DESCRIPTION

The present disclosure relates in general to the sector of nanoparticles and in particular nanoparticles which have optical and magnetic properties and which may be used for biomedical applications, such as biomedical imaging and tumour therapy.

Even more particularly, the present disclosure relates to nanoparticles including a magnetic core and an external coating including gold.

The development of multi-functional nanoprobes, which permit new imaging methods able to overcome the intrinsic limitations of individual components forming the nanoparticles, is of considerable interest for many areas of research including molecular imaging and medical diagnostics.

Recent advances in the sector of nanotechnology have resulted in a variety of nanoparticles such as magnetic nanoparticles (MNPs) and metallic nanoparticles. The extraordinary electronic, magnetic and optical properties of these particles have been exploited in a wide range of biomedical applications, such as biomedical imaging and tumour therapy.

The aforementioned nanostructures have various advantages, but also certain drawbacks.

As regards the advantages, it may be noted that magnetic nanoparticles (MNPs) have become an important contrast medium in T2 magnetic resonance imaging (MRI) since magnetic resonance offers a high resolution and excellent depth of penetration into the tissues.

As regards the drawbacks, it should be noted that magnetic resonance is not sensitive like other techniques, such as optical imaging or X-ray tomography.

In connection with metal particles, nanostructures including gold are often used for biomedical applications, in particular for imaging. However, the depth of penetration into the living tissue of the electromagnetic radiation necessary for interacting with the gold nanoparticles is limited to a few millimetres.

As a result of the above, in order to improve the performance and advantages of magnetic nanoparticles and metallic nanoparticles, the idea has occurred to combine these nanoparticles in a single multi-functional nanoparticle, which could be used in a vast variety of applications, while maintaining the unique properties of each (magnetic and metallic) component on a nanometric scale.

In particular, nanoprobes with multiple functions allow the use of new imaging methods which cannot be used for the single separate (magnetic and metallic) components, increasing the specificity and resolution of the techniques. In fact, properties such as absorption in the near-infrared range, magnetization or high scattering efficiency are extremely useful for biological applications of these nanostructures.

However, the production of nanoprobes which contain multiple integrated functions, while retaining compact dimensions is complicated.

In fact, during recent years, many attempts have been made to obtain nanometric gold coatings directly on magnetic nanoparticles. The resultant nanostructure have a low absorption in the near-infrared range (NIR), while maintaining compact particle dimensions. This fact is particularly critical for biomedical applications such as in vivo and therapy imaging.

The inventors of the present disclosure have in fact noted that this requirement is due to the fact that magnetic particles with thin gold coatings absorb mainly in the visible spectrum. For an efficient absorption in the NIR the nanoparticles should have larger dimensions (200-500 nm).

The present disclosure is based on a recognition by the inventors of the present disclosure that a possibility of covering inorganic magnetic surfaces with soluble, anti-fouling, biocompatible polymers are crucial for obtaining nanoprobes which can be used in clinical practice.

Even more particularly, the present disclosure is based on a recognition by the inventors of the present patent application that it is possible to optimize optical absorption of magnetic nanoparticles coated with an intermediate polymeric layer and with an external gold metallic layer in order to obtain particles with a compact size, optimum magnetic properties and an optimum absorption in the range of interest for biological applications (680-900 nm).

In the light of the above recognition, a technical problem forming the basis of the present disclosure is that of providing nanoparticles including gold which have optimum magnetic properties and an absorption in the range of 680-900 nm.

Said technical problem is solved by means of a nanoparticle as defined in the independent claims, by a method for synthesizing nanoparticles as defined in claim 14 and by a use of nanoparticles as defined in claim 21.

Secondary characteristic features forming the subject of the present disclosure are defined in the respective dependent claims.

In particular, with reference to the nanostructure as defined in the independent claims, the inventors of the present disclosure have recognized that, in order to obtain optimum optical properties, without modifying the magnetic properties, it is possible to use nanostructures including gold, with a non-spherical form, which absorb mainly the light in the following range of 650-900 nm.

Even more particularly, with reference to claim 1 , anisotropic nanoparticles including gold, for example with dimensions of about 60 nm, containing a core of magnetic nanoparticles, and an external coating of gold with an irregular or non- spherical, for example, branched form, are provided.

In one embodiment of the present disclosure, the non-spherical form is obtained by means of the presence of protuberances on the surface, which have an optical behaviour similar to gold nanorods. Anisotropy, or more specifically the presence of branches, allows correspondence or matching of the absorption frequency depending the size of the branches present in the particle. In particular, it has been found that the presence of the branches results in an increase in the absorption of the radiation in the red-infrared range of the spectrum associated with plasmon movements localized on the tips of the protuberances.

Even more particularly, in accordance with one aspect of the present disclosure, the synthesized final nanoparticle consists of a core of magnetic nanoparticles which are incorporated or embedded in a gold structure having what may be defined as a star shape. In the present disclosure, the nanoparticle may be viewed as being the combination of a central sphere and outwardly directed cones on the surface which have a classical "star-shaped" form. The optical properties of the resultant nanoparticle are determined by its form. In fact, not all the anisotropic nanoparticles have efficient absorption in the NIR range of 600-900 nm. Differently from other documents cited in the literature (such as the patent application US201 1/206619A1 and WO201 1/006002 A2), the nanoparticles of the present disclosure include a plurality or several magnetic cores which are trapped inside the gold matrix. It therefore consists of a multi-core. In this case, therefore, there is no growth of a thin film of gold on the surface of the polymer with a spherical or anisotropic form as in the abovementioned patent applications, but the formation of an agglomeration of magnetic nanoparticles inside a gold matrix.

The confirmation that each star-shaped nanoparticle contains several magnetic cores arises from the possibility of separating magnetically the nanoparticles in a time interval much shorter than that which is used to separate single magnetic cores. For example, by way of demonstration of this statement, the suspension of single magnetic cores in water subjected to the magnetic field of a commercial magnet requires more than 7 days in order to separate the magnetic cores from the solvent. This is due to the fact that the small dimensions of the magnetic cores (about 10 nm) limits the size of the magnetic domains which results in a low magnetic moment value. On the other hand, the star-shaped nanoparticles of the present disclosure may be separated in the same conditions in a much shorter time, for example of about 4 hours. This fact indicates the presence of numerous magnetic cores inside each star-shaped nanoparticle. The presence of several magnetic cores inside the same nanoparticle increases the magnetic moment of the material owing to the increase in the dimensions of the magnetic domains due to the presence of several magnetic cores. Therefore, in the system presented in the present disclosure, the final nanoparticle obtained, differently from the aforementioned documents published in the literature, has magnetic characteristics substantially different from those of the magnetic starting cores.

The magnetic cores of said plurality of magnetic cores are to be understood as being magnetic cores covered by the polymeric coating, namely there are a plurality of cores covered by the polymeric coating.

In other words, differently from the aforementioned patent applications in which each single magnetic nanoparticle is covered by a gold shell, in accordance with a number of embodiments of the present disclosure, the aforementioned experiments show that each single star-shaped nanoparticle contains several (unknown number of) magnetic cores (understood as being plurality of cores plus polymeric layer). Essentially, the final nanoparticle consists of a certain number of magnetic cores, grouped together and covered by the gold, so as to be embedded in a gold mass, which has a star-shaped structure. Unfortunately, there do not exist experimental methods which are able to indicate the number and the manner in which these magnetic cores are arranged inside the final nanoparticle. However, as mentioned above, considering the separation conditions, the presence of such a plurality of magnetic cores is certain.

In accordance with the present disclosure, the possibility of obtaining stable nanoparticles with a high absorption in the NIR region is due to the extensive protuberances present on the surface. In one embodiment of the present disclosure, the average dimensions of the cones are about 10-15 nm, measured by means of a transmission electron microscope.

These nanoparticles differ substantially from those cited in the aforementioned patent applications since, in accordance with certain aspects of the present disclosure, it is not possible to define clearly the resultant nanoparticle as consisting of a gold shell which covers the single magnetic nanoparticle, but it consists instead of an interpenetrating structure consisting of a plurality of magnetic nanoparticles and gold. In other words, differently from the nanoparticles described in the aforementioned patent applications where each single magnetic core is covered by a gold shell of uniform thickness, in the nanoparticles according to the present disclosure, the arrangement of the magnetic cores inside the nanoparticle is unknown and random. A structure such as this may be defined as interpenetrating in the sense that the magnetic cores are incorporated inside the gold structure. As a result it is therefore not possible to define a dimension of the gold shell since the dimension varies from one point to another inside the nanoparticle.

Moreover, the effective absorption in the NIR region does not depend, as in the case of the aforementioned patent applications, on the absorption due to the gold shell, but on the plasmon resonance localized on the tips of the nanoparticle. This allows effective localization of the surface plasmon resonance with a high absorption in the NIR.

In particular, the shell structures, such as those of the prior art, have a plasmon frequency which is dependent on the thickness of the gold shell. The thinner the shell, the more the plasmon absorption frequency moves into the near-infrared range. In accordance with the present disclosure, on the other hand, the plasmon resonance of the star-shaped particle depends on the dimensions of the protuberances. The longer and thinner the protuberance, the more the frequency moves towards the near-infrared range.

In addition, it is pointed that, differently from the gold nanoshell of the aforementioned patent applications, where the plasmon surface resonance is determined by the difference between the dielectric properties of the shell and the (metal-dielectric) core and depends principally on the thickness of the gold shell, in the case of the star-shaped nanoparticles according to the present disclosure, the plasmon resonance is localized on the protuberances of the nanoparticles. The dimensions and the form of these tips are a fundamental parameter for defining the plasmon absorption of the nanoparticle. It has been noted in fact that even small variations in the form of tip (length and width) may result in variations in the absorption spectrum.

The present disclosure also relates to a method for synthesizing nanoparticles including anisotropic gold, for example in some embodiments, with a star-shaped form. In particular, the nanostructures are produced by means of a controlled reduction of a gold precursor in a basic aqueous environment using magnetic nanoparticles, for example, consisting of iron oxide and covered by a polymeric coating as a growth core, or multi-core, for the gold.

The synthesis method according to the present disclosure is moreover much faster and simple to perform compared to other methods described in the literature relating to the synthesis of nanoparticles with magneto-optical properties. Moreover, the synthesis may not envisage the use of surfactants, growth agents or stabilizing polymers which may be difficult to eliminate at the end of synthesis.

In one embodiment of the present disclosure, the nanostructures are produced by means of the controlled reduction of a gold precursor, using for example hydroxylamine, in an aqueous basic environment, for example with a pH of at least 12. The nanostructures produced have a plasmon surface resonance with a maximum in the NIR range and magnetic properties derived from the magnetite core.

According to the synthesis procedure described, the gold is directly reduced on the magnetic particles, resulting initially in their agglomeration and subsequently the formation of a structure larger than the single magnetic cores, formed by a star- shaped gold matrix containing internally the magnetic nanoparticles. In other words, in accordance with the present disclosure, it may be reasonably assumed that, during initial reduction of the gold on the polymer of the magnetic core, gold nanoparticles of nanometric size are formed. The formation of these gold nanoparticles on the surface of the magnetic cores destabilizes the magnetic core since the polymer (which is principally responsible for the stability of the magnetic cores in a solution) is partially covered by the gold. As a result, a certain number of magnetic cores aggregate, forming a small group of magnetic cores (multi-core). At this point the gold is reduced rapidly on the group of magnetic cores, covering the entire multi-core and forming the star-shaped nanoparticle.

In other words, it is reasonable to consider that, during the initial stages of synthesis, it is likely that some magnetic cores will form aggregates (consisting of a certain number of attached magnetic cores), the multi-core being then covered by the gold which acts as a bonding agent for the structure.

This process differs significantly from other processes described in literature (C. M. Barnett et al. J Nanopart Res (2012) 14: 1 170 and Hoskins et al. Journal of Nanobiotechnology 2012, 10: 27) which refer to the use of preformed nanoparticles attached to the surface of the magnetic core and the subsequent reduction of the gold around the nanoparticle. The reduction process performed using the hydroxylamine reducing agent in the present disclosure was carried out at very basic values of the pH (pH 12). With this pH value it is possible to obtain star- shaped magnetic nanoparticles in the aforementioned conditions. At lower pH values (up to 10) there is no reduction of the gold on the metallic nanoparticles, while at intermediate pH values (between 10 and 1 1) there is the formation of nanoparticles which do not have a star-shaped form and with large dimensions (greater than 300 nm) such that they precipitate irreversibly.

This is due to the incomplete reduction of the gold on the nanoparticles which results in aggregation of the components which eventually precipitate. The reason for which - differently from the aforementioned patent applications - in the present disclosure it is required to have pH values which are highly basic is that, in accordance with the present disclosure, there are no preformed nanoparticles bonded to the magnetic core. In fact, it is known in the literature that gold may be reduced by the hydroxylamine only if colloidal gold is already present in the solution. In the synthesis process according to one embodiment, the pH value at least equal to 12 is fundamental for obtaining the cited nanoparticle with a star- shaped form in a single reaction step. A further difference between the method proposed and the methods of the aforementioned patent applications consists in the possibility of synthesizing magnetic nanoparticles with optical properties in a single step.

On the other hand, the methods described in the aforementioned patent applications envisage numerous steps for obtaining the final nanoparticle, which involve therefore a complicated synthesis procedure and long synthesis times. The reason for formation of a star-shaped nanoparticle consists in the fast reduction of the gold by the reducing agent. The reduction speed increases with an increase in the basicity of the solution; therefore, in order to obtain a star-shaped form, solutions with a pH of at least 12 are required.

The nanoparticles may have dimensions of about 60 nm and contain magnetic cores consisting of nanoparticles of magnetite with dimensions, for example, of 10- 13 nm. These nanoparticles have optimum magnetic properties and an intense plasmon absorption band in the region of 700-800 nm.

The nanoparticles according to the present disclosure have important magnetic and optical properties which, together with the small dimensions, make them ideal for a vast wide range of applications.

The present disclosure also relates to the use of nanoparticles as described in the present disclosure. In particular, the nanoparticles may be used for biomedical applications such as MRI imaging, contrast means for photoacoustic tomography or magneto- photoacoustics, nanoparticles for photothermal therapy and/or substrates for SERS (Surface Enhanced Raman Scattering) applications.

Further advantages, characteristic features and modes of use forming the subject of the present disclosure will become clear from the following detailed description of examples of embodiment thereof, provided by way of a non-limiting example.

It is evident, however, that each example of embodiment may have one or more of the advantages listed above; in any case it is not required that each example of embodiment should have simultaneously all the advantages listed.

In particular, with reference to the attached figures:

- Figure 1 illustrates in schematic form a synthesis method according to an embodiment of the present disclosure;

- Figure 2 shows an absorption spectrum of a nanoparticle according to an embodiment of the present disclosure;

- Figure 3 shows an image, obtained under a transmission electron microscope, of a nanoparticle according to an embodiment of the present disclosure;

- Figure 4 shows an image, obtained under a transmission electron microscope, of a nanoparticle according to an embodiment of the present disclosure;

- Figure 5 shows a schematic representation of a nanoparticle according to the prior art;

- Figure 6 shows a schematic representation of a nanoparticle according to an embodiment of the present disclosure.

With reference to the following examples and the attached drawings, a nanoparticle 10 according to an embodiment of the present disclosure, a method for synthesizing the nanoparticle 10 or nanoparticles according to an embodiment of the present disclosure, and a number of possible uses of a nanoparticle 10 or nanoparticles according to respective embodiments of the present disclosure are described.

In the examples described below, the following chemical products produced by Sigma-Aldrich were used below without further purification. In particular, magnetic nanoparticles of iron oxide (Fe ₃ 0 ₄ ) stabilized by oleic acid in toluene, with a nominal dimension of 10 nm, were used as magnetic cores. Phospholipid- polyethylene glycol with methoxy (-OCH ₃ ) and amino (-NH ₂ ) terminations, indicated also as (PL)-PEG-X (DSPE PEG-2000 X=- OCH ₃ , - NH ₂ ) produced by Avanti Polar Lipids, was used for example the polymeric coating. In order to obtain the shell containing gold, an aqueous solution of HAuCI4 was used.

EXAMPLE OF SYNTHESIS

Initially the magnetic nanoparticles are solubilised in water.

In particular a solution of iron oxide nanoparticles (Fe ₃ 0 ₄ ) coated by oleic acid in toluene (3 ml, 1 mg / ml) is added to a quantity of 4.8 ml of chloroform, obtaining a suspension.

A solution of DSPE-PEG (4.56 ml to 5 mg / ml) and DSPE-PEG-amine (240μΙ to 5 mg/ml) in chloroform are added to the suspension, obtaining a homogeneous mixture.

After stirring, the mixture is dried with argon and left inside a vacuum drier for 48 hours in order to remove all traces of organic solvents, obtaining a substantially dried film.

The dried film is suspended again in de-ionized water.

This method may be regarded as being a variation of a process described by Dubertret et al. (Benoit Dubertret, Paris Skourides, David J. Norris, Vincent Noireaux, AN H. Brivanlou, Albert Libchaber In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles Science 29 November 2002: Vol. 298 No. 5599 pp. 1759-1762).

The concentration of NH ₂ groups in the final solution may be measured using the fluorimetric method. In some embodiments of the present disclosure, the maximum number of NH ₂ groups in each MNP may be within a range of 30-60. The excess lipids were purified from the solution of MNPS with repeated ultracentrifugation (25,000 rpm for 1 h, 3 times) and removal of the supernatant, which is replaced with Milli-Q water.

The purified nanoparticles (32μg) were dissolved in 0.5 ml of aqueous solution with pH 12 by means of NaOH. After incubation for 1 minute, with continuous stirring, 60 μΙ of aqueous solution containing HAuCI ₄ (10 mM) are added and the solution stirred for 5 minutes.

A reducing agent, such as NH ₂ OH (200 mM in H ₂ 0, 50 μί) was then introduced into the solution in order to start reduction of the gold precursor, as illustrated schematically in Figure 1. It has been noted that, at the moment of mixing, the colloidal solution changes colour from pale orange to blue-green within the space of a few minutes, indicating the formation of colloidal gold. The solution is mixed for a further two minutes.

The suspension was purified by means of centrifuging/washing (for example 12,000 rpm for 5 minutes, which may be repeated three times).

Finally, in order to isolate the nanoparticles obtained, the latter were further purified by means of magnetic separation using, for example, commercial magnets.

In connection with the example of the synthesis method described above, the following comments may be made.

The method in accordance with an embodiment of the present disclosure, such as that described here, allows functionalization of the iron oxide nanoparticles by means of intercalation of phospholipid units in the hydrophobic polymeric coating which covers each of the iron oxide nanoparticles.

The hydrophobic shell therefore includes chains of eighteen carbon atoms, two of which are unsaturated due to the presence of the stabilizing agent (oleic acid). The hydrophobic part of the DSPE-MPEG polymer consists of saturated carbon chains and a phosphoric group. Owing to the hydrophobic interaction between the chains it is possible to intercalate the hydrophobic tail of the polymer inside the magnetic nanoparticles or magnetic core. The section containing the polyethylene glycol is thus exposed on the outside of the nanoparticle, imparting to it hydrophilic properties and allowing solubilisation in water of the nanoparticles.

In relation to the example described above it should also be noted that two different terminations were used for the PEG: methoxy (-OCH ₃ ) and amino (-NH ₂ ).

A sample consisting entirely of an amino termination (-NH ₂ ) may have a strong tendency towards agglomeration of the nanoparticles as a probable consequence of intra-particle acid-base interactions.

In some embodiments of the present disclosure, the concentration of PEG with an amino termination (-NH ₂ ) used may be 10% w/w.

Moreover, the presence of the PEG may allow the nanoparticles to be effectively stabilized in an aqueous solution, also in the highly basic conditions used for reduction of gold precursor (pH 12).

In basic pH conditions moreover, the amino terminations are deprotonated, allowing therefore the coordination of the gold precursor by means of the electron doublet of nitrogen.

The addition of the reducing agent finally starts the reduction reaction of the gold, which takes place completely within the space of one minute. It has been found moreover that the basic pH of the solution is a fundamental parameter for obtaining the protuberances. These protuberances arise from the rapid formation and clustering of gold particles on the surface of the magnetic nanoparticles which produce a non-smooth irregular surface with the formation of protuberances and branches.

The colour of the solution gradually becomes darker, passing from the pale orange of the magnetic nanoparticles to reddish brown and finally to blue green. Figure 2 shows the absorption spectrum of the sample in water.

The spectrum has a broad absorption peak at 690 nm which extends up to 900 nm. The nanoparticles may be easily separated from the solution using commercial magnets which may be moved towards the containers containing the solution of nanoparticles.

After about an hour, the nanoparticles are deposited quantitatively on the glass of the container in the proximity of the magnet located alongside the container so as to be physically separated, while the solution appears colourless.

It may therefore be stated that the steps of purification and separation of nanoparticles according to the present disclosure may be very rapid.

With reference to Figures 3 and 4, these figures include images, obtained under a transmission electron microscope, of the sample obtained. The images show the presence of non-spherical "branched" nanoparticles with a size of about 60 nm. The basic analysis obtained using the same instrument revealed the presence of iron in the sample (7% in moles), confirming the presence of nanoparticles of Fe ₃ 0 ₄ inside the gold coating. In particular, in accordance with the present disclosure, each single star-shaped nanoparticle contains several (unknown number) of magnetic cores (understood as being a plurality of cores covered by a polymeric layer). Basically, the final nanoparticle consists of a certain number of magnetic cores, grouped together and covered by gold, so as to be embedded in a gold mass, which then forms a star-shaped structure. With reference to Figures 5 and 6, these show, by way of comparison, in schematic form, forms of star-shaped nanostructures according to the prior art (Figure 5) and according to an embodiment of the present disclosure. In particular, the shell structures, such as those shown in Figure 5, have a plasmon frequency which is dependent on the thickness of the gold shell. The thinner the shell, the more the plasmon absorption frequency moves into the near-infrared range. In the nanoparticles according to the present disclosure, including a plurality of polymer-coated cores incorporated in a gold mass, the plasmon resonance of the star-shaped nanoparticle depends on the dimensions of the tips. The longer and thinner the tip, the more the frequency moves towards the near-infrared range.

EXAMPLES OF USE

The nanoparticles according to the present disclosure may be used for biological applications which envisage the use of media containing salts, proteins and other organic molecules.

In the case of biological applications a further stabilization of the nanoparticles may be performed by means of conjugation with further different polymers containing functional groups interacting with the gold surfaces (such as, for example, carboxyl groups, amines and thiols).

For applications as sensors using the plasmon absorption band or for applications as substrates for SERS (Surface Enhanced Raman Scattering), the nanoparticles may be used directly after synthesis without any further functionalization.

In order to obtain a Raman spectrum of an analyte, this analyte may be mixed with nanoparticles directly in an aqueous solution. It is possible to obtain, by means of the micro-Raman technique, the spectrum of the sample of interest, analysing directly the colloidal suspension.

In the case of water-insoluble molecules/analytes, the magnetic characteristics of the nanoparticles may be used to deposit a homogeneous film on different substrates.

The substrate obtained may then be used for micro-Raman analysis in dry conditions. The system for magnetic deposition of the nanoparticles may be used also for decorative purposes such as obtaining coloured glass.

Another important application of the nanoparticles according to the present disclosure may be the use of these nanoparticles as contrast agents for biological imaging. Owing to the combination of the magnetic and optical properties, the nanoparticles may be used as contrast means in magnetic resonance imaging techniques, using the magnetic relaxation times of the magnetite cores, or in photoacoustic tomography, using the plasmon properties of the nanoparticles, in particular their high impact cross-section in the transparency range of the biological tissues (700-900 nm) and the capacity of the nanoparticles to convert the photon impulses into heat.

It should be noted that the capacity of the nanoparticles to absorb the wavelengths in the near infrared region and the capacity of said nanoparticles to transfer the energy absorbed is one of the properties forming the basis of the analysis and imaging techniques described above.

In the SERS technique, the nanoparticle absorbs the radiant energy, transferring it to the molecule to be analysed, increasing significantly the emission intensity of the Raman spectrum, thus allowing the analysis of molecules also with low concentrations. In photoacoustic tomography, the capacity of the nanoparticle to convert the radiant energy absorbed into heat is exploited such that spatial detection thereof may be performed by means of an ultrasound detector.

In connection with the technique of magneto-photoacoustic tomography, it is pointed out that the spatial resolution of the photoacoustic tomography is increased by means of the application of a magnetic field able to induce translation phenomena in the nanoparticles. In this way a major reduction in the background noise present in conventional photoacoustic tomography is obtained, improving significantly the spatial resolution and the performance features of the technique. The nanoparticles produced according to the aforementioned embodiments of the present disclosure, since they have optimum magnetic properties and a high light absorption capacity in the range of interest, are potential candidates for use as agents for separating and detecting biological components in the blood such as proteins or circulating tumour cells.

The subject-matter of the present disclosure has been described hitherto with reference to examples of embodiment thereof. It is to be understood that other embodiments relating to the same inventive idea may exist, all of these falling within the scope of protection of the claims which are attached below.