Ming Sun

Isabelle Ly

Wilfrid Neri

Ahmed Bentaleb

Jinkai Yuan
email: jinkai.yuan@crpp.cnrs.fr

Systematic Investigation of the Influence of Experimental Conditions on TiO 2 Nanosheet Structures

Keywords: TiO2, solvothermal synthesis, morphology controlling, liquid crystal phase

The morphology of TiO2 nanoparticles has great influence on their physical/chemical properties. Controlled synthesis of TiO2 with adjustable morphologies is of utmost importance for their target applications. Herein, a series of TiO2 with continually adjustable shape, size and crystal structures were synthesized via the hydrolysis of titanium isopropoxide, followed by the self-assembly using P123 as structure-directing agent and ethylene glycol as co-surfactant under different solvothermal conditions. The effect of the diverse experimental conditions on the TiO2 nanostructures was systematically investigated. Particularly, an optimum TiO2 nanosheet was achieved and the formation of liquid crystal phase was observed. This work provides useful enlightenment for the fabrication of TiO2 nanostructures, which can be potentially employed in diverse fields, such as dielectric materials and photocatalysts.

Introduction

TiO2 has a long history, and its application as white pigment can date back to ancient times [START_REF] Hashimoto | TiO2 Photocatalysis: A Historical Overview and Future Prospects[END_REF]. Nowadays, the application of TiO2 nanoparticles as additives in fine chemicals (coatings, inks, plastic, rubber, and cosmetic) is advanced. In scientific community, TiO2 has been long known as an outstanding photo-catalyst for environmental restoration [START_REF] Sun | Preparation and electrorheological properties of eggshell-like TiO2 hollow spheres via one step template-free solvothermal method[END_REF][START_REF] Sun | Surface modification of TiO2 with polydopamine and its effect on photocatalytic degradation mechanism[END_REF]. The excellent physical and chemical properties of TiO2 materials endow them rich applications, and this spurs enormous research interest for decades [START_REF] Bavykin | The effect of hydrothermal conditions on the mesoporous structure of TiO2 nanotubes[END_REF][START_REF] Chen | Titanium dioxide nanomaterials: synthesis, properties, modifications, and applications[END_REF][START_REF] Zhang | Recent advances in the synthesis of hierarchically mesoporous TiO2 materials for energy and environmental applications[END_REF]. Since ten years ago, at least 10000 papers per year involving TiO2 have been published (based on the search result of Web of Science by using TiO2 as keyword). It is noted that, in the early stage, the attention was particularly paid on the exploration of different preparation methodologies of TiO2, including sol-gel method, hydrothermal/solvothermal method and hydrolysis method.

By contrast, recently the main interest seems shifting to the modification and application of diverse TiO2 nanostructures.

The physical and chemical properties of TiO2 are actually dominated by the morphology/structure, which is determined by the preparation strategy. Therefore, the morphology engineering of TiO2 has been long a hot spot in the community [START_REF] Katal | A review on the synthesis of the various types of anatase TiO2 facets and their applications for photocatalysis[END_REF].

One of the milestones is the discovery of anatase TiO2 with a large percentage of reactive (001) facets reported by Qiao et al. in 2008 [START_REF] Yang | Anatase TiO2 single crystals with a large percentage of reactive facets[END_REF]. Among the rich morphologies of TiO2, nanosheet outperforms its counterparts because of the following two aspects:

(1) two-dimensional (2D) materials can be used as effective building blocks to develop three-dimensional (3D) composites; (2) 2D materials have large surface area and rich active sites, which eventually serve as the perfect catalyst [START_REF] Sun | Atomically-thin two-dimensional sheets for understanding active sites in catalysis[END_REF]. Several studies have focused on the controlled synthesis of TiO2 nanosheets. The most-used process is delaminating layered protonic titanate, which is processed via a high-temperature solid-state reaction followed by an acid treatment [START_REF] Xiong | Two-Dimensional Unilamellar Cation-Deficient Metal Oxide Nanosheet Superlattices for High-Rate Sodium Ion Energy Storage[END_REF][START_REF] Sohn | Novel Flexible Transparent Conductive Films with Enhanced Chemical and Electromechanical Sustainability: TiO2 Nanosheet-Ag Nanowire Hybrid[END_REF][START_REF] Li | Two-Dimensional Layered Ultrathin Carbon/TiO2 Nanosheet Composites for Superior Pseudocapacitive Lithium Storage[END_REF]. However, such process is time-and energy-consuming. The second method is the so called "hard-template", using graphene oxide as template [START_REF] Li | General strategy to synthesize uniform mesoporous TiO2/graphene/mesoporous TiO2 sandwich-like nanosheets for highly reversible lithium storage[END_REF]. Recently, Zhao et al. [START_REF] Lan | Uniform Ordered Two-Dimensional Mesoporous TiO2 Nanosheets from Hydrothermal-Induced Solvent-Confined Monomicelle Assembly[END_REF] and Sun et al. [START_REF] Sun | Generalized self-assembly of scalable two-dimensional transition metal oxide nanosheets[END_REF] developed a surfactant-templated strategy using Pluronic P123 as soft template. As compared to the first two methods, such "soft-template" method has merits of tunable morphologies and easy processing. Whereas, the product is highly sensitive to the reaction conditions [START_REF] Zhang | Recent advances in the synthesis of hierarchically mesoporous TiO2 materials for energy and environmental applications[END_REF]. Inspired by the aforementioned progress, herein, we made a comprehensive and systematic investigation on the fabrication of TiO2 nanosheets in a reaction system composed of titanium isopropoxide as the source of Ti, Pluronic P123 as surfactant and ethylene glycol as co-surfactant under different solvothermal conditions. Our results showed that different synthetic parameters can allow for TiO2 materials with diverse geometries and crystal phases. The engineering of architectural diversity of TiO2 is achieved. The resultant nanosheets can form liquid crystalline structures after appropriate treatments.

Experimental

Preparation of TiO2 nanosheets

The TiO2 nanomaterials were prepared using a method similar to that reported by Sun et al. [START_REF] Sun | Generalized self-assembly of scalable two-dimensional transition metal oxide nanosheets[END_REF]. Two solutions were firstly prepared individually. Solution A: 1.1 mL of titanium isopropoxide (TTIP) was added into 0.63 mL concentrated hydrochloric acid under magnetic stirring. Solution B: 0.2 g Pluronic P123 (Mw=5800) was mixed with 3.8 mL pure ethanol until completely dissolved. Then, solution B was added into solution A under continuous stirring for at least 30 min. Afterward, 2.5 mL of the above mixed TTIP solution with 20 mL ethylene glycol (EG) were transferred into a Teflon-lined stainless steel autoclave (40 mL), which was then heated at 150 o C for 20 h. The products were centrifuged (8000 rpm) and then washed with ethanol, and eventually dried at 60 o C overnight in order to collect TiO2 powder.

A series of parallel experiments were performed based on the above synthetic protocol to evaluate the effect of different parameters on the TiO2 nanostructures.

For investigating the temperature effect, five reaction temperatures (120, 140, 170, 190, and 210 o C) were used respectively with all the other conditions unchanged. For the concentration of acid, different volumes of HCl acid were applied individually: 0 (0.63 mL of water), 0.4 mL (0.10 mol L -1 ), 0.63 mL (0.15 mol L -1 ), 1.3 mL (0.28 mol L -1 ), 2.3 mL (0.42 mol L -1 ), 3.3 mL (0.53 mol L -1 ) and 4.3 mL (0.62 mol L -1 ); For the type of acid, different acids (such as hydrochloric acid, sulfuric acid, nitric acid, and acetic acid (HAc)) were used respectively. The corresponding samples are denoted as TiO2-HCl, TiO2-HS, TiO2-HN, TiO2-HAc, respectively. For the concentration of P123, 0, 0.1 g, 0.2 g and 0.3 g were used, which corresponds to concentration of 0, 2 mg mL -1 ,4 mg mL - 1 , and 6 mg mL -1 , respectively. For the concentration of TTIP: 0.55 mL (0.041 mol L -1 ), 1.1 mL (0.075 mol L -1 ), 1.25 mL (0.082mol L -1 ), 1.65 mL (0.10 mol L -1 ) and 2.2 mL(0.12 mol L -1 ) TTIP.

Characterizations of TiO2 nanostructures

The transmission electron microscopy (TEM) images of achieved TiO2 nanostructures were recorded on a Hitachi H600 microscope operating at a voltage of 75 kV. The high-resolution transmission electron microscopy (HRTEM) images were obtained on a FEI Talos F2005 microscope operating at 120 kV. X-ray diffraction (XRD) tests were performed on a Rigaku Nanoviewer (XRF microsourcegenerator, MicroMax007HF) with a 1200W rotating anode coupled to a confocal Max-FluxOsmic Mirror (Applied Rigaku Technologies, Austin, USA) and a MAR345 image plate detector (MARResearch, Norderstedt, Germany). The liquid crystal phenomena were observed by optical microscopy under crossed polarizers (Leica DM 2500P).

Results and discussion

Influence of the reaction temperatures

To investigate the effect of reaction temperature, the autoclaves were heated at different temperatures for 20 h. As for the synthesis at 120 o C, we are failed to obtain any deposit after the centrifugation, indicating that this temperature is too low to trigger reactions to produce TiO2 materials. At 140 o C, very thin sheet-like samples are achieved, as demonstrated in Fig. 1a. When the temperature increases up to 150 o C, larger nanosheets could be achieved (Fig. 1b). As shown in Fig. 2, the surface of the nanosheet is not smooth and wrinkled. Two sets of lattice fringes could be detected, showing two interplanar spacings of 0.32 nm and 0.35 nm, which respectively correspond to the (110) plane of rutile and (101) plane of anatase TiO2 [START_REF] Li | General strategy to synthesize uniform mesoporous TiO2/graphene/mesoporous TiO2 sandwich-like nanosheets for highly reversible lithium storage[END_REF][START_REF] Sun | Continually adjustable oriented 1D TiO2 nanostructure arrays with controlled growth of morphology and their application in dye-sensitized solar cells[END_REF]. It should be also noted that, at this temperature, the produced nanosheets are poorly crystallized with many distorted lattices. At 170 o C, the sheet-like samples start to selfassemble into 3D nanostructures (Fig. 1c). At 190 o C, great changes in morphology occurs. As shown in Fig. 1d, the product is branched-like and dotted with very tiny sheets. [START_REF] Li | General strategy to synthesize uniform mesoporous TiO2/graphene/mesoporous TiO2 sandwich-like nanosheets for highly reversible lithium storage[END_REF][START_REF] Sun | Morphologycontrolled synthesis of TiO2/MoS2 nanocomposites with enhanced visible-light photocatalytic activity[END_REF], and the latter belongs to the (110) lattice of rutile (JCPDS 21-1276) [START_REF] Dai | Preparation of Highly Crystalline TiO2 Nanostructures by Acid-assisted Hydrothermal Treatment of Hexagonalstructured Nanocrystalline Titania/Cetyltrimethyammonium Bromide Nanoskeleton[END_REF].

With increasing the temperature, the XRD peaks become sharper, confirming that the crystallinity is raised. The fraction of rutile, fr, in the final product can be calculated using the following equation. [START_REF] Wu | Microemulsionmediated hydrothermal synthesis and characterization of nanosize rutile and anatase particles[END_REF] 𝑓

𝑟 = 1.26𝐼 𝑟 (𝐼 𝑎 + 1.26𝐼 𝑟 ) ⁄ (1)
where Ir and Ia stand for the intensity of the ( 110) and ( 101 Solvothermal/hydrothermal temperature has great influence on the morphology, crystal size and catalytic activity of TiO2. [START_REF] Burungale | Studies on effect of temperature on synthesis of hierarchical TiO2 nanostructures by surfactant free single step hydrothermal route and its photoelectrochemical characterizations[END_REF][START_REF] Lopez Zavala | Synthesis of stable TiO2 nanotubes: effect of hydrothermal treatment, acid washing and annealing temperature[END_REF][START_REF] Santara | Impact of reaction temperature, stirring and cosolvent on the solvothermal synthesis of anatase TiO2 and TiO2/titanate hybrid nanostructures: Elucidating the growth mechanism[END_REF] The reaction temperature could affect the properties of the reaction solvent, such as viscosity and dielectric constant and the pressure of the reaction system, which in turn determines the growth rate/process of nanomaterial and consequently the physical and chemical properties of TiO2. Herein, the reaction solvent is composed of a large amount of EG, small amount of ethanol and water. Using chemical analysis software, we made a rough estimation on the bubbling point of the mixture, which is round 115 o C. Apparently, 120 o C is too low for the reaction to completely profit the merits of solvothermal synthesis. This is why no deposit after reaction at 120 o C was achieved. By contrast, with the increase of temperature, the reaction rate arises, and more powders could be produced. As for the nanosheet morphology, it is the P123 that plays a vital role, which will be discussed in detail in the following section. At high temperature (190 o C), the nanosheet transforms into nanorod, due to the fact that the large surface of nanosheet cannot maintain at high temperature and split into nanorod. nanostructures. In detail, as there is no acid, the product presents many aggregations (Fig. 4a). With 0.10 mol L -1 of HCl acid, small sized nanosheets are produced (Fig. 4b).

As rising to 0.15 mol L -1 , big-sized nanosheets with good dispersion can be found in Fig. 4c. With 0.28 mol L -1 , the nanosheet starts to self-assemble (Fig. 4d). With 0.42 mol L -1 or even more acids, 3D hierarchical structures composed of nanosheets are obtained (Fig. 4e-f).

The crystal structure of selected products are studied and their XRD patterns are displayed in Fig. 5. Without adding any acid, the product is basically amorphous, which is evidenced by an extremely wide peak. By contrast, with the presence of acids, the resultant products show a mixture of anatase as the major phase and rutile as the The influence of HCl acid is realized by controlling the hydrolysis and polycondensation of titanium alkoxides. [START_REF] Wu | Sol-Hydrothermal Synthesis and Hydrothermally Structural Evolution of Nanocrystal Titanium Dioxide[END_REF][START_REF] Mahshid | Synthesis of TiO2 nanoparticles by hydrolysis and peptization of titanium isopropoxide solution[END_REF] In the presence of water, TTIP can proceed the following reactions:

Ti(OR) 4 + 4H 2 O → Ti(OH) 4 + 4ROH (hydrolysis) (2) Ti(OH) 4 → TiO 2 • xH 2 O + (2 + O)H 2 O (condensation) (3)
Where R stands for iso-propyl. If no acid, the formed product via Equation ( 3) is amorphous hydrous oxide. This agrees well with the XRD results (Fig. 5). During the preparation of the mixture of Solution A and Solution B, it turns even more transparent when more acid is added. The precipitation can transform into different crystal structures and different morphologies in response to the detailed experimental conditions. Lillo-Ródenas et al. [START_REF] Cano-Casanova | One step hydrothermal synthesis of TiO2 with variable HCl concentration: Detailed characterization and photocatalytic activity in propene oxidation[END_REF] reported that the TiO2 phase composition changes with the HCl acid concentration. The percentage of anatase present a U curve with the rise of HCl acid, while the percentage of rutile and brookite behaves in an opposite way. Sakai et al. [START_REF] Dai | Preparation of Highly Crystalline TiO2 Nanostructures by Acid-assisted Hydrothermal Treatment of Hexagonalstructured Nanocrystalline Titania/Cetyltrimethyammonium Bromide Nanoskeleton[END_REF] have made similar conclusions. Our results agree partly with the above two cases, but agree well with that reported by Wu et al. [START_REF] Wu | Sol-Hydrothermal Synthesis and Hydrothermally Structural Evolution of Nanocrystal Titanium Dioxide[END_REF].

Influence of the type of acid

The type of acid can affect the microstructure of TiO2. As reported, the TiO2 colloids prepared in acetic acid contain more [101] face than that in nitric acid [START_REF] Zaban | The effect of the preparation condition of TiO2 colloids on their surface structures[END_REF].

Besides HCl acid, three other common acids: sulfuric acid, nitric acid and HAC were also used to investigate the effect of acid types. The TEM images and XRD patterns were respectively shown in Fig. 6 and Fig. 7. Great difference could be found in terms of the morphologies, as changing the acid. TiO2-HAc is a mixture composed of small nanosheets and nanorods. TiO2-HS exhibits layered structure and TiO2-HN displays a shape of tiny nanosheet. The type of acid can also influence the crystal structure of TiO2. Wu et al. [START_REF] Wu | Sol-Hydrothermal Synthesis and Hydrothermally Structural Evolution of Nanocrystal Titanium Dioxide[END_REF] reported that in the reaction system composed of titanium n-butoxide and acid under hydrothermal conditions, the product was rutile in HCl, a mixture of rutile and anatase in HNO3, anatase in H2SO4 or HAc. Khalil et al. [START_REF] Khalil | Titania nanoparticles by acidic peptization of xerogel formed by hydrolysis of titanium(IV) isopropoxide under atmospheric humidity conditions[END_REF] also made a similar conclusion that H2SO4 or HAc stabilizes anatase, while HNO3 benefits the formation of rutile. In our case, as shown in Fig. 7, H2SO4 leads to amorphous state of TiO2, HNO3 leads to anatase TiO2, and HCl or HAc results in the formation of a mixture of rutile and anatase. The diversity of these reports can be attributed to the differences and complexity of the experimental system/conditions. Fig. 7 XRD patterns of TiO2 samples prepared using different acid under 150 o C.

Influence of P123 concentration

The influence of the P123 concentration in the reaction system was also evaluated.

When there is no P123, some irregular plates or aggregation particles are formed (Fig. 8a). As introducing 2 mg mL -1 of P123, it is found that the dominated nanosheet morphology is dotted with few particles (Fig. 8b). With continuing raising P123 content up to 4 mg mL -1 , as shown in Fig. 1b, nanosheets are produced. When 6 mg mL -1 of P123 is added, the nanosheet morphology is partly damaged into uneven flakes (Fig. 8c). In the reaction system, the Ti-oligomers are confined in the inverse lamellar micelles stabilized by the surfactant P123 and co-surfactant EG [START_REF] Sun | Generalized self-assembly of scalable two-dimensional transition metal oxide nanosheets[END_REF], both of which work as the structure-directing agent. To form the lamellar micelle, it is required that the concentration of P123 should be around the critical micelle concentration (CMC).

Therefore, if the amount of P123 is too low (<0.2 g), the nanosheet cannot be obtained.

P123 plays an important role in the formation of the nanosheet structure of TiO2.

Fig. 8 TEM images of TiO2 samples prepared using different concentrations of P123:

(a) no P123, (b) 2 mg mL -1 , (c) 6 mg mL -1 . When 0.041 mol L -1 of TTIP is added, the obtained TiO2 presents a 3D hierarchical structure (Fig. 9a). 0.075-0.082mol L -1 of TTIP (Fig. 1b and Fig. 9b) leads to nanosheet morphology, and the latter nanosheet seems even more uniform. Aggregations of nanoflakes and nanowires are produced with 0.10 mol L -1 of TTIP (Fig. 9c). Finally, the long nanowires and nanoparticles dominate when 0.12 mol L -1 of TTIP is used (Fig. 9d).

Influence of the concentration of TTIP

The precursor concentration has multiply effect on the prepared TiO2. It can affect the crystal phase. According to Wu's study [START_REF] Wu | Sol-Hydrothermal Synthesis and Hydrothermally Structural Evolution of Nanocrystal Titanium Dioxide[END_REF], a lower concentration of titanium produced the sample with higher fraction of rutile phase of TiO2. As reported in literature [START_REF] Akhavan Sadr | In situ sonosynthesis of nano TiO2 on cotton fabric[END_REF][START_REF] Muniandy | Green synthesis of mesoporous anatase TiO2 nanoparticles and their photocatalytic activities[END_REF], more TTIP benefits the crystalline structure of TiO2. Djouadi et al. [START_REF] Chelbi | Effects of Ti-precursor concentration and annealing temperature on structural and morphological properties of TiO2 nano-aerogels synthesized in supercritical ethanol[END_REF] reported that the rise of sol concentration could affect the TiO2 cell parameters. The precursor concentration can also affect the morphology. Joo et al. [START_REF] Moon | Controllable one-pot synthesis of uniform colloidal TiO2 particles in a mixed solvent solution for photocatalysis[END_REF]reported that the average diameter of TiO2 particle could be adjusted from 120 nm to 900 nm by controlling the concentration of titanium butoxide . Besides shape, the surface area and pore volume can be modified by the precursor concentration. [START_REF] Chelbi | Effects of Ti-precursor concentration and annealing temperature on structural and morphological properties of TiO2 nano-aerogels synthesized in supercritical ethanol[END_REF] In our case, with the variation of TTIP concentration, great changes occurs in the morphology. The reaction temperature and time is the same for these samples, therefore, such change in shape can only be induced by the kinetics. As aforementioned in Section 3.2, when different concentrations of TTIP is introduced, the reaction (2, 3) could undergo at different rates. With less TTIP, the reaction rate might be slow, and with the help of P123, the nanosheet morphology can be maintained. With more TTIP, in one aspect, the reaction rate might be fast , and hydrolysis-condensation could occur in preferential growth direction, forming nanowires [START_REF] Cozzoli | Low-temperature synthesis of soluble and processable organic-capped anatase TiO2 nanorods[END_REF]. In the other aspect, more nuclei breaks the balance of the P123 micelle, induces the aggregative growth of TiO2 [START_REF] Wang | Kinetics and Mechanisms of Aggregative Nanocrystal Growth[END_REF], and leads to the aggregation or transformation from nanosheet to nanowire. To gain a better understanding of the formation process of the nanosheet structure, time-dependent experiments were also performed. The shape evolution was studied by TEM (Fig. 10). The tiny nanosheet product forms within 4 h. With another 4 h, the tiny nanosheet continues to grow into big nanosheet. From 12 h until 24 h, there is little change in the morphology, indicating that the dominate parameters that affect the shape is the self-assembly with the assistance of P123 and EG. With up to 30 h, the big nanosheet disappears, which is decomposed/dissolved into small particles with lateral size of about 11 nm. This might be caused by the high surface energy of the large surface area at high temperate for long time. In general, the morphology of TiO2 changes from tiny/thin nanosheet to big/thick nanosheet, to curved nanosheet, and finally to small nanoparticles with prolonging the reaction time.

Influence of the reaction time

The formation of the tiny nanosheet at the initial stage confirms the key role of P123 surfactant. It is with the help of P123 that the TiO2 nucleation can grow with the pattern of orientation and consequently form nanosheets [START_REF] Hu | Controllable synthesis and formation mechanism of 3D flower-like TiO2 microspheres[END_REF]. Principally, relatively larger nanosheet could be formed based on the growth and assembly of primary tiny nanosheets by reducing their total surface energy. At final stage, the nanosheet is too huge to maintain its morphology. Again, to maintain the minimum energy of the reaction system, thick/curved nanosheet dissolved. Based on the above analysis, it can be concluded that the whole synthesis process of TiO2 nanosheets includes selfassembly, orientation growth, and Ostwald ripening [START_REF] Tsai | Effects of size and shell thickness of TiO2 hierarchical hollow spheres on photocatalytic behavior: An experimental and theoretical study[END_REF].

Brief summary of preparation

Based on the above experimental results, we could obtain TiO2 with adjustable crystal phase and morphology in the reaction system composed of titanium isopropoxide as titanium source, P123 as structure-directing agent, ethylene glycol as co-surfactant and solvent under different solvothermal conditions. From the aspect of crystal structure, all the synthesized samples have a mixed phase of rutile and anatase.

Their difference lies in the variation of molar ratio of the two phases, which is determined by the reaction temperature and the amount of acid. As photo-catalyst, the mixed phase of TiO2 gives rise to a better activity than single phase. From the point of crystallinity, the prepared TiO2 showed relatively low crystallinity according to the XRD patterns, indicating that they might have oxygen vacancies. In terms of the morphology, we can control the dimension of TiO2 from 2D to 3D simply by changing the concentration of HCl acid. The best 3D TiO2 is the one obtained with the HCl acid concentration between 0.28 mol L -1 and 0.42 mol L -1 . In some cases, the 3D TiO2 constructed using nanosheets as units can exhibit superior structure stability. We can also adjust the size of the TiO2 nanosheet, some are very tiny (the sample prepared at 150 o C for 4 h), some are very large (the sample prepared at 150 o C for 20 h). The samples in Fig. 1b and Fig. 9b are the best nanosheets with proper size. The richness of the products under different conditions endows them good candidates for various target applications. Therefore, our results provide good reference for the future controlled synthesis of TiO2 nanostructures.

Formation of liquid crystal phase

The liquid crystal (LC) phase based on inorganic material especially metal oxides is an interesting topic. The phenomena of liquid crystal dates back to the last century.

From the aspect of shape, up to now, nanorod (Fe3O4 [START_REF] Constantin | Lyotropic lamellar phase doped with a nematic phase of magnetic nanorods[END_REF][START_REF] Klöckner | Liquid crystalline phases from polymer functionalized ferri-magnetic Fe3O4 nanorods[END_REF], FeOOH [START_REF] Wang | Magnetically actuated liquid crystals[END_REF][START_REF] Vroege | Smectic liquid-crystalline order in suspensions of highly polydisperse goethite nanorods[END_REF], SiO2 [START_REF] Xu | Liquid crystalline phase behavior of silica nanorods in dimethyl sulfoxide and water[END_REF],

TiO2 [START_REF] Kanie | Organic-Inorganic Hybrid Liquid Crystals: Hybridization of Calamitic Liquid-Crystalline Amines with Monodispersed Anisotropic TiO2 Nanoparticles[END_REF][START_REF] Meuer | Liquid Crystals from Polymer-Functionalized TiO2 Nanorod Mesogens[END_REF][START_REF] Dessombz | Design of liquid-crystalline aqueous suspensions of rutile nanorods: evidence of anisotropic photocatalytic properties[END_REF], ZnO [START_REF] Kubo | Surface-assisted unidirectional orientation of ZnO nanorods hybridized with nematic liquid crystals[END_REF][START_REF] Srikantharajah | From evaporation-induced self-assembly to shear-induced alignment[END_REF]) and platelet (clay [START_REF] Hemmen | The Isotropic-Nematic Interface in Suspensions of Na-Fluorohectorite Synthetic Clay[END_REF][START_REF] Paineau | Liquid-crystalline nematic phase in aqueous suspensions of a disk-shaped natural beidellite clay[END_REF][START_REF] Gabriel | Observation of Nematic Liquid-Crystal Textures in Aqueous Gels of Smectite Clays[END_REF], Al(OH)3 [START_REF] Verhoeff | Liquid crystal phase transitions in systems of colloidal platelets with bimodal shape distribution[END_REF][START_REF] Kleshchanok | Lyotropic smectic B phase formed in suspensions of charged colloidal platelets[END_REF][START_REF] Ohno | Polymer-brush-decorated colloidal platelets: precision synthesis and self-assembly[END_REF], Ni(OH)2 [START_REF] Qazi | Alignment of dispersions of plate-like colloidal particles of Ni(OH)2 induced by elongational flow, The journal of physical chemistry[END_REF][START_REF] Brown | Ordered phase of platelike particles in concentrated dispersions[END_REF],

niobate [START_REF] Miyamoto | Liquid Crystalline Nature of K4Nb6O17 Nanosheet Sols and Their Macroscopic Alignment[END_REF][START_REF] Nakato | Electrooptic Response of Colloidal Liquid Crystals of Inorganic Oxide Nanosheets Prepared by Exfoliation of a Layered Niobate[END_REF]) are the most reported building blocks for LC. Compared with nanoplatelet, the nanosheet has even larger aspect ratios. Therefore, we made a preliminary study on the LC formed by the prepared TiO2 nanosheet. The sample was first under ultrasonic in a bath-type instrument for 5 min, then centrifuged at 3000 rpm for 5 min to remove the bottom precipitate. The supernatant was centrifuged again at 8500 rpm for 30 min to obtain the precipitation. The morphology was observed by TEM as shown in Fig. 11a-c. From the image, we can clearly see the uniform thin film assembled by the TiO2 nanosheet, which is different to the morphology before acid treatment (Fig. 9b). As demonstrated by the HRTEM image (Fig. 11d), the anatase and rutile phase are observed.

Conclusion

Through rational design, a series of TiO2 nanostructures with continually adjustable shape, size, and crystal structures were synthesized via solvothermal process. The crystalline phases and morphologies of TiO2 were proved to be affected by reaction temperature/time, the amount of acid, the type of acid, the amount of P123 and TTIP. The variation of the experimental conditions can lead to significant change in the morphology, dimensionalities and crystal phase of TiO2. The nanosheet TiO2 was successfully fabricated, and further treated using nitric acid to form LC phase.

This family of well-controlled TiO2 nanosheet has potential to be used as dielectric filler in polymer composites, or as photocatalyst/heterocatalyst.

Fig. 1

 1 Fig. 1 TEM images of TiO2 samples at different reaction temperatures.

Fig. 2

 2 Fig. 2 HRTEM images of TiO2 prepared at 150 o C.

  ) reflections. The variation of fr as a function of temperature follows the order of 0.30 (TiO2-140 o C) > 0.24 (TiO2-150 o C) > 0.15 (TiO2-170 o C), indicating that the reaction temperature can indeed affect the crystal structures of TiO2.

Fig. 3

 3 Fig.3 XRD patterns of TiO2 samples achieved at different reaction temperatures.

[22] 3 . 2 4 .

 324 Fig. 4 TEM images of TiO2 samples prepared using different concentrations of HCl acid: (a) no acid; (b) 0.10 mol L -1 ; (c) 0.15 mol L -1 ; (d) 0.28 mol L -1 ; (e) 0.42 mol L -1 ; (f) 0.53mol L -1 .

Fig. 5

 5 Fig.5 XRD patterns of TiO2 samples achieved with different concentrations of HCl acid.

Fig. 6

 6 Fig.6 TEM images of TiO2 samples prepared using different acids at 150 o C.

Fig. 9

 9 Fig. 9 TEM images of TiO2 samples prepared with different concentrations of TTIP: (a), 0.041 mol L -1 ; (b) 0.082mol L -1 ; (c) 0.10 mol L -1 ; (d) 0.12 mol L -1 .

Fig. 10

 10 Fig.10 TEM images of TiO2 samples prepared during different reaction time at 150 o C.

Fig. 11 (

 11 Fig.11 (a-c) TEM and (d) HRTEM images of TiO2 nanosheet used for liquid crystal formation study.

Fig. 12

 12 Fig.12 Optical images between crossed polarizers with magnification of (a) 4, (b)10, and (c) 40.

Acknowledgements

This work was partially funded by the CNRS Energy unit (Cellule Energie) through the project MicroPower. Ming Sun would personally thank the financial support from Chinese Scholarship Council (201808440556).


R Seghir
email: rian.seghir@iemn.univ-lille1.fr

S Arscott
email: steve.arscott@iemn.univ-lille1.fr

Extended PDMS stiffness range for flexible systems

Keywords: polydimethylsiloxane, process parameters, mechanical properties, mixing ratio, curing, wetting contact angle, rutpure

The use of polymers in the context of flexible systems such as flexible sensors leads to an incompatibility issue: On one hand, the flexibilty of the polymer must not be to the detriment of the fabrication process, e.g. excessive thermal expansion leading to process failure and on the other hand, certain applications will require high flexibility and also a specific mechanical stiffness, e.g. artificial skin, smart clothes, flexible screen . . . In other words, a compromise is necessary between rigidy for processing and controlled flexibility for applications. In this context it is crutial to be able to tune the mechanical properties of such polymers. Polydimethylsiloxane (PDMS) is a very versatile and useful soft polymeric material -Elastic modulus typically ≈1 MPa. This paper investigates the stiffness tunability of PDMS by varying the hardening agent to PDMS base ratio over 19:1 to 2:1, and using two extreme curing processes, i.e. 120 minutes at 100 o C and 2 days at 165 o C. It was observed that the stiffness of PDMS can be accuratly controlled from 800 kPa to 10 MPa with a rupture limit higher than 20 %. To our knowlegde this is the highest reported elastic modulus in PDMS by combining mixing ratio and curing temperature. The impact of such a stiffness variation on potential functional properties such as the rupture limit, Poisson's ratio and material's wetting contact angle is also analysed. We observe that the wetting contact angle depends on the bulk mechanical properties of the PDMS. The observations will be of use to all technological communities who are engaged in using PDMS-type polymers for their specific applications.

Introduction

Although the first organosilicon was first synthesized over one hundred years ago [1], it is only in the last 20 years that polymeric organosilicon compounds -such as polydimethylsiloxane (PDMS) -have found applications in areas such as flexible electronics [START_REF] Lacour | Mechanisms of reversible stretchability of thin metal films on elastomeric substrates[END_REF][START_REF] Kim | Large-scale pattern growth of graphene films for stretchable transparent electrodes[END_REF][START_REF] Hocheng | Study of novel electrical routing and integrated packaging on bio-compatible flexible substrates[END_REF][START_REF] Nathan | Flexible electronics: the next ubiquitous platform[END_REF][START_REF] Romeo | Elastomeric substrates with embedded stiff platforms for stretchable electronics[END_REF], moulding and soft lithography [START_REF] Kumar | Features of gold having micrometer to centimeter dimensions can be formed through a combination of stamping with an elastomeric stamp and an alkanethiol ink followed by chemical etching[END_REF][START_REF] Jackman | Fabrication of submicrometer features on curved substrates by microcontact printing[END_REF][START_REF] Xia | Soft lithography[END_REF][START_REF] Schmid | Siloxane polymers for high-resolution, highaccuracy soft lithography[END_REF][START_REF] Choi | A photocurable poly (dimethylsiloxane) chemistry designed for soft lithographic molding and printing in the nanometer regime[END_REF][START_REF] Barbero | High-resolution nanoimprinting with a robust and reusable polymer mold[END_REF][START_REF] Jothimuthu | Photodefinable pdms thin films for microfabrication applications[END_REF][START_REF] Hsieh | A soft photo-mask with embedded carbon black and its application in contact photolithography[END_REF], microelectromechanical systems (MEMS) [START_REF] Takao | A mems microvalve with pdms diaphragm and two-chamber configuration of thermo-pneumatic actuator for integrated blood test system on silicon[END_REF][START_REF] Schneider | Mechanical properties of silicones for mems[END_REF][START_REF] Schneider | Process and material properties of polydimethylsiloxane (pdms) for optical mems[END_REF], self-healing materials [START_REF] Cho | Polydimethylsiloxane-based self-healing materials[END_REF], laboratoryon-a-chip and microfluidics [START_REF] Jo | Threedimensional micro-channel fabrication in polydimethylsiloxane (pdms) elastomer[END_REF][START_REF] Eddington | An organic selfregulating microfluidic system[END_REF][START_REF] Mata | Characterization of polydimethylsiloxane (pdms) properties for biomedical micro/nanosystems[END_REF][START_REF] Bhagat | Photodefinable polydimethylsiloxane (pdms) for rapid lab-on-a-chip prototyping[END_REF][START_REF] Kim | Solventresistant pdms microfluidic devices with hybrid inorganic/organic polymer coatings[END_REF]. The physical and chemical properties of PDMS, such as its usability over a wide temperature range (T ∈ [-100,200] o C), its flexibility (Elastic modulus E ≈ 1 MPa [START_REF] Lötters | The mechanical properties of the rubber elastic polymer polydimethylsiloxane for sensor applications[END_REF]), its low chemical reactivity as well as its relativelly hydrophic behavior, its transparent nature, its biocompatibility [START_REF] Peterson | Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidics devices: Cell culture and flow studies with glial cells[END_REF], its low cost. . . have made it useful in such a wide range of application fields. In such a context a clear understanding of its mechanical behavior and its ability to reach specifics properties, for example a specific rigidity useful for a specific application can be of primary importance. Much work has been done into the investigation of the mechanical properties of PDMS and their potential tunability [START_REF] Lötters | The mechanical properties of the rubber elastic polymer polydimethylsiloxane for sensor applications[END_REF][START_REF] Eddington | Development of process protocols to fine tune polydimethylsiloxane material properties[END_REF][START_REF] Mata | Characterization of polydimethylsiloxane (pdms) properties for biomedical micro/nanosystems[END_REF][START_REF] Wilder | Measuring the modulus of soft polymer networks via a buckling-based metrology[END_REF][START_REF] Fuard | Optimization of poly-di-methyl-siloxane (pdms) substrates for studying cellular adhesion and motility[END_REF][START_REF] Schneider | Mechanical properties of silicones for mems[END_REF][START_REF] Khanafer | Effects of strain rate, mixing ratio, and stress-strain definition on the mechanical behavior of the polydimethylsiloxane (pdms) material as related to its biological applications[END_REF][START_REF] Liu | Thickness-dependent mechanical properties of polydimethylsiloxane membranes[END_REF][START_REF] Schneider | Process and material properties of polydimethylsiloxane (pdms) for optical mems[END_REF][START_REF] Cheng | Note: Mechanical study of micromachined polydimethylsiloxane elastic microposts[END_REF][START_REF] Hocheng | Study of novel electrical routing and integrated packaging on bio-compatible flexible substrates[END_REF][START_REF] Wang | Polydimethylsiloxane mechanical properties measured by macroscopic compression and nanoindentation techniques[END_REF][START_REF] Crevoisier | Volume changes in a filled elastomer studied via digital image correlation[END_REF][START_REF] Yoo | Direct physical exfoliation of few-layer graphene from graphite grown on a nickel foil using polydimethylsiloxane with tunable elasticity and adhesion[END_REF][START_REF] Johnston | Mechanical characterization of bulk sylgard 184 for microfluidics and microengineering[END_REF]. The largest observed tensile stiffness, using 10:1 PDMS, to date is less than 4 MPa [START_REF] Fuard | Optimization of poly-di-methyl-siloxane (pdms) substrates for studying cellular adhesion and motility[END_REF] after 7 days post-baking at 100 o C and less than 3 MPa for post-baking at 200 o C during 18 minutes [START_REF] Johnston | Mechanical characterization of bulk sylgard 184 for microfluidics and microengineering[END_REF]. It is known that the stiffness of PDMS can be tuned by modifying three main parameters: (1) the cross-linker agent concentration, (2) the curing temperature and (3) the curing time. Other parameters also affect stiffness, e.g. film thickness [START_REF] Liu | Thickness-dependent mechanical properties of polydimethylsiloxane membranes[END_REF], micromachined object dimensions [START_REF] Cheng | Note: Mechanical study of micromachined polydimethylsiloxane elastic microposts[END_REF] and the loading strain rate [START_REF] Schneider | Mechanical properties of silicones for mems[END_REF]. Nervetheless the major part of these studies have focused only on a specific parameter at one time, frequently in a limited range and no work has been done to harden PDMS material beyond the classical rigidity range, i.e. [0.1 -4] MPa [START_REF] Fuard | Optimization of poly-di-methyl-siloxane (pdms) substrates for studying cellular adhesion and motility[END_REF], that would increase the applications spectrum of the material. As a consequence it remains very difficult to find a complete study where fabrication recipies and mechanical properties, over a large domain of applications, are brought together. In this way, we propose here to investigate a large range of cross-linker agent concentrations, from 5 % to 33 %, meaning mixing ratios by weight ranging from 19:1 to 2:1 and two curing processes, i.e. 2h at 100 o C and 48h at 165 o C including different curing methods: conventional oven and hot plate. In the first part of the paper, the fabrication process is presented, then uniaxial mechanical tests are detailed and elastic modulus, rupture strain as well as Poisson's ratio are presented. Finally the impact of the stiffness on the wetting contact angle is also discussed.

Material and methods

Fabrication

All chemicals used in this work were used unmodified and off-the-shelf. All processing was performed in a class ISO 5/7 cleanroom. PDMS was purchased from Dow Corning Corporation, USA as a two liquid component kit, Sylgard R 184 Elastomer, containing the vinyl-terminated base and the curing agent (methyl hydrogen siloxane). We define the PDMS mixtures as the ratio A:B where A is the vinyl-terminated base weight content and B is the cross-linker agent weight content. PDMS mixtures were first prepared by mixing the base and the curing agent at the following mixing ratios by weight: 19:1, 10:1, 9:1, 8:1, 7:1, 4:1 and 2:1 in order to understand the mechanical behavior of low (5 %) to high (33 %) curing agent fraction. Note that the Sylgard R 184 data-sheet [START_REF] Corning | Sylgard R 184 silicone elastomer kit web page[END_REF] advises producing PDMS following a "by weight" ratio of 10:1 (i.e. 9 % of curing agent). Commercial 8.5x15 cm Teflon R oven dishes were initially cleaned using acetone, IPA and deionized water followed by a dehydration bake. Mixtures were then carefully poured into the dishes using a specific volume of the mixture in order to form 1 mm thick uniform films. Prior to moulding, the preparation involves mixing two liquids of different viscosities -5x10 -3 m 2 s -1 (vinyl-terminated base) and 1.1x10 -4 m 2 s -1 (curing agent) which incurs the formation of trapped bubbles. Thus, the samples were finally degassed (5 pumping cycles in a lowvacuum chamber) in order to remove the air bubbles formed during mixing. The prepared PDMS mixtures were then placed onto a level hotplate during 2 hours at 100 o C. We refer to this first step heating process as "curing" subsequent heat treatments are refereed to as "post-curing. Note that it was not known exactly how the increase of curing agent could affect the curing process, thus we decided to increase the curing time, compared to data-sheet information (e.g. 45 minutes at 100 o C [START_REF] Corning | Sylgard R 184 silicone elastomer kit web page[END_REF]), such that every sample was well cured. Half of each of the resulting PDMS films was then post-cured, in a conventional oven at 165 o C during 48 hours. The objective of the last curing step is to understand the impact of an upper curing time limit on material stiffness and rupture elongation while simply cured samples constitute our reference base. Finally, flat 4 mm wide, 60 mm long and 1 mm thick tensile bone shaped samples are produced from the PDMS films using a specially made dogbone sample cutter. Figure 1 shows the bone samples and the dimensions and table 1 We will focus, in the mechanical measurements part of this work, on the measurement of the initial tangent (elastic) modulus, the Poisson's ratio and the rupture elongation. To achieve such a mechanical investigation, increasing cyclic tests has been preferred to monotonic tensile ones since they allow one to identify material hysteresis due, in part, to viscosity [START_REF] Brieu | Material directional based constitutive law for mullins induced anisotropy[END_REF]. This is crucial complementary information towards an understanding of how manufacturing processes affect the sample's mechanical behavior. Thus, PDMS samples have been submitted to increasing cyclic loadings from 0 N to 5 %, 10 %, 15 %, 20 %, 30 %, 35 %, 40 %, 60 % of uniaxial strain, finally up to rupture (see figure 2). Tests were conducted on an Instron 5882 uniaxial testing machine, using a 2 kN cell-force at a constant crosshead speed of 0.5 mms -1 which corresponds to an average strain rate close to 10 -2 s -1 (see figure 2). Notice that the choice of such a strain rate, in the identification of material properties, has been validated by the lack of any hysteresis in the cyclic PDMS behavior (see figure 3). Such cyclic response commonly reveals that the material is not significantly affected by viscosity at such strain rate [START_REF] Brieu | Material directional based constitutive law for mullins induced anisotropy[END_REF].

Then, we note the nominal stress Σ = F S 0 with F the force measured by the cell and S 0 being the initial sample cross-section. The sample thicknesses are measured using a Vernier calliper -this introduces a small measurement uncertainty, of the order of 10 -1 mm, due to sample stiffness variations; implying an uncertainty on the order of 0.25 N on the stress estimation.

Strains are measured, within the sample gauge section (see figure 1), by video extensometry. Four painted droplets are put on the sample surface, two for axial displacement and two Notice that although the Poisson's ratio is generally assumed to be constant, near to 0.5 (incompressibility), some research has shown that volume changes occur within elastometric materials during deformation processes and are attributed to damage mechanisms [START_REF] Jones | Dilatometer studies of pigment-rubber systems[END_REF][START_REF] Ramier | In situ sals and volume variation measurements during deformation of treated silica filled sbr[END_REF][START_REF] Crevoisier | Volume changes in a filled elastomer studied via digital image correlation[END_REF][START_REF] Merckel | Effects of the amount of fillers and of the crosslink density on the mechanical behavior of carbonblack filled styrene butadiene rubbers[END_REF] -so the Poisson's ratio decreases with deformation, depending on the damage. Thus, for the sake of clarity we define the initial and the ruptured Poisson's ratios, meaning the undamaged and damage ratio.

As the linear and reversible domain is very different from one sample to another, and the domain size decrease as the material stiffness increases, a systematic and clear definition of the tangent (elastic) modulus is problematic. One should note at this point that, unlike for metals, it is not pertinent to speak of "Young's modulus" in the context of hyperelasticity. One must refer rather to tangent modulus which is defined as the slope of the stress-strain curve which can evolve with strain. Here we define the tangent modulus (E) as the rigidity of the material at low strain. Thus, one can employ the hyperelastic theory and invoke a Mooney-Rivlin model [START_REF] Mooney | A theory of large elastic deformation[END_REF][START_REF] Ogden | Non-linear elastic deformations[END_REF] to analytically estimate the initial tangent modulus. We can derive the nominal stress from the generalized Mooney-Rivlin strain energy potential (see equation 1).

W = n i, j=0 C i j (I 1 -3) i (I 2 -3) j - 1 2 k (I 3 -3) 2 (1)
with

I 1 = λ 2 y + λ 2 x + λ 2 z , I 2 = λ 2 y λ 2 x + λ 2 y λ 2 z + λ 2
x λ 2 z and I 3 = det F the strain invariants, (x; y; z) the transversal, the axial and the orthogonal space directions respectively, F is the deformation gradient, λ i is the strain ratios, n is the order of the Mooney-Rivlin model, k is the bulk modulus and C i j is the hyperelastic constants. Assuming the uniaxiality of the strains (λ y = λ, λ

x = λ z = 1 √ λ )
, the incompressibility (I 3 = 1), and a 2 nd order Mooney-Rivlin model (n = 2 (see equation 1)) one obtains :

W = C 10 (I 1 -3) + C 01 (I 2 -3) + C 20 (I 1 -3) 2 + C 02 (I 2 -3) 2 + C 11 (I 1 -3) (I 2 -3) (2)
One notes Σ = ∂W ∂λ , so :

Σ = 2 C 10 λ - 1 λ 2 + C 01 1 - 1 λ 3 + 2C 20 λ - 1 λ 2 λ 2 + 2 λ - 3
+ 2C 02 1 - 1 λ 3 2λ + 1 λ 2 -3 +3C 11 λ 2 - 1 λ 4 -λ - 1 λ 2 -1 - 1 λ 3 (3)
with λ = ε yy + 1. In equation 3, Σ is linearly dependant on the following vector α i = C 10 C 01 C 20 C 02 C 11 and the associated shape functions:

N i = 2                         λ -1 λ 2 1 -1 λ 3 2 λ -1 λ 2 λ 2 + 2 λ -3 2 1 -1 λ 3 2λ + 1 λ 2 -3 3 λ 2 -1 λ 4 -λ -1 λ 2 -1 -1 λ 3                        
Thus the identification of hyperelastic parameters can easily be done through the classical Least Square Method as follows:

                 Φ = 1 m m k=0 Σ exp (λ) -α i N i (λ) 2 ∂Φ ∂α i = 0 (4)
with Φ the cost function to minimize, Σ exp (λ) the experimental stress value at elongation λ, Σ = α i N i (λ) the modelled stress in Einstein's notation and m the number of experimental data points. Figures 3 shows a stress-strain fit superimposed on the experimental values -this validates the use of the Mooney-Rivlin potential. Finally, one can simply define the initial (E) stiffness as follows:

E = lim λ→1 ∂Σ ∂λ = lim λ→1 2 C 10 1 + 2 λ 3 + 3C 01 λ 6 + 2C 20 3λ 2 -3 - 6 λ 3 + 6 λ 4 + 2C 02 2 + 2 λ 3 - 9 λ 4 + 5 λ 6 +3C 11 2λ -1 - 2 λ 3 + 4 λ 5 - 3 λ 4 = 6C 10 (5)
In addition to the mechanical testing, wetting contact angle measurements were performed on the different samples. Deionized water droplets having a typical diameter of 1 mm were deposited on each sample surface in order to investigate how a change of the material's composition affects the droplet contact angle between PDMS and liquid. The wetting contact angles were measured using a contact angle meter in a humidity/temperature controlled cleanroom environment. These tests have important implications if the materials are used for specific microfluidic applications for example. It is well known that the wetting contact angle of a liquid on PDMS can be modified surface chemistry, e.g. using a plasma [START_REF] Bhattacharya | Studies on surface wettability of poly (dimethyl) siloxane (pdms) and glass under oxygen-plasma treatment and correlation with bond strength, Microelectromechanical Systems[END_REF][START_REF] Tsougeni | Control of nanotexture and wetting properties of polydimethylsiloxane from very hydrophobic to super-hydrophobic by plasma processing[END_REF] and chemical immersion [START_REF] Mata | Characterization of polydimethylsiloxane (pdms) properties for biomedical micro/nanosystems[END_REF]. As we will see we show here that the wetting contact angle of water depends on the bulk mechanical properties of the PDMS -without modification of surface.

Results and Discussion

Mechanical characterization

According to the PDMS Sylgard R 184 datasheet [START_REF] Corning | Sylgard R 184 silicone elastomer kit web page[END_REF] and [START_REF] Eddington | Development of process protocols to fine tune polydimethylsiloxane material properties[END_REF][START_REF] Fuard | Optimization of poly-di-methyl-siloxane (pdms) substrates for studying cellular adhesion and motility[END_REF], where a PDMS baking time study has shown that the PDMS stiffness saturates for a curing time over 2 days at 100 o C, one assumes that both curing processes (detailed in section 1.1) constitute a lower and an upper limit of reachable PDMS stiffness. Within these curing limits, some identically made sample have been tested (see table 1) and allow us to present average trends and standard deviations.

Let us first deal with an analysis of the resulting stress-strain curves. Figure 3 presents experimental stress-strain curves obtained on the PDMS samples, including different cross-linker agent concentrations, from 5 to 33 %, i.e. ratios of 19:1 to 2:1, and different curing process -2h at 100 o C (curing) and 48h at 160 o C (post-curing). Additional information is also shown: the rupture strain and the Mooney-Rivlin fit (see equation 4). Loading strains and stresses reach 95 % and 10 MPa respectively. As already stated, one observes a very large range of behavior: from elastic-linear behavior, on the cured 19:1 sample, to a hyperelastic one -for example on the cured 4:1 sample and even quasi-elastic-linear and "brittle" one (up to 20 %) on post-cured 2:1 sample. In parallel, one observes a systematic sample hardening from cured (black in figure 3) to post-cured (orange in figure 3) samples. This phenomenon is reflected in a systematic increase of low strain and high strain stiffness and is a function of cross-linker agent concentration. Indeed, one clearly observes how the 19:1 and 10:1 samples slightly harden compared to the 4:1 and 2:1 ones where curing time and temperature drastically affect the mechanical response. Figure 3 emphases notably the mechanical stability as a function of temperature and the great stretchability of samples at the classic 10:1 mixing ratio. Let us now discuss, in a more detailed way, the low strain stiffness (tangent modulus) and rupture limit variations. 1). One observes on cured samples (O), that sample stiffness ranges from 800 KPa at 5 % curing agent, to 2.4 MPa at 11 % curing agent and then falls to 900 KPa at 33 % curing agent. An identical trend and levels could be seen in part in [START_REF] Hocheng | Study of novel electrical routing and integrated packaging on bio-compatible flexible substrates[END_REF] where the influence of some mixing ratios (6:1, 8:1, 10:1, 12:1) on PDMS stiffness have been studied. this confirms, for both baking time and temperatures lower than 8h and 150 o C respectively, the existence of a stiffness maximum for 8:1 materials while other mixing ratios invariably lead to softer materials. The following results (see figure 4) extend such observations from 5 % to 33 % curing agent which is quite interesting with regards to the post-cured behavior. One observes on post-cured samples ( ), a quasi-identical trend, slightly stiffer, from 5 to 11 % of curing agent. Then stiffness increase up to 10 MPa at 33 % curing agent -to our knowlegde this is the highest reported elastic modulus in PDMS by combining mixing ratio and curing temperature [START_REF] Fuard | Optimization of poly-di-methyl-siloxane (pdms) substrates for studying cellular adhesion and motility[END_REF]. Thus, one observes on one hand, that the post-curing process does not significantly affect the PDMS stiffness up to a cross-linker agent concentration near to 10 %, and on the other hand, that it reverses the trend over this value. Note on figure 4 the largest difference in stiffness between cured and post-cured occurs in the 2:1 material -thus this mixture enable the user to explore the whole range of stiffnesses using a single mixture over the temperature range study. Thus, the tangent modulus could be increased by a factor higher than 10 by modifying the post-curing time and mixing ratios. Obviously such an increase of the material stiffness drastically affects the rupture strain -let us now discusses this point.

Figure 5 presents the sample rupture strain (ε R ), defined as the ultimate strain before failure, as a function of cross-linker agent concentration for both curing and post-curing processes. It is well known that the mechanical failure depends primarily on the surface state of the material -especially edge regions where crack initiation sites could be introduced depending on sample cutter quality. This explains notably the increase of scatter in the results shown by the standard deviation error bars. Thus absolute values must be regarded carefully in favour of trend including errors bars. Indeed, as the same cutter tool has been used for the whole set of samples one can assume that results obtained from all samples can be realiably compared and can provide realistic failure trends. Results are clearly in line with previous observations done on PDMS stiffness (see figure 4). If we compare figure 4 and figure 5 we can see that there is a certain symmetry in the results. When the stiffness increases, the rupture strain drops and conversely, when the stiffness decreases the rupture strain rises. Considering cured samples, the rupture strain ranges from 70 % to 110 % with a minimum reached for 8:1 samples. Notice that the same behavior symmetry, between stiffness and rupture, is also observed for post-cured ( ) samplessee figure 5. For both cured and post-cured samples one can observes that at low cross-linker agent concentration (<12.5%) the rupture trends are quite similar. The rupture strain value falls down up to 8:1 samples (ε R ∈ [55 -70]). However, when the cross-linker concentration is greater than 12.5 % (up to 33 %) the rupture strain behavior diverges considerably -see figure 5. Rupture strain of the cured samples (O) increases up to a plateau at 95 % while, for post-cured samples, it falls to 20 %. To complete the analysis of material stiffness and rupture, figure 6 presents the evolution of PDMS rupture as a function of stiffness for both, cured and post-cured samples. A 2 nd order polynomial fit is also superimposed and underlines the previously observed relationship between, cured or post-cured material stiffness and associated rupture strains. Irrespective of the curing time and temperature (within the limits studied here) one observes that the rupture of samples follows a polynomial digressive law -identified, by Least Square Method, as: ε R (E) = 103 ±12 -13.5E + 0.5E 2 . The following relation and figure 6 are particularly interesting for applications where both high rigidity and good flexibility are required since they clearly evidence the limits of such pdms mixtures. One observes that the initial Poisson's ratio remains almost unchanged whatever the curing process and mixing ratio with a mean value of 0.5±0.05 as classically shown in the literature. Secondly, one observes a fall of the Poisson's ratio up to rupture. Table 2 presents, with more details, the change of the Poisson's ratio (in %). The results are quite similar for all samples, with a mean value of -48 % and a standard deviation of 7 %, and its physical origin is probably the material cavitation leading to volume changes [START_REF] Merckel | Effects of the amount of fillers and of the crosslink density on the mechanical behavior of carbonblack filled styrene butadiene rubbers[END_REF]. Nevertheless, one observes that post-cured samples are systematically less sensible to this phenomenon. According to the fact that these materials are stiffer, especially the 4:1 (Cross-linker: 20 %) and 2:1 (Cross-linker: 33 %) ratios, one could speculate that the increase of material rigidity limits the cavitation phenomenon.

Wetting contact angle

Table 3 and figure 8 presents results of wetting contact angle analysis done on PDMS samples as well as experimental pictures of droplets in both, lower and higher, wetting conditions. Thus, by not considering these points one can see a relationship between cross-linker agent concentration and surface contact angle on both, cured (O) and post-cured samples ( ). Considering first, post-cured samples, one observes a monotonic trend. Indeed, when cross-linker agent concentration increases, and thus material stiffness increases too, one observes a decrease of the wetting contact angle from 109.6 o to 96.75 o (see table 3), i.e. a decrease of 12 %. Considering cured samples (O), one observes an identical trend from 5 to 12.5 % of cross-linker agent concentration, meaning from 19:1 to 7:1 mixing ratios. Nevertheless, over 12.5 % of cross-linker agent concentration the wetting contact angle increases to 111.6 o . A previous study [START_REF] Brown | Evaluation of polydimethylsiloxane scaffolds with physiologically-relevant elastic moduli: interplay of substrate mechanics and surface chemistry effects on vascular smooth muscle cell response[END_REF] considering mixing ratios from 50:1 to 10:1 has demonstrated that the wetting contact angle changes very little -in good agreement with our results (see figure 8) for all samples (cured and post-cured) having a cross-linker agent concentration lower than 9% (PDMS 10:1). On the other hand, it is also shown in [START_REF] Mata | Characterization of polydimethylsiloxane (pdms) properties for biomedical micro/nanosystems[END_REF] that the wetting contact angle changes very little considering mixing ratios from 19:1 to near 1:1. These results are explanable with reference to the figure 4 -in the absence of high temperature curing step, the stiffness remains virtualy the same and relatively small over the whole range of mixtures, whereas a high temperature curing step leads to a significant increase of the stiffness for cross-linker agent concentration over 12.5% (PDMS 7:1). Our work here implies that in order to observe a significant change in the wetting contact angle, mixing ratios from 10:1 to 2:1 -and even greater -are necessary in addition to a high temperature/long time curing step. Considering our results it is interesting to notice that such an increase of contact angle is totally in keeping with the fall of material stiffness (cured samples) between 7:1 and 4:1 mixing ratios observed on figure 4. Secondly, the occurrence of a contact angle deviation between cured and post-cured samples is also in line with observation done on material stiffness. Indeed, over 12.5 % of cross-linker agent concentration (PDMS 7:1) the stiffness of cured samples (O) falls while the contact angle rises and inversely, the stiffness of post-cured samples ( ) rises while the contact angle drops. Such comparisons validate contact angle observations and allow the identifying of a relationship between PDMS stiffness and the material's wetting contact angle. Figure 9 shows the evolution of wetting contact angle of deionized water as a function of the measured PDMS stiffness on both cured and post-cured samples. Irrespective of the curing process, the observed trend is that the wetting contact angleof the water -reduces with increasing material stiffness, i.e. it would appear that the surface energy of the material increases with increasing stiffness. Then, as already observed in figure 8, the figure 9 allows one to define a relationship between both quantities in the form of a 2 nd order polynomial function such as: θ (E) = 108.5 ±3 + 2.6E -1.2E 2 . The figure demonstrates that the contact angle on both, cured and post-cured samples, follows an identical law governed by the material stiffness.

The following results can be understood by analogy with the process of PDMS surface oxidation during oxygen plasma exposure. Indeed it is well known that a significant reduction of wetting contact angle on PDMS surfaces can be achieved by exposing the PDMS to oxygen plasma, e.g. [START_REF] Bhattacharya | Studies on surface wettability of poly (dimethyl) siloxane (pdms) and glass under oxygen-plasma treatment and correlation with bond strength, Microelectromechanical Systems[END_REF]. It is also well known that such an exposure results in a chemical change of the PDMS surface whose macroscopic manifestation is the creation of a thin and stiff silica-like film with a stiffness about 70 GPa [START_REF] Bayley | Wavefront kinetics of plasma oxidation of polydimethylsiloxane: limits for sub-µm wrinkling[END_REF]. Thus, from a mechanical point of view, hardening the PDMS crust and increasing its surface energy reduces the wetting contact angle. In consequence, oxygen plasma treatment is a way to drastically but temporarily [START_REF] Morra | On the aging of oxygen plasma-treated polydimethylsiloxane surfaces[END_REF] affect the PDMS wettability by considerably modifying the surface stiffness. Here we show that the wetting contact angle can be modified by tuning the volume stiffness of the PDMS. The effect is less than in the case of an oxygen plasma treatment [START_REF] Bhattacharya | Studies on surface wettability of poly (dimethyl) siloxane (pdms) and glass under oxygen-plasma treatment and correlation with bond strength, Microelectromechanical Systems[END_REF] but more durable. Such result is obviously interesting concerning PDMS in microfluidic applications.

Conclusions

This work demonstrates that by modifying the cross-linker agent concentration (mixing ratio), the curing time and the curing temperature, one can tune the stiffness of 1 mm thick PDMS samples from 800 kPa to 10 MPa -this is the largest stiffness range reported to date. A set of mixing ratios from 19:1 to 2:1 and two curing processes, i.e. 2h at 100 o C and 48h at 165 o C, has been investigated and no problems associated with moulding have been observed. A great diversity of mechanical responses has been observed, from linear-to hyper-elastic including a "brittle" behavior in the stiffest materials (2:1). Indeed, from our results at two extreme curing processes we can speculate that the 2:1 ratio, combined with the appropiate curing process, enables the user to explore the whole stiffness range from 800 kPa to 10 MPa. The relationship between rupture strain and material stiffness as well as between wetting contact angle and material stiffness has been systematically and statistically investigated. Our results indicate that both the rupture strain and the wetting contact angle (i.e. the surface energy of the PDMS) evolve as the square of material stiffness. We believe that by increasing the range of mechanical properties of PDMS we render the material more compatible with technological processes, e.g. depositon of materials such as metals, and at the same time maintaining its flexibility for specific applications.

Figure 1 :

 1 Figure 1: Experimental setup using flat 4 mm wide, 60 mm long and 1 mm thick PDMS tensile test samples. ∆X and ∆Y correspond to transversal and axial length variations during test, while l 0 , L 0 and e 0 correspond to the initial width, height and depth respectively.

Figure 2 :

 2 Figure 2: Example of loading cycles applied to tested samples. Force levels correspond to the case of a 10:1 sample.

Figure 3 :

 3 Figure 3: Example of Stress-Strain curves for different mixing ratios (1:19, 1:10, 1:4, 1:2) and curing processes (cured and post-cured). Mooney-Rivlin fit and rupture strains are superimposed.

Figure 4 :

 4 Figure 4: Elastic moduli of PDMS as a function of mixing and curing process: cured (2h at 100 o C) and post-cured (48h at 165 o C)

Figure

Figure 5 :

 5 Figure 5: Rupture strains of PDMS as a function of ratio and curing process: cured (2h at 100 o C) and post-cured (48h at 165 o C)

Figure 6 :

 6 Figure 6: Variation of rupture strain of PDMS samples function of PDMS stiffness. 2 nd order polynomial fit equation: ε R (E) = 103 ±12 -13.5E + 0.5E 2

Finally, Figure 7

 7 presents Poisson's ratio of the samples tested as a function of cross-linker concentration for both cured and post-cured samples at both, initial and rupture state. Standard deviation ( 1) -+ Post-cured (48h at 165 o C) : initial Post-cured : rupture Cured (2h at 100 o C) : initial Cured :

Figure 7 :

 7 Figure 7: Variation of Poisson's ratio of PDMS samples as a function of cross-linker agent concentration and curing processes (cured and post-cured) at initial and rupture state.

  mixing ratios (Base by weight, Agent by weight) 19:1 10:1 9:1 8:1 7:1 4:1 2:1 2h 100 o C -54 -47 -45 -47 -46 -56 -57 48h 165 o C -57 -50 -51 -37 -45 -44 -32

  mixing ratios (Base by weight, Agent by weight) 19o C 109.7 107.25 100.8 105.65 105.15 111.6 ±0.85 ±0.78 ±1.13 ±0.07 ±0.92 ±0.57 48h 165 o C 109.6 108.2 105.05 112.55 105.5 96.75 ±0 ±1.13 ±0.92 ±2.05 ±0.28 ±0.35

Figure 8 :

 8 Figure 8: Variation of wetting contact angle between deionized water droplets and PDMS surface function of cross-linker concentration for both, curing and post-curing, process.

Figure 9 :

 9 Figure 9: Variation of the wetting contact angle (θ) between deionized water droplets and PDMS surface as a function of PDMS stiffness (E). 2 nd order polynomial fit equation: θ (E) = 108.5 ±3 + 2.6E -1.2E 2

Table 2 :

 2 Drop of Poisson's ratio from initial to rupture state (in %)

Table 3 :

 3 Mean value ( o ) and standard deviation of wetting contact angle between deionized water droplets and PDMS surfaces.

Acknowledgements

The authors would like to thanks Dr E. Charkaluk from the Laboratory of Mechanics of Lille for helpful discussions and the access to laboratory facilities.

Financial support is acknowledged from the EQUIPEX project "LEAF" (ANR-11-EQPX-0025) funded by the French government.


Roberta Dainese

Giuseppe Tedeschi

Thierry Fourcaud

Alessandro Tarantino
email: alessandro.tarantino@strath.ac.uk

Measurement of xylem water pressure using High-Capacity Tensiometer and benchmarking against Pressure Chamber and Thermocouple Psychrometer

The response of the shallow portion of the ground (vadose zone) and of earth structures is affected by the interaction with the atmosphere. Rainwater infiltration and evapotranspiration affect the stability of man-made and natural slopes and cause shallow foundations and embankments to settle and heave. Very frequently, the ground surface is covered by vegetation and, as a result, transpiration plays a major role in ground-atmosphere interaction. The soil, the plant, and the atmosphere form a continuous hydraulic system, which is referred to as Soil-Plant-Atmosphere Continuum (SPAC). The SPAC actually represents the 'boundary condition' of the geotechnical water flow problem. Water flow in soil and plant takes place because of gradients in hydraulic head triggered by the negative water pressure (water tension) generated in the leaf stomata. To study the response of the SPAC, (negative) water pressure needs to be measured not only in the soil but also in the plant. The paper presents a novel technique to measure the xylem water pressure based on the use of the High-Capacity Tensiometer (HCT), which is benchmarked against conventional techniques for xylem water pressure measurements, i.e. the Pressure Chamber (PC) and the Thermocouple Psychrometer (TP).

Introduction

The response of the shallow portion of the ground (vadose zone) and of earth structures is affected by the interaction with the atmosphere. Rainwater infiltration and evapotranspiration cause settlement and heave in shallow foundations and embankments and control the stability of man-made and natural slopes.

Very frequently the ground surface is covered by vegetation and, as a result, transpiration plays a major role in the mechanisms of water removal by the atmosphere. Transpiration is the process of water movement taking place from the soil through the plant up to the leaves, where water eventually evaporates through the stomata. The soil, the plant, and the atmosphere form a continuous hydraulic system, which is referred to as Soil-Plant-Atmosphere Continuum (SPAC) [1]. The SPAC actually represents the 'boundary condition' of the water flow problem. Understanding its response is crucial to model the hydraulic boundary condition of any geotechnical system.

Water flow in the soil and plant takes place because of gradients in hydraulic head triggered by the negative water pressure (water tension) generated in the leaf stomata. To study the response of the SPAC, water pressure therefore needs to be measured not only in the soil but also in the plant.

The most common techniques to monitor xylem water pressure are the Pressure Chamber (PC) [2] and the Thermocouple Psychrometer (TP) [3]. The working principle of the Pressure Chamber is the same as the axis-translation technique used to measure or impose matric suction in soils [4]. Air pressure is increased around the xylem/leaf until water pressure is 'translated' from negative values to zero. The PC is often considered the 'reference' for xylem/leaf water pressure measurement [1,[START_REF] Boyer | Measuring the Water status of Plants and Soils[END_REF]. However, this is a destructive technique as it requires the trunk or branch to be cut or leaves to be excised. As a result, the PC is not suitable for continuous measurement and/or for monitoring leaf water pressure when a relatively small number of leaves is available, which is generally the case in laboratory experiments.

The Psychrometer is based on the use of a thermocouple, similarly to the instruments used to measure total suction in soils [START_REF] Bulut | [END_REF]. The psychrometer is widely used in the field for continuous monitoring of xylem/leaf water pressure. However, it measures total and not 'matric' water pressure, i.e. its measurement is affected by the presence of solutes in the xylem water. The common assumption that the osmotic component of xylem water pressure is negligible [7] does not always hold. [8,9] have shown that the osmotic component of xylem suction may not be negligible and may vary over time. In addition, the Thermocouple Psychrometer is not accurate at low values of water tension (high relative humidity) and its response is strongly affected by temperature, which is a critical issue in field measurements [2].

The similarities between instruments used to measure water tension in soils and plants suggest that High-Capacity Tensiometers, which are widely used to monitor (negative) pore-water pressure in soils, could also be used to measure xylem water pressure in plants. Indeed, [10] have successfully attempted to measure xylem water pressure directly using a pressure probe made of a capillary tube filled with water and silicon oil. When the tip of the probe was inserted into a xylem channel, the xylem water pressure was transmitted through the liquid in the probe and measured by a pressure transducer. However, this pressure probe has never registered xylem water pressures below -0.65 MPa [11] and for no more than a few hours due to cavitation occurring in the probe [10]. This was probably due to the absence of a high-air-entry porous interface, which is actually incorporated into high-capacity tensiometer used to measure pore-water tension in soil [4]. This paper presents the use of High-Capacity Tensiometer (HCT) to monitor the negative xylem water pressure in plants. The measurement of the HCT was validated against the measurement of leaf water potential via the Pressure Chamber and the Transistor Psychrometer over a relatively wide range of xylem water pressures via field and laboratory experiments [START_REF] Dainese | Géotechnique[END_REF]. However, this paper only focuses on one single experiment carried out on a cherry tree in the laboratory.

Background

Plant Structure

Plants are composed of rooting system, stem, and crown of leaves (Fig. 1a). Water is taken up from the soil by roots and transported through the stem up to the leaves, where it is released into the atmosphere in the form of vapour.

The stem is composed of different tissues, each one having its own specific role and structure. These include the outer bark, the cortex, the phloem, the cambium, and the inner xylem (Fig. 1c). The phloem and the xylem are the main pathways for solutes and water within the stem. The phloem is the tissue of the plant that redistributes downward the sugars synthesised in the leaves. The xylem is dedicated to the transport of water from the roots to the leaves and is characterized by a porous structure formed by tubes of reduced dimensions called vessels (diameter Ø~300 ȝm) or tracheids (diameter Ø~40 ȝm), which are similar to capillary tubes [START_REF] Canny | [END_REF]. The water within this porous medium is continuous and under tension and this allows the ascent of water to considerable heights.

The leaf is the site of photosynthesis (Fig. 1b). The CO 2 and solar energy are converted into sugars and oxygen in the chloroplasts. The CO 2 enters the leaf through the stomata, apertures on the surface of the leaf. The guard cells can close or open the stomata, regulating the gas exchange and the water loss from the wet cells within the leaf, i.e. the transpiration. For most plants, stomata close during night-time, when no photosynthetic process and transpiration occur [START_REF] Salisbury | Plant Physiology[END_REF]. Plants are in a dilemma throughout their lives: photosynthesis requires intensive CO 2 ingress but in relatively dry conditions this exchange can result in excessive water vapour loss.

Transpiration

The loss of water through the stomata produces a movement of water up the plant to replace what is lost by evaporation. Transpiration in itself is neither an essential physiological function nor a direct result of the living process of the plants. It is mainly caused by a vapour pressure gradient between the normally saturated leaves and the often quite dry atmosphere. In fact, plants can thrive in a saturated or nearly-saturated atmosphere with very little transpiration [START_REF] Hillel | Applications of soil physics[END_REF]. In other words, it is the evaporative demand of the atmosphere in which the plants live that drives transpiration (and not the plant). In this respect, there is no difference between evaporation from bare ground and transpiration from plants. In bare ground, evaporation is triggered by the differential in vapour pressure between the ground surface and the atmosphere. The menisci forming at the ground surface due to the evaporation process generates negative pore-water pressures at the surface that uplift pore-water from the deeper layers (Fig. 2a). In a plant, it is the difference in vapour pressure between the leaf and the atmosphere that generates evaporation. The menisci forming in the leaf generate the negative water pressure that uplifts water from the soil through the xylem (Fig. 2b). The plant therefore moves the site of evaporation from the ground surface to the leaves.

Pressure Chamber

The working principle of the Pressure Chamber is analogous to the axis-translation technique used in soil testing [4]. Water in the leaf is under tension whereas the air surrounding the leaf is at atmospheric pressure (Fig. 3a). In order to measure the 'matric' water tension, the leaf is excised. Water tends to retract into the petiole and menisci form at the interface between the cut end of the petiole and the atmosphere (Fig. 3b). The leaf is inserted into a chamber and sealed inside it. Only the last part of the petiole is left outside in direct contact with the atmosphere (Fig. 3c). The air pressure in the chamber is then gradually incremented until water can be observed to form a flat meniscus on the excised end of the petiole. The air pressure is then taken equal to the water tension in the leaf before excision [START_REF] Boyer | [END_REF]. This technique is based on the assumption that the difference between the leaf water pressure and the surrounding air pressure is kept constant throughout the whole procedure (Fig. 3d-e) [2]. The measurement of the PC is discontinuous and destructive; the frequency of the readings is therefore conditioned by the manpower and the sampling leaves available. The PC is a commonly used and trusted technique in plant science to measure the 'xylem' matric water pressure in plants and has been used as a benchmark to validate other techniques [10,17,18].

Thermocouple Psychrometer

The psychrometer used for this study is produced by ICT international (PSY1 Stem Psychrometer). The working principle of the psychrometer is based on the measurement of the air relative humidity in equilibrium with the xylem water pressure, which is then converted to water pressure via the psychrometric law. The thermocouple is cooled until the temperature drops below the dew point and a drop condense on the junction. As soon as the thermocouple stops cooling, the drop starts evaporating into the surrounding air. The rate of evaporation is related to the relative humidity in the chamber: the higher the relative humidity, the longer the drop will take to evaporate [START_REF] Boyer | Measuring the Water status of Plants and Soils[END_REF]. The response of the psychrometer must be calibrated against solution of known vapour pressure and, hence, liquid total suction.

High-Capacity Tensiometer

The High-capacity Tensiometer (HCT) is shown in Fig. 5 and is composed of an integral strain gauge, a diaphragm 0.4 mm thick and a ceramic filter with nominal air-entry value of 1.5 MPa [START_REF] Tarantino | [END_REF].

When the instrument is put in contact with the xylem, the tension of the xylem water is transferred to the water reservoir, deflecting the diaphragm and deforming the strain gauge. HCT measures the 'matric' water pressure in the sample, thanks to the free diffusion of ions through the porous ceramic [START_REF] Tarantino | Proc. 3rd Int. Conf. on Unsaturated Soils[END_REF].

Materials

The experimental test presented in this paper was conducted on a cherry plant (Bigarrau Burlat), a broadleaved species. The plant was selected for its stem diameter (stem diameter needs to be >20mm to allow proper installation of the high-capacity tensiometers and transistor psychrometer) and a sufficiently large number of leaves for pressure chamber measurements.

The plant was provided by an external nursery. Prior to the beginning of the experiment, it was kept in the laboratory at constant temperature and relative humidity, watered regularly with solar radiation mimicked through the use of a growth lamp. The oak tree had a height of 1.3 m and a stem diameter of 25 mm at 100 mm height from the soil surface.

Experimental procedures 4.1 Pressure Chamber

The PMS 1515D Scholander Pressure Chamber [22] was used for the xylem water pressure measurement (Fig. 6a). Leaves were initially wrapped in aluminium foil for at least 2h. Leaf wrapping stops transpiration and allows water in the leaf to equilibrate with the branch. As a result, the water pressure recorded in the leaf is assumed to coincide with the water pressure in the branch at the base of the petiole. The leaf was then excised with a sharp blade and promptly inserted into the sealing ring (in less than a minute), apart from the end of the excised petiole that was kept outside the sealing ring (Fig. 6b). The sealing ring was then mounted on the pressure chamber (Fig. 6c), with the leaf within the chamber and the petiole end kept at atmospheric pressure. Air in the chamber was gradually pressurised until a flat meniscus formed at the end of the excised petiole [START_REF] Meron | E3S Web of Conferences[END_REF]. The air pressure in the chamber recorded when a flat meniscus appeared at the excised petiole surface was assumed to be equal to the negative water pressure in the leaf before excision. Each measurement was made in triplicate, with the leaves excised at time intervals of 3-5 min.

Thermocouple Psychrometer

Before installation, the psychrometer was observed at the microscope to check that the thermocouple S (Fig. 4) was in position and sufficiently extended to make contact with the xylem surface.

The psychrometer was then wrapped with Parafilm® to facilitate the removal (at the end of the test) of the silicone grease used to seal the sensor on the stem. Only the psychrometer chamber was not covered (Fig. 7a).

A small area of xylem was then exposed, cleaned with distilled water, wiped dry, and then wrapped with several layers of Parafilm® in order to avoid localized evaporation. A hole was made through the layers of Parafilm® to insert the psycrometer sensor (Fig. 7b). The psychrometer was mounted on a clamping device (Fig. 7c), which was in turn installed on the stem (Fig. 7d). The gap between the psychrometer and the stem was sealed with silicone grease. The thermocouple signal depends on two parameters, the cooling time and the waiting time. The cooling time is the time whereby the current is circulated in the thermocouple. This causes cooling of the junction in proximity of the plant xylem due to the Peltier effect and condensation of a drop on the junction. The higher the cooling time, the bigger is the drop the condenses on the cold junction. Once a water drop has condensed, the current is no longer circulated in the thermocouple. Because of the difference in temperature between the cold junction (carrying the condensed water drop) and the reference junction (at ambient temperature), an electrical potential is generated due to the Seebeck effect.

The water drop is subjected to an inward sensible heat flux because the temperature of the drop is lower than the air temperature surrounding the water drop. As a result, the temperature differential between the cold junction and the reference junction tends to decay and so does the electrical potential. This decay in temperature differential is faster for the case where the water drop is small in size. This can occur for either small cooling time or low relative humidity of the surrounding air (because faster evaporation reduces the water drop size more rapidly).

The response of the thermocouple psychrometer is shown in Fig. 8 for three different values of total suction (relative humidity) imposed via NaCl solution (0.45, 2.24, and 4.55 MPa respectively) and different cooling times (5, 8, 10, and 20 sec). The temperature depletion required to condense the water drop is lower when the relative humidity is higher (because the air temperature is closer to the dew point temperature). This explains why the initial signal is only about 2μV for the lowest 0.45 MPa suction (Fig. 8a) and in the range 14-16 μV for the highest suction 4.55 MPa (Fig. 8c). At given cooling time, the electrical signal drops more rapidly at higher suction (Fig. 8c). For the lower suction, the electrical signal drops so slowly that no significant variation is appreciated in the measurement time interval (Fig. 8a).

For a given suction, the higher the cooling time, the bigger is the water drop that condenses initially, and the slower is the decay in electrical potential due to the higher thermal inertia of the drop (Fig. 8c). Because the electrical signal depends on the cooling time and varies over time, a choice has to be made about the cooling time and the 'waiting time', i.e. the time lag between the start of drop evaporation and the record of the signal. The instrument was then calibrated for a waiting time of 6 s and the cooling time to 8 s and the same setting was maintained for the measurements.

High-Capacity Tensiometer

The HCTs were kept in the saturation chamber at 4 MPa bars for at least 24 hours; The tensiometers were then removed from the saturation chamber and placed in free water at atmospheric pressure. Once equalisation in free water was achieved, negative water pressure was generated by wiping the tensiometer porous ceramic filter. Water pressure was let to drop to about -1000 kPa and the tensiometer was then replaced in free water. This procedure was repeated twice [START_REF] Dixon | PSY1 Stem Psychrometer Manual[END_REF][START_REF] Tarantino | [END_REF]. The tensiometer reading was then zeroed.

The tensiometers were installed on the stem of the plant by exposing an appropriate section of the xylem by means of a cutter blade (Fig. 9a). Any material released from damaged cells was cleaned with distilled water and wiped using tissue paper. The xylem surface was kept moist during the installation to avoid excessive dehydration of the tissues. To ensure proper hydraulic contact between the tensiometer porous ceramic filter and the xylem, a small amount of kaolin clay paste was placed on the tip of the porous ceramic filter (Fig. 9b). The paste was prepared at a water content sufficiently high to give enough plasticity to the paste and ensure good contact between the HCT and the xylem. Excessive water content was avoided due to the longer time that would have been required for the paste to equilibrate with the xylem water pressure. A latex membrane was used to seal the tensiometers on the stem and to avoid measurement errors due to localised evaporation from the paste (Fig. 9c).

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Results

The xylem water pressure measurement was carried out for about 30 days. However, only the measurement carried over 2 days (from day 23 to day 25) is presented here. Day and night cycles were mimicked by switching on (from 6am to 8pm) and off (from 8pm to 6am) a growth lamp. As shown in Fig. 10, water pressure tends to decrease during day time (when stomata are open and evaporation is ongoing) and increase during night time (when the stomata tend to close and transpiration reduces significantly). The two High-Capacity Tensiometers show an excellent agreement with each other and, in turn, their measurement is consistent with the ones carried out using the transistor psycrometer and the pressure chamber on leaves excised from branches.

Conclusions

The paper has demonstrated the use of High-Capacity Tensiometer (HCT) for measuring xylem water pressure in plants. The use of HCT on plants is a step change in the understanding and modelling the effect of plant transpiration on suction and moisture regime in a vegetated ground. HTCs have been used for more than 20 years in geotechnical engineering and this instrument is available in many research laboratories. Installing HCTs on stems and branches is quite straightforward and this will allow analysing the soil and the plant as a continuum in the same experimental setup rather than borrowing transpiration models developed by plant scientists for their specific applications.

Fig. 1 .

 1 Fig. 1. (a) Structure of a tree: roots, stem, and crown. (b) Leaf structure: stomata and gas exchange; (c) xylem structure.

Fig. 2 .

 2 Fig. 2. Evaporation from bare soil and transpiration through plant.

Fig. 3 .

 3 Fig. 3. Working principle of the Pressure Chamber (PC) technique for measurement of leaf water pressure. (a) leaf on the tree (water pressure is negative); (b) leaf excised (curved meniscus forming at the end of petiole); (c) air pressure increased around leaf (meniscus becomes flat).

Fig. 4 .

 4 Fig. 4. PSY1 Stem Psychrometer schematic layout [18].

Fig. 5 .

 5 Fig.5. High-Capacity Tensiometer (after[10]).

Fig. 6 .

 6 Fig. 6. (a) PMS 1515D Scholander Pressure Chamber. (b) Leaf inserted into the sealing ring. (c) Sealing ring mounted on the pressure chamber.

Fig. 7 .

 7 Fig. 7. (a) PSY1 wrapping. (b) Xylem exposure and stem wrapping. (c) PSY1 screwed on the clamping device; d) PSY1 on the stem.

Fig. 8 .

 8 Fig. 8. (a) NaCl 0.1 mol (0.45 MPa suction. (b) NaCl 0.5 mol (2.24 MPa suction. (c) NaCl 1.0 mol (4.55 MPa suction).

Fig. 9 .

 9 Fig. 9. HCT installation on stem. (a) Exposure of xylem tissues. (b) Kaolin paste on HCT. (c) Application to stem and sealing with latex membrane.

Fig. 10 .

 10 Fig. 10. Measurement of xylem water pressure using HCT2, HCT4, and transistor psychrometer (at 115 mm, 250 mm 400 mm above soil surface respectively) and pressure chamber (on leaves excised from tree branches).

E3S Web of Conferences 195, 03014 (2020) E-UNSAT 2020 https://doi.org/10.1051/e3sconf/202019503014

Acknowledgements

The authors wish to acknowledge the support of the European Commission via the Marie Skłodowska-Curie Innovative Training Networks (ITN-ETN) project TERRE 'Training Engineers and Researchers to Rethink geotechnical Engineering for a low carbon future' (H2020-MSCA-ITN-2015-675762)