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FP7

SETNanoMetro Report Summary

Project ID: 604577
Funded under: FP7-NMP
Country: Italy

Final Report Summary - SETNANOMETRO (Shape-engineered TiO2 nanoparticles for metrology of functional properties: setting design rules from material synthesis to nanostructured devices)

Executive Summary:
According to European Commission [EC, COM (2012) 572, 3.10.2012], important challenges at European level are related to the establishment of validated method and instrumentation for production, detection, characterization and analysis of nanoparticles. In the framework of the SETNanoMetro project, the use of various measurement techniques for the determination of nanoparticles (NPs) properties has allowed to move from the currently used “trial and error” approach toward the development of well defined and controlled protocols for the production of TiO2 NPs. A particular care has been devoted to the establishment of correct metrological traceability chain for the determination of dimension and shape of TiO2 NPs..
The lack of international measurement standards for calibration is an aspect of particular relevance in nanotechnologies as it is difficult to select a universal calibration artefact to achieve repeatability at nanoscale.
The TiO2 NPs, produced in the project according to such procedures, were characterised and their properties were considered homogeneous to become candidate Certified Reference Materials (CRM) and used in various applications where the lack of metrological traceability is encountered.
The project results are expected to lead to fundamental impacts on the following areas:
• Environment: the increased knowledge of TiO2 NPs allows to improve the photocatalytic properties for the treatment of pollutants in air and water.
• Energy: the better knowledge of dimension and electronic structure of TiO2 allows to improve the traceability of Dye Sentitize Solar Cells (DSSC) measurements.
• Health: the engineering of topographic and surface composition of TiO2 nanostructured coatings of orthopaedic and dental prostheses supported the design of rules for the production of devices exhibiting optimized interfacial properties for a better and quicker integration of the implants in the hosting bone tissues.
A number of challenges were faced and solved to achieve the targeted breakthroughs in producing TiO2 NPs and to ensure metrological traceability and reproducibility of measurement results of the functional properties of interest. To do this SATNanoMeto has:
• pursued the preparation of sets of TiO2 NPs, each highly defined and homogeneous in bulk structure, shape, size, surface structure, to be used as such and/or in highly controlled and reproducible aggregated or assembled forms on proper supports.
• carried out an extensive and comprehensive metrological research devoted to the characterisation and production of standardised TiO2, that allowed the attainment of products with defined properties and sufficient homogeneity to be considered as candidate CRM. These candidate CRMs, suitable for assuring metrological traceability to measurement results, have been used for measurements under traceability conditions of operational performances in some selected, actual technological applications such as DSSC, toxicology related to orthopaedic and dental prostheses and photocatalysis.

Project Context and Objectives:
Titanium dioxide (TiO2, titania) has been one of the most exploited semiconductor oxides in various technological fields for many years. This material has multisectorial application ranging from solar energy harvesting, photocatalysis for air and water remediation from pollution, production of smart materials with self-sterilizing, self-cleaning and superhydrophilic properties, healthcare sector for the development of implants or restoration materials with better prognosis, a longer durability and less failures.
These inputs are resulting in a flourish of both fundamental and technological studies devoted to titania, with a consequent increase in reports dealing with new functional behaviours and/or improved performances, with respect to what was previously known. Indeed, the number of papers dealing with TiO2 that appeared in the scientific & technological literature has exhibited an impressive growth since 1990, as well as the amount of patents based on this material. However, it can be difficult to attain an objective and comprehensive evaluation of the impact of the proposed novelties, because of the wide heterogeneity of:
i) types of TiO2-based NPs developed, each of them often exhibits a rich dimensional and morphological variety,
ii) surface and/or interfacial molecular states,
iii) assembly/aggregation states of the NPs to form nanostructured and nano-enabled systems,
iv) measurement conditions of the functional performances of these systems.

Moreover, in order to have NPs with engineered properties for specific applications, it is important to have flexible synthesis methods which can lead to NPs with different critical parameters (e.g. shape, crystallographic surface exposed) and critical dimensions.
With such scenario, one of the general objectives of the SETNanoMetro project was the evolution of TiO2 NPs synthetic procedures from a “trial and error” approach to one based on the possibility to predict the properties of the engineered nanoparticles.

MODELLING AND FORMULATION OF STANDARD SYNTHETIC ROUTES TO SHAPE AND SIZE CONTROLLED TiO2 NPs
A fundamental point will deal with the disclosure of the variables and parameters, and of the relationships among them, that rule the preparation of TiO2 NPs with pure anatase or rutile crystal structure, exhibiting defined shapes, exposed surface facets and surface composition (WP1, WP2, in loop with WP4). The goal has been pursued by using different synthetic approaches:

- hydrothermal methods (HT)
- sol-gel methods (SG)
- an innovative approach represented by Laser Ablation on liquids (LA)

In particular, pursued shapes for anatase have been: square-based bipyramids, square-based prisms, and square-based platelets. Dimensions ranged from 20 nm to 200 nm. A scheme of ideally pursued shapes is shown in Figure 1.1.



Fig 1.1 Ideal shapes of anatase TiO2 nanocrystals. The truncated bipyramid is the equilibrium shape in vacuum. Other shapes can be achieved by using shape controllers that adsorb selectively on a crystal facet, lowering its surface energy
The TiO2 based nanomaterials pursued in this project exhibit a defined geometry controlled by the lattice parameters and surface energy, enabling a new breakthrough in metrology of nanostructures.

METROLOGICAL CHARACTERIZATION OF SINGLE TYPE OF NPs

It was clear in this project that engineered TiO2 NPs present a complex and challenging system for measurement. To provide an adequate size-based metrological assessment of such samples, a number of factors have been considered.
- Firstly, it was important to determine the most appropriate measurand. For example, Critical Dimension (CD) in truncaded bypiramid was the height of the pyramids and the bases dimension of the pyramid. Playing with these dimesions has been possible to obtain the different shape depicted in fig 1.1
- Secondly, when the average particle size of the primary particles is measured, it is essential that the NPs system is adequately dispersed. This in itself is a non-trivial process and involved an independent study on each new sample encountered.
- Thirdly, it must be remembered that the addition of stabilizing agents to a sample in order to measure the primary particle size may change the state of the sample. This last point is particularly critical when considering risk assessment of engineered NPs in consumer products or for toxicological investigations.
In the project several fundamentally different measurement methods, like atomic force microscopy (AFM), dynamic light scattering (DLS), small-angle x-ray scattering (XRD), scanning electron microscopy (SEM) and scanning electron microscopy in transmission mode (T-SEM), were applied providing independent, reliable results of the selected measurands. Comparisons were carried out in order to verify the compatibility of these results. Each SETNanoMetro partner provided results and their measurement uncertainties.
Another innovative target of the project was constituted by the definition of standards for functional properties of TiO2 NPs, also in terms of the design of rules for reproducible and traceable measurements of surface and interfacial features defined at molecular level. To give some examples of the role played by these:

• surface hydroxy groups play a relevant role in the interfacial pathway of photocatalytic events;
• the size and location of the bandgap between the conduction band and the valence band of TiO2 is essential for the magnitude of the photocatalytic effect, and differ between e.g. crystal sizes;
• the effectiveness of the electron transfer from dye sensitizers to titania particles is significantly affected by the structure assumed by carboxylate moieties, the functional groups usually anchoring the dye molecules to the surface of TiO2 NPs, and this structure depends in turn on the local arrangement and electronic states of surface Ti4+ and O2- sites;
• the fate of the interaction of an orthopaedic or dental prosthesis with the cells of the hosting tissue is ruled in a large extent by the causal sequence: (i) biomaterial surface structure, (ii) states of adsorbed water molecules, and (iii) states of adsorbed proteins.
In SETNanoMetro, particular attention has been payed to participate in and organize interlaboratory comparisons at both national and international level. To ensure that TiO2 NPs sample quality requirements were met, we proceeded as follows:

• TiO2 NPs sample stability was verified before, during and after the comparison. Verifications during the comparison were performed at regular intervals;
• several feedback loops were set up simultaneously among the project partners. An important part of interlaboratory comparisons and proficiency testings was dedicated to the determination of the result value of the measurand and its associated uncertainty.

OBJECTIVES
In the area, resulting from the intersection between nanoscience/nanotechnology and metrology, several issues are emerging, and among them the following have been recognized as relevant in the European Research Area.

• need for characterization of the relationship between measurable key parameters through the nanoscale to the higher order scales that provide new insight into the performance of nanostructured and nano-enabled material;

• interest toward the generation of reference information to test and optimise new design rules derived from length scale models;

• development of methods for measuring properties for which currently no methods exist, also ensuring the traceability, or at least the reproducibility, of existing methods;
• establishment of mechanisms to integrate new design rules to existing modelling techniques and apply these to industrially relevant materials and devices, delivering concrete results of industrial relevance (for example, the ability to design nano-coatings or nano-enabled coatings with specific performance properties).


Project Results:
2.1 Preparation of sets of TiO2 NPs with defined shape and size
In WP1 we pursued the preparation of sets of TiO2 NPs, each highly defined and homogeneous in bulk structure, shape, size, surface structure, to be used as such and/or in highly controlled and reproducible aggregated or assembled forms on proper supports.
The work carried out in WP1 lead to the development of soft models that give design rules for the synthesis of engineered TiO2 NPs with controlled size, shape, specific surface area and surface properties. The approaches used were three:
• hydrothermal methods (HT)
• sol-gel methods (SG)
• an innovative approach represented by Laser Ablation in liquids (LA)
Hydrothermal Synthetic Methods and NPs Availability
Anatase TiO2 NPs
The bipyramidal and prismatic anatase nanoparticles were obtained by an hydrothermal synthesis starting from a water soluble precursor (a complex of Ti and triethanolammine 1:2) purposely synthesized and triethanolammine as shape controller. The variables of the process (titanium precursor concentration, temperature, shape controller concentration and pH) were identified and their influence in the final nanoparticles size was analyzed by an experimental modelling. Changing the synthesis conditions it is possible to obtain bipyramidal or prismatic nanoparticles, moreover it is possible to modulate the size and the elongation of the particles.
The flat anatase nanoparticles (platelets) were obtained by a solvothermal synthesis starting from Ti(IV) butoxide as precursor and hydrofluoric acid as capping agent. Changing the synthesis conditions is possible to modulate the size and the thickness of the nanoparticles. A treatment procedure was developed in order to selectively remove the fluorine from the surface or from the bulk of the nanoparticles.
The developed HT synthetic procedure can be summarized as follows:
HT synthetic procedure able to lead to low-dimensional dispersity bipyramidal TiO2 anatase NPs, with low truncation along c-axis, then mainly exposing (101) type facets, (40 to 100 nm).
HT synthetic procedure able to lead to low-dimensional dispersity bipyramidal TiO2 anatase NPs, with modulated truncation along the c-axis (platelets).
HT synthetic procedure able to lead to low-dimensional dispersity prismatic TiO2 anatase NPs, exposing (100) and (101) type facets.


Rutile Nanoparticles
Hydrothermal synthesis conditions for ensembles of prismatic rutile NPs were developed, with the procedure to modulate the shape factor and length along the c-axis. A variation of the length along c-axis between 150 nm and 1500 nm was obtained, with shape parameters ranging from 2.5 to 6. These sets of NPs have a relevant and predictable percentage of {110} facets. Typical results are reported in 2.1.3.

Sol Gel Synthetic Methods and NPs Availability
The sol-gel route is a versatile technology for obtaining TiO2 NPs with controlled crystalline ratio, surface area and sizes and one of the most interesting because of its simplicity, cheapness and high quality of final products. In SETNanoMetro project, two type of NPs were targeted:
• TiO2 anatase nanoparticles with optimized size <20nm
• TiO2 anatase nanoparticles with high specific surface area (>150 m2/g)
The low size anatase nanoparticles were obtained by water in oil (W/O) microemulsions, specifically, through reverse micelles microemulsions. The most significant variables of the process (water, oil, surfactant/co-surfactant concentration, Titanium precursor concentration and its ratio with respect to the water content, Sintering temperature and time) were identified and their influence in the final nanoparticle’s size were analyzed by an experimental modelling. A good repeatability of the synthesis method was observed.

The anatase TiO2 nanoparticles with high specific surface area were synthesized by a conventional sol-gel route. Many parameters including precursor, solvent type, solvent molar percent, water molar percent, reflux temperature, reflux time, sol drying method, calcination temperature and extraction-crystallization process were modelling to achieve the best synthesis process for titania nanoparticles with specific surface area of 163 m2/g and assize of 6-13 nm.

Laser Ablation Synthetic Methods and NPs Availability
The use of pulsed laser ablation (LA) on solid Ti plate (99.99%) targets in liquids (water, also in the presence of surfactants as shape controllers) led to the set up of procedures able to provide spherical anatase TiO2 NPs with controlled diameters between 25 nm and 100 nm.

TiO2 NPs commercialized by SETNanoMetro
The final output of SETNanoMetro regarding shape and size controlled TiO2 NPs is:

1. bipiramidal TiO2 anatase NPs with low truncation (30 nm - 100 nm hydrodynamic diameter), main surface exposed (101)
2. elongated TiO2 anatase NPs with shape parameters from 2 to 6, main surfaces exposed (101) and (100)
3. TiO2 anatase nanoplatelets with shape parameters from 0.15 to 0.30, main surfaces exposed (001) and (101)
4. Prismatic rutile NPs with length from 150 to 1500 nm and shape parameters from 2.5 to 6
5. Sol-gel TiO2 NPs with diameters < 20 nm
6. high surface area sol gel TiO2 NPs (specific surface area > 150 m2 g-1)
7. spherical TiO2 NPs from LA
The NPs can be purposely synthesized with the size and shape desired within the limits indicated above.
2.2 Modelling of the synthesis of TiO2 NPs
The proposed and achieved WP2 objective was the modeling of the processes for TiO2 NPs obtained by hydrothermal (HT) and sol-gel (SG) synthesis in view to predict process evolution and end product reproducible targeted characteristics.
As leader of WP2, RD was involved in the modelling activities, having as partners UNITO and IK4 TEKNIKER, which supplied the experimental data obtained in WP1, regarding anatase nanoparticles synthesis by hydrothermal and respectively sol-gel routes. Thus, a fundamental project point dealt with the theoretical modeling (WP2) in loop with the experimental activities of synthesis (WP1) and characterization (WP1 and WP4) performed by the partners UNITO, TEKNIKER, BAM and INRIM, in view to disclose the variable parameters and properties, and the relationships among them that rule the preparation of TiO2 NPs with pure anatase crystal structure, defined shapes, size and surface. This interplay between WP2, WP1 and WP4.

The modeling specific objectives, targeted to predict the characteristics of different NP types obtained by HT and SG synthesis and the applied modeling methods. The modeling methods were chosen to be suitable for the number of process parameters, their interaction during the synthesis processes and their influence on the end product characteristics.
The approach consisting of the use of different modeling methods for the same shape controlled anatase NP type obtained by hydrothermal synthesis, proved through the validation experiments that the computed outcomes with the developed models (i.e. experimental design and multiscale models applied for truncated bipyramids and prisms), are close - whichever modeling method is used, being also close to the experimental data.

The main S&T results of WP2 are:
- 9 SOFT MODELS for prediction the characteristics of anatase NPs obtained by HT and SG synthesis, related to relevant process parameters (4 of the models are WP2 milestones: MS 5, MS 6, MS 7, MS 8).
The developed mathematical models (soft models) are:
In the data bank tables, the process conditions giving the relevant shape and size controlled characteristics of each type of anatase NPs developed in the project are selected. From these data, it results that NPs with almost the same characteristics could be obtained with different combinations of process parameter values. It could be seen that the good operating conditions are not unique and also that the developed models could be used as tools for prediction and control of the NPs shape and size, because they give the correlation between certain product characteristics and the combinations of process parameters.

In WP8, the partner used, with good results, some combinations of optimal process parameters from the data bank, for the scale-up of sol-gel and hydrothermal syntheses.
In DoW the foreground for dissemination of WP2 was not provided, because WP2 work was strictly correlated with that of WP1 and WP4 for the synthesis experiments and NPs characterization. Nevertheless, the scientific results obtained through partners cooperation allowed RD to participate in dissemination activities (2 papers and 2 communications at project Erice workshop).
Through its close interplay with WP1 and WP4 work, modeling was a tool which contributed to the evolution of the preparation of predictive engineered TiO2 anatase NPs, from a “trial and error” approach to knowledge – based one, both at project beginning, by designing the experimental programs of WP1 for each synthesis route and each type of anatase NP, for the systemic investigations in the experimental research and optimization its time and cost, and finally, by processing the experiments data and development of mathematical models - soft models that can be used as design rules for synthesis of anatase NPs with defined and reproducible shape, size, surface, these specific characteristics being targeted for certain uses, well defined in the project, as energy (solar cells -DSSC type), environment (photo-catalysis in reactors for toxic effluents cleaning), health (nanocoatings for medical devices) and metrology (candidates of reference materials).
2.3 Assembly and aggregation of TiO2 NPs on proper supports
The thickness and physic-chemical characteristics of TiO2 coatings can greatly influence their final performance. In SETNanoMetro, different deposition procedures were set for applying films of TiO2 NPs with defined and homogeneous thickness on supports of interest for the application studied into project. The selected substrates were the following.
Silica glasses for photocatalytic measurements
• Conductive glasses (FTO) for dye-sensitized solar cells
• Ti-alloys for orthopedic and/or dental prostheses, for cell cultures.
Since it was found to be quite laborious to polish the Ti-6Al-4V substrates, a number of alternative substrates in the shape of Ti-coated glass slides was produced by Plasma Vapour Deposition (PVD).
The different film deposition procedures studied within the project were:
• Direct formation of films by using PVD sputtering and screen printing methodologies.
• Sol-gel technologies and conventional methods using TiO2 NPs (commercial and synthesized in the project).
• Self-assembly of TiO2 NPs in multiple layers (layer by layer deposition).
• Formation of films of TiO2 using PVD sputtering
As a result of the project PVD coatings of well-defined crystallinity (rutile, anatase and mixtures hereof) have been developed. A window of homogeneity was established and good batch to batch reproducibility was obtained. Furthermore, the heights in the deposition chamber resulting in a homogeneous sample thickness were carefully investigated. In addition, to perform and optimiz the PVD deposition process, basic analysis, XRD and measurements of photocatalytic activity of the CO2 generation originating from acetone degradation) on the PVD TiO2 coatings were done.

Formation of film of TiO2 fabricated by screen-printing
The choice of the right processing methods is crucial to increase the global efficiency and reduce the manufacturing costs of photovoltaic devices. In general, processing methods should allow the control of the main parameters of the deposited layer like morphology, thickness or geometry. These parameters were fine-tuned during the SETNanoMetro project in order to lead to the fabrication of an optimized device.
Screen-printing was considered for the deposition of the anode material. Screen-printing is a method that allowed us to optimize titania layer and to fine tune it depending on the targeted application. The layer thickness and its porosity are crucial for industrial applications where reproducibility is important for the success of the process. The relation between the ink concentration and the mesh type used for the printing process was studied, is now known and allows adjusting the layer thickness precisely. Porosity is in close relation with particle shape or size and sintering conditions of the resulting layer. A good compromise between porosity, surface area and efficiency was found using 18h heating time during hydrothermal growth.
Based on the optimized method to produce titania films with the required quality for dye solar cell applications, thin films of TiO2 nanoparticles, with defined size and shape produced in the project, were fabricated, on conductive glasses, by screen-printing technique with optimized thickness and porosity.
Titania nanoparticles were successfully incorporated into the organic binders and converted in printable ink. This ink was formulated to follow several requirements:
- The final viscosity should be compatible with the process
- Templating agents are incorporated in order to improve the layer rheology
- The dispersion of nanoparticle should be stable over the time
When using the optimum formulation, the use of two different processes allowed to improve the nanoparticles dispersion and reduced aggregation in order to get the best layer quality, which could be used in dye solar cells. All the printed layers were characterized using SEM and used by the project partners.

Formation of film of TiO2 NPs by using sol-gel technology
As the supports and NP’s functionalization was very challenging in the project, it was decided to use a sol-gel formulation to promote the adhesion of the NPs synthesized during the project to the supports and to slightly stick nanoparticles between them. The amount of sol-gel reactant was optimized with respect to the NPs concentration to maintain the porous structure between the nanoparticles.
For the preparation of mesoporous and mesostructured TiO2 anatase films obtained by using the NPs prepared in SETNANOMETRO two depostion technique were employed; dip-coating and spin-coating. By using spin-coating, thin layers were obtained (between 250 and 450 nm for coatings prepared with 1 or 3 layers, respectively). The dip coating deposition technique was employed for the controlled deposition of films with higher thicknesses, between 500 nm – 10000 nm as a function of the number of applied layers and the TiO2 concentration in the starting sol-gel solution. These coatings were deposited onto the 3 substrates of the project, and characterized by SEM.

Formation of supported films by self-assembly of TiO2 NPs
For the controlled assembly of TiO2 NPs, the layer-by-layer deposition method was selected. This technique of thin film fabrication consists of the deposition of alternating layers of oppositely charged/functionalized materials with wash steps in between. The technique offers an easy control over the film thickness and is a simple and inexpensive means for film preparation. In addition, one of the pros exhibited by this method is the stability of the functionalized surfaces when exposed to ambient air. However, the controlled assembly of TiO2 NPs on the surface of the selected supports requires a proper functionalization of the supports which will be employed in order to promote the adhesion of the film to the substrates. In this way, the functional groups on the substrates should react with the functional groups of the functionalized nanoparticles deposited on them.
In a first step, the two different functional groups to be attached to the NPs were selected: amine and aldehyde groups. These groups were also selected for the functionalization of the supports. The molecule chosen for the functionalization which provided the aldehyde-free groups was glutaraldehyde (GLUTA), a head-tail cross-linking molecule. In order to take advantage of the surface hydroxy groups which some supports could have, an alkoxysilane were employed in the functionalization as this linker was able to react with surface hydroxy groups of the support. 3-Aminopropyltriethoxysilane (APTS) was chosen as this alkoxysilane possesses an amine group which allowed the reaction with the aldehyde. In the case of Ti-alloys, instead of APTS, it was employed 3-aminepropyl phosphonic acid (3-APPA) as this was more reactive.
The activities carried out within the frame of WP3 allowed also to attain a step forward in extending the chemical strategies for the molecular surface functionalization of anatase TiO2 NP. The basic idea was to find an alternative to the exploitation of surface hydroxy groups as anchoring sites. The rationale was the limited amount of these groups on TiO2 anatase NP, resulting from the energy minimization of otherwise coordinative defective terminations and/or the dissociative adsorption of water molecules on surface facets with higher energy with respect to the {101} ones, which are the most stable, and typically the more abundant in usual TiO2 anatase NP. Conversely, {101} surfaces can strongly interact with phosphate/phosphate like groups, a behaviour typically observed when these nanoparticles, as such or in the form of coating of orthopaedic and dental implants, are used in cell-free tests or in-vitro and in-vivo experiments involving cells and/or tissues.
On this basis, the 3-APPA functionalization with two types of TiO2 NP mostly terminated by {101}, namely TiO2 P25 and UT001 was successfully attained. The presence of the amino group intended as functional group exposed toward the exterior by {101} surfaces reacted with 3-APPA was functional to the subsequent reaction with GLUTA, used to covalently bound TiO2 NP to supports or to other TiO2 NPs, both treated in order to expose amino groups.
The attainment of the scientific task constituted by the functionalization of TiO2 NP with 3-APPA required also the selection of proper characterization techniques, effective in probing the actual occurrence of the functionalization, in particular of the targeted type of surface terminations. This goal was successfully achieved too by using complementary technique. Among them, Auger Electron Spectroscopy (AES), combined with Transmission Electron Microscopy, where TiO2 NP functionalised with 3-APPA, and then reacted with glutaraldehyde, is displayed.
When the primary electron beam moves over the particles, the O and Ti signals (due to TiO2 NP) and C and P signals (due to 3-APPA) firstly increase, then reach a short plateau, and finally, decrease, whereas the Si signal coming from the Si wafer substrate, changes in a reciprocal manner. The variation of the C and P line scan intensities relates to the locations where the TiO2 NPs are present, monitors the successful functionalization with 3APPA.
Moreover, the anchoring of 3-APPA molecules to {101}surface terminations was proved by IR spectroscopy of CO adsorbed as a probe molecule on pristine and functionalised TiO2 NP: after anchoring 3-APPA molecules, the signal typical of CO in interaction with Ti4+ sites exposed by {101} appears significantly decreased in intensity.
Finally the layer-by-layer deposition was carried out with the functionalized NPs onto the functionalized supports. However, it was observed that the coverage of the support can attain almost to completeness, but only over limited areas, and the thickness of the multilayers of NPs appeared quite inhomogeneous. Conversely, PVD sputtering and screen-printing appeared methods suitable for coating supports using simpler technologies.
2.4 Characterization of individual TiO2 NPs and films resulting from their assembly/aggregation
The general aim of this WP was to improve existing methods for analysis of physico-chemical, mechanical, optical and electrochemical properties of TiO2 NPs and films so that these methods can be applied as reproducible measurement according to standard operation procedures (SOP). A number of 14 SOPs have been issued within WP4 reflecting the newest methodology of measurement of e. g. bipyramidal TiO2 NPs properties such as particle size and shape distribution by means of electron microscopy or elemental composition in-the-depth of TiO2 NP screen-printed layers loaded with ruthenium dye complex for solar cell applications. In most cases, significant methodical developments have been undertaken well beyond the state-of-the-art of the respective analytical methods for accurate measurement of the specific TiO2 NPs and layers fabricated in this project. Considerable part of the measurement procedures as developed under WP4 is either already published in scientific journals or submitted for publication or in preparation.
A special emphasis has been put on the metrological content of all WP4 SOPs with respect to a unitary structure including not only the exact description of the measurement procedure, but also the challenging aspects of method validation as well as the assessment of measurement uncertainties associated to the result obtained. Furthermore, there where it was possible, modelling and simulation of the signals have been performed with the main purpose to estimate the accuracy of the measurement result. Three extensive reports related to modelling and simulation of X-ray spectra (with electron and X-rays as excitation probes) from TiO2 NPs and layers as well as on multiscale modelling using Simulink/Comsol for electrochemical properties and Matlab and Monte-Carlo for X-ray spectroscopy including the relationship to critical parameters (CP) and critical dimensions (CD) have been issued as corresponding WP4 deliverables.
An inherent part for ensuring the traceability of the results obtained after following the procedures recommended in the SOPs developed in WP4 is the exploitation of reference materials for calibration purposes. Due to the lack of suited certified reference materials (CRMs) in the field of nano-properties, potential candidates as CRMs resulted from WP1 (NPs as well as layers) with respect to e.g. dimensional (size and – in premiere - shape) properties have been identified and metrological pre-characterization necessary for further certification process (e.g. by BAM) carried out. An inter-laboratory comparison with well-shaped (bipyramidal) TiO2 NPs manufactured within SETNanoMetro has been organized within ISO/TC 229 Nanotechnologies with 18 participants such as Evonik, BASF, Kronos, FDA, NIOSH, PTB, NIST, AIST with the scope of including this study case into the ongoing standardization project ISO/WD 21363 Nanotechnologies -- Protocol for particle size distribution by transmission electron microscopy (TEM). Further, three WP4 SOPs have been already prepared to be submitted as working drafts to national standardization bodies (see later).
The most reliable analytical method applied for the measurement of size distribution of nanoparticles also of more complex shapes is the electron microscopy, TEM and scanning electron microscopy (SEM). The special challenge in this project was to measure a large amount of newly prepared batches of TiO2 NPs of variable shapes in a short time as characterization accompanying the fabrication process for further optimization of the controlled parameters via supporting predictive models from WP2. Hence, the further development of a new variant of TEM, namely transmission in an SEM (TSEM), could be advanced considerably for the dedicated application of measurement of size and shape distribution of bipyramidal shaped TiO2 NPs.
The systematic dimensional characterization of the TiO2 NPs produced in SETNanoMetro has been complemented by the structural characterization carried out with different methods: X-ray diffraction (XRD), HR-TEM, selected area electron diffraction (SAED) at TEM and Raman Spectroscopy.
A special attention has been paid within a dedicated WP4 task to the accurate chemical analysis of the surface of TiO2 NPs as produced in SETNanoMetro. An Auger transition Ti LMV involving a valence level (V), as recorded by X-ray photoelectron spectroscopy (XPS), was demonstrated to provide templates for TiO2 phases (rutile vs anatase) and orientations that allow a quantitative analysis of TiO2 NPs. Additionally to the classical surface electron spectroscopies X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES), which are techniques sensitive to changes of chemical bonding from a TiO2 polymorph to another, a special form of infrared (IR) spectroscopy (in controlled atmosphere) has completed the systematical characterization of TiO2 surface chemistry: by means of molecular probes, such as CO, which are sensitive to structure of adsorbing sites, detailed information not only on the individual TiO2 NPs surface sites but also on facets distribution, i.e. particles morphology, can be extracted from the corresponding IR spectra supported by density functional theory (DFT) calculations.
A multitude of TiO2 films have been manufactured and tested within SETNanoMetro with respect to their performances for the main three application fields (energy, environment and health). One basic film parameter is its film thickness. Depending of the TiO2 film type (sputtered by PVD or screen-printed) several sample preparation procedures from simple, quick and cheap, such as (i) scratch and cross-sectional SEM analysis of peeled-off fragments of layer or (ii) fracture of the layered sample followed by SEM cross-sectional analysis, to more complex and expensive, such as (iii) cross-sectional preparation of the layered sample by focus ion beam (FIB) and cross-sectional SEM analysis, or (iv) preparation of a TEM lamella and T(S)EM analysis) have been recommended in form of a comprehensive SOP. The more complex procedures do not imply automatically a higher accuracy of the thickness measurement.
Other film parameters of crucial importance for the applications of the layered system as a product, but challenging to measure by traditional analytical techniques are porosity and the networking of the nanostructured TiO2 within the fabricated film, so that a quantitative evaluation of the inner surface of the porous TiO2 layer becomes possible.
Additionally to the results of the 3-D reconstruction of the porous TiO2 films by FIB slicing/SEM imaging, a new analytical technique, Transmission Kikuchi Diffraction (TKD) at SEM, could be applied successfully to extract quantitative information on orientation, size and shape of individual TiO2 NPs constituting the screen-printed layer.
The good accuracy of the results could be confirmed by independent AFM measurements of the same porous TiO2 films,. as well as by dimensional measurements with electron microscopy (TEM, SEM and TSEM) carried out on the TiO2 NP initial powder.
The characterization of SETNanoMetro TiO2 NPs and their assemblies undertaken in WP4 is completed by detailed investigations of their mechanical and electrochemical properties.
As far as the electrochemical properties are concerned a corresponding task has been dedicated to measurements providing information on the photocatalytic behavior of TiO2 coatings, such as: band gap, photocurrent / reaction kinetics, band bridging, and system capacitance (including surface, material and interfaces). Electrochemical measurements such as photo potential and photo current measurements, and impedance spectroscopy methods were used in combination with diffused reflection measurements. Using energy-loss electron spectroscopy (EELS), diffused reflection, and electrochemical methods, band gap at individual nano-particle level is compared with macro scale band gap. In this way, it was possible to identify the spread in band gap of nano-powders based on the synthesis methods.

2.5 Standardization rules for TiO2 NPs and films performances in photocatalysis
The main task of WP5 consisted of the development of traceable measurement methods of the photocatalytic activity of TiO2 crystals with focus on pre-normative recommendations and industrial application. The measurement methods encompassed macroscopic methods such as the standardized Methylene Blue decomposition, as well as newly constructed portable gas/solid and liquid/solid reactors. In addition, the photocatalytic activity has also been studied on the microscopic scale, down to the level of individual NPs. For this purpose, the existing measurement method of Scanning Kelvin Probe Microscopy (SKPM) has been made traceable to the work function property of metals. Several inter-laboratory comparisons have proven the various measurement methods to be in good agreement. Ultimately, in order to provide the same conditions for all measurement methods, and as a pre-normative approach for further standardization, prototypes of a new calibrated light source based on LED technology has been developed, built, and sent to the participants for best comparability of the measurement results.
The established measurement methods have been applied to TiO2 particles produced at an industrial scale, and the optimum design parameters for highest photocatalytic activity of the NP batches have been found. A reference standard for photocatalytic activity has been developed and is now commercialized by a consortium member. The work has led to 6 extensive major consortium reports on quantification, traceability and measurement uncertainty of photocatalytic activity at the nano- and micrometer scale, reference instrumentation and methods, as well as a comprehensive report on correlation and design rules. Outside the consortium, the work of WP5 has resulted in more than 5 peer reviewed publications, 2 under preparation, and more than 5 contributions to international conferences and workshops, and 5 prototypes/reference materials, out of which 4 are being made commercially available by the consortium.

Reference methods

Gas/Solid and liquid solid reactors
For gas/solid and liquid/solid experiments portable miniphotoreactors have been developed. They are equipped with the reference LED source a set of various sensors (photoionization detector, electrochemical NO, CO and NO2 sensors for gas/ solid, radiant power, pH, dissolved O2 for the liquid/solid) for continuous monitoring substrate. The gas/solid set-up employs a continuously stirred tank reactor (CSTR) which allows extrapolation to infinite turbulence, thereby obtaining an evaluation of the photocatalytic activity that is independent to mass transfer. For liquid/solid experiments a flow through recirculated photo reactor has been developed that employs a fluidic optimized in order to give standard and reproducible conditions of illumination and flow. The photocatalytic activity is measured as substrate disappearance rate. An important requirement is also the speed of measurement and the possibility to carry out in-situ and in-line measurement. The miniphotoreactors have been especially designed to allow fast measurements. The main source of measurement uncertainty for both miniphotoreactors is the repeatability. Other contributions to the uncertainty are the concentration of the substrate, the flow, the radiant power of the light source. Noteworthy, the relative standard uncertainty is always lower than 5%.
SKPFM
Photocatalytic activity at the molecular level results in charge separation by the electron-hole generation. This change in surface potential can be measured using Scanning Kelvin Probe Measurement technique (SKPM) which responds to changes in the contact potential difference (CPD) between the nanometer sharp probing tip and the probed surface. SKPM images of sub-micrometer areas have been taken while exposing the same to standardized UV radiation and the CPD studied in detail. The relative standard measurement uncertainty on CPD changes caused by photocatalytic activity is in the order of 10%, dominated by the repeatability of the experiment.

Reference materials and instrumentation
Lights sources
A portable light source based on LEDs has been designed for multi-purpose applications in the project. Compared with a 20 W halogen and 25W deuterium lamp, the 1W LEDs outperform the broadband light source regarding the spectral power with a factor of 1 000 to 10 000. While being individually narrow banded, the combination of different wavelengths together with digitally controlled drivers allow a sample irradiation at selectable and reproducible spectral regions. This allows maximum versatility, while at the same time providing robustness which minimizes shipping requirements during transportation among the partners. The spectral irradiances of the light source have been measured and are traceable to the SI-system of units. The expanded measurement uncertainty of the irradiance is approximated to 5%

Reference standard for traceability of photocatalytic activity at molecular level
Traceability at the molecular level has been established to SKPM for the measurement of the photocatalytic activity during UV activation. The choice of using nanometer-flat gold coated substrates Au has been based on the extensive studies during the project. Together with two other metals, the changes of TiO2 CPD during UV illumination can be related to the unaffected metal surface which established a potential scale using the work functions of the metals as a reference for calibration purposes.

Reference NPs materials
Two well-characterized candidate reference materials for the photo catalytic activity are chosen, TiO2 in bipyramidal form and nano-platelets. These different shapes and surfaces exposed, they encompass various aspects of the photocatalytic mechanism (OH radical like oxidation, direct hole transfer). Their photocatalytic activities were measured reproducibly both on the molecular level and at a macroscopic scale, also within different NPs batches, ensuring the suitability of these materials as candidate reference materials for the photocatalytic activity. The production of the bipyramids has been scaled up to the industrial level, and thus led to the standard reference sample for photocatalytic activity, which is commercialized by project partner. The reference sample exhibits a change of CPD.


Correlating results on the molecular and macroscopic scale Optimum coating
We have performed an inter-laboratory comparison (ILCs) on TiO2 PVD thin films comprising of 4 different laboratories and measurement methods, as well as an ILC on TiO2 NP coatings down to individual NP level with 2 different methods. Furthermore, one ILC for the calibration task of coupling the macroscopic photocatalytic activity to the molecular level has been performed.
In the ILC on PVD thin films, the 4 utilized measurement methods were: 1) gas/solid acetone degradation, 2) standardized liquid/solid methylene blue, 3) liquid/solid OCP, and 4) SKPFM. The first three methods acquire the photocatalytic activity at the macroscopic scale, and the last on the molecular scale. Four TiO2 thin films deposited at different temperatures were analysed. All four measurement methods unanimously identified those thin films with the highest photocatalytic activity. Consequently, a correlation between measuring the photocatalytic activity at the macroscopic and the molecular level has been established, including an already standardized method.
A correlation between the photocatalytic activity at the macroscopic and molecular level was also attained for coatings comprising of TiO2 NPs. Both the results from the gas/solid CSTR and from SKPFM indicated that the best photocatalytic performing NPs were UT001.
To establish traceability between the macroscopic and molecular level, TiO2 PVD thin films deposited on three different substrates (Au, Ni and Cu), where studied in an ILC between OCP and SKPFM [3]. Both techniques demonstrate that the substrate influence the achieved photocatalytic activity, and preferably the TiO2 thin film should be deposited onto Au due to an improved electron transfer when evaluated with SKPM.


Polymer embedded TiO2 particles

The established correlation between the macroscopic and the molecular photocatalytic performance allowed for using SKPFM to determine the photocatalytic performance of TiO2 NPs embedded in polymer pellets. Above is displayed the topography showing embedded TiO2 NPs and the result from SKPFM.
2.6 Standardization rules for TiO2 NPs and films performance in DSSC
The photovoltaic performance of a Dye Sensitized Solar Cell (DSSC) relies on the characteristics of its photoanode, which plays a central role in converting light into electrical energy. A DSSC photoanode typically consists of a mesoporous oxide film on a transparent conducting glass substrate. Dye molecules that capture photons from light during device operation are attached to the surface of oxide film. Photoexcitation of the dye molecules leads to the injection of electrons into the oxide film. A key fabrication factor for high efficiency and reproducible DSSC is the dye loading of the film, which determines the quantity and the nature of the adsorbed dye molecules. The dye loading should be optimized so that the interfacial surface of the oxide film is completely covered with a monolayer of dye molecules.
Preparation of dye sensitized TiO2 films.
Staining conditions were optimized, in terms of solvent, concentrations and duration in order to reach the maximum dye loading of titania layer used to build laboratory high efficiency dye sensitized solar cell. These staining conditions were recorded in a Standard Operation Procedure (SOP) for dye loading (see D6.1) that defines the procedure for efficient and reproducible dye loading of the films.
State-of-the art devices were produced using nanocrystalline titanium oxide, having particle sizes around 18 nm as the support electrode (anode), on which a sensitizer (organic or ruthenium metal organic complex) was attached as monomolecular layer, thanks to its anchoring groups (carboxylic acids). The electrolyte contains the redox couple iodide/tri-iodide in an ionic liquids mixture, and acts as the charge carrying media to contact each dye molecule with the electric circuit.
Characterization of the TiO2 films loaded with the dye sensitizer.
After application of the reproducible procedure of loading of the screen-printed TiO2 NP films with the Ru-complex dye ‘N719’ (Solaronix 535-Bis TBA) as described in D6.1, the distribution of elemental composition in-the-depth of the porous, µm-thick TiO2 layer has been investigated by energy-dispersive X-ray spectroscopy (EDX), Auger electron spectroscopy (AES) and time-of-flight secondary ion mass spectrometry (ToF-SIMS).
The through-the-depth analysis by AES and ToF-SIMS is carried out in form of semi-quantitative analysis, which is well suited for the purpose of assessing in-depth homogeneity of the Ru-complex dye throughout the TiO2 layer; quantification of the in-depth elemental distributions through the ~7.5 µm TiO2 layer by means of the two analytical methods is not recommended due to large errors caused by various sources such as matrix effects, sample surface morphology effects (AES), or stability of the chemistry of the analysed surface during ion bombardment (SIMS).
The best AES results have been obtained on samples prepared for analysis after breaking them carefully and selecting a relative plane surface where an AES line scan could be collected. By taking into account the effect of surface morphology on the Auger electron intensities a relative homogeneous distribution of the dye sensitizer through the thickness of the porous TiO2 screen-printed layer can be concluded.
That the dye is homogenously distributed over the entire TiO2 film thickness was confirmed by ToF-SIMS analysis. Similarly to AES, the method is very sensitive to surface and the methods work in the depth-profiling mode (by alternating sputtering and analysis) for depth up to 100-200 nm. However, a dedicated ToF-SIMS measurement session (by individual overnight measurements) in the depth-profiling mode has been carried out on the same sample.
No significant gradients along the thickness of the loaded TiO2 layer could be detected also by using EDX analysis on broken samples – similarly to the AES analysis. However, because the Ru X-ray signals were close to the limits of detection, mapping of three different depth areas has been carried out in order to collect X-rays with better signal-to-noise ratio.
N719 dye’s concentration profile obtained on films by confocal Raman spectroscopy was described. Raman analyses were performed in order to consider the low thermal stability of the dye and the quantification of dye loading has been developed using a calibration model based on peak analysis. Due to its high spatial resolution, lower than 1 μm, Raman spectroscopy allows to analyse thin film sections. Raman analyses on film section were conducted in mapping mode. Using low laser power during Raman analysis has allowed accurate detection of analytes in restricted areas, and in Raman resonance condition the enhancement of dye signals. However, the main problem encountered was still the thermal degradation of N719 dye.

Characterization of the laboratory cells.
DSSC devices were produced using high stability state of the art electrolyte. The efficiency is correlated to the specific surface area and the dye loading of the titania layer. The reference material is still the best in terms of pure efficiency. The particles produced in WP1, UT001, show an efficiency that is really close from the reference material and a higher open circuit voltage in all the cases. It can be noted that this result is interesting as among all the titania nanoparticles that were tested in the past by SOLAR, few of them were able to compete with the reference (Ref-SX).
Efficiency is one of the criteria to evaluate to viability of a system but stability is critical in order to determine the usefulness of these nanoparticles in a potential device.
The stability of two types of DSSCs, made using the most promising titania nanoparticules, was investigated. Various ageing conditions were used like: (a) temperature: -27,5 °C; 50 °C; 70 °C, 85 °C (b) light soaking at 35°C and 70°C: light intensity 1000W/m2, 400 nm UV filter in order avoid DSSC damage by UV (c) humidity: 80% (25°C)
On the basis of these investigations, it was found that:
1. Temperature treatment at 50° C doesn‘t cause considerable change of DSSCs efficiency but treatment at 70° C causes a decrease of efficiency while heating at 85° C leads to a fast degradation of the cells.
2. Low temperature treatment (-27° C) doesn’t influence the characteristics of DSSCs.
3. Light soaking (1000 W/cm2, UV cut off) at 35°C doesn’t influence efficiency of DSSC. Decrease of efficiency during light soaking treatment at 70°C is attributed only to the heating of samples during experiments.
4. Humidity (80 %) doesn’t influence characteristics of DSSC
5. There is no influence of the nanoparticle type as the behavior is similar for all samples independently from the nature of the titania used in the device. The devices were found to be stable under these conditions. The control of the device temperature seems critical to ensure a good stability under working condition.
2.7 Standardization rules for TiO2 NPs and films performances and toxicity in biomedical applications
Standard Operating Procedure for assessing the toxicity of various TiO2 nanoparticle coatings
We focus our work on test if TiO2 NPs that formed a surface coating on titanium/titanium alloy substrates would be present a low toxicity profile to cells relevant to tissue response to orthopaedic medical implants. The initial activities focussed on developing a Standard Operating Procedure for assessing the toxicity of various TiO2 nanoparticle coatings. This used two relevant cell lines, human osteoblast-like MG63 cells, and mouse macrophage RAW 264.7 cells, and utilised coated substrates that, when combined with an adhesive 8-chamber, allowed multiple independent cultures on the same substrate, which was designed to minimise sample-to-sample variability. Most of the studies were focussed on TiO2 NP coatings produced by magneton sputtering; these were initially on polished Ti6Al4V substrates but similar coatings were subsequently produced by first depositing a titanium substrate layer on glass slides, followed by deposition of the TiO2. The main observations were that all magnetron sputtered coatings, with different anatase: rutile ratios, exhibited negligible cell death, using the two different assays described in the SOP. Subsequent experiments with TiO2 NP coatings produced by screen printing of NPs and dip coating in a NP suspension showed similar results. Together these results indicate that when NPs form a fixed coating they do not present a toxic response, relative to controls.
Another aspect that we investigate was the response of human mesenchymal stem cells (hMSCs) to various TiO2 NP coatings produced by magneton sputtering, to determine if these coatings supported osteogenic differentiation of the cells (medium supplemented with osteogenic supplements) and assess if any coatings appear to induce osteogenic differentiation (in the absence of osteogenic supplements in the medium). Initial activities developed an SOP that described the procedures for characterisation of the hMSCs use in such a study and the procedure for evaluating the cell response Results demonstrated that all TiO2 NP coatings supported the differentiation of the hMSCs to an osteogenic lineage, with positive upregulation of alkaline phosphatase (ALP) activity after 7 days and osteocalcin (OC) expression after 21-28 days culture in osteogenic medium (Deliverable 7.4); these studies used multiple donors, to eliminate the possibility of a donor-specific response. Although all coatings studied exhibited an increase in ALP activity when hMSCs were cultured with osteogenic supplements, coatings composed mostly of anatase produced the strongest results, whereas the coating composed mostly of rutile exhibited lower levels of ALP activity. These results were also supported by quantifying expression of COLIa1 and VEGF by hMSCs, which both showed higher expression for cells on anatase rich coatings, compared to rutile. The opposite was observed for TGF-β1 expression which showed a peak expression for hMSCs cultured on a rutile rich coating. No coatings appeared to induce osteogenic differentiation of hMSCs, with no significant differences between controls and coatings when no osteogenic medium was present.
The different TiO2 NP coatings produced by magneton sputtering that were characterised using a range of techniques to provide information of phase composition (XRD), surface roughness (AFM), surface morphology (SEM) and water contact angle. These properties were used to determine if any correlations existed with ALP activity, collagen (COLIa1) expression, VEGF and TGF-β1.
We observed some material property correlations, in particular the positive correlation between increasing anatase content with increasing surface roughness of the coatings. Additionally, we observed a strong negative correlation between increasing anatase content (and increasing surface roughness) and decreasing TGF-β1 expression. Anatase-rich coatings appeared to show stronger osteogenic differentiation of hMSCs, so expression of TGF-β1 may be suitable as a predictive marker of hMSC differentiation on such substrates. Such correlations ultimately must be validated through tissue responses, in this case bone deposition, in pre-clinical models.
Evaluation of toxicity in lung and intestinal cells
Since inhalation and ingestion are the two most important uptake routes for nanoparticles, the human lung epithelial cell line A549, the human colon epithelial cell line Caco-2 as well as the rat alveolar macrophage cell line NR8383 were chosen as three well-established test models. The initial activities focused on the developed of a SOP for toxicity profiling of the various TiO2 NPs. Evaluation of cell death, induction of markers of oxidative stress and inflammation were included since these are considered as the key biological pathways in nanotoxicology. Important aspects in the SOP include an accurate description of the preparation of the TiO2 suspensions, using a Cuphorn-sonication method, and additional measures to avoid assay read out artefacts in the toxicity testing. The SOP was then used to assess the effects of the three TiO2 commercial samples (SX001, P25, and PC001), alongside with samples designed and developed within the project. Amorphous silica NPs and a fine crystalline silica sample, were included in an assay specific manner, to allow for a hazard comparison with the TiO2 NPs. Cytotoxic cell death was determined with water-soluble tetrazolium (WST-1) assay. It was demonstrated that all tested TiO2 NPs had negligible toxicity in all cell lines, when compared to the SiO2 NPs. As marker of oxidative stress induction, the mRNA expression of heme oxygenase-1 was determined by semi-quantitative real-time polymerase chain reaction (qRT-PCR). The TiO2 NPs caused mild oxidative stress only in the NR8383 macrophages. The inflammatory properties were evaluated by mRNA expression analyses of interleukin (IL)-1β, IL-6 and IL-8 (using qRT-PCR) and measurement of protein secretion of IL-1β and tumour necrosis factor-α (TNF-α) using enzyme-linked immunosorbent assay. Moreover, the activation of the NALP3 inflammasome cell signalling pathway activation was assessed in the NR8383 macrophages by evaluation of IL-1β secretion in lipopolysaccharide (LPS)-primed cells. Overall, the pro-inflammatory properties of the TiO2 NPs were minimal in comparison to the SiO2 samples. The activation of the NALP3 inflammasome pathway emerged as a potentially discriminating effect for the “biological activity” of various TiO2.
The toxicity testing also included an in-depth evaluation of oxidative related DNA damaging effects of TiO2 NPs under varying illumination conditions. DNA damage was evaluated in the A549 cells by the comet assay, and revealed that specific TiO2 NPs could cause mild DNA strand breakage, but no oxidative DNA damage induction. Interestingly, however, after photocatalytic activation (using pre-illumination of the samples with UV-B) DNA damage and oxidation became more pronounced for a specific sample (p25). Subsequent intervention experiments with the antioxidant status modifying compounds BSO and NAC indicated that reactive oxygen species generated during UV-illumination could be responsible.
Another aspect that was investigated was the assessment of the oxidative potential of TiO2 NPs produced within the project by cell-free tests. The first part of the activities has been dedicated to the development of protocols based on Electron Paramagnetic Resonance (ESR) spectroscopy and UV/Vis spectrophotometry, ending with the definition of a Standard Operation Procedure. By using TiO2 samples (anatase, rutile and anatase/rutile mixed phase) of known photo-activity a set of protocols to assess the generation of Reactive Oxygen Species (hydroxyl radicals, superoxide anion radicals, singlet oxygen) and the reactivity toward lipids and nucleic acid moieties in a solution simulating the physiological pH and osmolarity were considered. Among the probes tested TEMPONE-H and DMPO in the presence of sodium formate appear the most suitable to predict the oxidative potential of the powders (Marucco A., Carella E. and Fenoglio I. RCS Advances 2015). These protocols were therefore chosen for the Standard Operation Procedure development.
In the second part, the SOP was used to assess the oxidative potential of four TiO2 samples, one developed in the project (UT001) and three commercial specimens (SX001, P25, PC001). The oxidative potential was evaluated in different illumination conditions, i.e. dark, indoor illumination, simulated sunlight and pre-illumination.
The results indicated that the intensity of illumination is an important parameter in vitro testing of photo-active semiconducting NMs. A significant photo-activation of the powders was in fact detected also in normal indoor light. Interestingly, pre-irradiation resulted in a small but significant generation of H2O2 for P25 and UT001 samples.
The effect of illumination on nanoparticles dispersion has been also evaluated by Dynamic Light Scattering (DLS). In this case, small effect of the light on the aggregates/agglomerates size distribution was observed in water. However, when proteins were present, a clear light-induced agglomeration was observed.
Overall, the results indicate that the control of illumination during toxicological testing is necessary to improve the inter-laboratory comparability of toxicological data.
Another aspect that was investigated is the hazard evaluation of the TiO2 NPs to contribute to safer-by-design strategies in the nanomaterial engineering field. This has been achieved by modelling of the relationship between the generated toxicological data and the physicochemical properties of the TiO2 NPs.
The availability of structure-activity relationships (SARs) is a mandatory pre-requisite for the design of safe chemicals. The principal component analysis (PCA) has been applied to find correlation among a set of physico-chemical properties, the oxidative potential, and the oxidative-stress related cellular response of a series of TiO2 nanomaterials prepared. The results of this analysis indicated a clear correlation among some toxicological outcomes, suggesting that a possible reduced number of parameters is sufficient to define the toxic response of the cells for TiO2 NPs. A correlation between the oxidative potential of the powders and their ability to induce Interleukin-1β release without or with LPS-priming activation was found. This indicates that the oxidative potential of TiO2 NPs is related to their ability to activate the NALP3 signalling pathway. Moreover, a relationship between the NP shape factor and NALP3 “activation” was found. Taken together, the chemical-physical variables analyzed seem to be a good set of variables to predict the toxicity (biological activity) with a soft model.
The toxicological data generated in the project with the various TiO2 NPs indicate that the hazard for this type of nanomaterial can be considered as low. Induction of inflammation and oxidative stress are considered as key “modes of action” that drive the adverse health effects of nanoparticles. The available literature reveals that, in comparison to other types of nanomaterials, the reactivity of TiO2 NPs towards cells is generally low (in studies that performed side-by-side comparison of various types of NPs). This was also specifically observed in the toxicity studies performed within this project. When tested on equal mass dose, the majority of effects of the TiO2 NPs, was lower than that of the amorphous SiO2 NPs and, especially, in comparison to the fine crystalline SiO2, which is a known toxic material. Notably, specific findings on DNA damage induction observed in this project align with a growing number of in vitro genotoxicity studies were small albeit statistically significant effects are shown for specific TiO2 NPs. In this regard, a major outcome from present WP is that the oxidative DNA-damaging properties of TiO2 NPs can be enhanced under illumination conditions. At which extent this is relevant for inhaled TiO2 NPs remains to be investigated, since lung tissue is not exposed to light. However, the observed findings can be of importance with regard to the specific handling of TiO2 NPs in toxicity testing protocols, as unintended, uncontrolled light exposures could result in false positive results (assay artefacts).
Finally, the toxicity data for the TiO2 NPs established within this WP should be viewed in relation to the applied treatment doses, since health risk is a function of exposure and hazard. This is also important regarding to the observed SARs for the TiO2. The findings from the PCA analyses indicate that it could be possible to lower the “biological reactivity” of this material “by design”. This would further reduce the hazard of this relatively low toxic type of nanomaterial, and, under (non-controlled) high exposure situations, possible adverse health risks.
2.8 Standardization and scale-up of TiO2 nanoparticles at industrial level
WP8 was devoted to the scale-up of the TiO2 nanoparticles prepared in previous WPs, and in the study of the feasibility of their industrial scale-up and applications.
Scale-up of synthesis: sol-gel, hydrothermal and laser ablation.
Laser ablation worked at very low concentration, in order to reach the nanoparticles with the expected quality assessed in previous WPs. Using the existent laser technology in UAB-MET, it was impossible to achieve kilograms scale, however different possibilities to achieve the process were explained and suitable equipment were proposed to achieve the scale-up.Hydrothermal technologies needed special reactors at high pressure to achieve the production.
This methodology has gathered very good results in terms of controlling the morphology of the nanoparticles.In Setnanometro, AVANZARE has studied the preparation of hydrothermal nanoparticles in a 7 litres reactor. Different optimizations were proposed such as use of commercial precursors, low quality reagents, and even other techniques for purification of solid nanoparticles such as lyophilisation. Sensors for controlling these processes were not possible due to the pressure and temperature needed for the reactions.
Using sol-gel technologies, AVANZARE has prepared kilograms of the nanoparticles. AVANZARE has implemented different online monitoring system such as pH and temperature control, and also a surveillance camera in order to control the reaction in the long times needed.
Regarding high area nanoparticles by sol-gel technologies, the process was scaled-up in 400 times, reaching kilogram scale and aiming to obtain comparable results to laboratory scale. AVANZARE has scaled-up the process, only introducing slight changes, such as an additional step of filtration and washing to eliminate some steps that create corrosion. Also, the recovery of the solvent by fractionated distillation will reduce the cost of the preparation of the product. The most time consuming step was the ageing of sol-gel that using high volumes, implies the use of longer times.
Low size nanoparticles by sol-gel technologies have also scaled-up to kilograms scale in 300 times bigger than laboratory scale. AVANZARE has proposed a different point within the selected from TEKNIKER. In this way, the characteristics are very similar, but the required amount of reagents was optimized and the product was more likely to be scaled-up in a profitable way.

Recommendations of standardization procedures
Multiple informations was gathered along the project, in order to standardize procedures and create reference materials. Different protocols were proposed to characterize photocatalytic activity, based on all the techniques studied along the project to measure photocatalytic activity and characterize nanoparticles. The most interesting found along the project in liquid-solid systems were:
• Methylene-blue evolution using UV-VIS procedures
• HPLC coupled to the photoreactor system developed in the project
• Open circuit potential measurements using electrochemical equipment
Regarding gas-solid systems, the continuous stirred tank reactor developed in the project for measuring photocatalytic activity offered the best results. Two candidates for references were selected: hydrothermal nanoparticles for solid nanoparticles, and steel substrate coated with hydrothermal nanoparticles for coated surfaces. These rules were applied in the photodegradability of polymers such as varnishes or plastic parts. AVANZARE and TEKNIKER have prepared different samples loaded with nanoparticles such as varnishes, injected plastic parts or paintings.
The samples were aged in QUV and were characterized using different techniques such as Raman spectroscopy, SEM-EDX or XRD. Using these techniques is possible to characterize the photodegradation capability of the nanoparticles, showing the following order: P25 > SOL-GEL HIGH AREA > SOL-GEL LOW SIZE > HYDROTHERMAL > PP VIRGIN.
AVANZARE has also prepared injected samples with nanoparticles and graphene in order to obtain conductive samples. In this case, it is possible to compare the photodegration activity by means of conductivity measurements. The obtained results were compared with the measurement of photocatalytic activity developed under the rules established in Setnanometro.
DFM has measured different polymer parts, and due to the low concentration in SKPFM, they have not shown photocatalytic activity. However using samples prepared by the direct deposition of the nanoparticles over steel surface is possible to see the same tendency in photocatalytic activity, although is worthy to note that high area sol-gel nanoparticles increase their activity in the highest concentration, obtaining better results than P25 nanoparticles

DSSC prototyping
The results collection obtained in task 6.5 has allowed to select the Dye Sensitized Solar Cell exhibiting the best functional performances and to be proposed as a standard, in terms of current, voltage and efficiency, with the fabrication of a prototype and the drafting of a protocol for its reproducible preparation.
W-type modules were investigated to build up a prototype, which consists in serially interconnected dye sensitized solar cells. Following the optimized and standardized procedures developed during the SETNanoMetro project, state of the art W-modules (10 x 10 cm, 65 cm2 active area) were produced and fully characterized. The reproducibility of the performances was found to be good when comparing the modules to the test cells (7.86% for modules compared to 7.70% for lab cells) studied in WP6, which demonstrate the scalability of the process. The prototype stability was assessed as well, found to be good and compatible with real outdoor use that is in agreement with TRL7 definition.
The reproducibility of the preparation for efficient DSSC modules was demonstrated on 10x10 cm scale according to the procedure that was developed during to project. Performance traceability was achieved by having the control on the whole characterization chain.

Potential Impact:
3.1 Impacts
Materials
The activities in WP1, in combination with WP2 and WP3, led to the formulation of design rules for the synthesis of size and shape controlled Anatase and Rutile nanoparticles, namely:

1 – bipiramidal TiO2 anatase NPs with low truncation (30-100 nm hydrodynamic diameter)
2 – elongated TiO2 anatase NPs with shape parameters from 2 to 6
3 – TiO2 anatase nanoplatelets with shape parameters from 0.15 to 0.30
4 – Prismatic rutile NPs with length from 150 to 1500 nm and shape parameters from 2.5 to 6
5 – Sol-gel TiO2 NPs with diameters < 20 nm
7 – high surface area sol gel TiO2 NPs
8 – spherical TiO2 NPs from LA

These particles can be exploited as shape and size standards in nanometrology, model materials for functional application (energy conversion in dye sensitized and perovskite solar cells, photocatalysis, ostheointegration of prostheses in the health sector, nanotoxicology) and standards for macroscopic functional properties in the sectors cited. These NPs will be synthesized by UNITO and commercialized by Solaronix. In Figure 3.1.1 is showed an example of the commercialized anatase nanoparticles.


Figure 3.1.1. Size and shape controlled Anatase NPs produced and commercialized by SETNanoMetro partners.




Devices
Traceable and reproducible measurement of functional properties of nanoparticles assemblies and films is of paramount importance in order to establish correct correlation between properties at nanoscale and molecular level and the macroscopic functional properties.
In WP5 were developed two devices for fast, accurate and traceable measurement of photocatalytic activity of oxidic nanoparticles and films, both in gas-solid and liquid-solid regime.

Portable gas-solid photoreactor
The device developed has the following characteristics:

➢ Gas-Solid photoreactor for testing the abatement of gaseous pollutants on photocatalytic materials
➢ Turbulent reactor, extrapolation to infinite turbulence allows to measure intrinsic photocatalytic performances of the material, without problems of mass transfer
➢ Portable for in situ measurement, proprietary gasket assures very good seal on rough surfaces like concrete, possibility to control in situ the durability of materials
➢ Very Fast Response: 15 mL reactor chamber allows a complete measurement in minutes
➢ Option for different LED sources
➢ Control and regulation of Radiant Power with traceable calibration
➢ Option for different sensors and substrates (NO, Toluene, Acetaldehyde)
➢ Option for mounting 45 mm glass or metal disks for in lab testing of new materials and films
➢ Humidity and temperature control
➢ Automatic and manual operations with user friendly control software and a Standard Operating Procedure

The Beneficiary of this exploitable foreground is UNITO, and the Licence to equipment manufacturing was appointed to FONDERIA MESTIERI, Turin, Italy.
In Figure 3.1.2 is showed the running prototype of the portable gas-solid photoreactor and in Figure 3.1.3 and example of measurement.




Figure 3.1.2. Gas Solid portable photoreactor, gas generating unit, control software user interface and control electronics.

Figure 3.1.3. Example of a measurement of photocatalytic activity on SETNanoMetro NPs film (NO abatement).

Miniature photoreactor for fast, accurate and traceable measurement of photocatalytic activity in the liquid-solid regime
For the traceable measurent of photocatalytic activity of NPs suspensions and films was developed a flat recirculated miniphotoreactor. Particular care was devoted to the design of the fluidodynamic of the reactor in order to attain a homogeneous liquid flow in the illuminated region.
A standard operating procedure for its traceable use was developed, with a proper suite of substrates (melamine, phenol, terephthalic acid and formic acid). Also in this case there are option for different LED sources and control and regulation of Radiant Power with traceable calibration.
In figure 3.1.4 there is a sketch and a picture of the device. Figure 3.1.5 shows the particulars of the fluidic used to attain homogeneous flow in the irradiated area.


Figure 3.1.4. Picture and sketch of the flat recirculating miniphotoreactor for photoactivity testing in the liquid-solid regime. The photocatalyst can be in suspension or as a film deposited on the bottom window.


Figure 3.1.5. Particulars of the two stage binary flow distributor (left) and injection comb (right) to attain homogeneous flow.
The Beneficiary of this exploitable foreground is UNITO, and the Licence to equipment manufacturing was appointed to FONDERIA MESTIERI, Turin, Italy.



3.2 Dissemination and Exploitation

Dissemination and exploitation activities were covered by publishing articles in journals, contributing to national and international workshops and conferences, training of young scientists, performing of inter-laboratory comparisons and contributing to standardization. In addition, specific products were prepared for commercialization.
SETNanoMetro published 19 articles in scientific journals and additional 12 articles are under preparation. In addition, 34 oral presentations and 16 poster presentations were given at workshops and conferences. A publicly available website was created, where information about and created by the Consortium is available.
Clustering and training activities were also at the core of the dissemination tasks of SETNanoMetro. Even in the present global availability of information through the web, meetings offering to scientists the possibility to discuss, exchange ideas, debate, seed collaborations represent high added value actions. This target was pursued also by SETNanoMetro by organizing the international workshop “NANOSCIENCE meets METROLOGY: size and shape engineering of nanoparticles towards improved technologies for energy, environment and health”, held at the Ettore Majorana Foundation and Centre for Scientific Culture in Erice (Sicily, Italy) from 27th to 31st July 2015. The response of the European scientific community resulted in the presence of 54 participants, including the representative of 3 companies, Photocat A/S (Denmark) and the Italian branches of Thermofisher and Bruker (this last is a stakeholder of SETNanoMetro). The ensemble of participants gathered representatives from 8 European countries: Denmark, France, Germany, Italy, Netherlands, Switzerland, Spain and United Kingdom. In summary, the programme listed an introductory plenary lecture, 36 oral presentations, organized in 7 sessions, each opened by a keynote lecture, and in a poster sessions. The topics were dealing with emerging issues relevant for the European Research Area:

• need for characterization of the relationship between measurable key parameters through the nanoscale to the higher order scales
• interest toward the generation of reference information to test and optimise new design rules derived from length scale models;
• development of methods for measuring properties for which currently no methods exist, also ensuring the traceability, or at least the reproducibility, of existing methods;
• establish mechanisms to integrate new design rules to existing modelling techniques and apply these to industrially relevant materials and devices, delivering concrete results of industrial relevance
Programme and abstracts of the contributions are freely available at the Events page of the website of the project (www.setnanometro.eu), in the form of the digital copy of the Book of Abstract published with the ISBN code 978-88-97862-05-05.
The action in the field of training of young scientists was represented by the international summer school “NANOSCIENCE meets METROLOGY: synthesis, characterization, testing and applications of validated nanoparticles” held in Torino (Piedmont, Italy) on 4th-9th September 2016. The aim of the school was to offer to the young researchers international community the possibility to update and discuss on relevant topics of modern nanoscience and nanotechnology, augmented by their relationship with metrology. To this aim, two series of lectures were offered to participants. The first series was composed from wide-ranging lectures dealing with the results of the meeting of nanoscience with metrology in the fields of energy and environment, medicine and biology, upscale of applications from the lab to the industrial level. The second set was constituted by more specialized lectures on synthetic methods for validated nanoparticles (modeling the synthesis, controlling the morphology and guiding the aggregation), characterization methods for validated nanoparticles (electron microscopies, electronic and Vibrational spectroscopies, IR spectroscopy of surface probes), testing methods for validated nanoparticles (chemical and photochemical properties, biocompatibility and toxicology), applications of validated nanoparticles in catalysis, photocatalysis, medicine and biology. In total, the school was attended by 31 students, from Denmark, Finland, Ukraine, Spain, France, Germany, Italy, and from Brazil. Twenty-seven of them presented the results of their research projects in in the form of an extended abstracts, and among them nineteen accepted to act also as speaker in special sessions devoted to them, exploiting the opportunity to present their results to an international audience. In addition, these sessions were intended as a room for peer-to-peer discussion among students, with some inputs from more experienced researchers (the lecturers). Also, the ensemble of the abstracts of the lectures and of the students contributions are freely available at the website page indicated above (pdf of the Book of Abstract published with the ISBN code 978-88-87380-60-6)
Two inter-laboratory comparisons (ILCs) have been carried out within SETNanoMetro on most suited analytical methods for physico-chemical characterization of TiO2 nanoparticles (NPs) or their assemblies. From the list of fourteen deliverables generated by WP4 as standard operation procedures (SOPs) for the public domain on the measurement of physico-chemical parameters of titania (NPs or layers) the most suited procedure to be tested as an ILC was the measurement of TiO2 NP size and shape by electron microscopy. Two ILCs have been organized within JWG 2 "Measurement and characterization" of ISO/TC 229 "Nanotechnologies":
• Size and shape distribution of irregular primary titania crystallites by TEM,
And
• Size and shape distributions of bipyramidal titania nanoparticles by TEM.
In the first ILC SETNanoMetro participated with two labs (TEM and TSEM, both situated at BAM), and the second ILC has been coordinated by BAM and has engaged 16 participants from 8 countries. The first ILC was included as a case study into the ongoing ISO/TC 229 standard ISO/WD 21363 "Nanotechnologies — Protocol for particle size distribution by transmission electron microscopy".
The second ILC organized by BAM with the SETNanoMetro samples UT001 followed - according to the recommendations of ISO/TC229/JWG2 group of experts – similar guidelines as the first ILC on titania. The same measurement and reporting protocol has been distributed by BAM to all participants together with the uniformly prepared TEM grids. Furthermore, clear descriptions regarding specimen handling, descriptors to be measured or calculated, instrumental parameters, tracing the particle shape (see Figure 3.2.1), number of particles to be measured, resolution of the captured image, analysis software, reporting data format and time scale have been written in the protocols.
An xls-template with the descriptors and instrumental parameters to be reported has been also distributed to all participants. The received data are being evaluated by BAM in a unitary way, according to the same general procedural steps used in the ILC #1. The first results will be presented at the next ISO/TC229 meeting (June 2017). Similarly to ILC #1 the final results of this ILC on bipyramidal TiO2 NPs are planned to be published. Further, the data will be exploited to initiate the procedure of certification (of size and shape distribution) of the reference material at BAM.



Figure 3.2.1 Example of data analysis (image processing) of the bipyramidal TiO2 NPs (UT001) containing manual particle shape tracing (see yellow contours) and fulfilling the criterion roundness >0.7 (see rejected NPs in red).


As given above, SETNanoMetro was in close connection to the standardization organizations. SETNanoMetro was supporting the development of ISO/TC 21363 "Nanotechnologies – Protocol for particle size distribution by transmission electron microscopy" by providing nanoparticles for an inter-laboratory comparison and participating in the comparison. SETNanoMetro was also supporting the development of ISO 19749 "Nanotechnologies – Measurements of particle size and shape distributions by scanning electron microscopy" by participating in the inter-laboratory comparison. In addition, SETNanoMetro was preparing two New Work Item Proposals for CEN/TC 352 "Nanotechnologies". A Project Liaison with CEN/TC 352 has already been established in May 2014. The two proposals "Structural bulk characterization of the TiO2 nanoparticles by IR and Raman spectroscopy" and "Characterization of the surface of TiO2 nanoparticles by FT-IR in controlled atmosphere" were announced to CEN/TC 352 during its last meeting on 23 March 2017 in Verneuil-en-Halette and will be presented in detail during the next CEN/TC 352 meeting in October 2017. The complete working drafts and application forms were prepared and sent to the Italian Standardization Organization UNI who will act as the official proposer of the proposals. Another proposal "3-D characterization of layers built from the deposition of nanoparticles using focused ion beam technique and scanning electron microscopy" was announced to ISO/TC 202 "Microbeam analysis" and will be presented in detail during the next ISO/TC 202 meeting in September 2017 in London. The complete working draft and application form was prepared and will be discussed on 4 July 2017 during the next mirror committee meeting of the German Standardization Organization DIN who will act as the official proposer of the proposal.
Last but not least SETNanoMetro developed some products which are prepared for commercialization. A specific bipyramidal titania will be purchased as certified nanoparticles, a second kind of TiO2 nanoparticles showing photocatalytic activity will also be purchased as a certified material. While these materials will be purchased in 1 g containers, a sol-gel derived TiO2 with photocatalytic activity developed by SETNanoMetro will be purchased in larger amounts. Another product derived from SETNanoMetro will be a Standard reference substrate for measurement of the photocatalytic activity of TiO2 films. And finally a photocatalytic reactor developed within SETNanoMetro can already be ordered.

List of Websites:
www.setnanometro.eu

Relevant contact details:
ISTITUTO NAZIONALE DI RICERCA METROLOGICA INRIM ITALY a.rossi@inrim.it
DANSK FUNDAMENTAL METROLOGI DFM DENMARK kdi@dfm.dk
BUNDESANSTALT FUER MATERIALFORSCHUNG UND -PRUEFUNG BAM GERMANY dan.hodoroaba@bam.de
DIN DEUTSCHES INSTITUT FUER NORMUNG E.V. DIN GERMANY Michael.Schmitt@din.de
R&D CONSULTANTA SI SERVICII SRL RD ROMANIA doinastanciu09@yahoo.com
SOLARONIX SA SOLAR SWITZERLAND frederic.oswald@solaronix.com
AVANZARE INNOVACION TECNOLOGICA SL
AVANZARE SPAIN julio@avanzare.es
UAB MODERNIOS E-TECHNOLOGIJOS
MET LITHUANIA galdikas@protechnology.lt
METEC INNOVATION CONSULTING SRL
METEC ITALY marco.mangiantini@metecnetwork.com
UNIVERSITA DEGLI STUDI DI TORINO
UNITO ITALY gianmario.martra@unito.it
UNIVERSITE PIERRE ET MARIE CURIE - PARIS 6
UPMC FRANCE jupille@insp.jussieu.fr
THE UNIVERSITY COURT OF THE UNIVERSITY OF ABERDEEN
UNIABDN UNITED KINGDOM i.r.gibson@abdn.ac.uk
DANMARKS TEKNISKE UNIVERSITET
DTU DENMARK ram@mek.dtu.dk
TEKNOLOGISK INSTITUT
DTI DENMARK csje@dti.dk
INSTITUT FUR UMWELTMEDIZINISCHE FORSCHUNG AN DER HEINRICH-HEINE-UNIVERSITAT DUSSELDORF GMBH
Roel.Schins@uni-duesseldorf.de
FUNDACION TEKNIKER
IK4-TEKNIKER SPAIN miren.blanco@tekniker.es

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ISTITUTO NAZIONALE DI RICERCA METROLOGICA
Italy
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