A Le Postollec

M Dobrijevic

Ph S Incerti

H Moretto

L Seznec

G Desorgher

P Santin

L Nieminen

O Dartnell

Vandenabeele-Trambouze

Ph Moretto

H Seznec

L Desorgher

G Santin

P Nieminen

L Dartnell

O Vandenabeele-Trambouze

G Coussot

DEVELOPMENT OF A BIOCHIP DEDICATED TO PLANETARY EXPLORATION. FIRST STEP: RESISTANCE STUDIES TO SPACE CONDITIONS

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Detecting life in the Solar System is one of the great challenges of new upcoming space missions. Several techniques are proposed to detect traces of organic matter on extra-terrestrial objects. A new and promising technique based on biochips, and recommended by space agencies (ESA and NASA), is under study in several countries (Bada et al. 2005;Sims et al. 2005;Steele et al. 2001). A biochip is a miniaturized device composed of biological sensitive systems fixed on a slide. In space conditions, a lot of constraints might alter the efficiency of this analytical tool. Designing a biochip that will be efficient in space conditions requires testing the resistance of all the components of this instrument to space conditions.

With the help of the French space agency (CNES), our team currently develops a biochip especially designed for planetary exploration. The BiOMAS (Biochip for Organic Matter Analysis in Space) project, in progress for two years, has an interdisciplinary dimension bringing together specialists evolving in different area (planetologists, physicists, chemists, biologists and materials specialists) and developing complementary competences. Our aim is to optimize the design of a biochip (including the slide, the ligands and the chemical bonds) that meets the space requirements. As a first step, we have begun several experiments aiming to select the best constituents for our "space biochip", to determine its resistance under several conditions and define, if necessary, some well-adapted protections.

In this paper, we briefly present our current work. Focus is made on the methodology; first results will be presented in forthcoming papers. In section 2, we describe biochip concept and functioning, underlying the effort that must be done to adapt it to the harsh space environment. In section 3, we present the methodology used to start studies on the biochip resistance under several space constraints.

A biochip for space exploration

Description: what is a biochip?

A biochip is a miniaturized device composed of biological sensitive systems fixed on a solid substrate. It allows the quantification of hundreds to thousands of target molecules simultaneously (Maule et al. 2005). The biological systems used are called ligands. Antibodies are the most common on biochips. Each ligand is produced to recognize a specific molecule or family of molecules. The main characteristics of a ligand lie in the high affinity and the high specificity toward its target. In our case, target molecules are organic molecules, which can sign the presence of extent or extinct life: they are commonly called biomarkers (Tang 2007).

Geometrically, a biochip looks like a microscope slide. Ligands are fixed on a substrate, which dimensions are typically: 75×25×1 mm 3 . Each type of ligand is spotted on the slide and the density of 600 spots per cm 2 can be reached, proof of the high level of miniaturization. A spot measures approximately 90 to 200 µm in diameter and contains 300 to 400 pL of ligands solution. Ligands are linked to the substrate by chemical reactions, mainly by the creation of a covalent link between the ligand and the slide surface.

Principle: how does it work?

To function, a biochip needs fluids. The first step, previous to the analysis itself, consists in using solvents to extract organic molecules from the solid sample of interest. For the most efficient extraction possible, the sample is first crushed to improve the contact surface with the solvent.

The solution, containing the potential organic molecules extracted, is filtered and placed into contact with the biochip. One important point is to be sure that the solution will pass through all the ligands spots in order to have a chance to detect the molecules of interest (if it contains some). The incubation time is mainly function of the affinity constants of the ligands-biomarker complexes and the experimental conditions (temperature, etc).

The biochip is then washed and analyzed. The detection of the complexes ligand-biomarker can take several forms. One of the most current techniques consists of a fluorescent detection. Each ligand is labeled with a fluorescent dye. The biochip is submitted to a laser excitation at the characteristic wavelength of the dye. If the ligand is linked to its target molecule, a fluorescent signal appears. With this technique, we obtain a visual signal to determine which kinds of molecules are contained in the sample tested. The fluorescence intensity of each spot allows a quantitative analysis.

Using a biochip for space exploration

Nowadays, biochips are mainly used in the medical field. However, the space community feels more and more interested by this new technology which could be an interesting alternative to traditional techniques usually used to search for organic matter.

Advantages

Obviously, one of its main advantages for space missions lies on its high level of miniaturization. This is a very powerful tool, which weights only 4 g for a volume of less than 2 cm 3 . Another advantage, compared to actual techniques, is the very high sensitivity of the biochip, which relies on the particularity of ligand/biomarker binding. The detection limit can go down to a very low-level concentration.

Our approach

Instead of developing a biochip by adapting current biochip to space requirements, we have decided to study a new concept of biochip by optimizing all the constituents of this analytical tool in respect to space constraints. This work is in progress and will be presented in depth in forthcoming papers. In particular, analytical developments are not discussed in the present paper, which focus only on space constraints.

Space mission constraints and potential damaging effects on a biochip

Interplanetary space is a hazardous environment which combines large thermal cycles, extremes temperatures, microgravity, vacuum, radiations, etc. Moreover, a whole space mission adds new constraints such as contamination avoidance, long storage time, sterilization procedures, vibrations and shocks due to launching, landing and transportation. A biochip dedicated to space should take into account all this panel of constraints and its design, obviously, should be adapted compared to actual biochip used on Earth. All those constraints bring a lot of questions.

Extreme temperatures can destroy ligands, modify substrate geometry (expansion/shrinkage) and create residual stresses in the material. The problem of fluids state is also crucial. Vibration and shocks can produce cracks on the substrate and complicate the detection procedure, which needs a very precise positioning. Vacuum can be damaging from a contamination point of view. It can also weaken the substrate.

Considering all those potential damages, we have engaged works on different aspects to determine and quantify, if possible, the impact of harsh conditions on the biochip components.

3 Resistance studies: ongoing work

Substrate specifications

Different types of material can be used as a biochip substrate: glass, thermoplastics, elastomers, etc. Therefore, one of our first works was to define which criteria would be relevant to select the best substrate for a "space biochip".

A specification folder was established, presenting analytical and space constraints criteria. For each criterion, we tried to give the level to reach and the degree of flexibility that can be acceptable. For instance, we consider that the material should resist a temperature range between -70 • C and 70 • C. However, this criterion is relatively flexible as we can imagine an insulating system protecting the biochip and reducing the temperature variability.

Here after, we detail works we have already begun on four of these criteria: outgassing behavior, resistance to solvents, thermo-mechanical behavior and radiation resistance.

Outgassing behavior

A majority of materials outgas when they are placed under low pressure and/or high temperatures. As these two parameters are combined during a space mission, it seems relevant, in the design process of a biochip dedicated to space exploration, to take into account the outgassing properties of the substrate. Outgassing process consists in a loss of matter, which evaporates from the solid: gases encapsulated into the material escape, the most volatile elements desorb, etc. This loss of matter can be critical for the functioning of the biochip from a contamination point of view. Elements outgassed can alter ligands performances. They can link with the ligands and create a false signal, they can modify pH conditions necessary to the survivability of the ligands, they can deform ligands structure, etc.

Therefore, in collaboration with the French space agency, we are performing experiments on different types of potential substrate materials. To better know their outgassing behavior will help us to select the most adapted material for our biochip. Moreover, as outgassing rejects can not be completely avoided, our better understanding of the phenomenon will give us the means to lower contamination risks.

Resistance to solvents

To analyze a sample of soil, we need to extract organic molecules that could be present in this sample. The principle consists in crushing the sample, mixing it with a solvent (organic or not) and getting back the solution to analyze it. Therefore, the substrate has to resist organic solvents that could be necessary to extract molecules of interest. We performed a first battery of experiments in IMM (Institut Max Mousseron, Montpellier) to compare the resistance of different materials to solvents generally considered as relevant for analytical chemistry in the context of exobiological investigations.

Several parameters, such as temperature, solvents concentrations, mixing proportions, contact time, etc, can have an influence on the material resistance. Several damages should be checked after the immersion in the solvent: surface degradation, porosity modification, variation of the autofluorescence level, etc. As everybody SF2A 2007 knows, thermoplastics are especially sensitive to organic solvents and their behavior, after an exposure to some of them, must be carefully investigated.

Effects of radiations

One of the most critical parameter during a space mission should be the resistance to cosmic rays irradiation. Even if some shielding could be implemented around the device, it is impossible to completely stop all the charged particles: protection can not be perfect. This is the reason why, studying the resistance of the biochip under different levels of irradiation is essential.

In a first step, we decided to simulate the radiation environment a biochip would face during a typical mission to Mars. The objective is to determine particle types and energy ranges the most relevant for experiments on dedicated irradiation beam facilities. With this simulation work, collaboration is born with the Nuclear Studies Center of Bordeaux-Gradignan (CENBG). We worked on a Monte-Carlo simulation tool which allows the simulation of the particles interaction with matter. We focused on ionizing dose calculations and on determining particles fluences around the biochip.

Simulations strategies

We divided our simulations into two distinct phases.

The first phase was the simulation of the interaction between particle fluxes and a rover traveling from Earth to Mars. During this phase, the primary incident spectra interact directly with the rover.

The second phase consisted in simulating irradiation received on Mars. We considered a rover exploring Mars surface during one month. This second phase was more complicated than the first one because of the interaction of the particles with Mars atmosphere and soil. To simplify, and clarify, we adopted a two-step strategy. First, we aimed to define the particle environment the rover would encounter on Mars surface. Therefore, we simulated the interaction of primary particle fluxes with Mars environment, defining atmosphere and soil parameters. We generated primary incident particles on top of the atmosphere and analyzed particle fluxes above the surface. Both, downward and upward (backscattered) fluxes were taken into account. Then, we used those recorded fluxes as primary fluxes into a new simulation to calculate the ionizing dose received by the biochip during a one month stay on Mars surface. This two-step strategy allows us to free from the scale effect. Simulating interactions with a planet needs kilometers square area whereas our biochip is only few centimeters square large. If the simulation is down at once, you need to generate a lot of primary events to hit the biochip at least once: simulations are really too slow and statistics are really poor. A two-step simulation is a good way to save time and improve clarity.

Incident particles

A special care was taken to choose primary incident particle fluxes to use on our simulations. We considered two different types of primary fluxes: Galactic Cosmic Rays (GCR) and Solar Energetic Particles (SEP). GCR consist of charged particles coming from outside of our solar system. Therefore, in our case, the flux can be considered as isotropic. The radiation energy is proportionally opposite to solar activity: when the Sun activity is minimum, GCR energy is maximum, which represents 4 years of the 11 years solar activity cycle. SEP events consist mainly of protons fluxes due to solar flares or coronal mass ejections. They are punctual in time and can last few hours to few days. They constitute one of the most severe environment that space systems should face (particle fluxes are especially deleterious for electronics). The radiation flux is clearly directional and obviously correlated to the Sun activity. It is also function of the distance between the Sun and the location of interest.

As Monte Carlo simulations can be run with only one type of primary particle, we had to select the most abundant particles in the primary incident spectrum in order to be the most representative with the fewest number of simulations. Thanks to integration calculations, on energy and on flux, of CREME96 particle fluxes (Tylka et al. 1997), we chose to consider protons, alphas, carbon ions and oxygen ions as a representative sample of the global spectrum of GCR. For SPE, protons and alphas are largely majority.

Details and results of these simulations will be published in a forthcoming paper (Le Postollec et al., in preparation).

Irradiation experiments objectives

Simulations give us a precise idea of the radiation environment the biochip will encounter. Therefore it drives our future experiments on dedicated beam facilities and helps us to reproduce in the best way fluxes that can be deleterious to the whole system. Our objectives are to determine how the biochip, and especially the biological systems, will evolve under irradiation. Which constituents are the most sensitive? Which modifications will be induced on ligands or substrate properties? What are the thresholds not to overcome to be still functional? Which shielding could be efficient? A lot of questions have to be investigated and weve already managed to obtain beam time in a French laboratory to test some particular conditions.

Thermo-mechanical studies

A biochip placed on space exploration mission will surely face temperature variations and high vibration and shock levels. Studying the thermo-mechanical characteristics of the substrate appears clearly inescapable. We have started thoughts on this point to determine crucial experiments to perform to compare the different materials in the best way, still with the objective to select the most adapted. Minimize expansion, avoid cracks, keep planarity, for instance, appear as key points.

We would like to thank the French space agency (CNES) and the Région Aquitaine for their support.

1

  Université Bordeaux 1, CNRS, OASU, Laboratoire d'Astrophysique de Bordeaux (LAB), UMR 5804, Floirac, F-33270 2 (the same as A. Le Postollec listed above) 3 Université Bordeaux 1, CNRS / IN2P3, Centre d'Etudes Nuclaires de Bordeaux Gradignan, UMR 5797, Chemin du Solarium, BP 120, F-33175 Gradignan Cedex France 4 (the same as S. Incerti listed above) 5 (the same as S. Incerti listed above) 6 Physikalisches Institut, University of Bern, Sidlerstrasse 5, CH-3012 Bern, Switzerland 7 ESA-ESTEC, Keplerlaan 1, 2200 AG Noordwijk, The Netherlands 8 (the same as G. Santin listed above) 9 Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX), University College London, London,UK 10 Institut des Biomolcules Max Mousseron (IBMM), Université Montpellier II, Montpellier, France 11 (the same as O. Vandenabeele-Trambouze listed above) c Société Francaise d'Astronomie et d'Astrophysique (SF2A) 2007 SF2A 2007

Author manuscript, published in "Journées SF2A 2007 Semaine de l'Astrophysique Française 2007 2 au 6 juillet 2007, Grenoble :

France ( 2007)"


D Li

S Bulou

N Gautier

S Elisabeth

A Goullet

M Richard-Plouet

P Choquet

A Granier

Nanostructure and photocatalytic properties of TiO2 films deposited at low temperature by pulsed PECVD

Keywords: PECVD, pulsed plasma, TiO 2, anatase, TEM, photocatalytic activity

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

TiO 2 thin films have attracted intensive research as photocatalysts for their advantages in chemical stability, non-toxicity, commercial availability at a low cost and high photocatalytic activity [START_REF] Nasr | Recent progress on titanium dioxide nanomaterials for photocatalytic applications[END_REF][START_REF] Chen | Fabricating highly active mixed phase TiO 2 photocatalysts by reactive DC magnetron sputter deposition[END_REF][START_REF] Lu | Synthesis of photocatalytic TiO 2 thin films via the high-pressure crystallization process at low temperatures[END_REF]. The TiO 2 properties are due to its crystalline form which is mainly encountered as three phases including tetragonal anatase, tetragonal rutile and orthorhombic brookite [START_REF] Lee | Substrate effects on the oxygen gas sensing properties of SnO 2 /TiO 2 thin films[END_REF]. TiO 2 exhibits different band gap energies for its different phases, viz. anatase (3.2 eV), rutile (3 eV) and brookite (3.1 eV) [START_REF] Sang | TiO 2 nanoparticles as functional building blocks[END_REF][START_REF] Xu | Synthesis and photoluminescence of welldispersible anatase TiO 2 nanoparticles[END_REF][START_REF] Nagaveni | Solar photocatalytic degradation of dyes: high activity of combustion synthesized nano TiO 2[END_REF][START_REF] Yin | Preparation of visible light-activated titania photocatalyst by mechanochemical method[END_REF]. In addition to the effect of TiO 2 crystallization, some other essential factors such as grain size, preferred grain orientation, presence of structure defects and capability to absorb hydroxyl groups in the initial phase of excitation may also affect the photocatalytic activity [START_REF] Hurum | Explaining the enhanced photocatalytic activity of degussa P25 mixed-phase TiO 2 using EPR[END_REF][START_REF] Luttrell | Why is anatase a better photocatalyst than rutile? -Model studies on epitaxial TiO 2 films[END_REF][START_REF] Ogawa | Growth of TiO 2 thin film by reactive RF magnetron sputtering using oxygen radical[END_REF]. Powders suspended in polluted solution due to their higher specific area may lead to more efficient photocatalytic properties, however films do not require filtering to be recovered after the volume to be depolluted has been treated. These films can be prepared by using different methods including sol-gel method [START_REF] Grätzel | Sol-gel processed TiO 2 films for photovoltaic applications[END_REF][START_REF] Trepalis | TiO 2 (Fe 3+ ) nanostructured thin films with antibacterial properties[END_REF][START_REF] Rachel | Comparison of photocatalytic efficiencies of TiO 2 in suspended and immobilised form for the photocatalytic degradation of nitrobenzenesulfonic acids[END_REF][START_REF] Senthilkumaar | Photodegradation of a textile dye catalyzed by sol-gel derived nanocrystalline TiO 2 via ultrasonic irradiation[END_REF][START_REF] Christensen | Photoelectrocatalytic and photocatalytic disinfection of E. coli suspensions by titanium dioxide[END_REF][START_REF] Sunada | Studies on photokilling of bacteria on TiO 2 thin film[END_REF][START_REF] Mills | Characterisation of the photocatalyst Pilkington Activ™: a reference film photocatalyst?[END_REF][START_REF] Mills | Thick titanium dioxide films for semiconductor photocatalysis[END_REF], which is the most often used to synthesize TiO 2 thin films for photocatalytic applications. However other strategies were also largely investigated, such as liquid phase deposition [START_REF] Yu | The effect of calcination temperature on the surface microstructure and photocatalytic activity of TiO 2 thin films prepared by liquid phase deposition[END_REF], magnetron sputtering [START_REF] Mardare | On the structural properties andoptical transmittance of TiO 2 r.f. sputtered thin films[END_REF][START_REF] Zhang | Photocatalytic property of TiO 2 films deposited by pulsed DC magnetron sputtering[END_REF][START_REF] Daviosdottir | Investigation of DC magnetron-sputtered TiO 2 coatings: effect of coatingthickness, structure, and morphology on photocatalytic activity[END_REF], atomic layer deposition [START_REF] Pore | Atomic layer deposition of photocatalytic TiO 2 thin films from titanium tetramethoxideand water[END_REF], spray pyrolysis [START_REF] Miki-Yoshida | Thin films of photocatalytic TiO 2 and ZnO deposited inside a tubing by spray pyrolysis[END_REF], chemical vapor deposition [START_REF] Mills | Novel TiO 2 CVD films for semiconductor photocatalysis[END_REF], metalorganic chemical vapor deposition [START_REF] Jung | Metallorganic Chemical Vapor Deposition and Characterization of TiO 2 Nanoparticles[END_REF] and plasma enhanced chemical vapor deposition (PECVD) [START_REF] Battiston | PECVD of amorphous TiO 2 thin films: effect of growth temperature and plasma gas composition[END_REF][START_REF] Baba | Significance of a noble metal nanolayer on the UV and visible light photocatalytic activity of anatase TiO 2 thin films grown from a scalable PECVD/PVD approach[END_REF][START_REF] Baba | Photocatalytic anatase TiO 2 thin films on polymer optical fiber using atmospheric-pressure plasma[END_REF][START_REF] Maeda | Evaluation of photocatalytic properties of titanium oxide films prepared by plasma-enhanced chemical vapor deposition[END_REF][START_REF] Rico | Wetting angles and photocatalytic activities of illuminated TiO 2 thin films[END_REF]. Each method may lead to the growth of particular structure and film morphology, which may be quite distinctive and very difficult to repeat with another method. These particularities, on the other hand, may have a substantial effect on the overall photocatalytic activity of the material.

In the literature, titanium dioxide in anatase is generally reported to be obtained at high temperature, in the range of 400-600 °C [START_REF] Dorian | Review of the anatase to rutile phase transformation[END_REF]. However, plasma assisted deposition (magnetron sputtering, PECVD) is known to allow decreasing the deposition temperature of many materials due to the bombardment of the growing film by energetic neutral and charged species. Nevertheless, in the case of TiO 2 , the deposition of anatase thin films by magnetron sputtering or PECVD below 150 °C was reported by only few authors [START_REF] Daviosdottir | Investigation of DC magnetron-sputtered TiO 2 coatings: effect of coatingthickness, structure, and morphology on photocatalytic activity[END_REF][START_REF] Kolouch | Photocatalytic TiO 2 thin Film Prepared by PE CVD at low Temperature[END_REF].

In our previous studies, PECVD processes were used to yield some nanocolumnar structures with different phases including amorphous, anatase and rutile [START_REF] Li | Effect of ion bombardment on the structural and optical properties of TiO 2 thin films deposited from oxygen/titanium tetraisopropoxide inductively coupled plasma[END_REF][START_REF] Li | Structural and optical properties of PECVD TiO 2 -SiO 2 mixed oxide films for optical applications[END_REF]. Actually these processes exhibit favorable features such as low deposition temperature, good film adhesion, high purity, good step coverage and easy control of reaction parameters [START_REF] Battiston | PECVD of amorphous TiO 2 thin films: effect of growth temperature and plasma gas composition[END_REF][START_REF] Lee | Preparation and properties of amorphous TiO 2 thin films by plasma enhanced chemical vapor deposition[END_REF].

Enhancement of chemical reactions by means of low temperature plasma has been known and used for many years. In particular, some new nanostructured materials that cannot be obtained under equilibrium conditions could be prepared using chemical reaction under non equilibrium conditions [START_REF] Martinu | Plasma deposition of optical films and coatings: A review[END_REF][START_REF] Hitchman | Chemical vapor deposition: principles and applications[END_REF][START_REF] Szymanowski | Optical properties and microstructure of plasma deposited Ta 2 O 5 and Nb 2 O 5 films[END_REF][START_REF] Larouche | Microstructure of plasma-deposited SiO 2 /TiO 2 optical films[END_REF], and in the area of property-altering surface coatings [START_REF] Wróbel | Surface free energy of plasma-deposited thin polymer films[END_REF][START_REF] Behnisch | Formation of hydrophobic layers on biologically degradable polymeric foils by plasma polymerization[END_REF].

For photocatalytic applications, in order to reduce the substrate temperature during deposition while maintaining a sufficient energy input to obtain TiO 2 in crystallized form, the PECVD deposition can be controlled in pulsed plasma mode. In this mode, the electrical power is periodically applied to the gas, so that two times are then defined: the discharge time, when the power is applied for a time denoted T on , and the post-discharge time, when the power is switched off for a time denoted T off . Varying the plasma-on time to the period time has two important effects [START_REF] Wang | Time-resolved measurements of ion energy distributions and optical emissions in pulsed radio-frequency discharges[END_REF][START_REF] Heintze | Mechanism of C 2 hydrocarbon formation from methane in a pulsed microwave plasma[END_REF]: (1) to vary the mean power coupled to the plasma and hence the deposition temperature; (2) to allow the control of the ion to neutral flux ratio, which strongly influences the nanostructure and crystallinity.

In this work, we investigate the influence of the duty cycle (DC), defined as the discharge time to the period ratio, on the structural and photocatalytic properties of the TiO 2 films deposited on silicon substrates in pulsed PECVD mode. The latter are also compared to the film deposited in continuous mode. The aim is both to gain insight into the influence of pulsed parameters on the structure of the deposited films by transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), Raman spectroscopy and to deposit TiO 2 films with high photocatalytic activity at low temperature (typically below 100 °C).

Experimental

In this study, TiO 2 films were deposited on silicon (100) substrate (Si, supplied by Codex International, purity > 99.9999999%, CAS Number: 7440-21-3) using a low pressure high density radiofrequency (13.56 MHz) plasma process which is made of a plasma source connected to the diffusion chamber where a substrate is positioned. The rf inductively coupled plasma (ICP) source was generated by applying an rf power (400 W) to an external helicon antenna. Oxygen was introduced at the top of the plasma source. Titanium tetraisopropoxide [Ti(OC 3 H 7 ) 4 , TTIP, purity 99.999%, CAS Number: 546-68-9, supplied by Sigma Aldrich

Ltd.], used as the titanium precursor, was injected into the diffusion chamber through a dispersal ring above the substrate holder. The flow rates of pure TTIP and oxygen were fixed at 0.24 sccm and 16 sccm, respectively, which corresponds to a 98.5:1.5 O 2 /TTIP mixture, while the total working pressure was fixed at 0.4 Pa. The O 2 /TTIP plasmas can be operated either in continuous (CW) or pulsed mode. In this study the frequency was fixed at 1 kHz and the duty cycle (DC) was varied from 10 to 75% by tuning the T on time. The thickness lies between 250 and 300 nm for all deposited films. The substrate temperature was measured using temperature sensitive labels stuck on the back side of the Si substrates [46]. It was measured to be around 120 °C in continuous (CW) standard plasma conditions and was shown to be decreased in the pulsed mode down to 80 °C for DC=50% and below 50 °C for DC = 25%. The deposition parameters are summarized in Table 1. The film morphology was observed by SEM using a JEOL-type JSM 7600F, operating at 10 kV. TEM analysis was performed using a Hitachi H9000-NAR microscope (LaB 6 filament, 300 kV, point to point resolution: 0.18 nm). Cross-section TEM samples were prepared by tripod polishing and afterwards by Ar + ion polishing using Gatan precision ion polishing system (PIPS). The crystalline nature of all the films was determined by X-Ray diffraction (XRD) and Raman spectroscopy. XRD measurements were carried out on a Siemens D8 using Cu Kα radiation (Kα =1.5406 Å) operating in θ/2θ configuration, in the 2θ range of 20-60 º, with a step of 0.03 ° and a dwell of 15 s/step. TiO 2 anatase peaks were attributed thanks to JCPDS file number 89-4921. The Raman spectra were recorded with a Renishaw inVia micro-Raman spectrometer at an excitation wavelength of 532 nm with a laser power of approximately 0.44 mW focused on a 1 μm2 spot. The film surface topography was observed by atomic force microscopy (AFM, NanoWizard II, JPK Instruments) in the contact mode.

The photocatalytic activity of the as-deposited TiO 2 films was studied by measuring stearic acid (SA) decomposition under UV exposure (Herolab UV-8 SL, 254 nm, 8 W). In order to deposit stearic acid on the entire surface of the sample, it was diluted in methanol (0.02 mol L - spin-coated for 30 seconds at 10000 rpm. The samples were then stored in dark in a black box for at least 3 hours before being exposed to the light source. The samples were then illuminated by an UV lamp. The UV lamp (Herolab UV-8 SL, 254 nm, 8 W) was positioned 20 cm above the samples, corresponding to a calculated irradiance of ca. 0.38 mW cm -2 (i.e.

4.9×10 14 photons.s -1 cm -2 ) illuminating the samples surface [START_REF] Boscher | Photocatalytic anatase titanium dioxide thin films depositionby an atmospheric pressure blown arc discharge[END_REF].

The decomposition of the stearic acid was assessed using FTIR spectroscopy (Bruker VERTEX 70 in transmission) by measuring the integrated area of absorption bands in the range 2800-3000 cm -1 (C-H stretching modes of the stearic acid). The photodegradation efficiency was evaluated through the monitoring of this absorption band area as a function of illumination duration. The linear regression of the corresponding degradation curves gives a photocatalytic efficiency (cm -1 h -1 ).

Besides, the FTIR absorption in the range 2800-3000 cm -1 is linked to the quantity of SA by a conversion factor , which gives an equivalency between a number of molecules of SA per cm 2 and an integrated FTIR area value of 1 cm -1 . A conversion factor value of  = 9.7×10 15 molecules of SA cm -2 per cm -1 of FTIR absorption is used in this article [START_REF] Mills | Simultaneous monitoring of the destruction of stearic acid and generation of carbon dioxide by self-cleaning semiconductor photocatalytic films[END_REF]. This allows us to calculate an UV light activity R SA defined as the number of molecules cm -2 of SA degraded per second. SEM micrographs including side-views and top-views of the TiO 2 films deposited in continuous mode and in pulsed mode for different duty cycles are displayed in Fig. 1. All the films exhibit columnar structures, but it seems that the column size can be decreased as the duty cycle is reduced which can be identified by TEM in details. The CW mode leads to a typical morphology on Si substrate in agreement with previous studies [START_REF] Li | Effect of ion bombardment on the structural and optical properties of TiO 2 thin films deposited from oxygen/titanium tetraisopropoxide inductively coupled plasma[END_REF], which is the gradient columnar structure including a dense sublayer at the interface and a disordered columnar top layer. The morphologies for the films grown in the pulsed mode with DC ≥ 40% retain the same gradient columnar structure, but the degree of homogeneity in the growth direction is improved as DC is decreased. Moreover, when DC ˂ 40%, the columns appear more organized and the whole morphology seems to be homogenous along the growth direction.

Results

Film morphology

The evolution of cross-sectional morphology is also reflected in the surface topography (see The TEM image (in Fig. 3) of a 300 nm-thick TiO 2 film deposited in the CW mode on Si substrate clearly shows a highly crystallized film with a columnar morphology. The interface between the Si (100) substrate with a ~4 nm native amorphous SiO 2 layer and the polycrystalline TiO 2 film was identified in Fig. 3(a). The SAED pattern (Fig. 3(b)) is typical for the anatase phase. The presence of a dense layer topped with a columnar crystallized film is quite consistent with SEM pictures which indicated the presence of a dense film topped with a gradient layer [START_REF] Li | Effect of ion bombardment on the structural and optical properties of TiO 2 thin films deposited from oxygen/titanium tetraisopropoxide inductively coupled plasma[END_REF]. The TEM study of the TiO 2 film deposited in the pulsed mode (DC = 50%) displayed in Fig. 4 shows that using pulsed plasma allows decreasing the deposition temperature (measured to be less than 80 °C) while conserving anatase crystallites (in Fig. 4(b)). Compared to the CW mode, a similar columnar morphology was observed, but a slight decrease in the column diameter from ~30 to ~15 nm was observed for the pulsed mode.

Pulsing the plasma decreases the mean energy transferred to the substrate over a whole period (T on + T off ) (due to a decrease in the heat and ion fluxes during T off ), which affects the surface

Si

of the growing film and causes a reorganization of the surface atoms. Thus, the reduction of plasma deposition time by decreasing the duty cycle for pulsed mode leads to a more homogenous columnar morphology with a decreased column size and a decrease in the surface roughness. As can be seen in the XRD patterns displayed in Fig. 5, the as-deposited film in the continuous mode crystallizes in the anatase variety as demonstrated through the appearing of diffraction peaks at 25.5°, 37.8°, 38.6°, 48.1° and 55.1°, corresponding to lattice planes (101), (004), ( 112), ( 200) and (211), respectively. There is a preferential orientation along the (200) direction in the CW mode, while polycrystalline anatase is produced in pulsed mode. The intensities of the anatase peaks were weakened as DC decreased, and disappeared at DC = 25%, where no peak was detected meaning that the film is amorphous. The crystallization of the films was further investigated by Raman spectroscopy which is very sensitive to low amount of anatase and in our conditions provides more local information than XRD. Fig. 6 shows the evolution of the Raman spectra of the TiO 2 films deposited with different duty cycles for pulsed mode and continuous mode. For the TiO 2 films grown in the CW mode and the pulsed mode with DC ≥ 40%, the spectra exhibit five peaks at 144, 198, 395, 516 and 640 cm -1 which are characteristic of the anatase phase and assigned to E g , B 1g , A 1g and E g modes [START_REF] Ohsaka | Raman spectrum of anatase, TiO 2[END_REF]. However, in the case of DC = 25%, the characteristic peaks of anatase are very weak in intensity, but the peak at 144 cm -1 is clearly observed which suggests that the film may consist in a small amount of anatase dispersed in an amorphous matrix. For the films deposited at DC = 10%, the Raman spectra did not show any indication of crystallized TiO 2 .

Film crystallization

In the case of CW mode, ions in the plasma can be continuously generated to participate in the crystallization of the growing film with a simultaneous release of energy which is helpful for the anatase formation. However, in the pulsed mode, the flux and energy of the ion bombardment are no longer generated in post-discharge, which may lead to a reduction in the crystallization rate by increasing the plasma-off time. In addition, the probable phenomenon of hydrolysis of TTIP during post-discharge can also hamper the crystallization, as this reaction is thus carried out in thermodynamic equilibrium and tends to create amorphous TiO 2 [START_REF] Hanaor | Single and mixed phase TiO 2 powders prepared by excess hydrolysis of titanium alkoxide[END_REF].

Therefore, a reduction of the amount of the crystallized anatase phase is observed when the duty cycle is decreased. This statement can indicate that the growth of anatase crystalline nanocolumns is directly linked to the deposition period during the plasma on time, independently of the substrate temperature.

Photocatalytic activity

According to the above discussion concerning the influence of pulsed plasmas on the TiO 2 film morphology and crystallization, some interesting points about the anatase content, columns size and organization, surface state were demonstrated, which may affect the photocatalytic properties of these TiO 2 films. In this work, the photocatalytic activity of thin films was evaluated by monitoring of the degradation of stearic acid under UV light. photocatalytic performance (with the same measuring device) of bare silicon substrate, PECVD-grown rutile films [START_REF] Li | Effect of ion bombardment on the structural and optical properties of TiO 2 thin films deposited from oxygen/titanium tetraisopropoxide inductively coupled plasma[END_REF] and 2 μm TiO 2 film obtained by magnetron sputtering (PVD) at 250 °C [START_REF] Maurau | Atmospheric pressure, low temperature deposition of photocatalytic TiO x thin films with a blown arc discharge[END_REF]. One can note that the silicon substrate has no photocatalytic activity (as expected), whereas the films presented in this work all exhibit a decrease of the SA quantity when illuminated by UV light.

UV irradiation time (h) C-H normalized absorption area

In order to compare with available literature data, the formal quantum efficiency (FQE) for SA is calculated [START_REF] Mills | Simultaneous monitoring of the destruction of stearic acid and generation of carbon dioxide by self-cleaning semiconductor photocatalytic films[END_REF] and presented in Fig. 7(b). The FQE is calculated as the number of SA photodegraded by the incident photons (see eq. 1). The slope of the degradation curve of SA, R SA and FQE for the films deposited under different conditions are shown in Table 2.

(eq. 1) The reproducibility of photocatalytic activity measurements was confirmed for several samples deposited for DC = 100, 75 and 50%.

The photodegradation efficiency of TiO 2 films containing anatase deposited at DC = 100%, 75% and 50% exhibits very high values, with FQE equal to 390×10 -5 , 473×10 -5 and 385×10 -5 molecules/photon respectively. These values are very good as they surpass the FQE of some reference films such as P25 TiO 2 and Pilkington Activ thin films, which exhibit FQE values of 256×10 -5 and 10×10 -5 respectively [START_REF] Mills | Characterisation of the photocatalyst Pilkington Activ™: a reference film photocatalyst?[END_REF].

Nevertheless, photocatalysis is somehow dependent on the coating thickness. Indeed, thicker films induced a higher probability of producing reactive species that participate to catalytic processes. In this article, thin films have a thickness of ca. 250 nm, whereas in the article of A.

Mills et al [START_REF] Mills | Characterisation of the photocatalyst Pilkington Activ™: a reference film photocatalyst?[END_REF], P25 TiO 2 and Pilkington Activ have a thickness of 90 nm and 15 nm respectively. One can note the slightly higher photocatalytic activity of the samples obtained at DC = 50%, 75 % and 100% which might be due to more anatase content and higher surface area, induced by smaller column size and higher roughness. It is then noted that the photocatalytic activity decreases when DC decreases reaching FQE = 236×10 15 , 115×10 15 and 55×10 15 molecules per photon for DC = 40 %, 25 % and 10 % respectively. Although these films have lower photoactivity, their efficiency is still good especially when considering the very low substrate temperature (˂ 50 °C) during deposition.

TiO 2 layers deposited for DC ≥ 50% contain a significant amount of anatase and present the highest photocatalytic activity. Although the TiO 2 layer grown at DC = 40% has a quite rough top surface, the crystalline anatase content detected by Raman scattering and XRD is high enough to lead to a lower but still significant photocatalytic activity. In the cases of DC = 25 and 10%, TiO 2 layers have a reduced but still significant conversion rate which is higher than the PVD-deposited anatase at 250 °C [START_REF] Maurau | Atmospheric pressure, low temperature deposition of photocatalytic TiO x thin films with a blown arc discharge[END_REF]. Indeed, the R of the film obtained at DC = 10%, at a temperature < 50 °C is comparable to other films obtained using an atmospheric pressure PECVD process with substrate temperature around 100 °C [START_REF] Chemin | Transparent anti-fogging and self-cleaning TiO 2 /SiO 2 thin films on polymer substrates using atmospheric plasma[END_REF]. Moreover, the rutile TiO 2 film deposited by PECVD shows a rather poor photodegradation efficiency.

This study has demonstrated that it is possible to deposit TiO 2 layers with high photocatalytic activity at temperatures below 100 °C (50% ≤ DC ≤ 75%). This therefore opens up interesting prospects for the deposition of photocatalytic layers, for example on flexible polymer substrates.

Conclusions

Highly efficient photocatalytic anatase nanocolumnar TiO 2 thin films were successfully deposited by pulsed-PECVD process at relatively low temperature less than 100 °C on Si substrate. The effect of plasma-off time by tuning the duty cycle of pulsed mode was investigated on TiO 2 thin films in view of improving their photocatalytic properties. The efficient photocatalytic property of such a film depends on the presence of the anatase phase at its surface and also its roughness and microstructure. At a duty cycle between 40% and 100%, the bombardment of the particles at the substrate is strong enough for inducing mobility of surface particles, promoting the formation of crystalline anatase TiO 2 films with disordered columnar morphologies. The photocatalytic activity for these films is very high.

As DC is decreased below 40%, the accumulation of impact energy and heat is insufficient, leading to the formation of a surface including only a small amount of anatase embedded in an amorphous film, which has relatively smooth surface and homogeneous columns along the growth direction. Thus the photocatalytic activity is reduced, but is still significant regarding the low substrate temperature.

Fig. 1 .

 1 Fig. 1. SEM micrographs of the TiO 2 films deposited with different duty cycles for pulsed and continuous mode (100%). (a, b) continuous mode, (c, d) DC = 75%, (e, f) DC = 50%, (g, h) DC = 40%, (i, j) DC = 25%, (k, l) DC = 10%, (a, c, e, g, i, k) SEM side-views, (b, d, f, h, j, l) SEM top-views.

Fig. 2 )Fig. 2 .

 22 Fig.2), for the films deposited at DC > 40%, the top surfaces are quite rough because of the disordered columns leading to some clustering of grains till the surface. Moreover, the RMS roughness value decreases with DC, from 9.0 nm for CW mode to 6.0 nm for DC= 50%. But in the case of DC = 40%, the surface topography marks a transition with the gradually disappearance of the clusters leading to an abrupt increase in roughness up to 11.2 nm. The images of the surfaces of the TiO 2 films reveal a change from granular clusters to rice-shaped grains as DC is further decreased below 40%, and the grain size is also reduced with DC leading to smoother top surfaces, the roughness is even decreased down to 4.0 nm for DC=25%.

Fig. 3 .

 3 Fig. 3. TEM on the TiO 2 film deposited in the continuous mode (CW). (a) The whole film morphology, (b)selected area electronic diffractions (SAED): experimental diffraction of the film and simulation for anatase using JEMS software[49].

Fig. 4 .

 4 Fig. 4. TEM on the TiO 2 film deposited with the duty cycle of 50% for pulsed mode. (a) The whole film morphology, (b) selected area electronic diffractions (SAED): experimental diffraction of the film and simulation for anatase using JEMS software.

Fig. 5 .

 5 Fig. 5. XRD patterns of the TiO 2 films deposited with different duty cycles for pulsed mode and continuous mode.

Fig. 6 .

 6 Fig. 6. Raman spectra of the TiO 2 films prepared with different duty cycles for pulsed mode and continuous mode (CW).

Fig. 7 .

 7 Fig. 7. Evolution of photocatalytic activity for the TiO 2 films grown with different duty cycles for pulsed mode and continuous mode (100%), and compared with the reference TiO 2 film deposited by PVD and PECVD-rutile films. (a) C-H absorption integrated area (2800-3000 cm -1 ) versus UV illumination time, (continuous lines are just guidelines to get a better view of the evolutions) (b) Formal Quantum Efficiency for SA molecules calculated from SA degradation rate as a function of the duty cycles.

Fig. 7

 7 Fig.7presents the evolution of the integrated area of the FTIR absorption band in the range 2800-3000 cm -1 as a function of the exposure duration to UV illumination on the TiO 2 films deposited in continuous and pulsed modes. These measurements were also compared to

Table 1 .

 1 Growth conditions of TiO 2 films using the pulsed PECVD system.

	rf power coupled to the ICP source	400 W
	Working pressure	0.4 Pa
	Flow rate of TTIP	0.24 sccm
	Flow rate of O 2	16 sccm
	Pulsed mode frequency	1 kHz
	Duty cycle (DC) for pulsed mode	10%, 25%, 40%, 50%, 75%, CW
	Substrate temperature (±5 °C)	˂ 50, ˂ 50, 50, 80, 90, 120
	Substrate	Si (100)

Table 2 .

 2 Evolution the SA degradation rate, R SA and FQE for the TiO 2 films grown with different duty cycles for pulsed mode and continuous mode (100%).

	Duty cycle	SA degradation rate	R SA (SA, 254 nm)	FQE (SA, 254 nm)
		(h -1 )	(molecules cm -2 s -1 )	(10 -5 molecules/photon)
	100 % (CW)	0.71 ± 0.7	(1.9 ± 0.2)×10 12	390 ± 39
	75 %	0.86 ± 0.9	(2.3 ± 0.3)×10 12	473 ± 47
	50 %	0.7 ± 0.7	(1.9 ± 0.2)×10 12	385 ± 39
	40 %	0.43 ± 0.4	(1.2 ± 0.1)×10 12	236 ± 24
	25 %	0.21 ± 0.2	(5.7 ± 0.6)×10 11	115 ± 12
	10 %	0.1 ± 0.01	(2.7 ± 0.3)×10 11	55 ± 6

) and deposited by spin coating. 10 microliters were deposited on the coatings(1 cm

) and

Acknowledgments

Acknowledgements for the master students: Theo Leclerq (mainly) for this paper. The work of Dayu Li is supported by the National Natural Science Foundation of China (51602279), High-end Talents and Young Backbone Teacher Project of Yangzhou University.


Sandra Bigot

Guy Louarn

Nasreddine Kébir
email: nasreddine.kebir@insa-rouen.fr

Fabrice Burel

Facile grafting of bioactive cellulose derivatives onto PVC surfaces

Bioactive methyl cellulose and hydroxyethyl cellulose were successfully grafted onto PVC films using a two-step pathway. PVC surface was first modified with isothiocyanate in DMSO/water medium. Then, unmodified polysaccharides were directly grafted onto the thiocyanated surface in acetonitrile/DMSO mixture, in presence of DBTL as catalyst. The polysaccharide grafting onto the PVC surface was evidenced by contact angle measurements, AFM and XPS.

Introduction

Biofilm formation and infection is a problem of major concern covering several applications fields, especially the Biomaterials one. In order to prevent this phenomenon, several strategies were developed in the past such as: antibiotic impregnation, surface coating by some metals like copper and silver or by some specific polymers like hydrogels. However, these techniques exhibited some disadvantages such as antibiotic exhaustion, appearance of bacteria resistance phenomena, physical instability and lost of the biological activity in the long term.

During the last decades, a promising strategy consisting in surface modification was developed. This strategy employed either physical treatment such as plasma, or direct chemical treatment such as grafting of specific species and functionalities. In this context, the wet-chemical process has been found to be the best pathway to control the surface chemical and physical properties, in particular when active polymers are grafted onto the surface. Indeed, comparing to small molecules, polymer grafting offers numerous advantages such as surface stability and longevity, high surface coating and high bioactivity.

Polyvinyl chloride (PVC) is one of the most important polymers thanks to its versatile physico-chemical and biological properties. PVC is employed in various applications such as construction, packaging and medical devices. Many medical articles are based on PVC such as: blood bags, indwelling catheters, urinary catheters etc.

Polysaccharides represent a major natural, renewable and available resource in the polymer field. Their applications vary from textile [START_REF] Shang | Preparation of durable hydrophobic cellulose fabric from water glass and mixed organosilanes[END_REF][START_REF] Huang | Enhanced washing durability of hydrophobic coating on cellulose fabric using polycarboxylic acids[END_REF] to medical systems [START_REF] Rinaudo | Main properties and current applications of some polysaccharides as biomaterials[END_REF][START_REF] Costa | Biological activities of sulfated polysaccharides from tropical seaweeds[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Damonte | Sulfated seaweed polysaccharides as antiviral agents[END_REF][START_REF] Azevedo | Heparinoids algal and their anticoagulant, hemorrhagic activities and platelet aggregation[END_REF][START_REF] Goddard | Polymer surface modification for the attachment of bioactive compounds[END_REF][START_REF] Chen | Improving blood-compatibility of titanium by coating collagen-heparin multilayers[END_REF][START_REF] Zha | Self-assembled hemocompatible coating on poly (vinyl chloride) surface[END_REF][START_REF] Jin | Fabrication of PPgPEGMAgheparin and its hemocompatibility: from protein adsorption to anticoagulant tendency[END_REF][START_REF] Mussard | Facile and efficient control of bioadhesion on poly(dimethylsiloxane) by using a biomimetic approach[END_REF][START_REF] Mao | A photochemical method for the surface modification of poly(vinyl chloride) with O-butyrylchitosan to improve blood compatibility[END_REF][START_REF] Asadinezhad | Polysaccharides coatings on medical-grade PVC: a probe into surface characteristics and the extent of bacterial adhesion[END_REF][START_REF] Zhou | Preparation and characterization of polymeric coatings with combined nitric oxide release and immobilized active heparin[END_REF]. They are non-toxic, biocompatible, often biodegradable and water soluble. Such properties make them suitable for many uses in the biomedical field, health care, or drug delivery systems [START_REF] Rinaudo | Main properties and current applications of some polysaccharides as biomaterials[END_REF]. As example, heparin and chitosan, are used to improve biocompatibility and/or to prevent attachment of bacteria, blood platelets or proteins onto biomaterial surfaces [START_REF] Costa | Biological activities of sulfated polysaccharides from tropical seaweeds[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Damonte | Sulfated seaweed polysaccharides as antiviral agents[END_REF][START_REF] Azevedo | Heparinoids algal and their anticoagulant, hemorrhagic activities and platelet aggregation[END_REF][START_REF] Goddard | Polymer surface modification for the attachment of bioactive compounds[END_REF][START_REF] Chen | Improving blood-compatibility of titanium by coating collagen-heparin multilayers[END_REF][START_REF] Zha | Self-assembled hemocompatible coating on poly (vinyl chloride) surface[END_REF][START_REF] Jin | Fabrication of PPgPEGMAgheparin and its hemocompatibility: from protein adsorption to anticoagulant tendency[END_REF][START_REF] Mussard | Facile and efficient control of bioadhesion on poly(dimethylsiloxane) by using a biomimetic approach[END_REF][START_REF] Mao | A photochemical method for the surface modification of poly(vinyl chloride) with O-butyrylchitosan to improve blood compatibility[END_REF][START_REF] Asadinezhad | Polysaccharides coatings on medical-grade PVC: a probe into surface characteristics and the extent of bacterial adhesion[END_REF][START_REF] Zhou | Preparation and characterization of polymeric coatings with combined nitric oxide release and immobilized active heparin[END_REF]. Recently, cellulose derivatives, especially methyl celluloses, have been described as potent bacteria repellents when they are grafted onto a silicone surface [START_REF] Mussard | Facile and efficient control of bioadhesion on poly(dimethylsiloxane) by using a biomimetic approach[END_REF].

Nevertheless, in the literature only few examples describe the grafting of polysaccharides onto PVC surfaces. Mao et al. [START_REF] Mao | A photochemical method for the surface modification of poly(vinyl chloride) with O-butyrylchitosan to improve blood compatibility[END_REF] grafted O-butyrylchitosan (OBCS) onto PVC surface in order to improve blood compatibility. OBCS was first bound to 4azidobenzoic acid, coated on the PVC film surface and then, it was covalently immobilized by ultraviolet irradiations. In order to prevent bacteria attachment, Asadinezhad et al. [START_REF] Asadinezhad | Polysaccharides coatings on medical-grade PVC: a probe into surface characteristics and the extent of bacterial adhesion[END_REF] functionalized PVC surface by a multistep procedure. In a first step, the surface was activated by discharge plasma in air. Then, acrylic acid was polymerized from the surface via a radical graft copolymerization and in a final step chitosan layers were grafted onto the surface by amidification. With the aim of mimicking the nonthrombogenic properties of the endothelial cell, Zhou et al. [START_REF] Zhou | Preparation and characterization of polymeric coatings with combined nitric oxide release and immobilized active heparin[END_REF] reported the development of a new generation of NO release polymeric coatings that employ NO release chemistry in combination with surface immobilized active heparin. Heparin was grafted onto aminated PVC after modification of the polysaccharide chains with N-hydroxysuccinimide, using 1ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent.

All these examples support the need of alternative procedures to graft polysaccharides onto PVC.

Herein, we describe a simple and an alternative procedure for the grafting of polysaccharides onto PVC surfaces. First, isothiocyanate groups (NCS) were introduced onto PVC surfaces using potassium thiocyanate in a water/DMSO mixture. These groups are analogue to isocyanate (NCO) groups, and are commonly present in fluorescent probes for protein labelling. Then, cellulose derivatives exhibiting bacteria repellent properties [START_REF] Mussard | Facile and efficient control of bioadhesion on poly(dimethylsiloxane) by using a biomimetic approach[END_REF] were directly grafted onto PVC-NCS surfaces in presence of a catalyst. The grafting efficiency was assessed by mean of contact angle measurements, XPS and AFM analysis.

Experimental

Materials

Commercial bulk polymerized PVC was obtained from Sigma-Aldrich, Lyon, France. The average molecular weights determined by SEC were Mn = 47000 g/mol and Mw = 80000 g/mol. Methyl cellulose (DS = 1.5-1.9), hydroxyethyl cellulose (DS = 1.6) and tetrabutylammonium hydroxide (TBAH, 40 wt% in water) were purchased from Sigma-Aldrich. Potassium thiocyanate was purchased from Merck.

Characterizations

NMR spectra were recorded on a Bruker Avance (300 MHz) Spectrometer.

IR spectra were recorded on a FTIR (Perkin-Elmer) equipped with a diamond ATR device (attenuated total reflection).

XPS measurements were carried out at room temperature. An Axis Nova instrument from Kratos Analytical spectrometer with Al K␣ line (1486.6 eV) as excitation source has been used. The core level spectra were acquired with an energy step of 0.1 eV and using a constant pass energy mode of 20 eV, to obtain data in a reasonable experimental time (energy resolution of 0.4 eV). The pressure in the analysis chamber was maintained lower than 10 -7 Pa. The background spectra were considered as Shirley type and curve fitting was achieved with a mixture of Gaussian-Lorentzian functions [START_REF] Berresheim | A standard form of spectra for quantitative ESCA-analysis[END_REF]. The error in defining the position of peaks was estimated at about 0.1 eV. No surface cleaning, using Ar sputtering for example was made. We know from experience that carbonaceous atmospheric contamination on material usually occurs, but in our case, ion sputtering can be suspected of changing chemical composition and inducing structural damage.

Surface energy of the materials were evaluated using Digidrop Goniometer (GBX, France), by static contact angle (Â i ) measurements on the sample surfaces with 3 liquids: water, diiodomethane and glycerol. The static contact angles were measured at the equilibrium time. At least, five measurements on different surfaces were performed to calculate the mean contact angle and its standard deviation. According to the Owens-Wendt relationship (1) the dispersive d s and polar p s components of the surface energies ( s = d s + p s ) of the samples were determined.

(

+ cos  i ) i 2 d i = p s × p i d i + d s 1
where i , d i , p i are the solvent superficial tension parameters. Surface polarities (P) of the materials are calculated using Eq. ( 2):

P = p s s × 100 (2)
An AFM microscope (JPK instruments, NanoWizard, Berlin, Germany) was used for imaging and roughness measurements.

AFM images were obtained by using the intermittent contact mode AFM in air. Classical silicon cantilevers were used (NanoWorld Pointprobe ® NCHR). The average force constant and resonance were approximately 42 N/m and 320 kHz, respectively. Topographic images were taken at different locations on the surface, in order to account for any heterogeneity in the coverage of the polysaccharides. AFM data were analyzed by the software SPM Image Processing v.3 from JPK Instruments, to extract the surface roughness (calculated on a 10 m 2 area).

Preparation of PVC films

About 1 g of PVC powder was dissolved in 40 mL of dichloromethane and poured into a Teflon mould. PVC films were then obtained by slow solvent evaporation under nitrogen atmosphere.

Preparation of PVC-isothiocyanate surfaces (PVC-NCS)

In a round bottom flask, KSCN (9.0 × 10 -2 mol) and TBAH (4.5 × 10 -2 mol) were dissolved in a 60 mL of DMSO/water (4/1) mixture. PVC sheets (1 cm × 1 cm) were immersed and the medium was heated at 60 • C for 17 h under slow-stirring. Then, the PVC sheets were thoroughly washed with water, rinsed with diethyl ether and dried at 60 • C. The same reaction mixture could be recycled 4-5 times.

FTIR: max /cm -1 . . .2150 (S CN), 2051 (N C S), 1600 (C C).

Grafting of polysaccharides onto PVC-NCS surfaces

In a round bottom flask equipped with a magnetic stirrer, 12 mg of polysaccharide were dissolved in 2 mL of DMSO at room temperature. Then, 4-12 mL (Table 1) of acetonitrile were added and the mixture was heated to 60 • C. Then, 0.05 equivalent of DBTL with regards to the polysaccharide hydroxyl groups were added. After, PVC sheets were immersed in this solution under gentile stirring for 24 h. The treated PVC films were washed several times with water and then put in water bath under ultra sound (30 min), rinsed with diethyl ether and dried at 60 • C.

Results and discussion

Preparation of grafted and ungrafted PVC-NCS surfaces

The common strategy to directly graft a polysaccharide onto polymer surfaces consists in esterification reaction between the polysaccharide hydroxyl groups and surface carboxyl groups, in presence of coupling agents such as N,N-dicyclohexylcarbodiimide (DCC) or N,N -carboimidazole (CDI). In this study, an original procedure involving isothiocyanate groups was developed since these groups are easier to introduce onto the PVC chains compared to carboxyl groups. The chemical grafting of polysaccharides onto PVC surfaces was achieved in two steps (Scheme 1).

In a first step, isothiocyanate groups were introduced onto PVC surfaces. Actually, the reaction of thiocyanates with an alkyl halide is a nucleophilic substitution (SN2) that results in the formation of the corresponding alkyl isothiocyanate. The reaction proceeds in homogenous or heterogeneous conditions and may be applied to polymers as well. Thus, it has be shown [START_REF] James | Surface thiocyanation of plasticized poly(vinyl chloride) and its effect on bacterial adhesion[END_REF] that thiocyanation of PVC surfaces can be achieved in a solid/liquid two phase system in the presence of a phase transfer catalyst in aqueous media. A similar heterogeneous procedure was used herein to apply the reaction to the PVC surface. Chloride groups were substituted by isothiocyanate groups (-NCS) using KSCN, in the presence of TBAH as phase transfer catalyst in a DMSO/water (4/1) mixture. In a second step, methylcellulose and hydroxyethylcellulose with different molecular weights were dissolved at a concentration of 1 mg/mL in appropriate acetonitrile/DMSO mixtures in which the PVC films was not dissolved or damaged (Table 1). Then, an optimal amount of a catalyst, i.e., dibutyltin dilaurate (DBTL), was added (Table 1). After, the PVC-NCS samples were immersed in the polysaccharide solution during 24 h at 60 • C, to allow the chemical grafting. The obtained PVC grafted polysaccharide surfaces were thoroughly cleaned before characterizations.

Surface characterisations

Contact angle measurements

The contact angle measurement was a quick, simple and efficient method to control the chemical grafting of polysaccharides onto the PVC-NCS surface and to optimize the experimental conditions.

Static contact angles with water as well as surface energies and polarities of the modified and unmodified PVC surfaces are reported in Table 1.

Comparing to PVC surface, the PVC-NCS surface showed a significant decrease of the static contact angle with water, i.e., from 89 • to 44 • , indicating that thiocyanation increased considerably the hydrophilicity of the PVC surface. In addition, the polar component of the surface energy increased from 1 to 19 mN/m and the surface polarity increased from 2.3% to 32.7% (Table 1, entries 1 and 2).

Static contact angles with water of PVC-NCS surfaces that were in contact with polysaccharides were dependent on the amount of DBTL with regards to polysaccharide hydroxyl groups. For 0 and 0.02 equiv. of DBTL/OH (Table 1, entries 3, 4, 6, 8, 9 and 11), one can observe that the contact angle as well as surface energy and polarity were very close to those of the PVC-NCS surface (Table 1, entry 2), which suggest that chemical grafting did not happen in these conditions. For 0.05 equiv. of DBTL/OH (Table 1, entries 5, 7, 10 and 12), the surface properties changed significantly, suggesting that the chemical grafting of polysaccharide onto the PVC-NCS surfaces were successful. Actually, the two methylcellulose surfaces exhibited similar properties, i.e., contact angles with water around 60 • and polarities around 23%. However, hydroxyethyl cellulose surfaces were different; the PVC-HEC1 surface exhibited higher contact angle (65 • against 55 • ) and lower polarity (14.6% against 26.5%) comparing to PVC-HEC 2. Overall, the grafted surfaces were less -but still-hydrophilic and less polar than the ungrafted PVC-NCS surface. These grafted surfaces were then characterized by FTIR, XPS and AFM.

Dynamic contact angle with water was also performed to assess the surface wettability (Table 2). The AA is characteristic of the wettability of the low-energy or hydrophobic part of the surface whereas the RA is more characteristic of the high-energy or hydrophilic part.

Native PVC surface exhibited AA around 90 • and RA around 43 • . After thiocyanation, the AA and RA decreased dramatically and the balance between the two parts was also reduced as suggested by the decrease of hysteresis value from 48 • to 33 • (Table 2, entries 1 and 2).

PVC-NCS surfaces grafted with polysaccharides exhibited AA very close to static angle, whereas RA was in the range of 15-20 • (Table 2, entries 3-6). Thus, hysteresis values ranged from 35 • to 47 • and were consequently higher than the PVC-NCS hysteresis (33 • ). This behaviour is likely to be associated with the hydrophobic backbone of the polymer chains that increase the hydrophilic-hydrophobic balance of the surface. Besides, hysteresis can also be explained by surface heterogeneity, texture and roughness.

Spectroscopic analysis

The thiocyanation of PVC surface was followed by ATR-FTIR. The appearance of a new band at ∼2050 cm -1 attributed to isothiocyanate groups, according to literature [START_REF] Kameda | Chemical modification and dechlorination of polyvinyl chloride by substitution with thiocyanate as a nucleophile[END_REF], indicated once again that the reaction was successful. The detection of this band by this technique showed that the NCS groups were grafted on a PVC surface zone of 3 m of depth at least. This probably resulted from surface swelling in the DMSO/acetonitrile medium, which allowed the penetration of the thiocyanate ions. However, because of their big hydrodynamic volume, polysaccharides were only grafted on the extreme surface and were therefore not detected by this technique.

The elemental compositions of the grafted surfaces and their controls (PVC and PVC-NCS), were determined by XPS analysis. Results are shown in Table 3.

As expected, XPS survey scans obtained from the PVC surface gave two signals corresponding to C and Cl with contributions of 66.6% and 33.4%, respectively. The elemental analysis of the PVC-NCS surface confirmed the presence of N (1.5%) and S (1.3%) coming from the isothiocyanate groups. However, the percentage of Cl decreased (15.5%) suggesting an elimination of HCl (PVC dechlorination), which was not observed by FTIR. Kameda et al. [START_REF] Kameda | Chemical modification and dechlorination of polyvinyl chloride by substitution with thiocyanate as a nucleophile[END_REF] also observed this side reaction and have shown that the presence of phase transfer agents, such as TBAH, largely favoured the substitution reaction over the elimination one. The elemental analysis of the grafted surfaces showed the presence of O with abundance around 15%, suggesting the presence of polysaccharides.

The XPS survey scans for the main components were analyzed to assign the peaks C 1s , N 1s and S 2p to chemical functions (Table 3). Typical examples of C 1s high resolution XPS spectra are presented in Fig. 1.

For PVC-NCS surface, the C 1s peak was decomposed into four peaks. The peaks ranging from 284 to 285 eV are ascribed to C of saturated and unsaturated hydrocarbon chains. The peak at 286.4 eV is attributed to C making single bonds with N, S or Cl. The contribution of this peak is weak (15.5%) confirming the elimination of Cl during chemical treatment. The high resolution XPS spectra of the N 1s region revealed three peaks. The peak at 394.8 eV is owing to residual thiocyante ions adsorbed onto the surface. The peak at 399.9 eV is ascribed to grafted isothiocyanate groups. The peak at 401.5 is due to ammonium groups of residual TBAH, which exhibited also a very weak signal at 532.2 eV, undetectable in the survey spectra (<1%). The high resolution XPS spectra of the S 2p region revealed two peaks; each peak corresponds to two spin-orbit transitions. The main spin-orbit transition 3/2 was used for quantification. The peak at 163.8 eV is ascribed to the S in thiocyante groups and residual thiocyante ions. The peak at 168.1 eV is ascribed to the S in isothiocyante groups.

The PVC-NCS surfaces grafted with polysaccharides exhibited a C 1s peak at 286. 27% where about 17% was due to the surface control (PVC-NCS). In addition, two supplemental peaks were observed in the C 1s region, the peak at 287.7 eV was ascribed to C in acetal or hemiacetal functions in polysaccharides. The peak at 288.9 eV was attributed to C in the thiocarbamate links (NH (C S) O). The XPS high resolution spectra of the N 1s and S 2p regions were similar to those of the PVC-NCS surface but with variable peak contributions.

AFM analysis

AFM images of PVC and PVC-NCS surfaces as well as typical examples of grafted surfaces are shown in Fig. 2.

One can observe that the surface treatment with NCS led to changes in surface morphology without significant variation in the surface roughness, which was around 3 nm.

In contrast, polysaccharide grafting onto the PVC-NCS surface changed dramatically the surface topography, and the surface roughness increased to around 20 nm.

Furthermore, according to the literature [START_REF] Eick | Thermodynamics of contact angles II[END_REF][START_REF] Olivier | Effects of solid surface roughness on wetting[END_REF], the surface being homogeneous and roughness values being lower than 100-500 nm, the hysteresis measured by dynamic contact angle with water would be exclusively due to the hydrophilic-hydrophobic balance.

Conclusions

In this work we have grafted bioactive cellulose derivatives onto PVC surfaces using a straightforward pathway. First, PVC-NCS surfaces were prepared by a heterogeneous thiocyanation of the PVC surface in a DMSO/water mixture. Then, the selected polysaccharides were directly grafted onto the PVC-NCS surfaces in a DMSO/actonitrile media, in presence of DBTL as catalyst. The impact of DBTL amount on polysaccharide grafting onto the surface was monitored by contact angle measurements. 0.05 equiv. of DBTL by polysaccharide hydroxyl group seemed to be an optimal ration. In these conditions, the grafting reaction was effective and the resulted surfaces exhibited modulated properties comparing to the ungrafted surfaces.

Scheme 1 .

 1 Scheme 1. The chemical pathway used to graft polysaccharides onto PVC surface.

Fig. 1 .

 1 Fig. 1. XPS high resolution spectra of the C1s region: (a) PVC-NCS and (b) PVC-MeC 2.

Fig. 2 .

 2 Fig. 2. IC-AFM height images (10 m × 10 m) of (a) PVC, (b) PVC-NCS and (c) PVC-HEC 1, (d) PVC-MeC 1.

Table 1

 1 Experimental conditions and static contact angle data relative to PVC surfaces.

	Entry	Surface	Mw (g/mol)	DBTL (equiv./OH)	CH3CN/DMSO (v/v)	Static Âwater ( • ) a	d (mN/m) a	p (mN/m) a	Polarity (%)
	1	PVC	/	/	/	89 ± 5	42.2 ± 0.2	1.0 ± 0.8	2.3 ± 1.8
	2	PVC-NCS	/	/	0/0; 2/1; 4/1; 6/1	44 ± 2	38.9 ± 1.3	18.9 ± 0.1	32.7 ± 0.7
	3	PVC-MeC 1	41,000	0.00	6/1	47 ± 2	36.9 ± 0.7	18.1 ± 0.1	32.9 ± 0.3
	4	PVC-MeC 1	41,000	0.02	6/1	45 ± 1	38.6 ± 3.2	19.9 ± 0.3	34.0 ± 2.1
	5	PVC-MeC 1	41,000	0.05	6/1	59 ± 2	39.0 ± 1.6	11.9 ± 0.2	23.4 ± 0.9
	6	PVC-MeC 2	88,000	0.00	2/1	48 ± 2	38.7 ± 0.4	17.6 ± 0.6	31.3 ± 0.6
	7	PVC-MeC 2	88,000	0.05	2/1	60 ± 4	37.4 ± 0.5	11.0 ± 1.4	22.7 ± 2.4
	8	PVC-HEC 1	720,000	0.00	2/1	43 ± 2	38.3 ± 0.4	19.4 ± 0.3	33.6 ± 0.2
	9	PVC-HEC 1	720,000	0.02	2/1	45 ± 1	37.4 ± 0.3	19.2 ± 0.2	33.9 ± 0.1
	10	PVC-HEC 1	720,000	0.05	2/1	65 ± 2	41.4 ± 0.4	7.1 ± 0.9	14.6 ± 0.9
	11	PVC-HEC 2	250,000	0.00	4/1	41 ± 3	40.2 ± 0.8	18.7 ± 0.5	31.7 ± 0.7
	12	PVC-HEC 2	250,000	0.05	4/1	55 ± 1	37.8 ± 0.8	13.6 ± 0.4	26.5 ± 1.4

a Âwater: contact angle with water; d : dispersive component of the surface energy; p : polar component of the surface energy.

Table 2

 2 Dynamic contact angle data relative to PVC-NCS surfaces grafted with polysaccharide in presence of 0.05 equiv. of DBTL.

	Entry	Surface	Dynamic Âwater ( • )
			AA	RA	Hyst
	1	PVC (control 1)	90 ± 3	43 ± 5	48 ± 2
	2	PVC-NCS (control 2)	46 ± 2	13 ± 2	33 ± 3
	3	PVC-MeC 1	60 ± 1	15 ± 1	44 ± 1
	4	PVC-MeC 2	60 ± 1	16 ± 1	45 ± 1
	5	PVC-HEC 1	66 ± 1	19 ± 1	46 ± 1
	6	PVC-HEC 2	53 ± 1	20 ± 1	33 ± 2

AA: advancing angle; RA: receding angle; Hyst: hysteresis.

Table 3

 3 XPS data of the prepared surfaces: elemental analysis; peak assignment and contribution.

	Chemical state	Binding energy (eV)	PVC	PVC-NCS	PVC-MeCell 1	PVC-MeCell 2	PVC-HEC 1	PVC-HEC 2
	% C	-	66.6	81.7	66.7	67.3	70.9	63.8
	C (C,H), C C	284-285	52.9	64.5	61.9	65.6	59.2	68.4
	C S, C O, C N, C Cl, C C C ring	286.4	47.1	17.2	25.5	23.0	26.8	21.1
	O C O	287.7	/	/	6.0	5.4	6.3	5.0
	(C O) O , (C O) NH, NH (C S) O	288.9	/	/	6.6	5.9	6.9	5.5
	% O	-	0	<1*	16.3	16.4	13.9	15.8
	(C O) NH, (C O) O	531.6	/	/	32.3	27.5	28.2	32.8
	C O C, C O H	532.2	/	100	35.3	45.0	46.4	34.5
	(C O) O	533.4	/	/	32.3	27.5	28.2	32.8
	% N	-	0	1.5	1.7	1.5	1.5	1.6
	SCN -	394.8	/	5.6	6.3	5.3	0.0	0.0
	N C S, (C O)NH S C N, (C S)NH	399.9	/	74.2	69.6	63.0	61.5	74.5
	C N +	401.5	/	20.2	24.6	31.7	38.5	25.5
	% S	-	0	1.3	1.4	1.6	1.2	1.6
	C S CN, SCN -	136.8	/	52.7	40.9	48.7	45.1	31.5
	>C S	168.1	/	47.3	59.1	51.3	54.9	68.5
	% Cl		33.4	15.5	12.0	10.4	10.3	15.9

Acknowledgements

This work was a part of the project "Green Intelligent Material (GIMs)" which belongs to the "Interreg IV France (Channel) -England" European programme. Authors thank Europe and the "Haute Normandie" Region for their financial and logistic supports.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsusc. 2013.06.123.


Elisabeth Conchon

Fabrice Anizon

Bettina Aboab

Roy Golsteyn

Stéphane Leonce

Bruno Pfeiffer

Michelle Prudhomme

    Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory properties of pyrrolo[3,4-a]carbazole-1,3-diones, pyrrolo[3,4-c]carbazole-1,3-diones, and 2-aminopyridazino[3,4-a]pyrrolo[3,4-c]carbazole-1,3,4,7tetraone


Konrad Hinsen

The magic of content-addressable storage

published or not. The documents may come

The magic of content-addressable storage

A BIT OF THEORY

Traditionally, we use two techniques in computing to refer to data, which I will call quoting and naming. Quoting is just writing out the data explicitly, such as writing the number literal 42, the text snippet Computing in Science and Engineering, or the few-thousand-bytesequence consisting of the JPEG encoding of a photo, which I won't in fact quote here because it is too long. The last example illustrates the major limit of quoting: it is convenient only for small data. Naming works by defining a name in some namespace and assigning the data to it. Names, namespaces, and assignment take different forms in different technology layers. In a Python script, I can write value = 42 to assign an integer value to the name value in the global namespace of the script. Modules, classes, and functions/methods are the other kinds of namespaces in Python. In my (Unix) computer's filesystem, the command echo "Computing in Science and Engineering" > $HOME/my-preferred-magazine.txt assigns a string to the name my-preferred-magazine.txt in the namespace of my home directory. In conventional jargon I'd say that I am writing a text string to a file, but semantically this is just the same as assigning a value to a variable, we just use different terms for historical reasons. On the Web, URLs are also names that live in namespaces that are managed by name servers (DNS) and Web servers. Assignment can take various forms, from copying a file to a server to on-line editing via a content-management system (CMS).

One reason for naming rather than quoting data is to define a shorthand for data that is too big to be quoted. Another equally important reason is that names are mutable references, i.e. the value assigned to a name can change over time. The URL for my blog is a name that remains constant in time, even though my blog changes as I add new posts and correct mistakes in older ones. A permanent reference to my blog is obviously useful. A third motivation for naming is that the name itself can be chosen to be descriptive. The URL of my blog is https://blog.khinsen.net, which is quite suggestive, at least for people familiar with Web habits.

However, the mutability of names is also a liability, in particular for names that are descriptive. Seeing the URL https://blog.khinsen.net, you may well suspect that it refers to my blog, all the more if you know that I use the handle khinsen on several social networks. However, you cannot really be sure of your assumption. Some evil hacker could have taken over my domain name and assigned different content to my blog's URL. While that's unlikely for my personal blog (it's not worth the effort), it is an issue for, say, my bank's Web server. Many risks in computing, be it Internet security or program correctness, are ultimately due to the impossibility to guarantee that the value assigned to a name is indeed what the manager of that name expects it to be. This is why functional programming [START_REF] Hinsen | The promises of functional programming[END_REF] bans mutable names: a single name can refer to different values in different locations in the source code, but the value at each location is fixed over the duration of program execution.

The risks associated with mutability are probably the most important problematic aspect of naming, but not the only one. When naming is used to define a shorthand for data that is too big to quote, coming up with a name can be a chore, as anyone who has had to work with temporary files has probably experienced. Programmers have to decide where to put a temporary file, how to name it in such a way as to avoid collisions with all other programs that are also busy making up names, and to remove the name reliably when it is no longer needed. Speaking of collisions, they can also be a problem when it's humans that make up descriptive names and great minds happen to think alike. That's why we see fights over the right to certain domain names on the Internet, for example.

Since neither quoting nor naming is without problems, wouldn't it be nice to have another way to refer to data? That's the starting point of the development of contentaddressable storage, which uses hashes as references in order to provide most of the advantages of quoting while keeping the size of the references constant, irrespectively of the size of the data itself. In IPFS, which I will explain in more detail later, you store data by passing it to the IPFS software, which gives you a hash in return. You can then transmit the hash to someone else, or archive it for later use by yourself. Whoever knows the hash can instruct the IPFS software to retrieve the corresponding original data.

A hash is to data what a fingerprint is to a person: a compact yet almost unique descriptor. Hashes are the result of computing a hash function whose input is a data stream, i.e. a sequence of bytes. A given hash function produces hashes of fixed size. For example, the popular (though by now a bit outdated) hash function called SHA1 produces 20-byte hashes, which are usually written as 40-digit hexadecimal numbers. There are Web sites such as http://www.sha1online.com/ where you can paste in some data and get its hash computed with a click. You can also easily find implementations of the most popular hash functions in lots of programming languages, because hash functions are so widely used, in particular in cryptography.

Like fingerprints, hashes cannot be truly unique. There are only 2 8×20 ≈ 1.46 × 10 48 different SHA1 hash values, which is exactly the same as the number of possible contents of 20-byte files. But files can be much longer than 20 bytes, so there must be many files that share the same hash, a situation known as a hash collision. The conclusion seems to be that a hash cannot be sufficient as a reference to data. And yet, hash-based content-addressable storage is in daily use for various applications without any major trouble. Git uses content-addressable storage for managing data inside repositories. In spite of its enormous popularity, you don't hear people complaining about data loss. How is that possible?

A first observation is that most possible file contents are of no interest. Data that actually carries information is structured. Think of what you keep stored on your computer: text in English, source code in Python, addresses in vCard format, numerical data in IEEE floating-point formats, etc. The data formats impose many constraints on which byte sequences actually make sense. Additional informal constraints further reduce the number of potential meaningful files. Most syntactically correct Python programs are invalid for some other reason, and even though QQQqqqqqqq is valid content for a name field in vCard, it is not very probable that someone actually has that name. SHA2-256, today's default hash function in IPFS, yields 256-bit hashes of which there are almost as many as the estimated number of atoms in the universe. It looks reasonable to assume that there are enough distinct hash values to uniquely label each file content that we might actually encounter in real life, if the hash function does a good job of assigning labels to contents. This has in fact been a very active field of research for a while. As I said, hash functions are very widely used, so finding good ones is an important job. There are two criteria that are particularly important in practice: even a small change in file contents should change its hash, because small changes are frequent, and it should be very hard (practically impossible) to construct a file for a given hash, because that would allow various forms of attack on hash-based systems. The main reason why I described SHA-1 as outdated above is that such an attack has been demonstrated recently [START_REF] Stevens | Announcing the first SHA1 collision, Google Security Blog[END_REF].

There is a vast literature on the question that I discussed in the last paragraph, which I won't even try to summarize. But one aspect of this question needs to be emphasized: no matter how good your hash function, hash collisions are never impossible but merely very improbable. All reasoning about hashes is of a probabilistic nature. For people ap-proaching computing from a mathematics background, this is often hard to swallow. You want to prove that your software works, not merely argue that it works most of the time! When coming from a physics or engineering background, probabilistic reasoning is more familiar. In fact, quantum mechanics says that all of nature is ultimately probabilistic. It is not impossible for me to fall through the chair I am sitting on, the tunnel effect would in principle permit it. But it's so enormously improbable that I don't lose sleep over the risk. Transferred to computing, which is done by physical machines, if a piece of software has a much lower risk of failure than the hardware it runs on, it is good enough in practice.

FROM THEORY TO PRACTICE: IPFS

The Inter-Planetary File System (IPFS, https://ipfs.io/) is an ambitious research and development project aiming at reinventing the World Wide Web with many fundamental improvements. One major difference to today's Web infrastructure is the use of content-based addressing as opposed to location-based URLs. This is the only aspect I will discuss here, please refer to the IPFS documentation for any other questions you might have. Before going on, let me stress that IPFS should at this time be considered an experiment. In my experience it is reliable though a bit slow, but your mileage may vary. Relying on IPFS as a data storage medium is probably not a good idea for now.

If you want to play with IPFS yourself, you should start by installing IPFS software on your computer. The most convenient entry point is IPFS Desktop (https://github.com/ipfs-shipyard/ipfs-desktop), but you can find other options at https://ipfs.io/. There are IPFS bindings for many programming languages, which are listed at https://github.com/ipfs/ipfs. They are all rather light-weight, as they connect to an HTTP server run by IPFS Desktop, which does all the heavy lifting. In the following I will use Pharo (https://pharo.org/), a modern Smalltalk derivate, because it is a particularly nice environment for explorative programming. In fact, the IPFS bindings for Pharo (https://github.com/khinsen/ipfs-pharo) are a byproduct of my own experiments with IPFS.

Let's start simple: I will store the list of numbers [1, 2, 3] in IPFS:

#(1 2 3) storeInIpfs

Pharo

prints the result as IpfsCid(bafyreib5ragfuond76dbywu7yubse73goit3h6v4dwf6tti2wfzdajo5py) The long string in parentheses is the IPFS content identifier (CID for short) for my list. An IPFS CID is essentially a hash, but it contains some additional information. The initial 'b' indicates that the CID has been encoded as a number in base 32. This is done to make sure it will survive all kinds of Internet transmission channels that might strip spaces and control characters or convert everything to upper case. Base 32 uses only the 32 characters abcdefghijklmnopqrstuvwxyz234567/ and ignores case. Decoding the base 32 string yields the real CID, a sequence of 36 byte which I'll show in hexadecimal notation:

#(1 2 3) storeInIpfs hex yielding: 017112203d880c5a39a3ff861c5a9fc503227f667227b3fabc1d8be9cd1ab1723025dd7e
The first byte is a version number, whose presence allows future extensions to the CID format without invalidating existing CIDs. The second byte indicates how the data itself is encoded. In our case, the encoding is CBOR, the Concise Binary Object Representation (https://cbor.io/), which is best summarized as a binary version of the popular JSON format. The choice of CBOR was ultimately made by my Pharo bindings, but it's today's standard encoding for linked data in IPFS. The third byte indicates the hash function, SHA2-256, and the fourth byte is the length of the hash, 32 bytes. The 32 remaining bytes are just that hash. IPFS can accommodate many hash functions, but SHA2-256 is today's standard. You can now go to https://explore.ipld.io, paste this base 32 string into the search field at the top, and click "explore". You should see something very similar to Fig. 1. Under "Data" you see the list, which shows that the data transfer from my machine to yours has worked fine. In the "CID Info" box you see a confirmation of what I explained above. Except that the CID is printed as zdpuApZdg27uJf8MQCMqHEhCmcc7fkX7UCb8mxhhJpu9z7D8y This is just another encoding, the initial 'z' indicating base 58 instead of base 32. This leads to a more compact but also more fragile representation, as upper and lower case letters are no longer equivalent. IPFS software accepts any of these encodings, but recommends the use of base 32.

If you have done this little exercise, you have a firsthand proof that you can retrieve on your computer data that I have stored on mine, using nothing but the CID which is essentially a hash of the data. You may have wondered where the data is actually stored, and how IPFS manages to find it. I won't say much about the second part, because that would require me to explain IPFS' sophisti-cated peer-to-peer communication system. The basic idea is to send out requests to other nodes in the network, and choosing the nodes to turn to as a function of the CID itself. As for the storage location, there may be many but there is one I can be sure of: the IPFS pinning service at https://temporal.cloud/, where I pinned this CID. Pinning is IPFS jargon for instructing a server to keep a piece of data unconditionally. Otherwise, the IPFS software treats its storage area as a cache, from which items are deleted after a while if they are not requested. You can pin data on any computer that runs IPFS software. Pinning data on your desktop or laptop machine guarantees you an off-line copy, whereas pinning data on a computer with a permanent network connection effectively turns it into a server hosting the data. Pinning services are the IPFS equivalent of file hosting services, with a crucial difference: nobody needs to know on which pinning service you keep your data, because data requests involve only the CID, not the location. And that means that you are free to move to a different pinning service at any time.

Note also that all data stored in IPFS should be considered public. Even if you don't give your CIDs to anyone else, current or future versions of the IPFS distribution algorithms may send the data to other nodes even without an explicit request. If privacy is an issue, encrypt your data or use a dedicated network [START_REF] Ober | Dedicated IPFS Networks[END_REF].

Location-independent data access is one of the big advantages of content-addressable storage. Another one is protection against accidental or malicious modification of data. Unlike traditional file systems, IPFS has no concept of write permissions, because it doesn't need it. There is no way to overwrite or replace any data because CIDs are not mutable names, but identifiers computed from the data. When you ask IPFS to retrieve the data for a given CID, you can recompute the hash to check that the data is indeed what you requested: cid := IpfsCid fromString: 'bafyreib5ragfuond76dbywu7yubse', '73goit3h6v4dwf6tti2wfzdajo5py'. data := cid loadRaw. hash := SHA256 hashMessage: data. (cid last: 32) = hash (The CID string is split into two parts to make the code fit into one column.) Pharo says true, so I know that I received the data exactly as its creator stored it. Note that loadRaw accesses the data at the lowest level: a sequence of bytes, which in this case stores the CBOR encoding of our three-element list. Hashes are computed at that level because that's how hash functions are defined. For most purposes, it is more convenient to use the two higher-level layers: the CBOR-encoding layer that I used above, or the file-system layer that most closely resembles traditional file systems.

LINKED DATA

There is one more feature of IPFS that I will explain in some detail, because it showcases another nice property of content addressable storage: the use of CIDs in data structures to create immutable linked data, as defined by the Inter-Planetary Linked Data specifications (see https://ipld.io). The idea is very simple: you can put CIDs into the lists or dictionaries you store in IPFS, and IPFS provides support for managing the resulting linked data structures and to access their elements. Let's see how this works in practice: cidForData := #(1 2 3) storeInIpfs. cidForDescription := 'List of three numbers' storeInIpfs. (NeoJSONObject with: #data->cidForData with: #description->cidForDescription) storeInIpfs

In this piece of code, NeoJSONObject is a Pharo implementation of JSON objects, from a library called NeoJSON. The result as shown by Pharo is IpfsCid(bafyreichcsaqqhxwgdpqvlni4p2z75arewt3yoesnvizgfxqkhiisbc52u), and by now you should know how to look it up using the explorer at https://explore.ipld.io. Please do so! The explorer will show a dictionary with the two keys data and description, each of which has a CID as its value.

And you can click on the CIDs to inspect the data they refer to. Now paste the string bafyreichcsaqqhxwgdpqvlni4p2z75arewt3yoesnvizgfxqkhiisbc52u/data into the explorer. As you will see, this will take you directly to the list. This notation is called a path, and most IPFS-based software will accept a path wherever it requires a CID. Paths work much like file paths on today's operating systems, except that they traverse dictionary-like data structures rather than directories.

It is no coincidence that my small example looks like data plus metadata. When data is stored in files, metadata requires an unpleasant trade-off. You can store the metadata along with the data in a single file, as it is habitually done in HDF5 [START_REF] Koziol | [END_REF], for example. But if the metadata contains execution-specific information, such as a time stamp or the name of the computer that did a calculation, the whole file becomes irreproducible because of the irreproducible metadata. Checking if two output files are equivalent in spite of the differing metadata is then laborious because it requires a detailed knowledge of the data format. Storing metadata in a separate file avoids such problems, and the raw data can be compared using generic tools such as Unix' cmp. But the link between data and metadata has to be made through filenames, and is therefore fragile when the data is transferred to a different computer where files live in a different namespace. Moreover, files being mutable, data-metadata associations are fragile even on the computer where they were established. With content-addressable storage, this problem disappears. In fact, checking if two computations produce the same result, assuming it is structured like my small example, requires no more effort than comparing the CIDs stored under data.

IPFS also helps with data management for linked data structures. As I mentioned, the elementary operation in data management is pinning, which means telling a specific node in the IPFS network to keep a piece of data indefinitely. IPFS can pin data recursively, which means that it also pins the data items referenced by any CIDs in the originally pinned data structure. A complex data assembly can thus be treated as a whole while still having well-identified substructures that can be shared with other data structures.

APPLICATIONS IN SCIENTIFIC COMPUTING

Let's assume we had a high-performance and widely adopted infrastructure for content-addressable storage. IPFS isn't quite there yet, but that could well change in a few years. How could we put it to good use for managing scientific data?

The most obvious use case is referring to data, including software source code, in an unambiguous and future-proof way. A journal article could simply state "the raw dataset has the CID ..." and both authors and readers could be sure to retrieve exactly the right data via this CID. URLs cannot guarantee the same level of permanence, nor can DOIs, whose permanence is no more than a moral obligation for the organizations that issue them. CIDs are therefore much more robust references for archiving, which is the reason why hash-based IDs very similar to IPFS CIDs are used in Software Heritage's archive of software source code [6].

Applied to software, content-addressable storage is the best approach we have today for ensuring reproducibility. The fundamental cause of irreproducibility is the complexity of the typical software assemblies we use in scientific computing. Both dependencies (mainly libraries) used by the software and build tools (compilers etc.) have an impact of a computation's result, and must therefore be recorded precisely. Storing all the files in IPFS and using the linked data approach I outlined in the preceding section would provide a permanent and unalterable record for future reconstruction. In fact, the package managers Nix and Guix use a very similar approach.

Pushing this idea further, we could combine the software dependency graph with a computational workflow graph and have a complete provenance graph for a computation, with everything stored as linked data in IPFS or an equivalent system. Yet another graph layer could provide the full history of the evolution of data and software. Since such graphs can become very large, and reference many and potentially large datasets, another welcome feature of content-addressable storage is the built-in deduplication: if you store the same data ten times in IPFS, only one copy will be kept, whereas a typical computer's file system contains many files with identical contents.

Another use case is the simplification of data communication between the different pieces of software used in a research project, e.g. the components of a workflow, or data processing software connected to visualization software. If all programs read and write data from and to contentaddressable storage, communication only involves CIDs. Distributed computations does not require any additional effort because CIDs, unlike file names, are not specific to one computer. Simplifying communication between programs is a big deal because it encourages the use of smaller tools that do only one thing and do it well, a principle often called the "Unix philosophy". Smaller tools are easier to understand and easier to maintain, and that's something most computational scientists appreciate very much.

Finally, the transition from files to linked data structures should bring us further simplification. Using immutable references, we can package data in smaller units. Where today we use file formats such as tar, zip, netCDF, or HDF5 to ensure the coherence of multiple datasets and metadata, we will instead use simple linked data structures that don't require elaborate libraries to be integrated in every single piece of software.

In the long run, content-addressable storage could thus contribute to better computational science. Isn't that just the kind of magic that scientific research could profit from?

Fig. 1 .

 1 Fig. 1. My list of numbers in the IPLD explorer


Jérôme Hourdain

Marie Annick Clavel

Jean-Claude Deharo

Samuel Asirvatham

Jean Francois Avierinos

Gilbert Habib

Frédéric Franceschi

Vincent Probst

Nicolas Sadoul

Raphael Martins

Common Phenotype in Patients With Mitral Valve Prolapse Who Experienced Sudden Cardiac Death

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.