WO2017011877A1

WO2017011877A1 - Detection of gold nanoparticles

Info

Publication number: WO2017011877A1
Application number: PCT/AU2016/050649
Authority: WO
Inventors: Agnieszka ZUBER; Heike Ebendorff-Heidepriem; Malcolm Stuart PURDEY; Andrew David Abell
Original assignee: Deep Exploration Technologies Crc Limited
Priority date: 2015-07-21
Filing date: 2016-07-21
Publication date: 2017-01-26

Abstract

A method for detecting gold nanoparticles present in an aqueous sample containing gold nanoparticles, the method including: a. mixing a fluorophore with the aqueous sample containing gold nanoparticles to form a fluorescent gold solution, the fluorophore being a boron-dipyrromethane (BODIPY) dye, the fluorescent gold solution having a BODIPY concentration in a range of from 1.0 nM to 500.0 nM; and b. exciting the fluorescent gold solution in an optical chamber with incident light to produce emitted fluorescent light, collecting the emitted fluorescent light, and determining the concentration of gold nanoparticles in the fluorescent gold solution and thus in the aqueous sample.

Description

DETECTION OF GOLD NANOPARTICLES RELATED APPLICATION

[0001 ] This application claims convention priority from Australian provisional patent application filed on 21 July 2015. The contents of that application are incorporated herein by reference

FIELD OF THE INVENTION

[0002] The present invention relates to methods for the detection and measurement of low concentrations of gold nanoparticles utilising fluorescence spectroscopy. It is envisaged that the methods of the present invention will find use in a wide variety of technical areas from mineral exploration through to biomedical and environmental research and to medical diagnosis and gene therapy.

BACKGROUND OF THE INVENTION

[0003] Gold is utilised in everyday life from jewellery, through electronics, to drug delivery. Global consumer demand for this precious metal is increasing, and in 2013 it reached above 4000t per year. The major consumers are China and India, demanding 1275 ton and 975 ton in 2013 respectively. However, the global discovery rate of gold deposits is rapidly declining, which is primarily a function of mineral deposits exposed at the Earth's surface already being found, and undiscovered deposits being buried by younger rock sequences.

[0004] Thus, new techniques are required to explore for mineral deposits, which in turn calls for the need to be able to quickly obtain geochemical analysis of rock samples, and the ability to detect economic elements such as gold at lower concentrations.

[0005] For example, an alternative method for gold exploration is to analyse the concentration of gold in the regolith, as well as in the leaves and twigs of trees growing in the area of gold deposits. In this respect, gold is first transported in an ionic (Au³⁺), water soluble form from roots to leaves, and then reduces to gold nanoparticles (in a non-oxidised state, represented as Au°) and accumulates within plant cells. Advantageously, the subsequent analysis of such plant samples to determine the concentration of gold nanoparticles in the samples can avoid the need for traditional exploratory drilling to recognise the signature of a nearby gold deposit.

[0006] Plants are not the only living organisms capable of accumulating gold nanoparticles. Bacteria such as Cupriavidus metallidurans can also take up gold ions (Au³⁺) and convert them into gold nanoparticles (Au°), which contribute to the formation of gold grains in soil. The ability to detect low concentrations of gold nanoparticles in soil can also potentially reduce the cost of searching for new gold deposits.

[0007] In addition to the potential benefits for mineral exploration, detection of very low levels of gold nanoparticles is advantageous for biomedical and environmental research. Gold nanoparticles conjugated with antibodies are used for drug delivery and sensing applications, and gold nanoparticles are also employed for gene therapy and gene specific sequence sensing.

[0008] Furthermore, photostability makes gold nanoparticles a competitive staining method in cellular imaging, where gold nanoparticles conjugated to a cell binding ligand are visible as a bright spots in a dark field image. Also, due to the enhanced absorption and scattering compared to bulk gold, gold nanoparticles can be utilised in photothermal cancer therapy.

[0009] However, these growing biomedical applications for gold nanoparticles require an ability to detect very low concentrations of gold nanoparticles. For example, cell culture studies indicate that small gold nanoparticles (1 to 2nm) are more toxic than larger gold nanoparticles (15nm) in fibroblasts, epithelial cells, macrophages and melanoma cells.

[0010] Sensitive methods for the detection of gold nanoparticles would thus facilitate tracking a potentially dangerous accumulation of gold nanoparticles in these cells. In this respect, detection at concentration levels in the parts per billion (ppb) range is required, noting that the accumulation of gold nanoparticles in rat organs after administration of 0.56mg of nanoparticles per gram of animal are known to range from 0.08ppb in brain, through 20ppb in muscle and 30ppb in bone, to 260ppb in blood. [001 1 ] Similar detection limits as for medical diagnosis can be beneficial for gold exploration purposes. The average crustal abundance of gold is about 1 .3ppb, while anomalous gold concentrations can be between 0.5 and 8ppm. Therefore, detecting anomalous levels of gold also requires instrumentation with low detection limits, again in the ppb range.

[0012] With this in mind, it is an aim of the present invention to develop a method for the detection of gold nanoparticles at levels in the ppb range. In this respect, it will be appreciated that "nanoparticles", according to the American Society for Testing and Materials (ASTM) standard definition, are particles with lengths that range from 1 to 100 nm in two or three dimensions, and that a "ppb range" is a range from 1 to 1000 pg/kg (or 1 to 1000 pg/L).

SUMMARY OF THE INVENTION

[0013] The present invention provides a method for detecting gold nanoparticles present in an aqueous sample containing gold nanoparticles, the method including: a) mixing a fluorophore with the aqueous sample containing gold nanoparticles to form a fluorescent gold solution, the fluorophore being a boron-dipyrromethane (BODIPY) dye, the fluorescent gold solution having a BODIPY concentration in a range of from 1 .0 nM to 500.0 nM;

b) exciting the fluorescent gold solution in an optical chamber with incident light to produce emitted fluorescent light, collecting the emitted fluorescent light, and determining the concentration of gold nanoparticles in the fluorescent gold solution and thus in the aqueous sample.

[0014] In one form of the method of the present invention, the fluorescent gold solution has a BODIPY concentration in a range of from 5.0 nM to 300.0 nM. In another form, the fluorescent gold solution has a BODIPY concentration in a range of from 10.0 nM to 150.0 nM. In another form, the fluorescent gold solution has a BODIPY concentration of about 60.0 nM.

[0015] The BODIPY fluorophore may be any of the fluorescent dyes composed of dipyrromethane complexed with a disubstituted boron atom, typically a BF₂ unit, and having a core of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene as below:

Formula A

[0016] Ideally, the method of the present invention will utilise a BODIPY fluorophore in the form of a bisiodinated derivative of BOBIPY, preferably one obtained by the introduction of iodine at the 2,6-positions of the BODIPY, and more preferably being one synthesised by the reaction of a fluorescent H-BODIPY with iodine to form a non-fluorescent l-BODIPY. In this respect, l-BODIPY shows specific and desirable reactivity towards gold nanoparticles, with fluorescence being observed as a result of a catalytical change of the non-fluorescent l-BODIPY to the fluorescent H-BODIPY upon excitation of the fluorescent gold solution.

[0017] In a preferred form, the synthesis of a suitable l-BODIPY is conducted via the addition of l₂ to H-BODIPY, while HIO₃ is added dropwise, ideally while stirring, until it turns a deep fluorescent red/brown colour. The compound may then be eluted through a column to provide an l-BODIPY fluorophore as a dark red solid.

[0018] The solid l-BODIPY fluorophore can then be dissolved in a polar aprotic solvent, being a solvent that dissolves salts but lacks an acidic hydrogen, and has both high dielectric constants and high dipole moments. Examples of suitable solvents are the organosulfur compound dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and hexarnethylphosphoric amide (HMPA), with DMSO being the preferred solvent.

[0019] The dissolved l-BODIPY fluorophore is then preferably diluted in an aqueous solution prior to being mixed with the aqueous sample to form the fluorescent gold solution. The preparation of the fluorescent gold solution in this manner is thus ideally conducted in the presence of a 100% water solvent, rather than an alcohol-based solvent, as diluting the l-BODIPY in water instead of alcohol broadens the range of potential applications, especially the biological applications, where cells die and proteins denaturate in the presence of alcohol. [0020] In this form of the method of the present invention, the l-BODIPY fluorophore is preferably in the form of:

Formula B

[0021 ] In a preferred form, the method of the present invention thus includes the step of mixing a fluorophore with the aqueous sample to form a fluorescent gold solution, the fluorophore being an l-BODIPY dye of Formula B dissolved with a polar aprotic solvent and water, the fluorescent gold solution having an l-BODIPY concentration in the range of 1 .0 nM to 500.0 nM, or in a range of from 5.0 nM to 300.0 nM, or in a range of from 10.0 nM to 150.0 nM, or of about 60.0 nM.

[0022] In terms of the preferred concentration of the fluorophore in the fluorescent gold solution, it has been found that its final concentration in the fluorescent gold solution is able to be as low as 1 .0 nM to 500.0 nM as the intensity of the fluorescence is higher in that range, resulting in lower achievable detection limits for the method of the present invention.

[0023] As mentioned above, the method of the present invention includes the addition of the fluorescent gold solution to an optical chamber. In this respect, the optical chamber can be any suitable form of optical chamber, suitable for holding samples for spectroscopic experiments, such as a cuvette or an optical fibre sensor. Suitable cuvettes include optical glass cuvettes, plastic cuvettes, fused quartz cuvettes, or UV, visible or IR quartz cuvettes. Suitable optical fibre sensors include sensors that utilize evanescent field based sensing, such as tapered fibre sensors, D- shaped fibre sensors, microstructured optical fibres, photonic crystal fibres, and nanowires, and also include sensors that do not rely on the use of an evanescent field, such as capillary tubes, fibre tip sensors and hollow core photonic bandgap fibres. [0024] One particular form of optical fibre sensor envisaged to be beneficial for use with the method of the present invention is the optical fibre sensor described in International patent publication WO2009/012528A1 , the full content is herein incorporate by reference in order to include a description of one suitable form of optical fibre sensor. In this form, an optical chamber for use in the method of the present invention would be such an optical fibre sensor, and would include an elongate central core for propagating incident light, an interaction region capable of receiving the fluorescent gold solution for excitation by the incident light to produce emitted fluorescent light, and an interface region (at least one elongate chamber) located between the elongate core and the interaction region. In one form, the interface region can be provided by three elongate chambers configured symmetrically along the fibre sensor about the elongate core.

[0025] As mentioned above, the method of the present invention includes the excitation of the fluorescent gold solution in the optical chamber with incident light to produce emitted fluorescent light, and the subsequent collection of the emitted fluorescent light and the determination of the concentration of the gold nanoparticles in the fluorescent gold solution and thus in the aqueous sample, all being steps based upon fluorescence spectroscopy, a measurement technique reliant on the analysis of fluorescence from a sample. In broad terms, fluorescence spectroscopy utilises the excitation of a fluorescent sample (containing a fluorophore) with incident light to produce emitted fluorescent light, and the subsequent collection of the emitted fluorescent light to permit the determination of the concentration of the fluorophore in the sample.

[0026] Fluorescence occurs when a fluorescent capable material (the fluorophore) is excited into a higher electronic state by absorbing an incident photon and cannot return to the ground state except by emitting a photon. The emission usually occurs from the ground vibrational level of the excited electronic state and goes to an excited vibrational state of the ground electronic state. The energies and relative intensities of the fluorescence signals give information about structure and environments of the fluorophores.

[0027] In a preferred form of the invention, the fluorescent gold solution is allowed to incubate in the optical chamber during excitation with incident light for a period of between 40 and 60 minutes before the emitted fluorescent light is collected for the determination of the concentration of the gold nanoparticles. In a further preferred form, this incubation period is about 50 minutes. In this respect, the peak fluorescence intensity increases during this incubation time and then becomes saturated, indicating that after the incubation time, equilibrium for the transformation of l-BODIPY into H-BODIPY is reached, with further incubation being unlikely to further improve the fluorescence intensity.

[0028] In a further preferred form of the present invention, the method additionally includes a pre-treatment step where the concentration of the nanoparticles in the aqueous sample is increased without altering the concentration of the gold in the aqueous sample. In a preferred form, the pre-treatment step includes reducing the size of the nanoparticles without reducing the gold concentration, such as by known filtration or separation steps, including density gradient centrifugation, magnetic fields, chromatography, electrophoresis, selective precipitation, membrane filtration or extraction.

[0029] In this respect, it has been found that the detection limit of the method of the present invention improves with decreasing nanoparticle size, which is believed to be due to the larger number of nanoparticles present in a sample of smaller nanoparticles with the same gold concentration, noting that, for example, for the same gold concentration of 74ppb, the nanoparticle concentration is higher for 5nm nanoparticles (97.3pM) compared to 50nm nanoparticles (0.97pM). Without wishing to be bound by theory, it is envisaged that the catalytic effect of the gold nanoparticles increases with the increasing amount of the nanoparticles in a certain sample volume as more gold nanoparticles are then available to react with the BODIPY fluorophore. Of course, it will be appreciated that the need for such an additional pre-treatment step is more likely in applications where the range of sizes of nanoparticles in the aqueous sample is broader and somewhat random, as might be expected in mineral exploration applications.

[0030] It will be appreciated that the component parts typically necessary for fluorescence spectroscopy are a sample holder (referred to above as the optical chamber) and a spectrometer, an incident photon source, monochromators used for selecting particular incident wavelengths, focussing optics, a photon-collecting detector (single, or preferably multiple channel, and usually set at 90 degrees to the light source) and finally a control software unit. An emission monochromator or cutoff filters may also be employed.

[0031 ] In prior art laboratory systems, use is usually made of a fluorescence spectrophotometer with a built-in light source. However, with the method of the present invention, it has been found that suitably low detection limits can be achieved with the use of smaller equipment, such as hand-held spectrometers without built-in light sources, rendering the method of the present invention more adaptable to the use of portable equipment, such as would be useful for off-site applications, such as in mineral exploration, and particularly for use in determining the gold concentration of an ore body.

[0032] Indeed, also included within the scope of the present invention is a method for determining the gold concentration of an ore body, the method including preparing from the ore body an aqueous sample containing gold nanoparticles, and thereafter conducting on the aqueous sample the detecting method as described above.

BRIEF DESCRIPTION OF FIGURES

[0033] The method of the present invention will now be described with regard to experimental work illustrating several preferred embodiments, in conjunction with several Figures, noting that the following description is not to limit the generality of the above description. In the Figures:

[0034] Figure 1 shows the optical set-up for the measurement of BODIPY in the presence of gold nanoparticles in a cuvette, for the purpose of the following experimental work for preferred embodiments of the method of the present invention.

[0035] Figure 2 shows the optical set-up for the measurement of BODIPY in the presence of gold nanoparticles in an optical fibre sensor, for the purpose of the following experimental work for preferred embodiments of the method of the present invention.

[0036] Figure 3A shows a cross-sectional image of the optical fibre sensor used in Figure 2 (with the inset showing a magnified image of the fibre core). Figure 3B shows an example of a fibre core for the optical fibre sensor used in Figure 2, indicating the intensity of the evanescent field in the holes surrounding the fibre core with high intensity marked red and low intensity marked blue.

[0037] Figure 4 shows a fluorescence spectrum measured in the cuvette set-up of Figure 1 using a laboratory spectrometer at a 50m in incubation period for different concentrations of 5nm gold nanoparticles in water with a 60.0 nM l-BODIPY fluorophore.

[0038] Figure 5 shows the impact of incubation time on the fluorescence intensity for a range of gold concentrations measured in the cuvette set-up of Figure 1 , again using a laboratory spectrometer and 5nm (A), 20nm (B) and 50nm (C) gold nanoparticles mixed with a 60.0 nM l-BODIPY fluorophore.

[0039] Figure 6 shows fluorescence spectra for a range of concentrations of an I- BODIPY fluorophore with 197ppb of 5nm gold nanoparticles measured in the cuvette set-up of Figure 1 .

[0040] Figures 7A and 7B show the dependence of the fluorescence intensity at 510nm on gold concentration for 5nm, 20nm and 50nm gold nanoparticles with 50 minutes of incubation with a 60.0 nM l-BODIPY fluorophore, again measured in the cuvette set-up of Figure 1 , with Figure 7A using a laboratory spectrometer and Figure 7B using a portable spectrometer.

[0041 ] Figure 8 shows the detection limit for different sized gold nanoparticles in the cuvette set-up of Figure 1 , based on fluorescence measurements using a 50 minute incubation time and a 60.0 nM l-BODIPY fluorophore for both a laboratory spectrometer and a portable spectrometer.

[0042] Figure 9 shows the fluorescence spectra obtained with the optical fibre sensor set-up of Figure 2, again with a 60.0 nM l-BODIPY fluorophore and different gold concentrations of 5nm gold nanoparticles after a 50 minute incubation time using a high resolution laboratory spectrometer.

[0043] Figures 10A and 10B show the detection limit for three different sizes of gold nanoparticles (A-5nm, B-20nm, C-50nm) measured with the optical fibre sensor set-up of Figure 2 using a laboratory spectrometer (Figure 10A) and a portable spectrometer (Figure 10B). DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0044] The method of the present invention will now be described in relation to experimental work conducted as follows.

Gold Nanoparticles

[0045] Commercially available, monodispersed gold nanoparticles in three different sizes (4.8 ± 0.7nm, 20 ± 2.5nm and 51 ± 6.1 nm diameter, hereafter referred to as 5nm, 20nm and 50nm NPs) were acquired from Nanocomposix. The initial concentration of the nanoparticles in 2mM sodium citrate solution was 0.25mM. A range of concentrations was prepared by adding nanoparticulate solution to water with a final volume of 2ml.

[0046] The following gold concentrations were used:

- ratio of weight of gold to weight of sample in parts per billion (ppb), where 1 ppb equals 1 g of gold in 1 kg of sample; and

- molar concentration of gold nanoparticles in mol of gold nanoparticles per litre of sample.

[0047] Assuming that the gold nanoparticles were spherical in shape and that their density is the same as bulk gold (i.e. 19.3g/cm³), the concentration of gold nanoparticles was calculated using:

where c_s is the molar concentration of gold NPs, c_Au is the molar concentration of gold atoms/ions, M_Au is the molecular weight of gold, p_Au is the density of gold, D_s is the diameter of the gold NPs, and N_A is the Avogadro constant.

Synthesis of l-BODIPY

[0048] The fluorophore l-BODIPY was synthesized by reaction of H-BODIPY with iodine. Specifically, H-BODIPY (25mg, 0.08 mmol) was suspended in methanol (10 ml_), and l₂ (54 mg, 0.20 mmol) was added whilst stirring. A solution of HIO3 (30 mg, 0.16 mmol) in water (200 μΙ_) was added dropwise over 5 min. The reaction mixture was stirred at 25 °C for 30 min and the solution turned a deep fluorescent red/brown colour.

[0049] The solvent was then removed under reduced pressure and the compound was eluted through a silica column with 5:1 petroleum ether : chloroform, to give I- BODIPY as a dark red solid (21 mg, 46 %) with the following spectra: H NMR (CDCI₃): 7.53-7.49 (3H, m), 7.26-7.24 (2H, m), 2.65 (6H, s), 1 .38 (6H, s)

3C NMR (CDCI₃): 156.8, 145.4, 141 .4, 134.7, 131 .3, 129.5, 129.4, 127.8, 85.65, 16.9, 16.0

[0050] Using NMR (Supplementary data), the structure of the synthesized I- BODIPY was determined as follows (Figure B, also mentioned above):

Formula B

[0051 ] The structure of Formula B of the fluorophore was noted to differ from previously presented l-BODIPY fluorophores through the absence of two polyethyl ether groups on the phenyl ring.

[0052] For subsequent sensing experiments, l-BODIPY was first dissolved in dimethyl sulfoxide (DMSO) and then diluted in water as a solvent, for subsequent use in forming fluorescent gold solutions with the required l-BODIPY concentration.

Apparatus Set-Up

[0053] The fluorescence of BODIPY solutions was measured using a 473nm laser light in the optical set-ups shown in Figures 1 and 2. The fluorescence was measured in a cuvette (Figure 1 ) and an optical fibre sensor (Figure 2), hereafter referred to as a suspended core fibre (SCF).

[0054] For the cuvette measurements (Figure 1 ) the light was focussed with a 60x objective onto a 200pm core UV-VIS optical fibre (Ocean Optics) that was connected to the cuvette holder, thereby guiding the light to the cuvette situated in the holder (OceanOptics). The emitted light (perpendicular to the excitation light) was collected by a 200pm multimode fibre (Thorlabs) connected to both the cuvette holder and the spectrometer.

[0055] For the SCF measurements (Figure 2), the light focussed with the 60x objective was coupled into a solid fibre core of the SCF. Back propagating emission light passed through the 60x objective, reflected from the mirrors and was coupled by 10x objective into the 200pm multimode fibre (Thor labs), which guided the collected emission light into the spectrometer.

[0056] In this respect, the SCF was an optical fibre sensor of the type described in International patent publication WO2009/012528A1 , the whole content of which is incorporated by reference for the purpose of describing suitable optical fibre sensors. The SCF included an elongate central core for propagating incident light, an interaction region capable of receiving the fluorescent gold solution for excitation by the incident light to produce emitted fluorescent light, and an interface region located between the elongate core and the interaction region. The interface region in the SCF was provided by three elongate chambers configured symmetrically along the fibre sensor about the elongate core.

[0057] In such an SCF, part of the light guided along its fibre core is located outside of the core, in the elongate chambers, commonly referred to as its evanescent field. The portion of the light located in the elongate chambers can be used for light- matter-interaction, and hence sensing of an analyte situated in the elongate d chambers. Such SCFs offer a range of advantages for sensing applications, including that the analysis in such a SCF requires only small samples (nanoliter volumes), and that sensing with a SCF can be made highly specific by binding specific functional groups on the surface of the fibre core. Also, due to their compact size, such an SCF is particularly suitable for portable devices such that SCFs are useful for remote measurements, such as might be required for mineral exploration.

[0058] An SCF made from silica was used for these experiments since it provides several advantages for sensing applications. Silica is made in ultrahigh purity, enabling low fibre loss and thus the use of long fibre length. Also, the high thermal, mechanical and corrosion stability of silica allows use in harsh environments, and the relatively low refractive index of silica (1 .45 in the visible) leads to a larger fraction of light being located in the air holes, enabling higher sensitivity.

[0059] Furthermore, the power fraction of light in the elongate chambers of the SCF increases with decreasing core size, and hence the sensitivity of the SCF increases with decreasing core size. Therefore, a SCF was used with a small core diameter of 1 .5pm with a cross-sectional structure shown in Figure 3a. Additionally, the fraction of light being guided in the elongate chambers for sensing is schematically shown in Figure 3b. In this example, the fibre core is surrounded by three elongate chambers of 60 pm diameter, such that the large chamber size prevents blockage by particles present in a liquid, as well as reducing the required time to fill the SCF.

[0060] For the sensing experiments with the SCF, the fibre was mounted on a 3- axis stage and light was coupled into the core until maximal power at the end of the fibre was measured with a power meter. Approximately 50 cm lengths of fibre were used for the experiment.

[0061 ] In order to compare the achievable detection limits with different spectrometers, either a laboratory-based (benchtop), non-portable spectrometer iHR320 (Horiba Jobin Yvon) or a portable Ocean Optics QE65000 spectrometer was used for cuvette and SCF measurements.

[0062] For cuvette measurements, the fluorescence was measured using a 473nm laser with 8mW output power and an exposure time of 4 seconds. For SCF measurements, the laser power was decreased to yield similar signal intensity to that of the cuvette results.

[0063] l-BODIPY was added in a 1 : 1 ratio into the aqueous samples containing gold nanoparticles to form the fluorescent gold solution referred to above. The spectrum of a blank sample (a solution of l-BODIPY with no gold nanoparticles) was subtracted from all spectra measured for samples containing gold nanoparticles.

Detection Limit

[0064] The limit of quantification (LOQ) was used as a measure for the detection limit, and is defined as: LOQ = mean_b|_ank + 10 x SD_b|_ank where r?ean_biank is the mean of the blank sample and SD is the standard deviation of the blank sample.

[0065] The experimental detection limit is the lowest gold concentration used for which the fluorescence intensity is higher than the LOQ value. The theoretical detection limit is the gold concentration value at which the corresponding fluorescence intensity is equal to the LOQ value. The gold concentration is calculated using the linear regression of the concentration dependence of the fluorescence intensity.

Discussion of Results

[0066] With reference to Figure 4, the l-BODIPY fluorophore in the fluorescent gold solution, when tested in the cuvette set-up of Figure 1 , resulted in a fluorescence peak at 510nm (the vertical black line in Figure 4) in the presence of the gold nanoparticles, whereby the intensity of the peak increased with increasing concentration of gold nanoparticles in the solution. Using a laboratory spectrometer and these cuvette measurements, the impact of the incubation time of the fluorescent gold solution, and of the l-BODIPY concentration, on the fluorescence intensity and detection limit of 5nm, 20nm and 50nm gold nanoparticles was assessed.

[0067] Figure 5 shows the impact of incubation time on the fluorescence intensity. For 20nm and 50nm gold nanoparticles, and for all gold concentrations investigated, the peak fluorescence intensity increased until approximately 50 minutes and then became saturated, indicating that after an incubation time of 50 minutes, equilibrium for the transformation of l-BODIPY into H-BODIPY was reached. For the 5nm gold nanoparticles, even after an incubation time of 60 minutes, no clear saturation was observed, indicating that the reaction rate for small nanoparticles (5nm) is slower than than for the larger nanoparticles (20nm, 50nm). Due to this time dependence, it is apparent that it is preferred for the fluorescence intensity of the samples to be measured at the same incubation time regardless of nanoparticle size, which is preferably after an incubation time between about 40 and 60 minutes, but more preferably after an incubation time of about 50 minutes.

[0068] The investigations of a range of l-BODIPY concentrations from 1 .0 nM to 500.0 nM successfully revealed fluorescence spectrum peaks across that range, with higher intensity peaks at l-BODIPY concentrations in the range from 5.0 nM to 300.0 nM, and the highest intensity peaks at l-BODIPY concentrations in the range from 10.0 nM to 150.0 nM, with an optimal l-BODIPY concentration of about 60.0 nM. With regard to Figure 6, various fluorescent gold solutions, having concentrations of I- BODIPY in the range from 1 .0 nM to 500.0 nM, were prepared with 1 ml of an I- BODIPY solution being added into 1 ml of an aqueous solution of 5nm gold nanoparticles at a gold concentration of 197 g/L (or 197ppb). The intensity of the fluorescence of the samples was measured in cuvettes using a 473nm laser with 8mW power, with a 4s exposure time.

[0069] As can be seen from Figure 6, the intensity of the fluorescence was the highest with 60.0 nM of l-BODIPY. No peak at 510nm was detected with the highest of the analysed range of concentrations of 500.0 nM. It can thus be concluded that 5nm gold nanoparticles at a concentration of 197ppb can be detected using an I- BODIPY fluorophore concentration in the range of 1 .0 nM to 500.0 nM. Furthermore, it will be recognised by a skilled addressee that this finding can be extrapolated to other gold nanoparticle sizes at other ppb concentration levels.

[0070] Turning now to a description of Figures 7A and 7B, for an l-BODIPY fluorophore concentration of 60.0 nM and an incubation time of 50 minutes, the gold concentration dependence of fluorescence intensity was measured in a cuvette using laboratory spectrometer (Figure 7A) and a portable spectrometer (Figure 7B), for each of the 5nm, 20nm and 50nm gold nanoparticles. The experimental detection limits of these measurements are depicted in Figure 8.

[0071 ] The detection limit of cuvette measurements using the portable spectrometer was found to be identical to that using the laboratory spectrometer: namely, 74ppb for the 5nm gold nanoparticles and 1230ppb for the 50nm gold nanoparticles; and slightly different for the 20nm gold nanoparticles (392ppb and 492ppb). The similar detection limits achieved using two different spectrometers confirms consistency across the same optical set up, and the same amount of laser light coupled into the sample, and the same method of collection of fluorescent light measured, confirming the applicability of the method of the present invention to a portable, off-site, use, such as would be required for remote mineral exploration efforts.

[0072] The consistent detection limit is also reflected in a similar LOQ being obtained (using Eq (2) above) for both spectrometers (being 0.015 for the laboratory spectrometer and 0.032 for the portable spectrometer). These values are of course usually higher than the theoretical detection limit values presented in Table 1 (below) due to the range of analysed concentrations. In this respect, it will be appreciated that these theoretical detection limit values represent a concentration in the range between the detection limit (the LOQ value) and the lowest experimentally detected signal above the detection limit. The theoretical detection limit is the gold nanoparticle concentration that yields a fluorescence intensity that equals the LOQ value. This concentration was determined using a line between the lowest experimentally used gold concentration yielding a fluorescence intensity above LOQ and the highest experimentally used concentration yielding a fluorescence intensity below LOQ.

Table 1 - Detection limits achievable - gold (ppb) and nanoparticle (pM) concentration. [0073] With reference now to the fluorescence spectra of Figure 9, these fluorescence spectra indicate that measurements in an optical chamber of the optical fibre sensor set-up of Figure 2 yield similar results as for measurements in a cuvette. The peak at 510nm (marked with a vertical black line) depends on the gold nanoparticle concentrations used.

[0074] The gold concentration dependence of the fluorescence intensity in an SCF (the optical fibre sensor set-up of Figure 2) was measured using different nanoparticle sizes at a 50 minute incubation time with a 60.0nM l-BODIPY fluorophore for both laboratory and portable spectrometers. The experimental detection limits for these measurements are: laboratory spectrometer (see Figure 10A) - 74ppb for 5nm gold nanoparticles, 492ppb for 20nm gold nanoparticles, 738ppb for 50nm gold nanoparticles; portable spectrometer (see Figure 10B) - 74ppb for 5nm gold nanoparticles, 246ppb for 20nm gold nanoparticles and 492ppb for 50nm gold nanoparticles.

[0075] The results in Figures 10A and 10B demonstrate that the detection limits in the SCF are similar for the two spectrometers used, which is consistent with the fluorescence measurements in the cuvette. There is no difference between the detection limit using the laboratory and portable spectrometers for 5nm gold nanoparticles in the SCF (both 74ppb), and a small difference between the detection limit using the laboratory and portable spectrometers for 20nm and 50nm gold nanoparticles in the SCF (20nm - 492ppb and 246ppb respectively, 50nm - 738ppb and 492ppb respectively). As explained above, a similar detection limit regardless of the type of spectrometer used is related, at least in part, to the use of the same optical set up for the measurements, and is reflected in similar values of LOQ (0.059 for the laboratory spectrometer and 0.025 for the portable spectrometer).

[0076] Also, the conversion (using Eq (1 ) above) of the measured detection limits (in terms of gold concentration) from the experimental work, to molar concentrations of gold nanoparticles (see Table 1 above) reveals that the detection limit in fact decreases with increasing gold nanoparticle size. Indeed, both the cuvette and SCF studies show that the detection limit of fluorescence measurements actually decreases with decreasing gold nanoparticle size. This finding is attributed to the different number of gold nanoparticles in samples with the same gold concentration. For example, for the same gold concentration of 74ppb, the nanoparticle concentration is higher for 5nm nanoparticles (97.3pM) compared to 50nm nanoparticles (0.97pM).

[0077] In this respect, it is envisaged that the catalytic effect of the gold nanoparticles increases with the increasing amount of the nanoparticles in a certain sample volume as more gold nanoparticles are available to react with the l-BODIPY fluorophore. Additionally, for all nanoparticle sizes, the molar nanoparticle concentration at detection limits is considerably smaller than that of the l-BODIPY fluorophore, which is consistent with the gold nanoparticles acting as catalyser for the reaction of the l-BODIPY to H-BODIPY.

[0078] With further reference to Table 1 , the lower detection limit for the 50nm nanoparticles measured in the SCF compared to the cuvette measurement is attributed to the interactions between gold nanoparticles and the silica core of the fibre of the SCF. For small nanoparticles, the repulsion forces are stronger than for bigger nanoparticles. In turn, the 50nm nanoparticles thus attach to the fibre core of the SCF and interact with light longer than the 5nm nanoparticles do, resulting in a decreased detection limit in the SCF fibre in contrast to the cuvette.

[0079] Finally, previous reports have indicated that BODIPY fluorophores can provide detection of gold nanoparticles at nanoparticle concentrations as low as 500pM (around 30nm). However, the above work shows that the method of the present invention is capable of lower gold nanoparticle detection limits for all three nanoparticle sizes investigated, for example for measurements (in the SCF), using a portable spectrometer, such as 97.3pM for the 5nm gold nanoparticles, 5.05pM for the 20nm gold nanoparticles, and 0.97pM for the 50nm gold nanoparticles.

[0080] These results demonstrate that gold concentrations at ppb level, perhaps only previously detected using laboratory based spectrometer equipment, can actually be detected by the method of the present invention when using portable spectrometers. These detection limits for gold are also well below economic values, and shows the potential in being able to detect anomalous gold concentrations that are significantly above background (average crustal abundance) and within the range of what would be desired for measuring gold in an exploration environment. [0081 ] Biological studies could also benefit from such portable detection of gold. The accumulation of gold nanoparticles as low as few ng/g of animal has been shown to be beneficial (for example, in blood, bone and muscle). Also, gold nanoparticles conjugated with ligand, such as antibody, which binds specifically to a target such as a cancer cell or protein, could be quickly analysed without the need for time consuming analysis by ICP-MP, ICP-AES or TEM, leading to faster diagnosis of carcinogenesis or proteopathies.

[0082] While the invention has been described in conjunction with a limited number of embodiments, it will be appreciated by those skilled in the art that many alternative, modifications and variations in light of the foregoing description are possible. Accordingly, the present invention is intended to embrace all such alternative, modifications and variations as may fall within the spirit and scope of the invention as disclosed.

Claims

The claims are:

1. A method for detecting gold nanoparticles present in an aqueous sample containing gold nanoparticles, the method including:

a. mixing a fluorophore with the aqueous sample containing gold nanoparticles to form a fluorescent gold solution, the fluorophore being a boron-dipyrromethane (BODIPY) dye, the fluorescent gold solution having a BODIPY concentration in a range of from 1 .0 nM to 500.0 nM; and b. exciting the fluorescent gold solution in an optical chamber with incident light to produce emitted fluorescent light, collecting the emitted fluorescent light, and determining the concentration of gold nanoparticles in the fluorescent gold solution and thus in the aqueous sample.

2. A method according to claim 1 , wherein the fluorescent gold solution has a BODIPY concentration in a range of from 5.0 nM to 300.0 nM.

3. A method according to claim 1 , wherein the fluorescent gold solution has a BODIPY concentration in a range of from 10.0 nM to 150.0 nM.

4. A method according to claim 1 , wherein the fluorescent gold solution has a BODIPY concentration of about 60.0 nM.

5. A method according to any one of claims 1 to 4, wherein the BODIPY fluorophore is a dipyrromethane complexed with a disubstituted boron atom, having a core of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene as per Formula A:

Formula A and is a bisiodinated derivative obtained by the introduction of iodine at the 2,6-positions of the BODIPY.

6. A method according to claim 5, wherein the BODIPY fluorophore is synthesised by the reaction of a fluorescent H-BODIPY with iodine to form a non-fluorescent l-BODIPY.

7. A method according to claim 6, wherein, during the synthesis, a solid I- BODIPY is formed, which is then dissolved in a polar aprotic solvent.

8. A method according to claim 7, wherein the polar aprotic solvent is selected from the group of dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and hexarnethylphosphoric amide (HMPA).

9. A method according to claim 7, wherein the polar aprotic solvent is dimethyl sulfoxide (DMSO).

10. A method according to any one of claims 6 to 9, wherein the l-BODIPY fluorophore is diluted in a 100% water solvent prior to being mixed with the aqueous sample to form the fluorescent gold solution.

1 1 . A method according to any one of claims 6 to 10, wherein the l-BODIPY fluorophore is in the form of Formula B:

Formula B

12. A method according to any one of claims 1 to 1 1 , wherein the optical chamber is suitable for holding samples for spectroscopic experiments, and is either a cuvette or an optical fibre sensor.

13. A method according to claim 12, wherein the cuvette is either an optical glass cuvette, a plastic cuvette, a fused quartz cuvette, or a UV, visible or IR quartz cuvette.

14. A method according to claim 13, wherein the optical fibre sensors is a sensor that utilizes evanescent based sensing, including a tapered fibre sensor, a D- shaped fibre sensor, a microstructured optical fibre, a photonic crystal fibre, and a nanowire, or is a sensor that does not rely on the use of an evanescent field, including a capillary tube, a fibre tip sensor and a hollow core photonic bandgap fibre.

15. A method according to any one of claims 1 to 14, wherein the fluorescent gold solution is allowed to incubate in the optical chamber during excitation with incident light for a period of between 40 and 60 minutes before the emitted fluorescent light is collected for the determination of the concentration of the gold nanoparticles.

16. A method according to any one of claims 1 to 14 wherein the fluorescent gold solution is allowed to incubate in the optical chamber during excitation with incident light for a period of 50 minutes before the emitted fluorescent light is collected for the determination of the concentration of the gold nanoparticles.

17. A method according to any one of claims 1 to 16, additionally including a pre- treatment step where the concentration of the nanoparticles in the aqueous sample is increased without altering the concentration of the gold in the aqueous sample.

18. A method according to claim 17, wherein the pre-treatment step includes reducing the size of the nanoparticles without reducing the gold concentration.

19. A method for determining the gold concentration of an ore body, the method including preparing from the ore body an aqueous sample containing gold nanoparticles, and thereafter conducting on the aqueous sample the detecting method of any one of claims 1 to 18.