the quantum dots of the invention with a plurality of cells.

[0207] The step of contacting the quantum dots with the plurality of cells may be a step of mixing and / or incubating the quantum dots with the plurality of cells. When the quantum dots are incubated with the plurality of cells, then the incubation may be for at least 6 hours, preferably at least 10 hours.

[0208] The quantum dots typically comprise a surface-modifying agent, preferably a hydrophobic or hydrophilic surface-modifying agent.

[0209] The hydrophobic surface-modifying agent is preferably a ligand, such as two or more ligands. Each ligand may comprise a side chain, which is a hydrophobic group, such as described above.

[0210] It is preferred that the hydrophobic surface-modifying agent is a first ligand and a second ligand. More preferably, the first ligand is oleic acid or the conjugate base thereof (e.g. oleate anion) and the second ligand is oleyl amine.

[0211] The hydrophilic surface-modifying agent is preferably a polymer. The polymer may comprise a hydrophilic pyrrolidone moiety and a hydrophobic alkyl group.

[0212] More specifically, the polymer is polyvinylpyrrolidone.

[0213] The plurality of cells may be a plurality of eukaryotic cells, a plurality of prokaryotic cells or a mixture thereof. It is preferred that the plurality of cells is a plurality of prokaryotic cells.

[0214] In general, the plurality of cells may be a plurality of fungal cells, plant cells, bacterial cells or animal cells. It is preferred that the plurality of cells is a plurality of bacterial cells.

[0215] The invention also relates to a biofilm obtained or obtainable from the method.

[0216] Typically, the biofilm comprises a matrix of cells, such as a matrix of the plurality of cells described above.

[0217] The biofilm may further comprise the quantum dots.

[0218] A further aspect of the invention relates to the use of the biofilm in a bioreactor.

[0001] A further aspect of the invention relates to the use of the biofilm in a bioreactor.

[0219] A further aspect of the invention may relate to the use of the quantum dots in an optoelectronics device, such as a photodiode, a light emitting diode, a laser, a photovoltaic, etc.

[0220] A further aspect of the invention may relate to the use of the quantum dots in a photonics device, such as a quantum random number generator (QRNG).

[0221] A further aspect of the invention may relate to use of the quantum dots in quantumrepeaters, quantum memories, quantum nodes within quantum communication networks. Examples

[0222] The present disclosure will now be illustrated by the following non-limiting examples Characterization techniques Microscopy

[0223] A Perkin Elmer UltraView VoX (Perkin Elmer, Beaconsfield, UK) spinning disc confocal mounted to an Olympus IX-81 microscope fitted with a 60x / 1.4NA oil immersion objective (Olympus Keymed) was used to capture single plane fluorescence and bright-field images. A laser at 488 nm was used to excite green fluorescence and bright-field illumination was used for transmission imaging, with images collected sequentially on a Hamamatsu C9100-13 Electron Multiplying-CCD (EM-CCD) camera (Hamamatsu Photonics UK, Welwyn Garden City, UK) using the software package Velocity (Perkin Elmer). Post-acquisition processing, including generating merged images was performed using ImageJ / FIJI. All samples were prepared by adding a small amount of the biofilm to a glass substrate covered by a microscope slide glass.

[0224] An Olympus FluoView 3000 laser scanning confocal incorporating an Olympus IX-83 microscope stand fitted with a 60x / 1.4NA oil immersion objective (Olympus Keymed) was used to collect lambda scans in order to characterise the fluorescence emission characteristics of quantum dots when excited at different wavelengths. Quantum dots were excited at either 405 nm, 488 nm or 561 nm, and lambda scans were performed by collecting light in 10 nm bands from 420 to 600 nm, 490 to 690 nm and 570 to 760 nm for each excitation wavelength respectively using the native FluoView confocal software. Data analysis was performed by measuring fluorescence intensity at each 10 nm collection band using ImageJ / FIJI with the Olympus file format plug-in installed (Olympus Keymed). All samples were prepared by adding a small amount of the biofilm to a glass substrate covered by a microscope slide glass. Fluorescence system

[0225] Widefield fluorescence images were collected on a DeltaVision Core epifluorescence imaging system (Imsol, Preston, UK) using an Olympus (Olympus Keymed, Southend-on-Sea, UK) IX-71 microscope fitted with a 60x / 1.4NA oil immersion objective. Green fluorescence was generated by illuminating the sample at 488 nm using a Lumencor SpectraX light source (Lumencor, Beaverton, Oregon, USA) and bright-field transmission illumination was generated using a broad-spectrum white LED (Imsol). A Photometries CoolSNAP HQ2 cooled Charge Coupled Device (CCD) camera (Photometries, Tuscon, Arizona, USA) was used to sequentially capture fluorescence and bright-field images using the software package softWoRx (Imsol). Post-acquisition processing, including generating merged images was performed using ImageJ / FIJI (NIH, Bethesda, Maryland, USA). All samples were prepared by adding a small amount of the biofilm to a glass substrate and covered by microscope slide glass. UV-Vis-NIR spectroscopy

[0226] The optical absorption properties of all quantum dot samples were measured on a Cary Varian 4000 UV-Vis-NIR spectrophotometer. A typical arrangement of the instrument includes a light source, diffraction grating, aperture through which light travels to the sample cuvette and is captured by a detector (photomultiplier tube). The detector measures the resulting light intensity of a sample irradiated with a monochromatic beam of light. Liquid samples of diluted quantum dot solutions and pure organic ligands were loaded in quartz cuvettes. A baseline measurement was performed at 100 % transmission (with no sample) and 0 % transmission (blocking the light) before the analysis of the samples. Measurements were taken with respect to a reference sample containing the dispersant solvent. Photoluminescence

[0227] The 3D wavelength-dependent photoluminescence (PL) emission measurements were taken using an automated Agilent Cary Eclipse Fluorescence Spectrophotometer equipped with an Agilent xenon flash lamp allowing for fast acquisitions times (capturing data every 12.5 ms), highly sensitive optics and photomultiplier tube detectors connected to an Agilent Cary WinFLR software. Dilute dispersions of quantum dots were loaded in quartz cuvettes and excited with an excitation wavelength in the range of 350 nm to 500 nm (with 10 nm increments). The steady-state PL spectra was collected and processed using an Origin software.

[0228] Steady-state photoluminescent optical measurements were performed using a mode-locked Ti:Sapphore laser (Coherent Mira, operating at 76MHz with a pulse width of 150ps), with frequency doubled to 395 nm. The excitation was directed onto the sample through a 100x objective (0.7NA) giving an excitation spot of approximately 800 nm. Emission was collected through the same objective and directed to a 0.3 m spectrometer (with available gratings of 300, 600 and 1200 lines / mm). Time-resolved PL measurements (TRPL) were carried out by directing the emission to an output slit on the spectrometer (to act as a monochromator) and passed to a photomultiplier tube (PMT) (Becker and Hickl PMH-100) with an instrument response function width of 150 ps. A fast-photodiode detector was used at the excitation laser to act as a start timer for time-correlated single-photon counting (TCSPC) measurements. Fitting of lifetime data was performed (mono-or dualexponential fits where appropriate) using an Origin software.

[0229] Samples were prepared by drop casting diluted solutions of quantum dots onto silica substrates. For the temperature-dependent TRPL measurement, thin-film samples prepared by drop-casting of SnS quantum dots, which were loaded onto liquid nitrogen / argon cryostat and left under vacuum for 10 h to remove excess solvents before performing the analysis. Transmission electron microscopy

[0230] Transmission electron microscopy (TEM) was used to provide information about the shape, size, composition and size-distribution of the quantum dots. The measurements were performed on a JEOL 2100 TEM operating at 200 kV along with an EDS detector connected to INCA software. Dilute dispersions of colloidal QDs were prepared, in which TEM grids were briefly dipped in and left to dry at ambient conditions. Mean particle sizes were calculated from an experimental size histogram with a Gaussian distribution curve generated from the counts of multiple quantum dots present in the TEM sample.

[0231] FFT images were obtained by selecting an area of interest (e.g. the area occupied by a single quantum dot) and performing an FFT analysis using an Imaged software. The resulting FFT images were composed of an array of dots, which correspond to the d-spacing values of specific lattice planes in the crystal structure, which were later compared to theoretical values to assign the correct crystal phase of the material.

[0232] TEM reconstruction images were obtained by selecting the diffraction dots from the FFT images and performing an inverted FFT analysis. This allowed precise confirmation of the d-spacing values to the values of the original HRTEM images as well as determine crystal faults or imperfections.

[0233] EDS analysis was performed to chemically characterise the elemental composition of the quantum dots. The EDS measurements were performed at low magnifications acquiring statistical average data of multiple quantum dots. INCA analytical software was used to process the EDS spectra for chemical identification and quantification. Example 1 Method for the colloidal synthesis of ligand passivated SnS QDs

[0234] The method is a hot injection synthesis method. A Sn-based precursor (2 mmol), 30 to 60 mmol of 1-octadecene (ODE) and a surface-modifying agent were loaded into a 50 mL three-neck flask. The surface-modifying agent may be an X-type ligand and / or an L-type ligand, and can be used in a molar ratio as set out in Table 1 below. Table 1: Molar ratios of reaction components Reagent Molar ratio (mmol) Sn-based precursor (e.g. SnCh anhydrous) 0.1-2.00 Ligand: X-type L-type 1.00-5.00 5.00-12.00 Solvent (e.g. 1-Octadecene) 30.00-60.00 Sulfur precursor (e.g. elemental sulfur) 0.1-1.00

[0235] The flask was then connected to a Schlenk line. The system was evacuated using a rotary pump and left on continuous stirring at 105°C for at least 1 h to maintain moisture free conditions. The Schlenk line was then purged with an Argon flow and the reaction conditions were maintained for another 1 h to aid the complete dissolution of the Sn-based precursor to yield a clear yellowish transparent organotin solution.

[0236] In this example, SnCh anhydrous was the Sn-based precursor. The surfacemodifying agent was oleic acid (“OA”; an X-type ligand) and oleylamine (“OLA”; an L-type ligand), which were used in the molar ratios shown in Table 1.

[0237] Two different sulfur precursors were used to prepare SnS quantum dots as a comparison. The first sulfur precursor was elemental sulfur. The second sulfur precursor was bis-(trimethylsilyl)sulphide (e.g. S(SiMe3)2j “(TMS)2S”). The sulfur precursors were injected, at 55°C, into separate three-neck flasks containing the Sn-based precursor.1 mmol of the sulfur (S) precursor was used in each instance.

[0238] Upon addition of (TMS)2S, there was a rapid colour change of the reaction solution from clear yellowish to a dark, redish / blackish colour, which is indicative of a fast nucleation rate.

[0239] Upon addition of the elemental S precursor to the flask, the reaction solution gradually changed colour from clear yellow to dark red and then eventually to a dark, blackish colour. The gradual change in colour is believed to be due to a decreased rate of nucleation compared to when the (TMS^S reactant was used. The colour changes observed after the addition of elemental S may result from multi-step nucleation events and slow QD growth. These results indicate that elemental sulfur has lower reactivity than (TMS)2S.

[0240] The QDs produced from elemental sulfur are referred to herein as “SnS-S” and the QDs produced from (TMS)2S are referred to as “SnS-TMS”. Results

[0241] The colloid of SnS quantum dots (QDs) that were produced from each sulfur precursor were characterised. UV-Vis absorption spectra

[0242] The UV-Vis absorption spectra of the QDs are shown in Figure 1 (A) and the calculated band-gap energy values are shown in Figure 1 (B). Figure 1 (A) shows a difference between the optical properties of the SnS QDs, which depends on the reactivity of the S precursor. As shown in Figure 1 (B), the optical band-gap of the SnS QDs prepared from (TMS)2S is narrower (2.55 eV; see “SnS (TMS)” in Figure 1)) than the band-gap of the SnS QDs prepared from elemental S (2.95 eV; see “SnS (S)” in Figure 1).

[0243] The reason for the band-gap difference between the two samples of SnS QDs may be a size-related phenomenon, as shown below. The narrower band-gap (2.55 eV) of the SnS QDs prepared from (TMS)2S reflects a larger average size of the QDs compared to the average size of the SnS QDs produced from elemental sulfur, which had a wider band-gap (2.95 eV). Transmission Electron Microscopy

[0244] The mean-particle sizes of the colloidal SnS QDs were obtained using TEM analysis. Figures 2 shows TEM micrographs of the SnS QDs. Figures 2 (A) and 2 (B) are low magnification images of the SnS QDs. Both the SnS QDs produced from elemental sulfur (see Figure 2(A)) and the SnS QDs produced from (TMS)2S (see Figure 2 (B)) appear to have sizes between 2-5 nm with semi-spherical shapes.

[0245] The size measurements obtained from the TEM analysis were in a good agreement with the corresponding band-gap values of the two samples as seen from the UV-Vis spectra in Figure 1.

[0246] There were noticeable variations in the chemical composition of the QDs, as can be seen from the EDS summary data in Table 2 below. The EDS data represents the % composition of Sn and S elements in SnS QDs and excludes any other elements present in the sample. Table 2: Mean elemental composition of QDs from EDS analysis Sample Element Atomic ratio Sn:S S Sn Weight % Atomic % Weight % Atomic % SnS-S 12.55 31.31 87.45 68.69 2.2:1 SnS-TMS 21.77 48.34 78.23 51.66 1:1

[0247] The SnS-S QDs have a Sn:S ratio that is significantly higher than the Sn:S ratio of the SnS-TMS QDs. It is believed that this is a result of the slower nucleation rate of SnS-S QDs, which is due to the reactivity of the sulfur precursor. Photoluminescence

[0248] The photoluminescence (PL) emission properties of the colloidal SnS QDs were measured and are shown in Figure 3.

[0249] When the high reactivity TMS precursor was used, QDs with excitation-independent emission properties were obtained (see Figure 3 (A)) regardless of the sample’s polydispersity. Other than the single PL emission peak at 370 nm, which is believed to be ligand-induced since this is the region of the electromagnetic spectrum where organic ligands are expected to emit light, the PL emission spectrum of SnS-TMS QDs showed no other significant emission features at longer excitation wavelengths (> 300 nm).

[0250] As can be seen from Figure 3 (B), the use of elemental S as a precursor resulted in the formation of QDs with multi-colour PL emission characteristics covering the ultraviolet, the entire visible and near-infrared region of the electromagnetic spectrum. Example 2 Reaction temperature study for the colloidal synthesis of ligand passivated SnS QDs

[0251] The method of Example 1 was repeated, except that the reaction was performed using elemental sulfur as the sulfur precursor and at a temperature of either 35°C or 45°C. The samples were labelled “SnS-1” (35°C) and “SnS-2” (45°C). Results

[0252] The colloid of SnS quantum dots (QDs) that were produced from each set of reaction temperature conditions were characterised. Transmission Electron Microscopy

[0253] The SnS-1 and SnS-2 samples were imaged under low and high-resolution TEM (HRTEM). The images for SnS-1 are shown in (A) and (C) of Figure 4. The images for SnS-2 are shown in (B) and (D) of Figure 4. The properties of the samples are shown in Table 3 below. The resulting QDs showed a semi-spherical morphology with well-defined lattice fringes corresponding to their high crystallinity. Table 3: Properties of SnS QDs as a function of reaction temperature SnS-1 SnS-2 Amax (nm) 486 528 Eg (eV) 2.55 2.35 Size distribution (nm) 1.14±0.32 1.34 ±0.24 Sn:S (atomic %) 2.2:1 2.2:1 Example 3 Wavelength-dependent emission studies

[0254] The method of Example 2 was repeated, except that the reaction was performed at a reaction temperature of 55°C. Absorption and Emission Properties

[0255] Figure 5 shows 3D (A) PL and (B) PLE spectra of SnS QDs. A PL bright point is reached at around 520 nm which is a general phenomenon found in semiconductor quantum dots during the growing step. This result suggests an optimal surface rearrangement of the QDs under given reaction conditions.

[0256] The sample appears to be composed of multiple populations of quantum dots with close absorption profiles depicted from the PLE spectra in Figure 5 (B).

[0257] Figure 5 shows an image of the SnS QDs excited at 405 nm, 532 nm, and 605 nm resulting in wavelength-dependent emission properties. Transmission Electron Microscopy Figure 6 shows low and high TEM images of SnS QDs with an average QD size of 4.8 nm. Time-resolved PL (TRPL) measurements

[0258] The smaller the quantum dot size, the higher its surface-to-volume ratio resulting in a higher number of surface trap states quenching the PL lifetimes.

[0259] The TRPL measurements in Figure 7 show a progressive increase in the PL decay lifetime as the PL emission wavelength is red-shifted to longer wavelengths. This result could suggest that the increase in the PL lifetimes might be attributed to weaker Coloumbic attractions between the charge carriers as a function of increasing exciton’s distance. The smaller the size of the QDs, the stronger the charge carriers attraction resulting in a fast charge recommbination rate (shorter PL decay lifetime). Example 4 Method for the colloidal synthesis of polymer passivated SnS QDs

[0260] The method is a hot injection synthesis method. A surface-modifying agent and solvent were loaded into a 50 mL three-neck flask. The surface-modifying agent may be a polyvinyl-based polymer and can be used in a molar ratio as set out in Table 4 below. Table 4: Molar ratios of reaction components Reagents Moles (mmol) Sn (II) chloride 0.38-2.00 PVP 1-20 Ethylene glycol 18.00 Thiourea 0.5-1.00

[0261] The flask was then connected to a Schlenk line. The system was evacuated using a rotary pump and left on continuous stirring at 105°C for at least 1 h to maintain moisture free conditions. The Schlenk line was then purged with an Argon flow.

[0262] In this example, SnChxhW was the Sn-based precursor. The surface-modifying agent was polyvinylpyrrolidone (PVP). The solvent was ethylene glycol (EG). The sulfur precursor was thiourea, which were used in the molar ratios shown in Table 4.

[0263] Sn and S precursor solutions were prepared by dissolving 0.38-2.00 mmol of SnCl2xH2O in EG (% w / v) and 0.5-1.00 mmol of thiourea in EG (% w / v).

[0264] The precursor solutions were loaded into a syringe and quickly injected into the PVP solution once the reaction temperature was stabilised at 140°C.

[0265] Upon addition of the Sn and S precursors to the flask, a progressive change of the solution colour from clear yellow to dark grey was observed as the reaction went to completion.

[0266] The QDs produced from PVP are referred to herein as “SnS / PVP”. Results

[0267] The colloid of SnS / PVP QDs that were produced were characterised. UV-Vis absorption spectra

[0268] Figure 8 (A) illustrates the optical absorption spectra of five SnS / PVP solution aliquots and the calculated band-gap energy values taken at different growing stages as shown in Figure 8 (B) and compares the results to the absorption spectrum of pure PVP at the same concentration used for the synthesis of the QDs.

[0269] An increasing trend in the absorption spectrum of the quantum dots is observed with time. This observation could be related to the higher number of SnS / PVP QDs synthesised at longer reaction times giving rise to increased optical absorption.

[0270] The appearance of an exciton peak at ~ 450 nm in the visible region of the electromagnetic spectrum compared to pure PVP is a clear indication of quantum confinement. The large blue-shift in the exciton peak position (band gap of 2.8 eV) of SnS / PVP QDs (see Figure 8 (A)) compared to the bulk band-gap energy value of SnS NCs (1.3 eV) as stated in the literature indicates that the QDs have entered a strong quantum confinement regime.

[0271] The intensity of the exciton peak progressively increases over time becoming more prominent at longer reaction times. This observation suggests a monodisperse sample composed of high-quality QDs with well-passivated surface atoms reducing non-radiative charge recombination processes. Emission Properties

[0272] Figure 9 illustrates the PL emission properties of SnS / PVP QDs measured by 3D (A) PL and (B) PLE analysis. SnS / PVP aliquots all show PL emission features even at early reaction stages (reaction time of >30 min).

[0273] The intensity of the PL emission peak progressively increases by more than ten times with increasing reaction time reaching maximum intensity at 120 minutes. This could also be observed by the image in Figure 9 (C) showing the increase of the PL emission intensity with longer reaction times from 30 to 120 minutes.

[0274] Significant narrowing of the highest intensity PL peak is observed in the first 40 minutes of reaction time followed by a further narrowing over time. This phenomenon could be related to optimum surface rearrangements and size-focusing processes during the growth of the quantum dots resulting in improved size distribution with reduced inter-particle distances. This could lead to fewer QDs aggregations and an overall improvement in sample monodispersity.

[0275] The results of the 3D PL and PLE measurements support the data from the UV-Vis analysis as shown in Figure 8 (A), which indicate an increase in the absorption spectrum at prolonged reaction times resulting in the formation of more quantum dots with identical optical and emission profiles and improved surface functionalisation giving rise to an excitonic absorption peak. Example 5 Reaction Temperature study for the colloidal synthesis of polymer passivated SnS QDs

[0276] The method of Example 4 was repeated, except that the reaction was performed at a temperature of either 125°C, 140°C or 160°C. Emission Properties

[0277] Figure 10 summarises the results from the 3D PL measurements and the corresponding changes in the PL emission intensity and narrowing of the size-distribution of the quantum dots at various reaction times as a function of temperature. Regardless of the drop in the PL emission intensity at reaction times longer than 60 min at 140°C, a progressive narrowing of the size distribution of the quantum dots is observed at longer reaction times at a growing time of 120 min.

[0278] The increase in reaction temperature resulted in a faster growth rate and surface rearrangement of atoms by enhancing the PL emission intensity of the QDs. Example 6 Precursor Concentration study for the colloidal synthesis of polymer passivated SnS QDs

[0279] The method of Example 5 was repeated, except that the reaction was performed at a temperature of 140°C and various precursor concentrations and reaction times. Emission Properties

[0280] The summary graph in Figure 11 shows a systematic increase in the PL emission intensity of the quantum dots with increasing Sn concentration, which is in good accordance with the PL data recorded from the initial precursor concentration studies. The results imply that the high Sn content within the crystal structure of the quantum dots improves the surface stabilisation as a source of Sn passivation ions. The higher the Sn concentration, the greater the degree of surface passivation of the QDs boosting the SnS PL emission.

[0281] However, increasing the molar ratio of the Sn precursor more than two times with respect to the S precursor shows a decrease in the PL emission properties of the quantum dots (Sn: S= 4). Therefore, the obtained PL results indicate that there is an optimum precursor molar ratio of Sn: S= 2 required in the synthesis of SnS / PVP CQDs in order to obtain a full surface coverage of the quantum dots resulting in high-intensity PL emission and a narrowed size-distribution. Transmission Electron Microscopy and Energy Dispersive X-Ray analyses

[0282] Figure 12 represents HRTEM micrographs of individual SnS / PVP QDs with distinctive interplanar distances. The QDs are highly crystalline and monodisperse having small average sizes of <5 nm.

[0283] Figure 12 (c, f, i) show HRTEM reconstruction images, which provide greater precision for measuring the interplanar distances (d-spacing) in each quantum dot. The d-spacing values of d= 0.40 nm measured in all three quantum dots closely match the standard d= 0.4035 nm for lattice plane (110) of the orthorhombic (Pbnm) phase of SnS. Close examination of the individual quantum dots reveals their highly ordered crystal atoms and absence of defects due to dislocations or twinning effects.

[0284] The performed EDS analysis on a larger area containing multiple quantum dots revealed the stoichiometric balance between the constituent elements (Sn: S= 1:1) summarised in Table 5 below: Table 5: Stochiometric balance of constituent elements Element Weight % Atomic % S 18.63 47.89 Sn 81.37 52.18 Total 100.0 Atomic ratio Sn:S = 1:1 This observation suggests that the surface of the quantum dots is well-passivated resulting in less non-radiative recombination sites at the surface vacancies (trap states).

[0285] TEM and EDS data support the PL results from the summary plots in Figure 12 indicating an optimised surface coverage resulting in improved PL emission properties and narrowed size-distribution features of SnS / PVP QDs. Time-resolved PL (TRPL) measurements

[0286] The TRPL measurements in Figure 13 show a gradual increase in the PL decay lifetime from 2.8 ns (40 min aliquot) reaching stable value of 3.0 ns (90-120 min aliquots) at longer reaction times. The small changes in the PL lifetimes at early reaction stages could be related to surface rearrangements and size-focusing processes of the QD as a function of growing time.

[0287] The fast QD charge recombination rate of 3.0 ns suggests a well-passivated surface for efficient radiative charge recombination processes. Example 7 Biofilm synthesis

[0288] SnS QDs were prepared as in Example 1 using elemental sulfur as the sulfur precursor. Depending on the required band-gap, the reaction temperature was controlled to be between 35°C and 55°C.

[0289] 1 mmol of elemental sulfur (S) precursor was injected into the three-neck flask at the reaction temperature. There was an immediate colour change from clear yellowish to dark within 15 s, indicating nucleation. After injection of the sulfur precursor, the solution was left on the heating mantle for another 5-20 min for growing of the quantum dots. The reaction vessel was then cooled to room temperature in a cold-water bath for 30 s. The resulting colloid of QDs is referred to herein as “SnS / OA-OLA” QDs. 35

[0290] In another method, SnS QDs were prepared as in Example 6 in the presence of polyvinylpyrrolidone (PVP), instead of the OA and OLA, using thiourea as a sulfur precursor and reaction temperature of 140°C. The resulting colloid of QDs is referred to herein as “SnS / PVP” QDs.

[0291] Both PVP and the OA-OLA ligand system modify the surface of the QDs. The OA-OLA ligand system is hydrophobic, whereas PVP is hydrophilic. Bacterial strains

[0292] Pseudomonas fluorescens (P. fluorescens) ATCC 13525 bacterial strain was used in all of the biofilm and bioimaging experiments. Liquid medium preparation

[0293] For liquid medium preparation, 10 g of Luria-Bertani (LB) broth powder was mixed with 500 mL deionised (DI) water and swirled until reaching homogeneous solution. The LB medium was autoclaved at 121°C for 15-20 minutes. The LB medium was ready for use after cooling down to room temperature. Bacterial culture and preparation

[0294] Bacterial cultures of P. fluorescens were prepared in 50 mL centrifuge tubes by transferring one colony grown overnight on LB plate into a 25 mL solution of fresh LB broth and incubated at 30°C and agitated at 250 rpm overnight (> 20 h). Aliquots of bacteria were removed from the culture solution with a sterile pipette to monitor growth by measuring optical density (OD). Once the OD (600 nm) was reached, aliquots (~ 1 mL) of bacterial culture were added to fresh LB medium (~ 24 mL) forming a total volume of 25 mL and incubated for another 5 h at 30°C reaching a mid-exponential (log) growing phase. Incubation of bacteria with quantum dots

[0295] Samples of each of the hydrophobic SnS / OA-OLA quantum dots and the hydrophilic SnS / PVP quantum dots prepared as described above were mixed with a culture of P. fluorescens. A small volume of the quantum dots was added to the bacterial cells forming a 0.8 % (v / v) solution concentration in each sample. Following overnight incubation (> 20 h) at 30°C, the changes to the cells were examined. Toxicity test

[0296] 100 pL of bacteria culture was added to a 96-well plate containing varying concentrations of the SnS / OA-OLA quantum dots. The concentrations ranged from 0.3 to 20 % (v / v). The plate was covered with a lid and incubated in a plate reader (Tecan instrument) at 30°C overnight. After the overnight incubation, the lid was removed and the dose-response growth curves of the bacteria were recorded using Spark control software. Results and Discussion Incubation of bacteria with quantum dots - biofilm formation

[0297] As mentioned above, after overnight incubation of the quantum dots with the bacteria, the changes to the cells were examined. A biofilm-like growth for the culture containing SnS / OA-OLA quantum dots was observed (see Figure 14 (c) in comparison to the other culture containing SnS / PVP quantum dots (see Figure 14 (b)), which showed no biofilm formation. A reference sample that does not contain QDs is shown in Figure 14 (a).

[0298] The biofilm was examined more closely, as shown in Figure 14 (d) and (f). The biofilm was observed to form on the walls of the centrifuge tube, specifically at the surface interface between the air and liquid medium. Uniform biofilm formation was observed at some parts of the walls, which could be explained by the angle at which the centrifuge tube was swirled by the mechanical agitation process during overnight incubation.

[0299] These observations appear to be related to changes in the degree of bacterial attachment to and growth on the surface of the quantum dots as a result of the type of organic capping agent used for the SnS surface passivation. The P. fluorescens show a preferential attachment to the hydrophobic surfaces provided by the SnS / OA-OLA quantum dots resulting in a biofilm formation, as shown in Figure 14 (c). Morphological studies of the biofilm

[0300] To gain a better understanding of the morphology of the biofilm, the sample was further examined under a fluorescent microscope. The biofilm sample was studied using an optical and spinning disc confocal microscope with a laser excitation wavelength of 488 nm. Figure 15 (a) shows a fluorescence microscope image of the biofilm. The image clearly shows the high surface roughness of the biofilm formed of layers of bacteria and quantum dots.

[0301] It was observed that there were mainly two ways in which the quantum dots were arranged and assembled within the biofilm. In the area where the resulting thickness of the biofilm is the highest (estimated by eye), bubble-like spheres were seen from the fluorescence image (see Figure 15 (b)). The quantum dots tend to assemble at the surface edge of the bubbles, as well as around the bacteria showing high-intensity green emission. The bacteria were still alive and moved throughout the entire biofilm regardless of its thickness and within the biofilm voids. This is indicative of SnS / OA-OLA quantum dots having low to no toxicity. Furthermore, in the area where the surface of the biofilm was thinner and smoother (e.g. composed of a thin biofilm layer), the quantum dots were well-dispersed and surrounded the bacteria, which suggested an interaction between the bacterial cells and SnS / OA-OLA quantum dots.

[0302] Since the bacteria was still alive after an overnight incubation with the quantum dots, the formation of the biofilm does not appear to be due to the toxicity of the quantum dots, but instead results from a change in their surface properties. Toxicity test - growth assay

[0303] To reveal whether the biofilm formation was induced by the toxicity of the quantum dots, the effect of different concentrations was studied, as described above.

[0304] Figure 16 shows growth curves for P. fluorescens. The optical density changes as a function of the bacterial population over time.

[0305] No significant impact on the bacterial growth was observed with different dosages of SnS / OA-OLA quantum dots compared to the control sample, which was not exposed to the quantum dots. The overall trend shows a relatively high consistency throughout the growing stages of the bacteria regardless of the concentration of quantum dots compared to the control growing curve of P. fluorescens.

[0306] These findings confirm that the quantum dots have no observable toxicity on the reproduction of P. fluorescens bacterial cells even at high loadings. The results in Figure 16 together with the results obtained from the surface chemistry study of SnS / OA-OLA and SnS / PVP quantum dots (as shown in Figure 15) suggest that the biofilm formation is a nontoxicity induced process. The process seems to be initiated by the hydrophobic nature of the quantum dots acting as a preferential surface for bacterial cell adherence, which causes no apparent changes to the cell surface. Example 8 Bioimaging of bacterial cells

[0307] P. fluorescence biofilms formed in the presence of SnS / OA-OLA QDs after 20 hours of incubation. Results and Discussion Lambda scans of SnS QDs with surface-modifying agent

[0308] Figure 17 shows lambda scan spectra were collected as a series of individual images within a wavelength range from 420 nm to 760 nm by collecting light every 10 nm in order to characterise the fluorescence emission features of SnS / OA-OLA QDs in the presence of bacteria when excited at 405 nm, 488 nm, and 561 nm. Data analysis was processed by measuring the fluorescence intensity of the QDs at each 10 nm collection band. Changes in the intensity of the emission characteristics of SnS / OA-OLA quantum dots excited at 405 nm (blue laser), 488 nm (green laser) and 561 nm (red laser) are 38 observed. The lambda scan spectra revealed four emission peaks in the visible region of the spectrum with bright point emission wavelengths at 470 nm, 530 nm, 610 nm, and 660 nm.

[0309] It should be noted that P. fluorescens emit light in the near-visible region of the electromagnetic spectrum and does not influence the interpretation of the emission properties of the quantum dots. These observations show that the bacteria did not alter the emission wavelengths of the quantum dots, which confirms their stability upon prolonged exposure to live cells. Thus, the SnS / OA-OLA quantum dots is an excellent candidate for use in bioimaging applications. Bioimaging of bacterial cells

[0310] Figure 18 shows confocal fluorescence images of SnS / OA-OLA quantum dots (0.8 % (v / v)) incubated with P. fluorescens recorded at regions with different biofilm thickness. The intense blue, green and red fluorescence were obtained at excitation wavelengths at 405 nm, 488 nm, and 561 nm, respectively. These images reveale the size-dependent emission characteristics of the quantum dots and demonstrate their imaging potential. The images illustrate that SnS / OA-OLA quantum dots were well-distributed within the biofilm and showed higher emission intensities at regions of thicker biofilm formation.

[0311] The quantum dots showed no photobleaching even at prolonged excitation times. This is a common problem associated with fluorescent dyes, due to photochemical alterations of the organic molecules, which limits the number of excitation cycles that can be performed.

Claims

1. A plurality of tin sulfide quantum dots having a mean atomic ratio of tin to sulfur of from 1.1:1 to 10.0:1, a mean diameter of 0.50 nm to 5.00 nm, and which are photoluminescent at wavelengths of (a) 360 to 470 nm, (b) 475 to 545 nm and (c) 550 to 740 nm.

2. The plurality of tin sulfide quantum dots as claimed in claim 1, wherein the meanatomic ratio of tin to sulfur is from 1.2:1 to 9.0:1, preferably 1.3:1 to 9.0:1, more preferably 1.5:1 to 2.5:1.

3. The plurality of tin sulfide quantum dots as claimed in claim 1 or claim 2, wherein the mean diameter is in the range 0.70 nm to 3.50 nm, preferably 1.00 nm to 2.50 nm.

4. The plurality of tin sulfide quantum dots as claimed in any of claims 1 to 3, wherein said quantum dots comprise a surface modifying agent which comprisesa) a first ligand covalently bonded to the tin of the quantum dot via an amine group, preferably oleylamine; andb) a second ligand covalently bonded to the tin of the quantum dot via a carboxylate group, or conjugate acid thereof, preferably oleic acid.

5. A hot-injection synthesis method for manufacturing a plurality of tin sulfide (SnS) quantum dots as defined in any of claims 1 to 4, wherein the method comprises:(i) dispersing a metallic precursor comprising tin (Sn) in an organic solvent at a first temperature of 50°C to 150°C to produce a reactant liquid; and(ii) mixing a precursor comprising sulfur (S) with the reactant liquid at a second temperature of 25°C to 55°C.

6. The hot-injection synthesis method as claimed in claim 5, wherein said method further comprises step (i)(a) increasing the temperature of the reactant liquid to a temperature in the range 125°C to 150°C under an inert gas, preferably nitrogen (N2) or argon (Ar), which takes place between steps (i) and (ii), preferably wherein step (i)(a) further comprises addition of a ligand comprising an amine group, preferably oleylamine.20 06 257. The hot-injection synthesis method as claimed in claim 5 or 6, wherein the first temperature is a temperature of 75°C to 125°C, preferably 85°C to 110°C, such as about105°C.

8. The hot-injection synthesis method as claimed in claim 5, 6 or 7, wherein the metallic precursor comprises SnCh, SnCh (Tin (II) chloride) anhydrous, SnChx H2O (Stannous chloride, dihydrate), SnCh (Tin (II) chloride) anhydrous, SnCh x H2O (Stannous chloride, dihydrate), SnCk (Tin (IV) chloride), SnBr2 (Tin (II) bromide), SnF2 (Tin (II) fluoride), Snh (Tin (II) iodide), Sn(N(SiMe3)2)2 or a hydrate thereof, preferably the metallic precursor comprises, or consists essentially of, SnCh anhydrous.

9. The hot-injection synthesis method as claimed in any of claims 5 to 8, wherein the precursor comprising sulfur is elemental sulfur, a thioether compound or a thiocarbonyl compound, preferably elemental sulfur or a thiocarbonyl compound (e.g. thioacetamide (C2H5NS) or thiourea (SC(NH2)2)).

10. The hot-injection synthesis method as claimed in any of claims 5 to 9, further comprising mixing a surface-modifying agent in the solvent, preferably either before or after the metallic precursor is mixed with the precursor comprising sulfur.

11. The hot-injection synthesis method as claimed in claim 10, wherein the surfacemodifying agent comprisesa) a first ligand comprising an amine group, preferably oleylamine; andb) a second ligand comprising a carboxylate group, or conjugate acidthereof, preferably oleic acid.

12. A monodisperse plurality of tin sulfide quantum dots having a mean atomic ratio of tin to sulfur of from 0.5:1 to 1.5:1, a mean diameter in the range of 2.00 nm to 7.00 nm, and which are photoluminescent at a wavelength from 450 nm to 550 nm, wherein said quantum dots comprise a surface modifying agent which comprises an organic polymer.

13. The plurality of tin sulfide quantum dots as claimed in claim 12, wherein the mean atomic ratio of tin to sulfur is 1.0:1.20 06 2514. The plurality of tin sulfide quantum dots as claimed in claim 12 or 13, wherein the mean diameter is in the range 3.00 nm to 6.00 nm, preferably 5.00 nm.

15. The plurality of tin sulfide quantum dots as claimed in any of claims 12 to 14, wherein said organic polymer is selected from the group consisting of polyglycolic acid, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid), poly(N-isopropylacrlyamide), polysiloxane, polyethylene glycol, polyvinyl alcohol and polyvinylpyrrolidine (PVP), more preferably wherein said polymer is PVP.

16. A hot-injection synthesis method of manufacturing a monodisperse plurality of tin sulfide (SnS) quantum dots as defined in any of claims 12 to 15, wherein the method comprises:dispersing an organic polymer in an organic solvent at a first temperature of 50°C to 150°C to produce a reactant liquid; andmixing a metallic precursor comprising tin (Sn) and a precursor comprising sulfur (S) with the reactant liquid at a second temperature of 125°C to 160°C.

17. The hot-injection synthesis method as claimed in claim 16, wherein the first temperature is a temperature of 75°C to 125°C, preferably 85°C to 110°C, such as about105°C.

18. The hot-injection synthesis method as claimed in claim 16 or 17, wherein the metallic precursor comprises SnCh, SnCh (Tin (II) chloride) anhydrous, SnChx H2O (Stannous chloride, dihydrate), SnCh (Tin (II) chloride) anhydrous, SnCh x H2O (Stannous chloride, dihydrate), SnCh (Tin (IV) chloride), SnBr2 (Tin (II) bromide), SnF2 (Tin (II) fluoride), Snh (Tin (II) iodide), Sn(N(SiMe3)2)2 or a hydrate thereof, preferably the metallic precursor comprises, or consists essentially of, SnCh or a hydrate thereof.

19. The hot-injection synthesis method as claimed in any of claims 16 to 18, wherein the second temperature is a temperature of 130°C to 150°C, preferably about 140°C.

20. The hot-injection synthesis method as claimed in any of claims 16 to 19, wherein the precursor comprising sulfur is elemental sulfur, a thioether compound or a thiocarbonyl compound, preferably elemental sulfur or a thiocarbonyl compound (e.g. thioacetamide (C2H5NS) or thiourea (SC(NH2)2)).

21. The hot-injection synthesis method of any one of claims 16 to 20, wherein the organic polymer is selected from the group consisting of polyglycolic acid, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid), poly(N-isopropylacrlyamide), polysiloxane, polyethylene glycol, polyvinyl alcohol and polyvinylpyrrolidine (PVP), more preferably polyvinylpyrrolidone (PVP).

22. A method of imaging a biological target comprising:administering the plurality of tin sulfide quantum dots as defined in any of claims 1 to 4 or 12 to 15 to the biological target; andimaging the biological target.

23. The plurality of quantum dots as defined in of any one of claims 1 to 4 or 12 to 15 for use in a diagnostic method practised on the human or animal body.

24. A method for manufacturing a biofilm comprising contacting the quantum dots as defined in any of claims 1 to 4 or 12 to 15 with a plurality of cells.20 06 25Application No: GB2411564.4Examiner: James HoggClaims searched: 1-25Date of search: 10 February 2025Patents Act 1977: Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance X 13-16 Journal of Solid State Chemistry, vol. 300, 2021, Diaz-Cruz Evelyn B. et al., Development of SnS / PVP core / shell quantum dots with tunable color emission synthesized by microwave heating, pages 54-62. See experimental, discussion and figure 1c. A - JP 2022131898 A (INSTITUTE PHYSICAL &CHEMICAL RES) A - Journal of the American Chemical Society, vol. 131, no. 44, 2009, Xu Ying et al., Synthesis of SnS Quantum Dots, pages 15990-15991. A - Journal of the American Chemical Society, vol. 137, no. 31, 2015, de Kergommeaux Antoine et al., Synthesis, Internal Structure, and Formation Mechanism of Monodisperse Tin Sulfide Nanoplatelets., pages 9943-9952. A - US 2017 / 0369779 Al (OWEN et al.) A - CN 115571906 A (SHANGHAI INST TECH)Categories:X Document indicating lack of novelty or inventive step A Document indicating technological background and / or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of same category'. before the filing date of this invention. & Member of the same patent family E Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:International Classification:Subclass Subgroup Valid From C09K 0011 / 08 01 / 01 / 2006 C01G 0019 / 00 01 / 01 / 2006 C09K 0011 / 66 01 / 01 / 2006

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