M Kucki

P Rupper

C Sarrieu

M Melucci

E Treossi

A Schwarz

V León

A Kraegeloh

E Flahaut

E Vázquez

V Palermo

P Wick

Interaction of graphene-related materials with human intestinal cells: an in vitro approach †

Keywords: Material characterization details, light microscopy images of Caco-2 cells, cell-free control tests

Graphene-related materials (GRM) inherit unique combinations of physicochemical properties which offer a high potential for technological as well as biomedical applications. It is not clear which physicochemical properties are the most relevant factors influencing the behavior of GRM in complex biological environments. In this study we have focused on the interaction of GRM, especially graphene oxide (GO), and Caco-2 cells in vitro. We mimiked stomach transition by acid-treatment of two representative GRM followed by analysis of their physicochemical properties. No significant changes in the material properties or cell viability of exposed Caco-2 cells in respect to untreated GRM could be detected. Furthermore, we explored the interaction of four different GO and Caco-2 cells to identify relevant physicochemical properties for the establishment of a material property-biological response relationship. Despite close interaction with the cell surface and the formation of reactive oxygen species (ROS), no acute toxicity was found for any of the applied GO (concentration range 0-80 µg ml -1 ) after 24 h and 48 h exposure.

Graphene nanoplatelet aggregates led to low acute toxicity at high concentrations, indicating that aggregation, the number of layers or the C/O ratio have a more pronounced effect on the cell viability than the lateral size alone.

Introduction

Graphene as the most prominent member of the family of 2D materials has attracted enormous attention due to its unique combination of properties which offers a high technological potential for a broad variety of applications. 1,2 Graphene and several Graphene-related materials (GRM) are commercially available in various degrees of quality. Generally GRM can be produced by top-down approaches, [3][4][5][6] bottom up approaches, [7][8][9] reduction or functionalisation of other GRM 10,11 and transformation of other carbon nanomaterials. 12 Graphene oxide (GO), a subclass of GRM exhibiting oxygencontaining functional groups, has been identified as a promising candidate for biomedical applications such as tissue engineering, drug delivery, cancer treatment and biosensing. [13][14][15] The presence of oxygen functional groups facilitates the dispersibility in aqueous environment and enables the covalent functionalization with several different drugs, polymers and fluorescence labels by various chemical routes. 16,17 However, the public acceptance of every new technology is always coupled to its benefits and safety. The rapid development of graphene-based technology and expected large distribution has raised a lively safety discussion. There is great interest and attention on graphene not only in the academic community, but also in social media. 18 Despite a steadily increasing number of publications regarding the biological effects of GRM, especially GO, there is little consensus on the effect on human and environmental health. 19 Both the inconsistency of experimental results and a generic use of the term graphene for several different GRM have led to confusion. 20 A recently proposed classification framework aims to group different GRM by three main parameters (the average lateral dimension, the number of layers and the amount of oxygen as an indicator for oxidation) and to understand the relationship between GRM-structure and biological effects. 21 Similar to other nanomaterials the physicochemical properties and quality of GRM can highly influence the type and strength of the biological response. Despite several in vivo studies, as summarized and analysed recently by Bussy et al. 22 a systematic investigation to correlate GRM properties and their biological effects with respect to the exposure route (inhalation, oral, dermal, intravenous) as well as their fate within the exposed organism is missing. From the occupational health perspective there are several exposure routes for workers towards GRM: inhalation, dermal contact and/or ingestion. Among these, inhalation is regarded as the most relevant way of unintended entrance of GRM into the human body. Therefore it is not surprising that a majority of published studies, except those addressing intravenous application for biomedical research, has addressed the possible impact of GRM on cells of the respiratory tract in vitro, as well as in vivo. 23,24 Nevertheless, information on the fate of these materials in the lungs and the possible induced biological effects is very limited. Next to the lungs, scant attention has been given to other biological barriers which are likely exposed to GRM. The gastro-intestinal (GI) tract can not only be exposed by direct ingestion of GRM, but also by indirect ingestion of formerly inhaled GRM. Trapped in the mucus of the respiratory system foreign bodies are transported upwards by ciliary movement, a process called the "mucociliary elevator", and can be finally either coughed out or swallowed down. In addition, potential application of GRM in food packaging and contamination of drinking-or surface water, are likely scenarios how GRM could gain access to the human GI-tract.

Nanomaterials that enter the alimentary tract are exposed to a series of different environments before they reach the intestine. Several factors such as pH, ionic strength, digestive enzymes, surface active compounds, physical forces, type and amount of food intake as well as microbes have the potential to induce changes to the physicochemical properties of the nanomaterial. Due to this complexity it is not clear which factors have the strongest influence on the nanoparticles. However, it is very likely that the acidic conditions within the stomach are among the most relevant factors. As every change in the physicochemical properties of the GRM has the potential to modulate the induced biological response, it is important to assess any possible change that can occur before the material reaches the cells or tissue of interest. Nevertheless, there is lack of information regarding GRM behaviour under physiologically relevant acidic conditions. Furthermore there has been no detailed investigation of the possible impact of GRM on the gastro-intestinal tract. Only very few studies with a small selection of GRM have addressed the interaction of GRM with intestinal cells in vitro. 25 However, the mucosal barrier of the intestinal tract is one of the most important biological barriers within the human and animal body. It serves multiple tasks, such as nutrient uptake, defence against pathogenic bacteria and maintenance of tolerance against commensal bacteria. Loss of intestinal homeostasis can result in uncontrolled entrance of pathogens and food antigens as well as in reduced nutrient supply, which in turn can lead to severe health complications and disturbance of the entire organism.

Therefore the aim of this study is to acquire a systematic understanding of the biological effects of GRM on cells of the human intestinal tract. For this purpose we selected and characterized graphene oxides (GO) of different sources and quality and explored their interaction with non-confluent Caco-2 cells. The Caco-2 cell line is derived from human colon adenocarcinoma and one of the gold-standards for in vitro studies regarding the intestinal tract with wide-spread application in pharmaceutical research. The selection of GO presented here offers a direct comparison of commercial and research grade materials, research-grade GO obtained by the same production method but with different lateral dimensions, as well as of commercial GO obtained from two different starting materials. To mimic stomach transition, exemplarily one selected GO, as well as graphene nanoplatelets aggregates (GNP) as benchmark GRM, were incubated under physiologically relevant acidic conditions in vitro followed by material characterization, especially focusing on determination of the surface functionalization (C/O ratio), introduction of defects and changes in material morphology. As there is no information available on the exposure dose of GO in the intestinal tract we selected a rather broad dose range up to a concentration of 80 µg GO per ml. It has to be noted that 80 µg GO per ml is already assumed to reflect an over-dose situation and that the expected exposure dose range is probably significantly lower. The obtained results will help to identify relevant physicochemical properties for the establishment of a material property-biological response relationship.

Results and discussion

Physicochemical properties of GO and GNP

To understand the correlation of the physicochemical properties of GO and the biological effects, we selected a set of four different GO samples produced by modified Hummers method. Two GO samples (GO1, GO4) were obtained from commercial sources in addition to two graphene oxides (GO2, GO3) produced in research laboratory. GO1, GO2 and GO3 were derived from graphite, whereas graphitic nanofibers (GANF©) were the starting material for GO4. As exfoliation is connected to the application of ultra-sonication which leads to random fragmentation of the GO sheets, 26 all GO samples exhibited poly-dispersed lateral sheet size. The commercial graphene oxide GO1 showed the largest lateral size distribution with a range of several tens of micro-meters. The commercial graphene nanoplatelets (GNP), applied as benchmark material for the digestion procedure, consisted of small and large flakes which were already fused to larger aggregates when received as powder from the manufacturer (see ESI †). Dispersion of hydrophobic GNP in water was improved by the application of sodium cholate, salt of a natural primary bile acid which is present in the human intestine and plays an important role in the digestive process, especially in the absorption of dietary fats. Bile acids are frequently used to disperse graphene in aqueous dispersions. 27,28 Short-time bath-sonication was applied to further improve dispersion. Nevertheless, short-time bath-sonication (<10 min) did not lead to dissociation of the graphene layers. Further sonication (long duration or high energy) was not applied in order to prevent significant changes in the material properties.

All graphene oxides did not require additional treatment and were either already obtained as aqueous dispersions or dispersed in endotoxin-free ultra-pure water. An overview of the physicochemical properties of the applied GRM is given in Table 1.

Physicochemical properties after in vitro digestion (acid-treatment)

Based on the fact that all nanomaterials entering the GI-tract have to pass through the stomach prior to any contact with intestinal cells, we explored the impact of physiological relevant acidic conditions as present in the stomach on the physicochemical properties of graphene-related materials in vitro. For this purpose, we selected two different commercially available GRM which highly differed in their physicochemical properties. GO1 was selected as representative for graphene oxides, whereas graphene nanoplatelet (GNP) aggregates were used to cover GRM with significantly different properties, especially carbon-to-oxygen ratio (C/O) and layer number. Selected GRM were incubated in 0.1 M hydrochloric acid (HCl) for 2 hours at 37 °C under mild agitation. As a control, aliquots of the same GRM were incubated in water instead of hydrochloric acid at similar conditions to exclude any changes in the material properties independent of the acidic conditions. After treatment, GRM were harvested by centrifugation, re-dispersed and neutralized in phosphate buffered saline (PBS, pH 6.8), washed and transferred to ultra-pure-water prior to material characterization. Physicochemical characterization of the acid-treated GRM and the respective control (water-treated) GRM was performed by SEM analysis, zeta-potential measurement, X-ray photoelectron spectroscopy (XPS) analysis and Raman spectroscopy. Results are summarized in Table 2. SEM analysis did not indicate major changes in the GRM morphology, neither for GO1, nor for GNP as shown in Fig. 1. Control samples and acid-treated samples showed no obvious differences. The zeta-(ζ)-potential of GO1 (as received & acid-treated) in ultra-pure water with potassium chloride (KCl) as background electrolyte showed identical values of -39 mV, giving no hints towards altered surface charge. The zeta-(ζ)-potential of GNP was between -50 and -62 mV, and therefore more negative than that of GO1. The presence of sodium cholate, which was used as surfactant a Measured in ultra-pure water with potassium chloride (KCl) as background electrolyte. b According to information given by the manufacturer; further details are given in the ESI. c Further characterization data for GO4 can be found in Kurapati et al. 30 to achieve dispersion of GNP in aqueous solution, had no to minor influence on the zeta-(ζ)-potential of the GNP (see Table S4b in ESI †) and is therefore not responsible for the highly negative values. After acid-treatment of GNP, the absolute zeta-(ζ)-potential was reduced to a slightly smaller value.

The elemental composition of the GRM, in particular the carbon to oxygen ratio (C/O) was characterized by XPS. For each of the two materials, GO1 and GNP, the surface of the material was characterized in the as received form, the control sample, as well as after acid-treatment. In addition, high resolution spectra for the elements carbon and oxygen were recorded in order to detect possible chemical changes. For the GO1 sample as received, the survey scan revealed the elements carbon and oxygen in significant concentrations, whereas small amounts (≤1 atomic%) of nitrogen, silicon and sulfur were also detected, probably resulting from the GO production and/or storage. For the GNP sample as received, only the elements carbon and oxygen were present. Table 2 summarizes the C/O ratios calculated from the oxygen and carbon concentrations determined from the XPS survey spectra. The C/O ratio for the GO1 sample as received resulted to 1.7, in agreement with literature values for commercial graphene oxide. [31][32][33] A small increase of the C/O ratio was found after GO1 was acidtreated as well as for the control sample. For further information see Fig. S5 in the ESI. † As expected, the C/O ratio was significantly higher (above 20) for the GNP samples. Only small concentrations of oxygen were present in the GNP. No significant differences were found between the GNP sample as received, the acid-treated GNP and the control GNP sample. The values are all well within the estimated uncertainty of ±2.5. Further information to the XPS measurements is given in the ESI. † Raman spectroscopy of GO1 and acid-treated GO1 did not give evidence for significant differences after treatment of GO1 under physiological relevant acidic conditions (cf. Fig. S6-c and S6-d in ESI †). However in the case of GNP, a decrease in the D/G intensity ratio, on average 25%, was evidenced after acidtreatment (cf. Fig. S6 in ESI †). Such a decrease of the ratio for a constant G peak width usually corresponds to a lower level of defects in the samples. 34 This could result of a selective degradation of the sites containing the highest density of defects, sites which should also have the highest chemical reactivity. But, according to the statistical tests performed on the measurements (cf. Experimental section), the possibility that this observation comes from the heterogeneity of the starting GNP material (evidenced by measuring the Raman signal at different places) cannot be excluded.

The probability that the GRM was not modified by the treatment is in fact about 8%, which is not negligible. Indeed, as presented in Table 2, the standard deviation on the measured D/G intensity ratio is very high, about 30%.

In summary, we can conclude that the acid-treatment of the two selected GRM, GO1 and GNP, did not result in major changes in the material properties. At least for the GO this is not surprising, as production of GO by classical methods such as Brodie, Staudenmaier and Hummers method involves sulfuric acid (H 2 SO 4 ) and nitric acid (HNO 3 ). 3 Nevertheless, our results demonstrate that pristine GO and GNP are relatively inert materials which appear to be not highly affected by the acidic conditions ( pH ≤ 2, fasted state, 2 h, 37 °C) during stomach transition.

Interaction of acid-treated GRM with the Caco-2 cell surface

In a next step we assessed the interaction of acid-treated and control GRM derived from the in vitro digestion procedure with the Caco-2 cell surface. As the physicochemical characterization of acid-treated and control GRM did not reveal major changes in material properties, we did not expect significant differences in the interaction with the cell surface after exposure to GRM. As shown in Fig. 2 the interaction of the acid-treated GRM and the cell surface showed no distinctive features in comparison to the respective control GRM. For GO1, in both samples (acid-treated and control) GO sheets were primarily found folded and wrinkled, reminiscent of crumbled paper. There were no visible hints towards a possible damage of the cell surface by the GO sheets. Cell morphology of Caco-2 cells after exposure to GO1 samples (acid-treated and control samples) did not show significant alterations in comparison of untreated Caco-2 cells. Similar observations were made for all GNP samples. GNP aggregates of different shapes but in the usual size range of 1-10 µm were found associated with the Caco-2 cell surface. For both GO1 and GNP a few observations were made which might be hints towards attempts of cellular uptake as shown in Fig. S7 and S8 in ESI. † If these events are real uptake events or rather overgrowth of the GRM has to be clarified by detailed analysis in future investigations. sulfophenyl)-2H-tetrazolium, inner salt) assay. Cytotoxic potential of a sample was reached when the metabolic activity decreased to less than 70% of the control cells. For GO1, exposure to the acid-treated sample or the respective control sample for 24 h resulted in a concentration dependent slight decrease in mean viability of Caco-2 cells as shown in Fig. 3. Nevertheless, even at highest GO concentration applied (80 µg GO per ml) the cell viability remained above 80% in relation to the unexposed control cells. For GNP, the decrease in cell viability was more pronounced. Mean cell viability decreased to around 60% for the highest concentration applied, indicating low acute toxicity. Comparison of the viability of Caco-2 cells exposed to acid-treated GRM and the respective water-treated control sample did not reveal any differences in metabolic activity. Light microscopy images also showed the absence of visible differences in cell morphology between control cells and cells exposed to water-treated or acid-treated GRM (see Fig. S9 and S10 in ESI †). Therefore, as confirmed by material characterization, incubation of GRM under acidic conditions reflecting the pH value in the stomach did not result in significant changes of the material properties or the cellular activity of exposed intestinal cells in vitro. Nevertheless, prior to this study it has not been demonstrated yet that the acidic conditions in the stomach do not affect graphene oxide properties. The results indicate that acid pre-treatment of pristine GO (without further functionalization) to mimic stomach transition is not mandatory, at least when the production method already involved treatment with acids.

Cell viability of Caco

Cell surface interaction of different GO samples

Detailed characterization and knowledge of the material properties of GRM enables a comprehensive correlation of the physicochemical properties and possible biological effects. To acquire a systematic understanding of the interaction of a certain GRM type with human intestinal cells, non-confluent Caco-2 cells were exposed to the above mentioned selection of different graphene oxides (GO1-4). In addition, untreated GNP was implemented for comparison. Based on the results obtained previously, the following investigations were performed without acid-treatment, as the involved GO samples were produced by modified Hummers method. Non-confluent Caco-2 cells were exposed directly to the untreated GO samples.

SEM analysis of Caco-2 cells after exposure to different GO samples showed a close interaction of the GO sheets and the cell membrane. Well exfoliated GO sheets showed the tendency to align in parallel to the cell surface, especially in areas where the cell surface was relatively smooth with a low amount of cell protrusions. In several cases GO sheets could only hardly be distinguished from the cellular membrane as shown in Fig. 4 for GO2. Similar observations were made by Russier et al. for GO with human and murine macrophages and described as so-called "mask-effect". 35 In addition to the parallel alignment of the GO sheets, several sheets comprising numerous folds and wrinkles were found on the cell surface, especially near the edges of Caco-2 islets. In this region Caco-2 cells showed numerous wave-like cell protrusions. All GO samples showed well exfoliated sheets. Nevertheless, GO4 fre-Fig. 2 Representative SEM images of Caco-2 cells after exposure to 20 µg ml -1 acid-treated GRM or the respective control-GRM for 24 h, as well as of untreated control cells. quently exhibited some puff paste structured sheet assemblies. As described before, larger GNP aggregates were frequently found attached to the cell surface, again predominantly near the edges of Caco-2 islets. Exposure of Caco-2 cells to the four different GO, as well as the GNP, in a concentration range from 10-40 µg ml -1 GRM for 24 h did not result in any visible morphological alterations of the cells or hints towards altered cell turnover. Furthermore cells frequently showed affinity to GO deposited on the glass substrate. As mentioned before, for GO1 and GNP few single events were observed which might indicate cellular uptake (Fig. S7 and S8 in ESI †).

Induction of reactive oxygen species by GO and GNP

Studies with other carbon-based materials have shown that these materials can induce the formation of reactive oxygen species (ROS). 36,37 ROS such as hydrogen peroxide (H 2 O 2 ) are highly reactive molecules which are involved in intra-and intercellular signalling. Up to a certain limit ROS-formation can be outbalanced by the cellular antioxidant defence. Nevertheless, imbalance due to elevated ROS levels can lead to severe damage of lipids, proteins and nucleic acids, which can in turn result in inflammatory reactions, genotoxicity or cell death. Therefore we assessed the possible GRM-induced formation of ROS by 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) assay, a common assay previously applied with multi-walled carbon nanotubes (MWCNTs) 36 and graphene shells. 37 Fig. 5 shows the results obtained for Caco-2 cells exposed to different GO and GNP for 2 hours. 3-Morpholinosydnonimine hydrochloride (SIN-1) served as qualitative positive control applied at a concentration of 150 µM in HBSS. In addition, sodium cholate in water was applied as vehicle control for GNP dispersions. The results clearly show that all applied GO, as well as the GNP, induced the formation of ROS in Caco-2 cells. Nevertheless, despite slight differences in the intensity of ROS formation found for individual GRM quantitative comparison is not possible due to concentrationdependent quenching of the DCF fluorescence in the presence of GO1, GO2, GO4 and GNP (see Fig. S14 and S15 in ESI †).

Similar decrease in fluorescence intensity was already reported for high concentrations of MWCNTs 36 and graphene shells. 37 In contrast, GO2 exhibited fluorescence enhancement activity at low and medium concentrations, as well as fluorescence quenching at high concentrations (see Fig. S15 ESI †). Interference by intrinsic fluorescence of the GO and GNP could be excluded (see Fig. S12 and S13 ESI †). Further control experiments showed that all GRM were able to transform the de-acetylated H 2 DCF molecule into a fluorescent product, indicating the production of ROS by the GRM in the absence of cells (see Fig. S16 ESI †). These results are again in line with findings previously reported for MWCNTs 36 and graphene shells. 37 Therefore, at least part of the ROS detected in the cellbased assay results from the activity of the GO or GNP alone.

Cell viability of Caco-2 cells after exposure to a whole selection of GO representing research grade and commercial GO

We further investigated whether the induced ROS formation can reach sufficient levels to have an impact on the cell viability of proliferating Caco-2 cells. Exposure to four different graphene oxides (GO1-4) for 24 h and 48 h resulted in a slight dose dependent decrease of the metabolic activity of Caco-2 cells, as determined by the application of the MTS-assay. Even at the highest GO concentration (80 µg ml -1 ) applied the mean cell viability was above 80% for 24 h as well as 48 h exposure time for all GO samples, again indicating no acute toxicity of the GO for Caco-2 cells. To assess possible loss of cells after exposure to GO due to an increase in cell death, the total number of adherent Caco-2 cells was measured by modified lactate dehydrogenase (LDH) assay. After exposure to GO cells were washed with phosphate buffered saline and lysed for 60 minutes by addition of 9% triton-X 100 in PBS. The cell number was determined by measurement of the total LDH release after complete cell lysis. Release of LDH was linear to the number of lysed cells. As shown in Fig. 6 no significant decrease in cell number indicating loss of cells after exposure to GO was found for any GO sample and concentration applied.

In summary the results indicate that acid pre-treatment of pristine GRM seems to be not mandatory. Nevertheless it is definitely advisory to perform equivalent acid pre-treatment in case the selected GO is further functionalized or loaded with drugs for drug delivery or cancer treatment. Detailed analysis of the stability of functionalization under gastric conditions is inevitable. It should be noted that the digestion procedure applied here can be seen as a starting point for further sys-Fig. 4 Representative SEM images of Caco-2 cells after exposure to different GRM for 24 h with an applied concentration range of 10-40 µg mL -1 GRM; images shown for 10 µg ml -1 GO1 and GO4; 20 µg ml -1 GO2 and GNP, as well as 40 µg ml -1 GO3 respectively. GO3 sheet assemblies are visible next to the cells on the glass substrate in the upper right corner. Black arrows point towards the respective GRM.

tematic investigation of relevant factors influencing the material properties. We considered the acidic conditions as an isolated factor. But in vivo gastric fluids consist of a large diversity of further factors, such as salts, proteins and enzymes, which alone or in combination can have an influence on the GRM properties. From the multitude of available simulated body fluids of the alimentary tract (e.g. saliva, gastric and intestinal fluids) appropriate protocols have to be selected resembling the in vivo situation under consideration (e.g. fasted state vs. fed-state).

Despite close interaction with the cell membrane and induction of ROS-formation, no GO, either with or without acid pre-treatment, led to acute toxicity towards non-confluent Caco-2 cells. These results are in line with those obtained for Caco-2 cells exposed to two graphene oxides of different lateral dimension 25 or single walled carbon nanotubes (SWCNTs), 38 which can be regarded as rolled up graphene sheets. Two graphene oxides with different lateral size distributions also showed no acute toxicity to the lung epithelial cell line A549 when applied in concentrations up to 100 µg ml -1 GO. 39 The physicochemical characterization showed that GO obtained from different sources but produced by the same top-down method (modified Hummers method) did not show large differences in the C/O ratio and layer number, but in the lateral size distribution. Expected differences in cell response of Caco-2 cells in dependence of the lateral size distribution of the GO, could not be observed. In addition, the starting material (graphite or GANF®) also had no influence on the acute toxicity towards Caco-2 cells. This is in a way surprising since other studies with different cell types reported size Fig. 5 H 2 DCF assay: ROS formation after exposure of Caco-2 cells to 5-40 µg ml -1 of the respective GO or GNP. Sin-1 served as positive control. Aqueous solution with 0.1 mg ml -1 sodium cholate served as vehicle control for the GNP samples.

dependent effects of GO. 39 But it should be noted that the size distribution of the applied GO is extremely large compared to other nanomaterials such as monodisperse silica nano-particles. It cannot be excluded that certain sub-populations within the huge size range have a stronger effect than others. Pre-requisite for further analysis of the influence of the lateral dimension would be the availability of GO with a very narrow size distribution, which is still a challenge to achieve. Large differences in the physicochemical properties, such as layer number and/or the C/O ratio, as it is the case between GO and GNP, led to visible changes in the level of acute toxicity towards Caco-2 cells but only at concentrations higher than 40 µg ml -1 . The influence of the layer number/thickness and the C/O ratio has to be further determined in future experiments.

Despite the lack of acute toxicity of GO and low acute toxicity of GNP at high concentrations, our results do not rule out that specific biological effects can be observed at sub-toxic GO levels. Further experiments regarding possible cellular uptake, loss of cellular functions and DNA damage have to be performed in future. Next to enterocytes the intestinal epithelium comprises several other types of cells (e.g. mucus-secreting goblet cells, antigen-sampling M-cells, entero-endocrine cells, Paneth cells, intestinal stem cells as well as intra-epithelial lymphocytes) with high variation in function and morphology which might show different interactions with GO. Even if these cell types occur in significant lower number compared to enterocytes which represent the majority of the cells of the intestinal barrier, further studies with these cell types (undifferentiated and differentiated, in vitro and in vivo) are needed to exclude any possible adverse effect of GO on the intestinal barrier. In addition, possible effects of chronic exposure of the intestinal barrier to GRM should be addressed.

Conclusions

The results of this study show that graphene oxides as well as graphene nanoplatelet aggregates are relatively inert towards physiological relevant acidic conditions and are likely to undergo stomach passage without significant changes in their physicochemical properties. Therefore acid pre-treatment of pristine GO is not required to investigate potential acute toxicity towards cells of the intestinal tract in vitro. Nevertheless, this should not necessarily be valid for GO functionalized with biomolecules or drugs for biomedical application. In this case careful assessment of the stability of the functionalization under relevant acidic conditions is recommended. The lack of acute toxicity of four different GO samples to non-confluent Caco-2 cells is very promising in respect to a possible biomedical application as well as important regarding unintended exposure. In contrast exposure of Caco-2 cells to high concentrations of GNP showed low acute toxicity, which indicate that physicochemical parameters such as layer number and C/O ratio seem to be critical for the level of biological response of Caco-2 cells towards GRM.

Experimental section

Materials, sources & production

Graphene oxide 1 (GO1; SLGO) and Graphene nanoplatelets (GNP; HDPlas™ GNPs Grade 4) were obtained from Cheap Tubes (Battleboro, 112 Mercury Drive, VT05301, USA; http:// www.cheaptubes.com). Research grade graphene oxide samples GO2 and GO3 were prepared from graphite flakes by a modified Hummers method as described by Treossi et al. 40 The lateral size of the GO flakes can be tuned by changing the sonication time of the starting solutions, in particular in this work we used two graphene oxide solution sonicated 2 h (GO2) or 20 h (GO3) respectively. GO4 was obtained from Grupo Antolin-Irausa S.A. (Ctra. Madrid-Irún, Km. 244,8 -E09007 -Burgos, Spain).

Digestion of GRM

GO1 and GNP were incubated in 0.1 M hydrochloric acid (HCl) for 2 h in 37 °C under slight agitation. In parallel, GO1 and GNP were incubated in ultra-pure water under similar conditions as controls for the digestion procedure. After incubation, all samples were precipitated by centrifugation (10 min at 5000 rpm). The clear supernatant was exchanged by phosphate buffered saline (PBS) with pH 6.8 which resembles the pH values in the small intestine and served for neutralization. GRM were re-dispersed and washed twice with ultra-pure water by centrifugation prior to further use.

Scanning electron microscopy (SEM) analysis of GRM

For SEM analysis of the GRM morphology GRM were applied as received from the manufacturer. GRM powders or dispersions in ultra-pure water were applied on clean glass cover glasses (10 mm Ø, # 1, Menzel GmbH, Braunschweig, Germany) fixed on the SEM sample holder by conductive carbon strips to prevent direct contact of the carbon film and the carbon nanomaterial. Dispersions were dried at room temperature. All samples were sputter coated with 5 nm goldpalladium (Au/Pd = 80/20). Analysis was performed with SEM (Hitachi S-4800).

Zeta-(ζ)-potential measurements

The zeta-(ζ)-potential was measured with a Zeta Sizer Nano ZSP (Malvern Instruments, Worcestershire, UK) using disposable capillary cells (DTS1070, Malvern Instruments, Worcestershire, UK). For measurements, GRM dispersions were diluted to a concentration of 100 µg ml -1 in ultra-pure water, containing potassium chloride (KCl) as background electrolyte (endconcentration: 1 mM), 0.1 mg ml -1 sodium cholate containing 1 mM KCl (for GNP only) or cell-culture medium (MEM + 10% FBS). Particle dispersions were diluted 1 : 10 in medium. The zeta potential of GO1 was also measured after incubation in cell-culture medium and separation of the medium, according to the following steps: 30 min incubation in cell-culture medium, centrifugation (centrifuge 5418 Eppendorf, Hamburg, DE) of the samples at 16 000g for 1 h, aspiration of the supernatant and re-dispersion (1 min vortex) in ultra-pure water. Each sample was measured three times.

X-ray photoelectron spectroscopy (XPS)

XPS measurements were carried out with a Scanning XPS Microprobe (PHI VersaProbe II spectrometer, Physical Elec-tronics) using monochromatic Al Kα radiation (1486.6 eV). The operating pressure of the XPS analysis chamber was below 5 × 10 -7 Pa under the measurement conditions presented here. The spectra were collected at photoemission take off angles of 45°(with respect to the surface). Survey scan spectra (0-1100 eV) were acquired with an energy step width of 0.8 eV, acquisition time of 160 ms per data point and analyser pass energies of 187.85 eV. Higher resolution narrow spectra for the elements carbon C1s (278 eV to 298 eV energy range) and oxygen O1s (523 eV to 543 eV energy range) were acquired with energy step widths of 0.125 eV, acquisition times of 3.2 s (carbon) and 2.56 s (oxygen) and analyser pass energies of 29.35 eV. Under these experimental conditions ( pass energies), the energy resolution (FWHM, full width at half maximum height) measured on silver Ag 3d5/2 photoemission line is 2.4 eV and 0.7 eV, respectively. The total acquisition times were approximately 4 min for survey scans and 16 min for the two high-energy resolution elemental scans together, which achieve an adequate signal-to-noise ratio without observable X-ray radiation damage to the samples. Each sample was analysed at a randomly chosen spot using a micro-focused, scanned X-ray beam with a diameter of 200 µm (operated at a power of 50 W at 15 kV). The 180°spherical capacitor energy analyser was operated in the fixed analyser transmission mode (FAT). The GRM samples (acid treated and control) have been attached to clean and sterile cover glasses, whereas the as received GRM were pressed onto an indium foil. The cover glasses as well as indium foil were then placed on a 2.5 cm diameter sample holder. In order to compensate possible sample charging, dual beam charge neutralization with a flux of low energetic electrons (1.4 eV) combined with very low energy positive Ar-ions (10 eV) was used. The binding energy is referenced to the C-C, C-H hydrocarbon signal C 1s at 285.0 eV. Curve fitting (least-squares fit routines) was carried out with CasaXPS software version 2.3.16. Thereby, a mixed Gaussian-Lorentzian product function (constant ratio of 70% Gaussian and 30% Lorentzian) was used to de-convolute the XP spectra. During the fitting process, the positions on the binding energy scale were constraint to corresponding literature values. 33 Atomic concentrations were calculated from XPS peak areas after subtracting a Shirley type background. Thereby, tabulated PHI sensitivity factors 41 corrected for our system's transmission function and spectrometer geometry (asymmetry function) have been used for quantification.

Raman spectroscopy

Raman spectroscopy was performed on a confocal Raman microscope Labram HR800 Horiba Yvon Jobin, at 2 different laser wavelengths (red: 633 nm; green: 532 nm; hole: 100 µm, 100× magnification, 5 accumulations of 5 s each, between 1000 and 3000 cm -1 ). The use of two different wavelengths is important because the shape and intensity ratios depend on the laser energy. Samples were prepared by drop casting on microscope glass slides. For each kind of treated GRM, 3 slides were studied, with about 7 measurements on agglomerates of different sizes. Significance of observed differences was then estimated with Student's t-tests supposing a normal distribution of measurements around the value really characterising each sample. Such a test is preconized to compare the means from two small and independent samples. Previously, the equality of the variances of each assay of measurements had been verified with F-test.

Cell culture

Caco-2 cells derived from human colorectal adenocarcinoma were obtained from ATCC (Product ATCC®HTB-37™, ATCC, Manassas, VA, USA). Caco-2 cells from cryogenically-preserved stocks were sub-cultured at least twice before the experiments. Cells were grown in Minimum essential medium (MEM, Ref. M2279, Sigma-Aldrich Chemie GmBH, Steinheim, Germany) supplemented with 10% non-heat inactivated fetal calf serum (FCS), 1% non-essential amino acids (NEAA), 1% L-glutamine, 1% penicillin-streptomycin-neomycin (PSN) and incubated in a humidified atmosphere with 5% CO 2 at 37 °C. Routinely cells were sub-cultured twice a week at 70-80% confluence by 0.5% trypsin-EDTA. To avoid pheno-and genotypic changes during cultivation of the cells, cells used for experiments were limited to 30 passages after thawing.

Scanning electron microscopy (SEM) analysis of Caco-2 cells

Caco-2 cells were seeded on clean and sterile cover glasses (15 mm Ø, # 1, Menzel GmbH, Braunschweig, Germany) in 12-well cell culture plates at a cell density of 1 × 10 5 cell per well, allowing attachment of the cells for 24 h prior to the experiments. Cells were exposed to 10-40 µg ml -1 GRM in supplemented cell culture medium for 24 h in parallel to control cells without GRM exposure. After exposure cells were washed twice with pre-warmed phosphate buffered saline (PBS). Cells were prepared by chemical fixation adding modified Karnovsky fixation solution (4 g paraformaldehyde (CAS 30525-89-4), 50 ml aqua bidest, 5 ml glutaraldehyde 50% (CAS 111-30-8, toxic if swallowed or if inhaled, causes severe skin burns and eye damage!), 45 ml phosphate buffered saline without glucose and pH 7.4) and incubation at room temperature for 1 hour. Samples were washed twice with PBS. Dehydration of the samples was performed by ascending ethanol series (50-100% ethanol) followed by hexamethyldisilizane (HMDS, CAS 999-97-3, harmful if swallowed or if inhaled, toxic in contact with skin!) treatment. Samples were dried overnight in a fume hood and stored in a desiccator until sputter coating with 10 nm gold-palladium (Au/Pd = 80/20).

Detection of reactive oxygen species (DCF assay)

The formation of reactive oxygen species (ROS) was determined by 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCF-DA, CAS 4091-99-0) assay as described in ref. 37 with small changes. In brief, Caco-2 cells were seeded with 1 × 10 4 cells in 100 µl of complete cell culture medium per well in a 96 well plate and grown overnight under standard cell culture conditions. On the following day, cells were loaded with 50 µM H 2 DCF-DA in Hank's buffered salt solution (HBSS) in a humidified atmosphere at 37 °C for 60 minutes. Cells were washed twice with pre-warmed HBSS. In each case Caco-2 cells were exposed to 100 µl of the GRM dispersions with concentration ranges from 0-40 µg ml -1 . 3-Morpholinosydnonimine hydrochloride (SIN-1, CAS 16142-27-1) served as positive control applied at a concentration of 150 µM in HBSS. After 2 hours incubation time fluorescence intensities were measured at 485 nm excitation and 528 nm emission wavelengths by FLx800 fluorescence microplate reader (BioTek Instruments). Control experiments in the absence of cells were performed to determine possible ROS generation by the GRM, intrinsic GRM absorbance and fluorescence, as well as fluorescence quenching (see ESI †).

Cell viability

Cell viability of Caco-2 cells after exposure to different GO was assessed by measurement of the metabolic activity via MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-cyrboxymethoxy-phenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt) assay obtained as Cell-Titer96 Aqueous One Solution from Promega Corporation (2800 Woods Hollow Road, Madison, WI 53711-5399, USA). The assay was performed according to the manufacturer's protocol with slight changes to consider intrinsic GRM absorbance. Cells were seeded in 96-well plates (tissue culture test plate 96F, Ref. 92096, TPP® Techno Plastic Product AG, Switzerland) at a cell density of 1 × 10 4 cells in 100 µl of complete cell culture medium per well. Cells were grown overnight at standard cell culture conditions. The day after, cells were exposed to respective GRM diluted in complete cell culture medium at a concentration range of 0-80 µg ml -1 GRM for 24 and 48 hours. Cadmium sulphate (CdSO 4 ) was applied as positive control in a concentration range of 0-1000 µM. After exposure the medium was replaced by 120 µl MTS working solution (20 µl MTS reagent plus 100 µl phenol-red free RPMI-1640). Directly after addition of the working solution absorbance was measured at 490 nm to obtain background absorbance values (t0 value) considering intrinsic absorbance of residual GRM. Cells were incubated under standard cell culture conditions for 60 min. Final absorbance (t1 value) was measured at 490 nm wavelengths. Absorbance values (t1 values) were corrected for intrinsic GRM absorbance by subtraction of the t0 values. Data is presented as mean and standard deviation of at least three independent experiments.

Analysis of total number of adherent cells (modified lactate dehydrogenase [LDH] assay)

Total number of adherent cells was assessed by the CytoTox96® Non-Radioactive Cytotoxicity Assay (Ref. G1780, Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399, USA) according to the manufacturer with slight modification to consider intrinsic GRM absorbance. For this purpose cells were seeded in 96-well plates at a cell density of 1 × 10 4 cells in 100 µl of complete cell culture medium per well and grown overnight at standard cell culture conditions. Cells were exposed to 0-80 µg ml -1 GRM diluted in complete cell culture medium for 24 and 48 hours. Cadmium sulphate (CdSO 4 , CAS 10124-36-4, toxic if swallowed, fatal if inhaled) served as positive control in a concentration range of 0-1000 µM. After exposure cells were washed twice with prewarmed phosphate buffered saline. All cells were lysed by the addition of lysis solution (9% Triton-X 100, CAS 9002-93-1) and incubation at 37 °C for 45 min. After complete cell lysis absorbance was measured at 490 nm wavelength to obtain background absorbance values (t0 value) considering intrinsic absorbance of residual GRM. Assay reagent was added to each well. The 96-well plates were incubated at room temperature for 30 minutes. After 30 min incubation time stop solution was added to stop reaction. Final absorbance (t1 value) was measured at 490 nm wavelength. Results were corrected for intrinsic GRM absorbance (t1 -t0). Data is presented as mean and standard deviation of three independent experiments.

  -2 cells after exposure to acid-treated GRM The possible impact of GRM exposure on Caco-2 cells was assessed by measurement of the metabolic activity via MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-

Fig. 1

 1 Fig. 1 Representative SEM images of the GRM morphology with or without acid-treatment.

Fig. 3

 3 Fig.3Cell viability of Caco-2 cells after exposure to acid-treated and control GRM samples for 24 h; left: GO1, right: GNP; all materials were tested in three independent experiments with three replicates for each concentration. Results are shown as mean and standard deviation.

Fig. 6

 6 Fig. 6 Cell viability and total cell number of adherent Caco-2 cells after exposure to different GO samples for 24 h and 48 h. Cadmium sulphate (CdSO 4 ) served as chemical control for the assay performance. Cell viability and total cell number are both presented as percentage of control cells not exposed to GO. Results are shown as mean and standard deviation of 3-6 independent experiments.

Table 2

 2 Physicochemical properties of GRM after acid-treatment

	Sample		C/O a	Zeta-(ζ)-potential b (mV)	Raman ID/IG bands (633 nm)
	GO1	As received	1.7 ± 0.1 -39.4 ± 1.3	N.D.
		Control	1.9 ± 0.1 N.D.	1.19 ± 0.08
	Acid treated GNP c As received	1.8 ± 0.1 -39.7 ± 3.7 24.0 ± 2.5 -62.6 ± 1.9	1.22 ± 0.12 N.D.
		Control	25.0 ± 2.5 -54.6 ± 4.1	0.37 ± 0.07
		Acid treated 24.4 ± 2.5 -56.7 ± 1.8	0.31 ± 0.11
	a Determined from the XPS survey spectra. The estimated error is ±0.1
	and ±2.5 for GO1 and GNP samples, respectively, and results from the

uncertainty of the determination of the elemental concentrations from the XPS in the order of 10% under our experimental conditions. b Measured in ultra-pure water with potassium chloride (KCl) as background electrolyte. c Contains primary bile salt sodium cholate (SC); N.D. = not determined.

Table 1

 1 Overview on the physicochemical properties of applied graphene-related materials (GRM)

						Aggregated graphene
		Graphene oxide				nanoplatelets
	Material type GRM	GO1	GO2 29	GO3 29	GO4 c	GNP
	Source	Commercial	Research	Research	Commercial	Commercial
	Preparation	Modified Hummers	Modified Hummers	Modified Hummers	Modified Hummers	Microemulsion
		method	method	method	method
	Starting material	Graphite	Natural graphite	Natural graphite	Graphite nanofiber	Natural graphite
					(GANF©)
	Size distribution/ lateral dimension	1-40 µm (SEM) (300-800 nm, AFM b )	360 ± 188 nm (AFM), some flakes larger than 1 µm	150 ± 44 nm (AFM)	20 nm-1.4 µm (TEM) Aggregate size 1-10 µm; mean ∼5 µm (SEM) (1-2 µm, AFM b )
	Number of layer/ thickness	Few to single layer (0.7-1.2 nm b )	Monolayer/ 1.1 ± 0.1 nm	Monolayer/ 1.1 ± 0.1 nm	Few to single layer	Aggregate thickness up to 5 µm; 5-10 layers (shape of
						the Raman 2D band) (<4 layers; <4 nm b )
	Raman ID/IG ratio	1.19 ± 0.08 (633 nm)	1.34 ± 0.06 (633 nm)	1.30 ± 0.06 (633 nm)	0.81 ± 0.05 (n = 5)	0.37 ± 0.07 (633 nm)
		0.96 ± 0.02 (532 nm)	1.06 ± 0.01 (532 nm)	0.97 ± 0.02 (532 nm)		0.17 ± 0.11 (532 nm)
	C/O ratio Zeta-potential (mV) a -39.4 ± 1.3 1.7 ± 0.1 (XPS)	1.9 -44.0 ± 0.2	1.9 -43.9 ± 1.4	2.61 (XPS) -37.7 ± 0.4	24.0 ± 2.5 (XPS) -62.6 ± 1.9

This journal is © The Royal Society of Chemistry 2016View Article Online

Acknowledgements

The research leading to these results has received funding from the European Union Seventh Framework Programme under grant agreement no 604391 Graphene Flagship. The authors thank Laura Neff (Empa) for technical support of the DCF assay and Nils Bohmer (Empa) for proof-reading of the manuscript.

Notes and references


Ambrosino Julien
email: ambrosino@aerospace-valley.com

Technopôle Estia

F-64210 Izaberl

Bidart

Daniel Maxime

Estia

Dimitri Masson
email: d.masson@estia.fr

Jérémy Legardeur
email: j.legardeur@estia.fr

IdeaBulb: A Smart and Tangible User Interface for Monitoring Ideation During Creative Sessions

Keywords: creativity, ideation, tangible interface, idea generation. I

In this paper, we propose a tangible user interface for the monitoring of new ideas generations (ideation phase) during creative sessions. Based on the analysis of different brainstorming, we highlight the dynamic of ideas production by different groups during ideation phase using electronic devices. We claim that this tangible user interface is designed in order to give live feedback concerning these dynamic of ideas production to the participants and the facilitator. Our work is the result of the interaction between two scientific approaches: the analysis of creative sessions and the design of tangible user interface. The result of our experiments show a creative cliff and propose a design concept for monitoring ideation phase.

INTRODUCTION

The use of creative processes is a factor increasingly recognized as essential in the emergence of new ideas or business opportunities [START_REF] Long | Mapping the New Venture Opportunity Identification Process[END_REF]- [START_REF] Detienne | Opportunity Identification and Its Role in the Entrepreneurial Classroom: A Pedagogical Approach and Empirical Test[END_REF]. These processes can be declined in a punctual way by involving groups of participants in a creative session, or a brainstorm session [START_REF] Osborn | Applied imagination principles and procedures of creative problem solving[END_REF].

Open-Innovation oriented creative sessions gather participants from different backgroundsservices, companies -from clusters are more likely to be competitive domestically and globally when their business is competitive and collaborative at the same time [START_REF] Christensen | Bridging community and economic development-A strategy for using industry clusters to link neighbourhoods to the regional economy[END_REF]. These ambivalent aptitudes create a context of coopetition [START_REF] Porter | Clusters and the New Economics of Competition[END_REF].

Creative sessions are day events led sometimes by a facilitator (internal or external) to help the generation and the evaluation of new ideas, also they follow a process characterized by a succession of convergent and divergent main phases according to the facilitation model proposed by Ambrosino et al. [START_REF] Ambrosino | STAR : An animation methodology to facilitate the emergence of interclustering projects[END_REF].

The generation of ideas called the ideation phase is a critical part of the innovation process [START_REF] Kohn | Building on the ideas of others: An examination of the idea combination process[END_REF] where participants are asked to produce as many ideas as possible in an allotted time. Time-constrained is thus important, because when deadline is fixed, it add an emotional power in the form of fear, lest participants may fall down [START_REF] Clark | Brainstorming[END_REF].

In this article, we propose first to analyze the ideation phase of creative sessions in order to highlight the dynamic of ideas production within the group.

Second, we propose a tangible user interface for the monitoring of ideation phase in order to improve creative sessions by giving to the participants and the facilitator a live feedback concerning these dynamic of ideas production.

II. BRAINSTORMING TECHNIQUE AND SUPPORT TOOLS

Classic brainstorming usually causes blocking and slows down the generation of ideas in groups [START_REF] Diehl | Productivity loss in brainstorming groups: Toward the solution of a riddle[END_REF]. Galuppe et al. [START_REF] Gallupe | Unblocking brainstorms[END_REF] show electronic brainstorming systems can improve creative sessions by acting on three classes of explanatory mechanisms which can cause productivity loss in brainstorming groups:

 Procedural mechanisms like production blocking [START_REF] Lamm | Group versus individual performance on tasks requiring ideational proficiency (brainstorming): A review[END_REF], when just only a person can speak at a time.  Social psychology mechanisms like drive-arousal [START_REF] Geen | Theories of Group Behavior[END_REF] and self-attention [START_REF] Mullen | Group effects on self-attention and performance: Social loafing, social facilitation, and social impairment[END_REF], which is due to the presence of the other people and individual membership in the group.  Economic mechanisms like social loafing [START_REF] Latané | Many hands make light the work: The causes and consequences of social loafing[END_REF] and free-riding [START_REF] Kerr | Dispensability of member effort and group motivation losses: Free-rider effects[END_REF], which correspond to an intentional withdrawal.

IdeaValuation [START_REF] Ambrosino | IdeaValuation : Favoriser les échanges lors d'un atelier de créativité (b) (a) par le vote qualitatif des idées à l'aide d'un outil numérique[END_REF] is a structured electronic brainstorming tool. It supports the ideation and evaluation through a discovering matrix [START_REF] Moles | The scientific creation[END_REF]. The matrix is composed of needs (rows) and means (columns). As advised by [START_REF] Diehl | Productivity loss in brainstorming groups: Toward the solution of a riddle[END_REF], the facilitator using IdeaValuation invites participants first to generate their ideas in individually in the relevant cells of the matrix.

Then, these ideas are discussed and evaluated in a group session, following the instruction of the facilitator to evaluate each idea in turns. For each idea, the evaluation is done along four criteria and is followed by a short discussion between participants.

III. CHARACTERIZING THE IDEAS PRODUCTION

A. Experimental setup and process

Simultaneously, two creative sessions were performed in an event dedicated to Innovation in France. Groups are composed of participants who are members of the regional Agency of Development and Innovation in Nouvelle-Aquitaine (ADI-NA). There are located in two different rooms and the rooms do not interact together.

First participants are introduced to IdeaValuation tool and in particular the discovering matrix used for this event. Then, participants are then invited to generate as many ideas as possible, following one of the 4 fundamental rules of brainstorming [START_REF] Osborn | Your Creative Power[END_REF]. They use a Samsung Galaxy Tab A tablet connected by WIFI. Some characteristics of these ideation phases, which last 20 minutes, are described in the matrix, in TABLE I.

Results of ideation phase

Number of participants who generate ideas 23 26

Total amount of ideas 79 79

While there were more participants registered to the workshop 1, only 23 of them submitted ideas through IdeaValuation, resulting in two groups of 23 and 26 idea producers.

B. Observations and analyses

The use of an electronic brainstorming tool such as IdeaValuation allows to monitor the production of ideas during the ideation phase. During 20 minutes of ideation phase, we compute every 2 min two metrics: the amount of ideas generated and the flow.

1) Evolution of amount of ideas generated Figure 1 indicates the evolution of the amount of ideas generated during the ideation phase for both workshops.

Participants slowly start to produce ideas. Then, 10 minutes after the beginning, the group reaches a peak followed by a sharp drop: we observe it in the amount of ideas proposed after 12 minutes for group 1 and after 16 minutes for group 2. In the case of group 2, no idea was proposed during the last three minutes. This part of the curve can be called the creative cliff.

An ideal ideation phase could see all participants produce regularly until the end of the session. Without any external solicitation, the participants seem to be more limited by their ability to generate ideas than by time.

2) Evolution of ideation flow

Figure 2 indicates the evolution of ideation flow acceleration during ideation phase. There are some positive and negative variations of values which reflect the acceleration or deceleration flow of amount of ideas proposed. It seems to be a boundary beyond which the production of ideas tends to a zero value. When the curves tend to intersect this boundary, ideation flow is alarming. This part of the curve can be called the "warning area for creativity".

C. Opportunities for a tangible support tool 1) Lack of efficiency: boost productivy As we have seen the time allocated to ideation phase is not fully used by all participant, the idea production cliff in the last quarter of the ideation phase. Individual generation lets them express and formulate ideas as they want, but the absence of external action, for example by giving to participants a live feedback about their progress may be a lever to increase their productivity.

In workshop 1, many participants do not generate any idea. Although, the absence of propositions by 19 participants in this workshop is notable, it is difficult to identify clearly the causes, but the use of a full digital solution may be at stake.

2) Lack of competition and collaboration

Despite the fact that competition benefits between intergroup are clearly highlighted as described in Introduction, participants need to have a live feedback of their progress. The only element which may be perceived as a competition one is the visualization of the other participants keyboarding during ideation phase. Currently, participants do not have any information about the real progress of the other participants.

Collaboration benefits are implied since participants accept to involve themselves in an open-innovation oriented creative session. However, they individually generate ideas and they cannot read the ideas of the others. So, collaboration may be perceived as limited during ideation phase.

In order to investigate these opportunities, we consider tangible user interfaces as a mean to give back control to participants on the productivity.

IV. IDEABULB: A TANGIBLE USER INTERFACE DESIGNED FOR IMPROVING CREATIVE SESSIONS

Tangible User Interfaces (TUIs) use physical objects to represent and/or manipulate digital information [START_REF] Ishii | Tangible bits: towards seamless interfaces between people, bits, and atoms[END_REF]. With the use of electronic brainstorming systems, we think TUIs to be adequate for enhancing collaboration and ideation in creative sessions thanks to a live feedback represented by a physical object, in order to make the ideation more tangible. They are known to support social interaction (e.g., collaboration) and to support thinking process (e.g., problem solving) through bodily actions, physical manipulation, and tangible representations [START_REF] Shaer | Tangible User Interfaces: Past, Present, and Future Directions[END_REF].

A. Peripheral Interaction and Ambient Interfaces

Because ideation phase during a creative session is a cognitive process that requires central attention and a lot of mental resources, we wish to design an ambient TUI working with peripheral attention and requiring little mental resources to interact with.

Ambient awareness makes human beings aware of surrounding information [START_REF] Wisneski | Ambient displays: Turning architectural space into an interface between people and digital information[END_REF]. Weiser and Brown [START_REF] Weiser | The coming age of calm technology[END_REF] defined calm technologies as technologies able to move from the peripheral attention to the central attention of users, and backwards. They affirmed that calm technologies enhance ambient awareness by bringing more details into the periphery: it makes users aware of what is happening around them, what is going to happen, and what has just happened. In line with calm technologies, Ambient Interfaces use perceptible artefacts (e.g., shape, motion, sound, color, light, smell, air) to represent unobtrusively digital information. Bakker et al. [START_REF] Bakker | Peripheral interaction: characteristics and considerations[END_REF] peripheral noted that recent studies have been conducted under the term peripheral interaction, aiming to broaden the scope of calm technology by designing not only for the perceptual periphery but also enabling users to physically interact with the digital world in their periphery.

B. Design concept: IdeaBulb

We think that giving live feedback to participants on their performance can mitigate ideation off-peaks. Therefore, we propose to design a smart interface to inform participants on the amount of idea they generated and their flow of ideation. Because we do not want to interrupt participants during their ideation process, we wish to design an ambient interface to subtly inform participant without being intrusive.

1) General design: metaphorical items

For designing IdeaBulb, we choose the metaphor of a bulb held by a hand, in Figure 3. Light bulb shape is part of the clichés to symbolize the ideation of an individual [START_REF] Kennedy | Metaphor in pictures[END_REF], while the hand holding it represents the group.

2) Feedbacks

The smart interface IdeaBulb will give in real-time two types of feedback during ideation phase.

a) Cumulative feedback on the amount of ideas submitted

Using a led strip, IdeaBulb lights on its "belly" according to the amount of ideas submitted. For instance, IdeaBulb informs participants that there is a total of 3 ideas submitted by turning on 1 led over 30 leds maximum, as shown in Figure 4 (a).

b) Immediate feedback on the flow and the submission of ideas

Using a servomotor, IdeaBulb opens its "mouth" according to the flow of ideation. For instance, a flow of 0 ideas by minute is represented by a mechanical dome widely opened meaning that IdeaBulb is "hungry" for ideas. In contrary, a flow of 4 ideas by minute is represented by a "mechanical dome" completely closed meaning that IdeaBulb is "satisfied", in Figure 4 Each time an idea is submitted, IdeaBulb quickly opens and closes its mechanical "mouth" meaning that an idea was eaten. Moreover, when IdeaBulb "eats" an idea, the led strip is animated informing participants that the idea is going down in the "belly" of IdeaBulb.

V. CONCLUSION AND PERSPECTIVES

Our work presented here is the result of the interface between two main scientific fields: analysis of live facilitation during creative sessions and the design approach for a tangible user interface in a context of competition and collaboration. Based on the analysis of our many experimental results, including the two workshops presented here, the monitoring of ideation phase highlights a creative cliff and a warning area for creativity, when generation of ideas becomes low. Then, we present a smart and tangible user interface, IdeaBulb, which could counteract the offpeaks observed.

The prototyping and testing during creative sessions could validate our assertions. The total amount of ideas, ideation flow and participant experience should be monitored. The context of competition and so creative performance could be increased by dividing the group into many sub-groups in order to enhance group creativity [START_REF] Baer | Intergroup Competition as a Double-Edged Sword: How Sex Composition Regulates the Effects of Competition on Group Creativity[END_REF]. An IdeaBulb could be assigned for each sub-group. The implementation of a physical interaction or rewarding in a non-game context, can also be a process to foster participant's performance, as gamification invite to in [START_REF] Deterding | Gamification: Using game design elements in non-game contexts[END_REF].

Figure 1 .Figure 2 .

 12 Figure 1. Highlighting the creative cliff

Figure 3 .

 3 Figure 3. IdeaBulb's metaphorical design

  (b).

Figure 4 .

 4 Figure 4. Opening mechanism (b) and lighting of leds (a)

TABLE I .

 I CHARACTERISTIC SETTINGS OF TWO WORKSHOPS

		Information according phase concerned	Workshop 1	Workshop 2
				Marine	Biomimicry for
			Thematics adressed	Renewable	Sustainable
				Energies	Housing
	Registrering	Number of participants registred "Expand my network" "Generate ideas for collaborative innovation projects" Initial motivation "Other"	42 10 25 7	26 6 14 12
	Discovering	matrix	Type of entries Size	Needs vs Means 4 x 5	Needs vs Means 5 x 5

ACKNOWLEDGMENT

The authors would especially like to thank the NEPTUNE project, coordinated by the Aerospace Valley competitiveness cluster and the New-Aquitaine Development and Innovation Agency, which support the work presented here.

The NEPTUNE project received funding from the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 691554.


Professor Etienne Sicard
email: etienne.sicard@insa-toulouse.fr

Introducing 10-nm FinFET technology in Microwind

This paper describes the implementation of a high performance FinFET-based 10-nm CMOS Technology in Microwind. New concepts related to the design of FinFET and design for manufacturing are also described. The performances of a ring oscillator layout and a 6-transistor RAM memory layout are also analyzed.

Technology Roadmap

Several companies and research centers have released details on the 14-nm CMOS technology, as a major step for improved integration and performances, with the target of 7-nm process by 2020. We recall in table 1 the main innovations over the past recent years.

Improved performances

The power, performance and area gains are an important metric for justifying a shift from older technology nodes to new ones.

Page 2/20 etienne.sicard@insa-toulouse.fr 21/06/17 As compared to 14-nm technology node, the 10-nm technology offers [Brain2017, Mistry2017]:

• 30 % less power consumption. This is a key feature for mobile industry for which the battery life is the top one problem. • 10 to 20 % increase in switching performance. This is equally important in server applications and smartphones, which use faster processors and higher resolution screens.

• 2 times higher density. This is a key advantage to produce the lightest and thinnest possible smartphones.

A comparison between 45nm, 32, 22, 14 and 10nm technologies in terms of density is proposed by [Brain2017] are reported in Fig. 2. The IC surface is shrunk by a factor of 13 between 45-nm and 10nm nodes. The giant cost of fab and IC design in 10-nm

The consequences of the fab and chip design cost explosion is the drastic decrease of foundries. While more than 20 foundries existed for 130nm technology, only four major companies are providing the 10-7nm processes (Table 2). One reason is the extraordinary fab and IC design cost, as illustrated in Some examples of processors used in mobile phones, laptops and servers are reported in Fig. 3, with complexity approaching 10 Billion devices. Within a same technology node, improvements are made over the years, such as Intel who plans to improve its original process "10nm" with the "10nm+" offering 10% higher drive current, followed by "10nm++" process with supplementary 10% higher drive current [Mistry2017]. Similar trends were observed for Intel's 14nm, 14nm+ and 14++ technology as illustrated in Fig. 5 [Mistry2017].

A continuous increase in switching performance has been made possible thanks to several innovations, as shown in Figure 6. Starting 14-nm, the MosFET is replaced by the FinFET, for improved current capabilities.

Page 5/20 etienne.sicard@insa-toulouse.fr 21/06/17 Within the same "10-nm" label, we may observe a wide variety of performances, depending whether the IC fabrication process is targeted to "high performance" (HP) devices (speed whatever the power consumption), "general purpose" (GP) or "Low Power" (LP) (lower speed but power-efficient), as summarized in Table 3. We implement in Microwind the general purpose 10-nm process.

Power supply operation

The supply voltage, both internal to the cores and the external I/O supply have been continuously

decreased due to the thinning of the gate oxide and faster switching rates thanks to reduced voltage swings. The 10-nm technology operates around 0.7V, while I/Os are supplied at 1.2, 1.5 or 1.8 V (Fig. 5).

Page 6/20 etienne.sicard@insa-toulouse.fr 21/06/17 elements from the previous 28-nm and 20-nm nodes, so that planar designs may be partially reused starting 14-nm and below. However, the Fin design is a totally new feature which contrasts with the traditional MOS design. This leads to new screens and new acronyms such as "Fin Pitch", "Fin thickness", etc, as shown in Figures 9 and10.

Lambda

In Microwind, we use an integer unit for drawing, which is fixed to 6 nm for 10-nm CMOS process (Table 4).

• The drawn gate length is 2 λ that is 12 nm.

• The fin width is 1 λ that is 6 nm.

• The lower metal pitch is 36 nm.

IO MOS devices

Table 5 gives an overview of the key parameters for the 14-nm technological concerning the Input/output MOS devices and associated supply voltage. In Microwind, we only consider 1.2V I/O supply and tune the "High-Voltage" (HV) MOS device on the median performances. Other standards usually supported in commercial 14-nm process include 1.2, 1.5 and 1.8V I/O supply.

Parameter

Transistor performances in 10-nm technology

Designing a 1µm-width FinFET

The evaluation of the equivalent FinFET channel width corresponds to the following formulation (Eq.

1). The evaluation of the current is usually expressed in mA/µm, meaning that a 1-µm equivalent width

FinFET design is needed to evaluate the current. the proposed 10-nm technology, HFIN is 40nm, WFIN 6nm, so one fin has an equivalent width of 86 nm (Figure 11). An equivalent width of 1µm, as it would be designed using MosFET's, corresponds to around 12 fins in 10-nm technology. As seen in Fig. 12, the FinFET with a 1-µm width is twice smaller than a MosFET with the same width. We get the following I/V characteristics (Fig. 12) for n-channel FinFET and pchannel FinFET.

Page 10/20 etienne.sicard@insa-toulouse.fr 21/06/17 The drawback associated with this high current drive is the leakage current which rises from 4 nA/µm (low leakage NMOS) to 30 nA/µm (high speed NMOS), as seen in the Id/Vg curve at the X axis location corresponding to Vg= 0 V. From a design view-point, the "option" menu in the MOS generator enables to switch from low leakage to high-speed. In terms of layout, the only difference is the option layer that contains the MOS option information which can be Low leakage (Regular Vt), High Speed (Low VT), or High Voltage.

Page 11/20 etienne.sicard@insa-toulouse.fr 21/06/17

P-channel MOS device characteristics

The p-FinFET drive current in 10-nm technology is quite similar to the n-FinFET thanks to the strain engineering for p-channel that nearly compensates the intrinsic mobility degradation of holes (Pchannel) vs. electrons (N-channel). The leakage current is around 2 nA/µm for the low-leakage device and nearly 12 nA/µm for the high-speed device (Fig. 15).

MOS Design for Manufacturing Process Variability

One important challenge in nano-CMOS technology is process variability. The fabrication of millions of MOS devices at nano-scale induces a spreading in switching performances in the same IC.

The effect of process variability on the MOS Ioff/Ion characteristics is plotted using the menu "Ioff vs.

Ion" under the "MOS I/V curve" menu (Fig. 16). It can be seen that the MOS devices have a wide

Interconnects Metal Layers

The number of metal layers in nano-CMOS technology usually ranges from 8 to 15, with a trade-off between integration and cost. In Microwind, only 8 metal layers are considered, according to Layers metal5 and metal6 are a little thicker and wider, while layers metal7 and metal8 are significantly thicker and wider, to drive high currents for power supplies (Fig. 17).

Interconnect Resistance

The resistance and capacitance of interconnects are an important metric for evaluating the RC delay and consequently the switching speed of the signals. The order of magnitude is 50 ohm/µm and 0.35 fF/µm.

Page 14/20 etienne.sicard@insa-toulouse.fr 21/06/17

Double Patterning for M1-M2 Interconnects

The double patterning is required for metal layers as the pitch between tracks is smaller than 80nm.

Half the patterns go on the first patterning and half go on the second patterning, as illustrated in Fig. 18. In order to ensure an easy selection of metal tracks for the first and second patterning, regular structures with straightforward orientation such as M1 east-west, and M2 south-north are requested.

The other solution is to relax the pitch constraints for an improved manufacturability, at the cost of an extended silicon area. M3-M8 with pitch of 80-nm and higher are still fabricated using simple patterning.

Ring Inverter Simulation

Performances

An improvement of switching performances as high as x 5 is obtained between 45nm and 14nm designs, although VDD is reduced from 1.1V to 0.7V. Microwind operates with lambda-based designs which are independent of the technology, but MosFET and FinFET designs are not compatible.

However, comparison remains possible at similar drive currents, circuits and design styles. As seen in The most usual simulation consists of simulating extreme situations (Min and Max), as compared to typical conditions (Table 7).

•

In Min situation, VT is high and the mobility U0 is low. The supply is minimum and the temperature is maximum. The ring oscillating is around 100 GHz.

Conclusions

This application note has illustrated the trends in CMOS technology and introduced the 10-nm technology generation, based on technology information available from manufacturers. The key features of the 10-nm CMOS technology have been illustrated, including the FinFET, design for Page 20/20 etienne.sicard@insa-toulouse.fr 21/06/17 manufacturing and double patterning. A 3-stage ring, FinFETs wih 1-µm equivalent width as well as interconnects with 1-µm length have been used for comparison purpose.

Figure 1 :

 1 Figure 1: The reduction of feature sizes from 45 to 7nm may induce drastic gains in power consumption and leakage power [Xie2015]

Figure 2 :

 2 Figure 2: The evolution of silicon area with the technology nodes as presented by Intel [Brain2017]

Figure 4 :

 4 Figure 4.

Figure 5 :

 5 Figure 5: Examples of multi-giga-transistor processors used in mobiles, laptops and servers from various companies [Mistry2017].

Figure 6 :

 6 Figure 6: Increased switching speed capabilities over technology nodes

Figure 7 :

 7 Figure 7: The core supply voltage in 10nm technology is 0.7 V, with typical IO voltage of 1.2 V

Figure 8 :

 8 Figure 8: FinFet technology from Intel [Mistry2017] with increased fin height to improve the current drive.

Figure 9 :

 9 Figure 9: The introduction of the FinFET in Microwind, and comparison with traditional MosFET

Figure 11 :

 11 Figure 11: The equivalent width of a multi-fin FET

Figure 12 :

 12 Figure 12: The 10-nm multi-fin FET with an equivalent width of 1 µm is twice more compact than the MosFET traditional design

Figure 13 :

 13 Figure 13: Id/Vd characteristics of the low leakage and high speed n-channel FinFET devices.

Figure 15 :

 15 Figure 15: Id/Vg characteristics of the low leakage and high-speed p-FinFET devices

  variability in performances. The 3 MOS types (low leakage, high speed, high voltage) are situated in well-defined space in the Ioff/Ion domain. The low leakage is in the middle (medium Ion, low Ioff), the high speed on the upper right corner (high Ion, high Ioff), and the high voltage is at the lower left side of the graphics (low Ion, very low Ioff). Note that the exact locations of the dots will change for each MOS characteristics plotted because it is a random process.

Figure 16 :

 16 Figure 16: Ioff/Ion calculated on 100 samples of n-FinFET with random distribution of VT and U0, with a Gaussian distribution around the nominal value

Figure 17 :

 17 Figure 17: The 8 metal layers proposed in Microwind's implementation of the 10-nm technology

Figure 18 :

 18 Figure 18: Single, double and quadruple patterning as a function of interconnect pitch [Brain2017]

Fig. 19 ,Figure 19 :

 1919 Fig. 19, a 3-inverter ring oscillator (RO) features a natural 43 GHz oscillation in 45-nm, which rises to nearly 250 GHz with 10-nm technology.

Figure 20 :

 20 Figure20: the voltage scaling depends on the operating scenario[Pinckney2017]

Figure 21 :

 21 Figure 21: Modify VDD to extract the minimum operating voltage of the ring oscillator.

Figure 22 :

 22 Figure 22: Typ/Min/Max simulation using PVT analysis

Figure 23 :

 23 Figure 23: Reduction of the 6T-SRAM memory from 45-nm to 10-nm process of Intel, reaching 0.03µm2 for the most compact design [Miustry2017]

Figure 25 :

 25 Figure 25: Quick access to MosFET and FinFET technologies

Figure 26 :

 26 Figure 26: Convert MosFET design into FinFET. Only vertical gates are considered.

Table 1 :

 1 Most significant technology nodes over the past 15 years

	Technology node	Year of	Key Innovations	Application note
		introduction
	180nm	2000	Cu interconnect, MOS options, 6 metal
			layers
	130nm	2002	Low-k dielectric, 8 metal layers
	90nm	2003	SOI substrate	[Sicard2005]
	65nm	2004	Strain silicon	[Sicard2006]
	45nm	2008	2nd generation strain, 10 metal layers	[Sicard2008]
	32nm	2010	High-K metal gate	[Sicard2010]
	20nm	2013	Replacement metal gate, Double	[Sicard2014]
			patterning, 12 metal layers
	14nm	2015	FinFET	[Sicard2017]
	10nm	2017	FinFET, double patterning	This application note
	7nm	2019	FinFET, quadruple patterning

10-nm Technology variant Pros Cons Target application Typical Ion/Ioff General purpose

		Medium speed	Medium	Laptops	1.4 mA/µm
			consumption		10 nA/µm
	High performance Fast speed	High	Servers	1.8 mA/µm
			consumption		100nA/µm
	Low power	Low speed	Low	Smartphones	1.1 mA/µm
			consumption		1 nA/µm
	Table 3: technology variants within the same node

Microwind parameter Unit Code Name in rule file 10-nm process

	Lambda	nm	λ	lambda	6
	Core supply V	VDD	Vdd	0.7
	Fin Width	λ	WF	R301	1
	Fin pitch	λ	FP	R308	6
	Fin Height	nm	HF	thdn	40
	Gate height nm	GH	thpoly	60
	Gate length λ	GL	R302	2
	Gate pitch	λ	GP		6
	Spacer width nm	SW		8
	Contact size λ	CS	R401	2
	EOT	Nm	EOT	b4toxe	0.8
	M1 pitch	λ		R501+R502	6
	Page 8/20		etienne.sicard@insa-toulouse.fr	21/06/17

Table 4 :

 4 key parameters of the 14-nm processes used to configure Microwind rule file Low Threshold Voltage causes more power consumption and switching timing is optimized. LVT are used in time critical functions.• RVT -Regular Threshold Voltage (sometimes called Standard VT or SVT) offers trade-off between HVT and LVT i.e., moderate delay and moderate power consumption.In Microwind, we only use 2 types of MOS devices for the core and reuse the same name as for previous

	Cmos10n.RUL
	Core MOS devices
	Table 4 gives an overview of the key parameters for the 10-nm technological node concerning the
	internal MOS devices and layers. Commercial 10-nm processes propose up to 3 threshold options,
	namely high-Vt (HVT), regular Vt (RVT), and low Vt (LVT).
	• HVT -High Threshold Voltage causes less power consumption, but switching is slow. HVT are
	used in power critical functions.
	• LVT -Parameter	In Microwind
	VDD core (V)	0.7
	Effective gate length (nm)	12
	MOS variants	2
	Ion N (mA/µm) at 0.7V	1.3 (LL) 1.7 (HS)
	Ion P (mA/µm) at 0.7V	1.1 (LL) 1.4 (HS)
	Ioff N (nA/µm)	1 (LL) 10 (HS)
	Ioff P (nA/µm)	1 (LL) 10 (HS)
	Gate dielectric	HfO2
	Gate stack	Al/TiN
	Equivalent oxide thickness (nm)	0.8

nano-CMOS technologies:

• Low leakage MOS (LL), close to RVT

• High Speed MOS (HS), close to LVT

Table 4 :

 4 Key features of the core devices proposed in the 10-nm technology

Table 5 :

 5 Key features of the I/O devices proposed in the 10-nm technology and corresponding

	High Voltage (HV) MOS in
	Microwind

table 6 :

 6 •

	Parameter Pitch	Pitch	Rules	Thickness	Thickness	Purpose
		(nm)	(λ)		(nm)	parameter
	M1	36	6	R501, R502	35	Thme	Short routing
	M2	36	6	R701, R702	35	Thm2	Short routing
	M3	48	8	R901, R902	70	Thm3	Medium routing
	M4	48	8	RB01, RB02	70	Thm4	Medium routing
	M5	96	16	RD01, RD02	120	Thm5	Long routing
	M6	96	16	RF01, RF02	120	Thm6	Long routing
	M7	276	46	RH01, RH02	400	Thm7	Supply, very long routing
	M8	450	75	RJ01, RJ02	640	Thm8	Supply, Coil inductance

M1 & M2 are at 6 λ pitch for local routing • M3 & M4 are at 8 λ pitch for medium routing • M5 & M6 are at 32 λ pitch for long routing and local supply • M7 & M8 are dedicated to power supply and coil inductance Page 13/20 etienne.sicard@insa-toulouse.fr 21/06/17

Table 6 :

 6 Key features of interconnects in the 10-nm technology implemented in Microwind

  Max situation, VT is low, mobility U0 is high and the channel is short (LINT<0). The supply is maximum and the temperature is minimum. The ring oscillation is around 250 GHz.

	Oscillating				150	240	290
	frequency
	Parameter	Parameter	Symbol	Unit	Min	Typ	Max
	class		(BSIM4)		(10-nm)	(10-nm)	(10-nm)
	Process	Threshold	VT	V	0.33	0.28	0.24
		Voltage
		Mobility	U0	m2/V.s	0.030	0.035	0.040
	Voltage	Supply	VDD	V	0.60	0.70	0.81
	Temperature	Temperature	TEMP	°C	125	25	-50

•

In

Page 17/20 etienne.sicard@insa-toulouse.fr 21/06/17

Table 7 :

 7 Variation of process parameters and effects on the oscillating frequency

Acknowledgements

The adaptation of Microwind to 10-nm FinFET technology and the development of an e-learning course on nano-CMOS cell design have been made possible thanks to the financial support of the ERASMUS+ program Knowledge Alliance "Micro-Electronics Cloud Alliance" 562206-EPP-1-2015-1-BG-EPPKA2-KA.