NANOPARTICLE DISTRIBUTION IN A SUBSTRATE

The present invention relates to the detection, quantification and identification of engineered nanoparticles (ENP) in a substrate, in particular a food product.

Background of the invention

The appearance of engineered nanoparticles (ENP) in food is a relatively new issue for public safety. Regulations exist limiting the type and amount of ENP permitted in food. The detection and quantification of ENP in food matrices is necessary to obtain information on the migration and distribution of the material in order to meet future regulatory requirements. Such analysis is of great importance in terms of health and quality assurance. Further, methods are required which do not further complicate food production lines.

There is no solution for food analysis in the nanoscale range yet. Food industry currently targets structures in fruits where the texture of the objects investigated or the hydration state of the organic cells are of interest. For example Mehta et al. (J FOOD SCI 74 E455 2009) reported the use of ultrasound for on-line quality assurance in the baking industry.

Non-invasive techniques are known from medical applications as for instance ophthalmologic applications. Optical coherence tomography (OCT) is one type of data acquisition used in such medical constrains. Chronakis et al describe this non-contact method used in biomedical diagnostic (Int J Adv Manuf Technol 47 2 010 963). A further development of optical coherence tomography is the Fourier domain optical coherence tomography (FD-OCT), which can give an insight into the dynamics of biological systems. (Koch et al, J Biomed Opt 2009 143 0340 27). Also Davis et al describe the coherent confocal microscopy (J Opt Soc Am A 25 2008 2102) as tool for medical investigation of nanoparticles. Oh et al. (Lasers Surg Med 2007 266 39) reported a new method for the investigation of macrophages using super paramagnetic iron oxide (SPIO) nanoparticles. The analysis method used here was a combination of differential-phase optical coherence tomography (DP-OCT) with an external oscillating magnetic field. More biomedical applications of analysis based on the optical properties of nanoparticles are nuclear magnetic imaging, MRI (Kim et al, Lasers Surg Med 40 2008 415; Monziols et al, Magn Resonance Imag 23 2005 745;. Ramaswamy et al. TISSUE ENGIN Part A 15 2009 3899), Hyperspectral Imaging (Vo et al, IEEE Eng Med Biol Mag 23 2004 40) or gyromagnetic resonance imaging (Wei et al. JACS VOL. 131 2009 9728). Imaging allows the visualization of multi-dimensional and multi-parameter data. It is also increasingly used to measure concentration, tissue- and surface characteristics (Hood et al., 2004 Science 306 640-3). Non-invasive methods of nanoparticle detection are increasingly employed in material sciences (D. Stifter, Appl Phys B 88 2007 337). Contrast reagents as used in medical analysis, can contribute to improve the resolution. Such contrast reagents are for example nano-gold shells or other nanoparticles used in analytical methods. Contrast reagents are used to enhance contrast and resolution in medical

applications. Such contrast reagents are for instance antibody conjugated nano-shell particles having a plasmon resonance at 800 nm. They specifically target breast cancer cells (Chen et al., Nano Lett 5 2005 473).

Gold nanoparticles have interesting optical properties. Therefore, they are combined with optical coherence tomography applications used in biomedical diagnostic (Ungureanu et al, J Appl Phys 105 2009 1020 32;. Sokolov et al, Cancer Res 63 2003 1999;. Argawal et al, JBO 11 2006 0411 2). Kah et al. (JBO 14 2009 0540 15; APPL OPT 48 2009 D96) reported the use of gold nanoshells in mouse in vivo experiments to provide better contrast in optical coherence tomography imaging.

Furthermore, the good contrast properties of gold nano-shells and titanium dioxide

nanoparticles were used for in vivo studies of skin tissue using optical coherence tomography (Kirillin et al. JBO 14 2009 0210 17). Zagaynova et al. reported (Phys Med Biol 53 2008 4995) the use of silica-gold nanoshells with a 150 nm silica core and a 25 nm thick gold layer as optical coherence tomography contrast agent in rabbits in vivo tests. None of the above mentioned approaches describes the quantitative determination of ENPs in food/feed matrices. The non-invasive techniques used in medical applications are meant to locate for instance tumour cells while using optically active nanomaterials. Nothing is apparent with respect to a material and apparatus enabling for traceable quantification. None of the above mentioned publication relates to the development of a method or apparatus being used to produce a set of comparable data. In addition, none of the state of the art techniques suits for online detection application providing information on spatial distribution of ENP, particle size Z/particle size distribution PDI, mass fraction W, identity X of ENP. Nothing is mentioned about data collection and to provide a comprehensive picture on the before mentioned characteristics. The prior art description of the application of the optical active ENP concentrates on in vitro or in vivo studies where as in the latter case the material is administered systemically. If ENP are described to be embedded, the used substrates are cross-linked and not further analysed with respect to e.g. particle size Z/particle size distribution PDI, mass fraction W. If solid micro-particles were under question, then solely destructive methods were employed and the digested product analysed. Such approaches are complex and therefore difficult to integrate in production lines. Despite that, the data generated depict only partially the true circumstances in food systems because any way of changing the matrix or digestion might result in one or the other change in ENP appearance. The latter can be for instance changed by aggregation or dissolution. Hence, the data gained are also questionable with respect to correctness. Accordingly, an object of the present invention is the provision of a simplified process giving more reliable data on ENP distribution. An object of the present invention is provision of a non-invasive or non-destructive technique for detection of nanoparticles. It is another object of the present invention to provide a system for analyzing distribution of nanoparticles within food.

To predict the migration and distribution of nano-materials, e.g. between packaging materials and food, fast and with high resolution working methods are favourable.

The variety of nanomaterials potentially applied in food processing also raises the question of particle stability during separation techniques proposed for destructive approaches. During these separation techniques it is most likely that the appearance of the nanoparticles is changed. No substrate material has been described so far as a food reference with assured characterisation, stability and homogeneity. The employment of well defined substrates loaded with ENP responding to electromagnetic radiation is described to enable for reference material production as well as method development/validation. Such a defined reference enabling for comparable data has not been published. An object of the present invention is therefore a reference material having a pre-defined nanostructure with a sufficient homogeneity and stability to act as a reference. A further object is the provision of an apparatus combining non-invasive and destructive analysis.

Nothing is mentioned in the prior art regarding using well-defined but simple model matrices serving as reference to be employed for future method development. This is crucial as no single analytical method exists for quantitative ENP online detection in food/feed production. A further object of the present invention is the provision of a standard substrate for calibrating ENP detection and characterization equipment.

Little is published about ENP detection in food. Issues tackled concern micron dimensional problems in food structures or investigations on surface topography. Further, destructive methods suggested for ENP analysis demand highly skilled personnel. Yet another object of the present invention is an apparatus combining a non-invasive imaging technique with size determination, nano-scale weighing and elemental composition analysis. Accordingly, the present invention provides a method for analyzing engineered nanoparticles (ENP) in a substrate comprising: (a) measuring the particle size distribution of the ENP; (b) quantifying the ENP in the substrate and (c) identifying the ENP in the substrate.

The present invention further provides apparatus suitable for analyzing engineered

nanoparticles (ENP) in a substrate comprising (a) means for measuring the particle size distribution of the ENP; (b) means for quantifying the ENP in the substrate and (c) means for identifying the ENP in the substrate.

The present invention further comprises a nanoparticle comprising a core surrounded by a substrate matrix. The present invention further comprises a nanoparticle comprising a shell with a hollow core.

The present invention further provides a film comprising a homogeneous distribution of electromagnetically active engineered nanoparticles (ENP).

The present invention further provides a food product produced by process which comprises a method as defined herein.

Figure 1 comprises the following elements:

100 - Apparatus

110 - Means for detecting ENP

120 - Means for measuring particle size distribution of ENP

130 - Means for quantifying the ENP

140 - Means for identifying the ENP

200 - Sample

210 - Solvent

310 - Data from means for detecting

320 - Data: Particle size Z/particle size distribution PDI

330 - Data: Mass fraction W

340 - Data: Identity X

350 - Data: Confirmation C

360 - Data bus

370 - Discarded sample

410 - Decision

420 - Dilution/Dissolution

430 - Homogenization 440 - Solute

450 - Eluent

500 - Data: collection As used herein a non-invasive method is a method which is not destructive for the ENP.

Preferably it is also not destructive for the substrate. As used herein particle size distribution defines the relative amounts of particles present, sorted according to size. As used herein an online process is a process which is carried out in cooperation with a(nother) continuous process. For example detection of ENP in a food product carried out in cooperation with a continuous food production process. As used herein a substrate is a material which supports or is in contact with an ENP analyte. As used herein food product includes processed and unprocessed food, both raw and cooked. It also includes food for human and non-human consumption. As used herein identifying means determining the chemical or physical nature, for example elemental composition, of an ENP. For the avoidance of doubt it does not mean determining the presence of an (unknown) ENP.

The invention is based on the idea to 'visualize' ENPs in both liquid and solid food matrix. In order to facilitate a fast and straightforward decision, first the presence or absence of ENP is preferably determined. Preferably this is by a non-invasive method. The decision on further characterization is done in a decision step based on the presence/absence of ENP. Preferably the screening for presence of ENP is by the non-invasive technique optical coherence tomography (OCT). If ENP is present in the sample, a technique such as dynamic light scattering technique is used to determine the particle size Z/particle size distribution PDI.

However, further characterization is needed to make the results traceable to SI standards. Such traceability may be obtained via nano-accurate weighing exploiting a quartz crystal

microbalance (QCM). As the chemical nature of the ENP is often not known the determination of the chemical composition of the nanomaterial of interest is crucial. This may be done using inductive couple plasma - mass spectroscopy. The method combines the above steps, carried out in a logical order. First, the substrate is inspected for ENP contamination. If a positive answer is obtained, the ENP are analysed with respect to their size. The latter is a major issue in the definition of nanomaterials. If particle sizes are indeed at the nanorange, the amount of ENPs within the substrate is determined. This quantification enables for evaluation of potential safety aspect, i.e. if a significant amount of ENPs is present and how it compares to legal threshold data. As a last step, the apparatus analyzes the quantitatively the chemical composition of eluent and solute derived from the weighing step. The results give raise to conclusions about specificity and recovery of the weighing step. ICP-MS may be used for this purpose. The sum of all data can be processed by a computer and correlations between the single results can be examined. The use of ENP reference materials enables not only for the development of further methods and their validation using the data obtained from the invented apparatus but also for traceability to the SI units.

An alternative method can be used if the ENPs in question have magnetic susceptibility. The apparatus can be equipped for non-destructive testing of the anisotropic magneto-resistance parameters as described in USH000585. The magnetic response can be detected in situ using the longitudinal magneto-optic Kerr effect (MOKE) and visualized. In order to manipulate the susceptibility of the ENPs the set up may enable for temperature and magnetic field variation. Based on the results of this screening step, the apparatus user can decide about further analysis with respect to particle size Z/particle size distribution PDI, mass fraction W and identity X of ENP For the determination of particle size Z/particle size distribution PDI the apparatus can be equipped with an electroresistance counting set up. In particular Coulter counters are interesting for non-conducting particles but also the use of a Scanning Ion Occlusion

Spectrometer can be used for ENP sizing. The determination of the mass fraction W can be done by performing surface plasmon resonance (SPR) measurements. An advantage of such measurements is the possible visualization of weighing results. The elemental analysis can be performed exploiting inductively coupled plasma - optical emission spectrometry (ICP-OES). This analytical technique can be used for the detection of elements in trace level.

The sum of all data can be processed by a computer and correlations between the single results can be examined. The use of ENP reference materials enables not only for the development of further methods and their validation using the data obtained from the invented apparatus but also for traceability to the SI units.

An alternative method can be used if the ENP are radioactively labelled. The apparatus can be equipped with Positron Emission Tomography (PET) for non-destructive testing on for instance Fluorine-18 positron emitters (radiotracer). The radiotracer labelled ENPs can be detected and visualized. Based on the results of this screening step, the apparatus user can decide about further analysis with respect to particle size Z/particle size distribution PDI, mass fraction W and identity X of ENP. For the determination of particle size Z/particle size distribution PDI the apparatus can be equipped with an ultrasound attenuation spectroscope. Such acoustic spectroscopy is able to determine the particle size distribution in colloidal systems. The determination of the mass fraction W can be done by performing surface plasmon resonance (SPR) reflectivity measurements. An advantage of such measurements is the possible visualization of weighing results. The elemental analysis can be performed exploiting inductively coupled plasma - optical emission spectrometry (ICP-OES). This analytical technique can be used for the detection of elements in trace level.

The sum of all data can be processed by a computer and correlations between the single results can be examined. The use of ENP reference materials enables not only for the development of further methods and their validation using the data obtained from the invented apparatus but also for traceability to the SI units.

Preferably step (a) of the method is non-invasive. The non-invasive imaging provides for easy costumer security/quality management where as invasive analysis build the link between measured intensity from non-invasive imaging and nanoparticle characteristics such as particle size Z/particle size distribution PDI, mass fraction W and identity X of ENP. Furthermore the combination provides for traceability for instance if ENPs matrices with known concentration and identity are used for calibration purposes. Thus it is of advantage to have an apparatus providing non-invasive and invasive analysis. The material under question is first evaluated using a non-invasive step. Non-contaminated material will be not further investigated. This results in the advantage over prior art systems in a reduced workload and easier protocol procedures. Additionally, such step is characterised by a fast data generation and the output can be an easy to interpret 3D-image. Typically, apparatus according to the present invention comprises the following parts:

- non-invasive:

- wavelength transmitting application which detection is based on e.g. reflection,

detraction, scattering, resonance and/or absorption of (electro)-magnetic matter (application examples: ultra-sound, infra-red, ultra-violet, X-ray, magnetic resonance tomography (MRT), positron emissions tomography (PET), dynamic light scattering

(DLS))

- destructive:

- nanoparticle specific assay, highly sensitive to low mass (low limit of detection), e.g. quartz crystal microbalance, surface plasmon resonance

- analysis on elemental composition

The employment of non-invasive techniques using for instance an IR-Laser beam enables for the detection of IR sensitive ENP's (e.g. gold nanocages) in solid food matrices at least in a semi-quantitative manner (or scanning). Due to the plasmon resonance and/or light scattering, the ENP's appear in high contrast to the food matrix. Using data imaging software, pictures can be prepared displaying intensity differences which correspond to the ENP distribution in food systems.

The combination with a destructive approach results in an apparatus able to build a

straightforward relation between measured light intensity and the identity of the nanoparticles. Preferably the apparatus further comprises (a') non-invasive means for detecting potential ENP in a substrate. More preferably in the process (a') the non-invasive means for detecting potential ENP in a substrate comprises optical coherence tomography; (a) the means for measuring particle size distribution of the ENP comprises dynamic light scattering; (b) the means for quantifying the ENP in the substrate comprises a quartz crystal microbalance and (c) the means for identifying ENP in a substrate comprises inductive couple plasma - mass spectroscopy. The employment of non-invasive techniques using for instance an IR-Laser beam enables for the integral detection (screening) of IR sensitive ENP's (e.g. gold nanocages) in solid food matrices at least in a semi-quantitative manner (or scanning). Due to the plasmon resonance and/or light scattering, the ENP's appear in high contrast to the food matrix. Using data imaging software, pictures can be prepared displaying intensity differences which correspond to the ENP distribution in food systems. The combination with a destructive approach results in an instrument able to build a straightforward relation between measured light intensity and the identity of the nanoparticles.

Currently the maximum axial resolution of non-invasive methods are seen as the limiting factor for many applications and the use for detection of nanomaterials in foods is not described.

However, these methods can be employed as screening tools to assure for instance regulatory compliance in the food industry by providing integral data on the distribution of nanomaterial in food matrices. However, besides the particle distribution there are other information to be gained in order to obtain a comprehensive picture. Such data concern particle size Z/particle size distribution PDI, mass fraction W and identity X of ENP.

Because of this advantageous approach the device is appropriate to produce data for a comprehensive 3D picture displaying the distribution of ENPs in various matrices. The device can be used to produce reference materials as it provides traceable data concerning

homogeneity, stability and characterization of the sample. Both, reference materials and device will enable food industry and regulatory bodies for food safety control and quality assurance. Further, the device is applicable for online detection due to fast production of data concerning the presence of ENPs. The calculated 3D structures allow for simplified and understandable displaying on ENPs in complex food/feed matrices.

Another preferred embodiment of the present invention are thin films comprising ENPs. Such films may be used as reference materials having pre-defined nanostructures with a sufficient homogeneity and stability. They are thus useful in calibrating an apparatus according to the present invention. They may be used in apparatus combining non-invasive and destructive analysis.

The ENPs used in the film may have the following characteristics :

Localized Surface Plasmon Resonance (LSPR) occurring at metallic ENP give raise to an

(electro)-magnetically response of the ENPs. Such response can be tuned via the shape, size and chemical composition but also via the electric constant of the ENP's environment. This characteristic can be employed to make nanoparticles in a food product visible using for instance OCT which can be equipped with a near-infrared laser. Near-infrared active gold nanoparticles (GNP) (NIR-GNP) can be synthesized (Liz-Marzan et al Langmuir 2006, 22, 32) and/or derivatized to become model nanoparticles (core/shell material) mimicking physico- chemical behaviour (e.g. Brownian motion, zeta potential) of 'real life' ENP. For instance, gelatin coated NIR-GNP, mimicking gelatin spheres of food additives, can be created via post-coating of NIR-GNP. An additional cross-linking step, using e.g. glutaraldehyde, assures the stability of the gelatin shell. Due to the nanoscale of the particles the surface properties as well as the physical-chemical properties of core/shell ENP and ENP made solely of the shell material are similar. However, the core acts as a contrast reagent enabling for non-invasive detection.

Also colloidal matter of natural origin (WO2009147209 A2, Lerche KG) can be used to synthesize ENP detectable via non-invasive techniques. The material can be used as crystallization substrates and modified by means of surface coating. Because of the formation of e.g. Gold coating having sharp edges, the material can gain localized surface plasmon resonance and gets detectable via OCT. Another advantage of the materials described in WO2009147209A2 is the highly defined and continuous structure making the material suitable as reference material.

The ENP can be enabled for sensing techniques such as surface plasmon resonance, quartz crystal microbalance via attaching (bio)functional groups at the ENP surface. Such conjugation may provide for chelation, D,L complexation, electrostatic interaction, hydrophobic interaction and/or Hydrogen bonding. Crucial characteristic of ENP derivatization is the ability to interact with the surface of the sensing device in a reversible manner.

Ultradispersed diamonds (UDDs) have a defined structure, high surface to volume ratio and are optical active. The high surface to volume ratio of the UDDs and the presence of reactive sites enable for surface conjugation (Mkandawire et al, J. Biophoton. 2 2009 596). Together with their commercial availability, UDDs are advantageous for the production of reference materials. The ENPs can serve as model nanoparticles in well defined polymeric thin films or can be loaded to artefacts or other phantoms made from e.g. gelatine, sugar, polyvinylpyrrolidone, polyurethanes) in successive concentrations. Characterized for homogeneity and stability, such ENP's imbedded matrices enable for the production reference material. Non-cured systems can be dissolved and used for liquid based analysis techniques.

Thin films having defined ENP embedded can be prepared by homogeneously wetting a glass plate by means of for instance spin coating or using spirally wound bars. The used coating formulation can be solvent- or water born. After dissolving preferably 1 -10% m/m of a film forming polymer like PVP, ENP like NIR active GNP can be dispersed for instance at concentration preferably at part per billion (ppb) to part per million (ppm) level.

The dried-out film holds to the glass plate by means of physical adsorption. Cohesion of the film is achieved by means of physical interaction of the polymer molecules.

Typically in a film according to the present invention, the ENP are optically active. Typically in a film according to the present invention the ENP comprise a radioactive isotope.

Contrast reagents such as GNPs can be used to develop non-invasive methods for detection of nanomaterials in foods. Such methods represent a significant improvement in quality assurance and consumer safety. Because GNPs are except to be stable in a wide range of conditions, such material are suitable as food reference with assured characterisation, stability and homogeneity. Additionally they provide for a high recovery rate.

Detailed description of the drawings Fig. 1 illustrates an apparatus (100), which is charged with a sample (200) comprising a substrate and optionally ENP. First, an ENP detecting step (110), for example optical coherence tomography, is performed in order to differentiate between a sample containing ENP and a sample absent ENP. This first step is characterized to be non-invasive. The resulting data (310), for example light intensities, can be transformed into 2-/3-D images at computer (500). The substrate is unchanged after the screening step and therefore can be further analysed.

As a result of a decision step (410) the sample absent ENP is discarded (370) while the sample containing ENP is processed further (420), for example by dissolution or enzymatic digestion. The process is further characterized through homogenization (430), for example by shaking, mixing, ultra-sonication or other means.

The homogenized sample is transferred to a particle size determination step (120). This step is preferably but not limited to dynamic light scattering (DLS). The extracted data give information on particle size Z/particle size distribution PDI (320). After a decision step (410), a sample with particles exceeding the nanoscale is discarded (370) while an ENP- containing sample is processed further. The ENP quantification method (130) comprises for example a quartz crystal microbalance (QCM). The sample passes a quartz crystal resonator facilitating for accurate quantification of the mass fraction W (330). An additional elution of the ENPs loaded on the quartz crystal microbalance with a solvent (210) is not only necessary to clean the resonator of the quartz crystal microbalance but also enables for the following identification step. After a decision step (410), a sample with an ENP quantity below a certain threshold is discarded (370) while the ENP- containing sample is processed further.

The identification step (140) is preferably but not limited to inductive couple plasma - mass spectroscopy (ICP-MS). The solute (440) and eluent (450) are analyzed with respect to their elemental composition. While the solute carries information on the ENP identity X (340), the analysis of the eluent provides for confirmation C (350) of the quantifying step (130).

All data get transferred by a data bus (360) and get processed in a computer (500).

Finally the remains of the sample are discarded (370). The present invention is illustrated by, but not intended to be limited to, the following examples.

Examples

Example 1

For investigation or calibration purposes, a piece of cheese is treated with a dispersion of a defined concentration of gold nanoshells for a defined period of time. The enrichment of gold particles in the cheese is analyzed by optical coherence tomography (OCT). The apparatus according to Figure 1 is used. The apparatus (100) gets loaded with the cheese sample (200). It first performs a screening step (110) in order to differentiate between cheese containing ENP and cheese absent ENP. This first step is optical coherence tomography (OCT): the sample gets irradiated with near-infrared laser light; its reflected entity is compared to a reference beam and light intensity information are acquired. The resulting data are values of light intensities (310), which can easily be transformed into 2-/3-D images.

Because of the non-invasive approach, high speed detection can be performed minimizing data generation time and work load. As the images display integral values, information on the homogeneity can be obtained. These matrices are unchanged after the screening step and therefore can be further analysed.

After a decision step (410), the cheese containing no gold nanoshells is discarded (370) while the cheese containing gold nanoshells is processed further by enzymatic digestion (420). The digested cheese is homogenized by ultrasonication (430). The homogenized sample is transferred to a particle size determination step (120), where it is subjected to dynamic light scattering (DLS). While correlating the Brownian motion of the ENP the extracted data give information on particle size Z/particle size distribution PDI (320). After a decision step (410), a sample with particle size not matching the nanoscale is discarded (370).

The ENP containing cheese sample is then subjected to an ENP quantification method (130), which comprises using a quartz crystal microbalance (QCM). The sample passes a quartz crystal resonator facilitating for accurate quantification of the mass fraction W (330). This step is crucial in order to provide traceability. The surface functionalization of the resonator corresponds to the surface characteristics of the ENP and enables for specific binding which increases the accuracy of the resulting data. An additional elution of the ENP loaded on the quartz crystal microbalance with a solvent (210) is not only necessary to clean the resonator of the quartz crystal microbalance but also enable the following identification step. After a decision step (410), the sample with a ENP quantity below a defined threshold is discarded (370) while the ENP- containing sample is subjected to an identification step (140).

The solute (440) and eluent (450) are analyzed with respect to their elemental composition. While the solute carries information on the ENP identity X (340), the analysis of the eluent provides for confirmation C (350) of the quantifying step (130).

All data get delivered by a data bus (360) to a computer (500) where they are processed. It is here, that the Intensity I (310) resulting from sample containing ENP of a certain particle size Z/particle size distribution PDI (320) and identity X (340) is correlated to the mass fraction W (330) of the ENP in the cheese sample. In such a way the measured intensity I (310) is traceable to the mass SI unit and a set of coherent data is generated. These data illustrate if there is a dependency between the dispersion concentration and particle concentration in the cheese. Finally the remains of the sample are discarded (370). Example 2

In order to mimic a nanoparticle containing packaging material, a film containing gold shells is used. This is brought in contact with a piece of cheese and the concentration of gold particles in both the cheese and in the film is analyzed preferably but not limited to optical coherence tomography (OCT). The sample is analysed by a method analogous to Example 1.

These data illustrate if and to what extent penetration of GNP from film into the cheese sample occurs. Finally the remains of the sample are discarded (370).

Example 3

In order to facilitate for a method development/validation, a reference material is needed. As such GNP embedded coatings can be employed. These can be prepared by homogeneously wetting a glass plate by means of for instance spin coating or using spirally wound bars The used formulation can be solvent- or water born. After dissolving preferably 1 -10% m/m of a film forming polymer like PVP, NIR active GNP can be dispersed at concentration preferably at part per billion (ppb) to part per million (ppm) level.

The dried out film hold to the glass plate by means of physical adsorption. Cohesion of the film is achieved by means of physical interaction of the polymer molecules.

As shown in Figure 1 , the apparatus (100) gets loaded with the coated glass sample (200). By displaying the results as integral values in 3D, information on the homogeneity of the GNP distribution can be obtained. Because of the non-invasive approach, high speed detection can be performed minimizing data generation time and work load. These matrices are unchanged after the screening step and therefore can be further analysed. The sample is processed further preferably but not limited to dissolution and rinsing off of the coating (420). The process is further characterized through homogenization by shaking, mixing, ultra-sonication or other means (430). The sample is then processed in the same way as Example 1. All data get delivered by a data bus (360) and get processed in a computer (500). It is here, that the Intensity I (310) resulting from sample containing GNPs of a certain particle size Z/particle size distribution PDI (320) and identity X (340) is correlated to the mass fraction W (330) of the GNPs in the coating. Such way makes the measured intensity I (310) traceable to the mass SI unit and a set of coherent data on homogeneity and stability of the reference material is generated. Finally the remains of the sample are discarded (370).

Example 4

In order to set up a reference material while mimicking complex liquid matrices like processed meat solutions being ENP contaminated, a better defined solution of synthetic polymers can be used. Such synthetic polymer is PVP being a good protein replacement. These solutions can be created by dissolving preferably 1 -10% m/m of PVP. The solution can be spiked with NIR active GNP at concentration preferably at part per billion (ppb) to part per million (ppm) level.

As shown in Figure 1 , the apparatus (100) gets loaded with the solution (200). The method according to Example 3 is then followed. A set of coherent data on homogeneity and stability of the reference material is generated. Finally the remains of the sample are discarded (370).