Benjamin Chollet

Loïc D ' Eramo

Ekkachai Martwong

Mengxing Li

Jennifer Macron

Thuy Quyen Mai

Patrick Tabeling

Yvette Tran
email: yvette.tran@espci.fr

Tailoring Patterns of Surface-Attached Multi-Responsive Polymer Networks

Keywords: polymer, hydrogel, responsive, thin film, grafting, pattern, photolithography 2

A new strategy for the fabrication of micro-patterns of surface-attached hydrogels with wellcontrolled chemistry is reported. The "grafting onto" approach is preferred to the "grafting from" approach. It consists in cross-linking and grafting preformed and functionalized polymer chains through thiol-ene click chemistry. The advantage is a very good control without adding initiators.

A powerful consequence of thiol-ene click reaction by UV irradiation is the facile fabrication of micro-patterned hydrogel thin films by photolithography. It is achieved either with photomasks using common UV lamp or without photomasks by direct drawing owing to laser technology.

Our versatile approach allows the fabrication of various chemical polymer networks on various solid substrates. It is demonstrated here with silicon wafers, glass and gold surfaces as substrates, and two responsive hydrogels, poly(N-isopropylacrylamide) for its responsiveness to temperature and poly(acrylic acid) for its pH-sensitivity. We also demonstrate the fabrication of stable hydrogel multilayers (or stacked layers) in which each elementary layer height can widely range from a few nanometers to several micrometers, providing an additional degree of freedom to the internal architecture of hydrogel patterns. This facile route for the synthesis of micrometerresolute hydrogel patterns with tailored architecture and multi-responsive properties should have strong impact.

INTRODUCTION

Micro-patterned polymer hydrogels increase attention in biomedecine as cell culture substrates, permeable membranes, microvalves or microactuators. [START_REF] Nikkhah | Engineering Microscale Topographies to Control the Cell-Substrate Interface[END_REF][START_REF] Beebe | Functional Hydrogel Structures for Autonomous Flow Control Inside Microfluidic Channels[END_REF][START_REF] Yu | Flow Control Valves for Analytical Microfluidic Chips Without Mechanical Parts Based on Thermally Responsive Monolithic Polymers[END_REF] Hydrogels are very attractive owing to their biocompability and high deformability. Additional advantage of polymer hydrogels is their tunable properties through their chemistry. [START_REF] Richtering | Gel Architectures and their Complexity[END_REF] In particular, responsive hydrogels can undergo high volume changes under external stimuli such as temperature or pH. [START_REF] Döring | Responsive Hydrogels -Structurally and Dimensionally Optimized Smart Frameworks for Applications in Catalysis, Micro-System Technology and Material Science[END_REF][START_REF] Stuart | Emerging Applications of Stimuli-Responsive Polymer Materials[END_REF] For example, poly(Nisoproprylacrylamide) (PNIPAM) hydrogel films show reversible and high thickness change with temperature by absorbing and expulsing water. [START_REF] Halperin | Phase Diagrams: Fifty Years of Research[END_REF] The change could be four-fold or more depending on the crosslinks density of the hydrogel. [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF][START_REF] Chollet | Multiscale Surface-Attached Hydrogel Thin Films with Tailored Architecture[END_REF][START_REF] Tokarev | Stimuli-Responsive Hydrogel Thin Films[END_REF][START_REF] Vidyasagar | Temperature Induced Volume-Phase Transitions in Surface-Tethered Poly(N-Isoprylacrylamide) Networks[END_REF][START_REF] Toomey | Swelling Behavior of Thin, Surface-Attached Polymer Networks[END_REF][START_REF] Harmon | Photo-Crosslinkable PNIPAAm Copolymers. 2. Effects of Constraint on Temperature and pH-Responsive Hydrogel Layers[END_REF][START_REF] Harmon | Photo-Crosslinkable PNIPAAm Copolymers. 4. Effects of Copolymerization in Cross-Linking on the Volume-Phase Transition in Constrained Hydrogel Layers[END_REF] Hydrogel micro-patterning can be performed using micro-molding and softlithography techniques. Soft lithography techniques are based on in situ polymerization of precursor monomer solutions against microstructure stamps (in general PDMS stamp) which are then removed by demolding. [START_REF] Koh | Molding of Hydrogel Microstructures to Create Multiphenotype Cell Microarrays Anal[END_REF][START_REF] Moeller | A Microwell Array System for Stem Cell Culture[END_REF] Kobel et al. proposed a different microfabrication technique called soft embossing to overcome the damage of soft and fragile polymer networks upon demolding. [START_REF] Kobel | Micropatterning of Hydrogels by Soft Embossing[END_REF] Alternatively, photolithography techniques enable hydrogel micro-patterning without the use of stamps. The photopolymerizable patches are obtained directly under UV light irradiation through photomasks. [START_REF] Revzin | Fabrication of Poly(Ethylene Glycol) Hydrogel Microstructures using Photolithography[END_REF] However, as radical polymerization is inhibited by the presence of oxygen, improved control on the chemical hydrogel formation can be performed by using preformed polymer chains instead of starting from monomers. Several groups have explored this photolithography approach with specific methodologies. For example, PNIPAM hydrogel patterns were immobilized on PTFE polymer substrate by low-pressure argon plasma treatment with a masking method. [START_REF] Schmaljohann | In situ Study of the Thermoresponsive Behavior of Micropatterned Hydrogel Films by Imaging Ellipsometry[END_REF] Using an alternate patterning technique, Chen et al. developed a grid pattern of two different nanothick microgels including poly(acrylic acid) pH-responsive microgel. [START_REF] Chen | Grid Pattern of Nanothick Microgel Network[END_REF] Szilagyi et al. demonstrated that micrometer-scale microrelief with a high aspect ratio could be formed on a thin hydrogel layer composed of poly(N-isoproprylacrylamide) and spirobenzopyran photoresponsive group. [START_REF] Szilagyi | Rewritable Microrelief Formation on Photoresponsive Hydrogel Layers[END_REF] We propose a new approach for the micro-patterning of surface-grafted hydrogels with wellcontrolled chemistry, which has the advantages of simplicity and versatility. The method is inspired from the "grafting onto" technique: preformed ene-functionalized polymer chains are cross-linked and surface-attached by thiol-ene click reaction. It enables a better control than the approach starting from monomers. Additional advantage is that thiol-ene reaction can be performed under deep UV irradiation (around 260 nm) without any addition of initiators. The proof-of-concept is achieved with responsive polymer hydrogels such as poly(Nisopropylacrylamide) for its temperature-responsiveness and poly(acrylic acid) for its pHsensitivity. But the extension to more general polymer networks can be envisaged depending on the properties targeted. The versatility of the method is illustrated with plane substrates such as silicon wafers, glass slides and gold surfaces. The thickness range of the hydrogel patterns is controlled by the precursor reactive polymer coating. Here, the spin-coating technique is chosen so that the thickness widely ranges from a few nanometers to several micrometers. The facile fabrication of micro-patterned hydrogels by photolithography is enabled with thiol-ene reaction as click chemistry. The versatility of our approach based on thiol-ene click chemistry owes to the commercial availability of many thiol molecules required for the cross-link reaction and the surface treatment. Bifunctional thiol molecules are used for the cross-linking and the treatment of gold surface and mercaptosilane for the functionalization of glass and silicon substrates.

In a previous publication, [START_REF] Chollet | Multiscale Surface-Attached Hydrogel Thin Films with Tailored Architecture[END_REF] we reported the development of surface-attached hydrogel films with various internal architectures such as multilayers, interpenetrating networks and nanocomposite hydrogel films using this "grafting onto" approach based on thiol-ene click chemistry. In this present article, we focus on the method for the fabrication of micro-patterned hydrogels owing to thiol-ene reaction performed by UV irradiation. The parameters that control the formation of patterned hydrogels with multi-responsive properties are investigated. The photolithography can be performed either with photomasks using a common 8 Watt UV lamp at 254 nm or without photomasks by direct drawing using a 50 mWatt laser at 266 nm, which provides micrometerresolute patterns. We also show that these responsive hydrogel patterns can be integrated in microfluidic systems, demonstrating a new route for the facile fabrication of micro-actuators.

EXPERIMENTAL SECTION

Thiol-modification of substrates. Prior to any chemical modification, silicon wafers and glass substrates are cleaned in a freshly prepared "piranha" solution (mixture of 70 vol% of sulfuric acid and 30 vol% of hydrogen peroxide (35 vol% in H 2 O) heated at 200°C). The solid substrates are then rinsed and sonicated in Milli-Q water and finally dried with nitrogen flow. Silanization with 3-mercaptopropyltrimethoxysilane is carried out on the freshly cleaned substrates to perform the thiol-modification of the surface. A solution of anhydrous toluene with 3 vol% of mercaptopropyltrimethoxysilane (ABCR Gelest) is put in contact with the substrates for 3 hours inside a reactor under nitrogen. The solid substrates are rinsed and sonicated in toluene and finally dried with nitrogen flow. The thickness of the thiol layer on silicon wafer which is measured by ellipsometry, is 1 nm with a standard deviation lower than 10% of the average thickness.

Gold surfaces are made of a gold film (thickness is typically 100 nm) evaporated on microscope glass slides. A chromium adhesion layer ( 3 nm) has to be first coated in order to improve adhesion of the gold film. Prior to metal deposition, the glass slides are cleaned in the "piranha" solution. Gold surfaces are modified with a thiol self-assembled monolayer. The substrates are immersed in a solution of dithioerythritol at 1 mM in chloroform. They are kept immersed for 2 hours, in a sealed reactor to prevent evaporation of chloroform. The gold surfaces are then rinsed and sonicated in chloroform and finally dried with nitrogen flow.

Synthesis of hydrogel films. The synthesis of ene-reactive polymers was described in detail in our previous articles. [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF][START_REF] Chollet | Multiscale Surface-Attached Hydrogel Thin Films with Tailored Architecture[END_REF] Briefly, ene-functionalized polymers are synthesized in two steps for PNIPAM and one step for PAA. The two-steps method consists in free radical copolymerization of NIPAM and AA and ene-functionalization of the copopolymer by peptide reaction. First, poly(AA-co-NIPAM) copolymer is synthesized in water by free radical polymerization initiated by (NH 4 ) 2 S 2 O 8 /Na 2 S 2 O 5 redox couple under nitrogen at room temperature. The amount of the (NH 4 ) 2 S 2 O 8 /Na 2 S 2 O 5 initiators depends on the molecular weight of polymer chains targeted. For example, a molecular weight of 250 kg/mol is obtained for the concentration of (NH 4 ) 2 S 2 O 8 and Na 2 S 2 O 5 in water equal to 10 -3 mol/L. The reaction is allowed to proceed for 24 hours. The final solution is purified by dialysis in water. Second, the P(NIPAM-co-AA) copolymer is enefunctionalized by allylamine in water at room temperature in the presence of EDC/NHS couple.

The reaction is allowed to proceed for 24 hours. The final solution is dialyzed in NaCl solution (to remove the unreacted EDC/NHS) and then in water. For ene-reactive PAA, only the step of funtionalization using allylamine is necessary.

Single-network films are prepared as follow. A solution containing ene-reactive copolymers and added dithioerythritol cross-linkers is dropped onto thiol-functionalized substrates for spincoating. The ratio of dithioerythritol to ene-reactive polymer units is 15 times. We chose to fix the conditions of spin-coating: the final angular velocity is 3000 rpm and the spinning time is 30 seconds. For thermal activation of thiol-ene reaction, the polymer films are annealed at 120°C for 16 hours under vacuum. For deep UV irradiation, either a common 8 Watt UV lamp ( = 254 nm) with photomasks or a 50 mWatt laser ( = 266 nm) is used.

Multilayers films (or stacked polymer network layers) are fabricated by successive coating of a new layer over former layer, the thickness of each layer being decided by the molecular weight of the polymer and the concentration of the solution for coating. The very first layer is synthesized like a single-network film. The next layers are made with the same process: polymer films are coated and cross-linked by thiol-ene reaction. For thermal process, the multilayers architecture is obtained after 16 hours annealing at 120°C as for single-network films. The schematic of synthesis of surface-attached hydrogel films, single network and multilayers, is shown.

Patterns of surface-attached hydrogels. Hydrogel patterns are achieved under UV light irradiation through photomask using a (8 Watt) common fluorescent lamp ( = 254 nm). The photomask is aligned over the coated polymer film for typically 2 hours irradiation. Photomasks are obtained by patterning a 100 nm-thick layer of chromium on quartz plates as quartz is transparent to UV contrarily to the chromium layer. The chromium layer is chemically wetetched with the use of a photoresist masking material (DOW Chemical). Another way to obtain hydrogel patterns is to perform the UV irradiation with a laser spot (diameter of 2 m or 20 m) focused on the polymer film. The laser spot is moved over the surface for the direct drawing of highly resolute patterns. This laser lithography is performed using a KLOE DILASE 650 device (respectively 1.53). Hydrogel film refractive index is 1.50 as it contains about 10% of water in air (water content lower than 10% was measured for humidity ratio between 20% and 60%). The water content is weak so that the thickness of PNIPAM gel film in air can be considered as the thickness of dry film. Measurements in water (in situ) are performed with a temperaturecontrollable liquid cell equipped with thin glass walls (fixed perpendicularly to the light path with the angle of incidence at 60°). The polymer hydrogel film is modeled as a single layer with the thickness and a constant refractive index between that of water (1.33) and of the polymer.

Since the hydrogel film is covalently attached to the substrate, The polymer amount is the same when immersed in water as the polymer is chemically grafted to the substrate. So we have: and with the volume fraction of polymer in water. The swollen thickness , the refractive index of the film in water and the dry thickness are deduced from the fitting of the experimental data. The two equations should provide the same value of to ensure that the fitting is reliable.

For Fourier transform-IR attenuated-total-reflection (FTIR-ATR) spectroscopy measurements, silicon substrates are used as infrared wavelength guides so that they are cut in special shape (70 mm × 10 mm × 1.5 mm trapezoidal crystal with an angle of 45°). The FTIR-ATR spectra are obtained using a Magna IR 550 (Nicolet) apparatus with a mercury-cadmium-telluride (MCT)

detector cooled with liquid nitrogen. The spectra are recorded with a resolution of 2 cm -1 and a 256-scan accumulation.

The 3D surface profile of hydrogel patterns are measured with a noncontact optical profiler Wyco NT 9100 employing white-light interferometry. The measurements were analyzed and the profiles are plotted using Vision for Profilers software (Veeco Instruments).

Microfluidic experiments. Microfluidic channels are printed in NOA (Norland Optical

Adhesive, photocurable monomer) according to the microfluidic stickers method. [START_REF] Bartolo | Microfluidic Stickers[END_REF] A drop of NOA 81 is deposited on a glass slide. A PDMS stamp is gently pressed onto the drop to create the channel structure. The NOA 81 is then insolated through the transparent PDMS stamp. The stamp is removed, a thin uncured adhesive layer remains thanks to the PDMS gas permeability.

Putting the sticker onto contact with the substrate containing the surface-attached hydrogel patterns seals the microfluidic chip. A second insolation guarantees the adhesion between the sticker and the substrate by curing the adhesive layer. For fluorescence imaging, pictures are taken using a Leica DMI 6000B inverted microscope equipped with a 5X dry objective, coupled with a sensitive Hamamatsu ORCA-ER CCD camera. To control the temperature on the microfluidic device, a Linkam Scientific PE120 Peltier stage is inserted in the microscope XY stage. For better thermal conductivity, thermal grease (Radiospares) is applied in between the heating/cooling area and the glass slide of the microfluidic device. Channels are filled with a 0.8 mM solution of fluorescein (Sigma-Aldrich) in Milli-Q water.

RESULTS AND DISCUSSION

Synthesis of surface-attached hydrogel films

The synthesis of surface-attached hydrogels is achieved with an inspired "grafting onto" strategy.

It consists in cross-linking and grafting preformed ene-reactive polymers by thiol-ene click chemistry. The ene-functionalized polymer is coated on thiol-modified substrate with dithiol cross-linkers, the thiol-ene click reaction allowing simultaneously the cross-linking of chains and their covalent grafting to the substrate. It is the same process for the fabrication of multilayers films in which single-network films are stacked as for polymer layer-by-layer assemblies.

As shown in our previous articles, [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF][START_REF] Chollet | Multiscale Surface-Attached Hydrogel Thin Films with Tailored Architecture[END_REF] the variation of the thickness of surface-attached polymer network films can be achieved with the change of the viscosity of polymer solution for coating.

Films of different thickness are then achieved by changing the viscosity with the variation of the polymer molecular weight and the polymer concentration. Figure 1 clearly demonstrates that surface-attached hydrogel films can be obtained with thickness ranging from a few nanometers to several micrometers using polymer concentrations from 1% to 10% and molecular weights from 70 kg/mol to 700 kg/mol. The low molecular weights provide submicrometer thin layers with high precision and the high molecular weights allow the fabrication of micrometer thick films.

Figure 1 also displays the dry thickness of the hydrogel films as function of the number of coated/cross-linked layers. The linear increase of the thickness with the number of layers is demonstrated for two thicknesses of the elementary layer: 15 nm and 65 nm. Each elementary thickness is controlled by the concentration or the molecular weight of the polymer solution for spin-coating . Any polymer hydrogel, PNIPAM or PAA, can be introduced in the multilayers, providing a degree of freedom to the internal architecture. It provides evidence of the simplicity and versatility of the approach to build stable polymer films with multi-responsive properties.

Compared to usual layer-by-layer polymer assemblies in which the elementary adsorbed layer is a few nanometers-thick, the advantage of multilayers hydrogel films is the chance to vary the thickness at will in addition to change the nature of polymer network. All hydrogel films are stable and durable due to covalent bonds between chains and with the surface. If there is no chemical (or covalent) cross-linking, the hydrogel films will not be stable and polymer (free) chains will be removed during samples washing. For multilayers hydrogel films, entanglements between peripheral chains of two adjacent layers probably allow the stability. It can also be attributable to chemical (thiol-ene) bonds between these peripheral chains. At short time heating, lower than 20 hours, the swelling ratio at 25°C decreases to reach the constant value of 3.2. It is likely due to the increase of the thiol-ene crosslinks reaction with time heating. At 40°C, collapsed PNIPAM hydrogel films have the same swelling ratio of 1.5, no matter the time heating. As discussed in our previous publications, water is not entirely expulsed from the PNIPAM collapsed hydrogel which contains about 30% of water. [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF][START_REF] Chollet | Multiscale Surface-Attached Hydrogel Thin Films with Tailored Architecture[END_REF] It was found for PNIPAM collapsed hydrogel films no matter the crosslinks density [START_REF] Vidyasagar | Temperature Induced Volume-Phase Transitions in Surface-Tethered Poly(N-Isoprylacrylamide) Networks[END_REF][START_REF] Toomey | Swelling Behavior of Thin, Surface-Attached Polymer Networks[END_REF][START_REF] Harmon | Photo-Crosslinkable PNIPAAm Copolymers. 2. Effects of Constraint on Temperature and pH-Responsive Hydrogel Layers[END_REF][START_REF] Harmon | Photo-Crosslinkable PNIPAAm Copolymers. 4. Effects of Copolymerization in Cross-Linking on the Volume-Phase Transition in Constrained Hydrogel Layers[END_REF] and also for PNIPAM collapsed brushes. [START_REF] Yim | Temperature-Dependent Conformational Change of PNIPAM Grafted Chains at High Surface Density in Water[END_REF][START_REF] Yim | Effects of Grafting Density and Molecular Weight on the Temperature-Dependent Conformational Change of Poly(N-Isopropylcrylamide) Grafted Chains in Water[END_REF] The collapsed state does not depend on the architecture of PNIPAM film, network or brush. It is only decided by polymer-solvent interaction or  Flory parameter.

The damage of hydrogel films with long time UV irradiation is supported by the variation of the swelling ratio at 40°C. The swelling ratio found is not 1.5 for supposedly collapsed PNIPAM films as expected, but it increases improperly with temperature. It can be attributable to oxidation and accelerated ageing with UV irradiation. As a result, the optimized UV irradiation time using the 8 Watt lamp at 254 nm is between 2 hours and 6 hours. In this time range, the dry thickness and the swelling ratio of hydrogel films are invariable.

activation (round markers of Figure A) is compared. The dry thickness of these hydrogel films is

shown in Figure 3.

(C) Swelling ratio at 25°C (blue markers) and 40°C (red markers) of PNIPAM hydrogel films made from reactive polymers with two different ratios of ene-

functionalization 2% (open markers) and 3% (filled markers) and with two thiol-ene crosslinking processes (UV irradiation and thermal activation).

Figure 4 also displays the swelling properties of PNIPAM hydrogel films obtained with UV irradiation and thermal activation of thiol-ene cross-linking reaction, the data being shown for hydrogel films made from reactive polymers with two different ratios of ene-functionalization (2% and 3%). The swelling ratio of collapsed hydrogel films at 40°C is about 1.5 for any crosslinking activation process and any ratio of ene-functionalization. It is independent of the crosslinks density as discussed above. The swelling ratio of swollen films at 25°C decreases with the ratio of ene-reactive units. It is lower for thermal activation than for UV irradiation corresponding to higher yield of thiol-ene cross-linking reaction for thermal process. The values of swelling ratio are comparable to those obtained by Toomey et al. with benzophenone photocrosslinkers. They demonstrated that the swelling ratio is a power law of the proportion of benzophenone photo-crosslinkable units (from 0.5 to 14.3%) with the exponent of -1/3. [START_REF] Toomey | Swelling Behavior of Thin, Surface-Attached Polymer Networks[END_REF] Our values of swelling ratio of 4 (respectively 3) with 2% (respectively 3%) ene-reactive polymers fit Toomey's results. Moreover, the comparison between the swollen and collapsed states shows phase transition with high amplitude (3 to 4). This high amplitude transition could be advantageously exploited for applications of hydrogel films using temperature-responsive properties. As silicon is used as infrared waveguides, it is difficult to quantitatively analyze the wave number range below 1600 cm -1 because of the high absorbance of the silicon substrates (O-H, Si-O, Si-Si bonds) so that information can only be extracted from 4000 -1600 cm -1 range. The absorption bands between 3100 and 2800 cm -1 are characteristic of asymmetric and symmetric stretching vibrations of CH, CH 2 and CH 3 groups. The absorbance is expectedly the same for all spectra. The loss of the peak at 2500 cm -1 attributable to dithiol cross-linkers (S-H stretch) for spectra of PNIPAM films after grafting and cross-linking is explained by the thiol-ene reaction and the removal of dithiol in excess. It is also consistent with the high increase of the absorption band at 3300 cm -1 characteristics of O-H (due to H bonds) found in the spectrum before crosslinking (because of the presence of dithioerythritol in excess), this broad and high band even hiding the peak at 3430 cm -1 characteristics of N-H amide groups. Unfortunately, it is not possible to quantify the thiol-ene reaction for two main reasons. First, the absorption peak characteristics of S-C bond is at very low wave number (around 700 cm -1 ). Second, the thiol-ene ratio is weak ( maximum at 2-3%), [START_REF] Nikkhah | Engineering Microscale Topographies to Control the Cell-Substrate Interface[END_REF] H NMR showing a ratio of ene-functionalization around 2-3%.

The spectra of PNIPAM network films after 2 hours and 10 hours UV irradiation are the same while the alteration of temperature-responsive properties is observed at long time irradiation (see Figure4). The same absorbance of the peaks between 3600 and 2800 cm -1 (characteristic of N-H amide groups, CH, CH 2 and CH 3 groups) is indicative of no major damage of PNIPAM chains.

In fact, the accelerated oxidation of the polymer network film with UV irradiation, even not obviously confirmed by FTIR-ATR, seems to be the most reliable assumption. It can be supposed that the formation of hydrophilic sulphonate groups (around 1300 cm -1 and 1100 cm -1 ) in the PNIPAM hydrogel films which is favored by UV irradiation, is responsible of the cancelation of the collapse behavior of PNIPAM hydrogel films at 40°C. The advantage of selective UV irradiation is the facility to fabricate hydrogel patterns. Figure 6 shows two examples of surface-attached hydrogel micro-patterns using a common 8 Watt UV lamp through photomasks, the patterning corresponding to a negative photolithography. 400 microns-wide squares with 50 microns-wide and 2.5 microns-thick walls are covalently grafted on glass susbtrate. The images of the patterns on the glass surface are observed using optical profilometer. The glass surface is dark grey and the hydrogel blocks appear in bright tones proportionally to the hydrogel thickness. Another example of photolithography using photomask is shown with 6 microns-wide and 250 nanometers-thick lines of hydrogels grafted on silicon wafer. The images on the silicon wafer are obtained with reflected light microscope. Here, the reflective silicon substrate is bright while the polymer patterns are in black tone. The same patterns with square enclosures as in Figure 6 can also be achieved from direct writing (without photomask) using a 50 mWatt laser at wavelength ( = 266 nm) integrated in the lithography device. Depending on the laser spot size, it provides an output power of 3 mW and 5 mW for a 2 µm-and 20 µm-size spot respectively. Figure 7 shows 10x10 square patterns drawn and grafted on glass substrate with micrometer-size high resolution. The optical profile of the hydrogel enclosures is obtained with the optical mire exposed to the laser spot. The optimal conditions of UV irradiation are determined as function of the laser power and the writing speed.

Micro-patterns of polymer network

The calibration is performed by using the optical mire of one hundred squares, each square being drawn by a unique crossing of the laser spot. For the lines of the mire, the laser power varies with a constant writing speed. For the columns of the mire, the writing speed varies with the same laser power. The square enclosures exposed to very weak irradiation (on the right upper corner) are eliminated due to partial cross-linking, the uncross-linked polymer chains being removed by rinsing. The square enclosures which are not sufficiently irradiated are a little ondulated due to the wrinkling of weakly cross-linked hydrogels. From the optical profilometer image, the thickness of hydrogel film of each square is deduced. Calibration cartography can be then determined with the hydrogel thickness as function of the parameters of the laser irradiation, the writing speed and the laser power as shown in Figure 7B for the laser spot of 20 m diameter. The UV energy received by the hydrogel patterns for the cross-linking is proportional to where P is the laser power, the spot diameter and v the writing speed. For the same laser power, the energy only depends on the product of the spot diameter and the writing speed so that the writing speed is ten times higher for the 2 m-size laser spot than for the 20 m-size laser spot. Calibration cartography can also be represented by the thickness of the hydrogel film as function of the UV energy received by the cross-linked patterns as shown in Figure 7C. The kinetics of polymer cross-linking through thiol-ene reaction using the 50 mWatt laser is the same as obtained with the common 8 Watt fluorescent lamp. The thickness of the polymer network increases and reaches its maximum. For lower energies, the increase of the film thickness is due to partial cross-linking, the uncross-linked chains being removed by washing. It means that the synthesis of surface-attached of hydrogel patterns using the 50 mWatt laser technology must be performed with UV energy (in arbitrary unit equal to

) comprised between 200 and 600. on the free height of the channel. At temperatures below the LCST (for example 25°C), the star hydrogel is swollen and then appears dark as the fluorescence intensity at this place is weak. For temperatures above the LCST (for example 40°C), the hydrogel pattern is collapsed and then appears bright as the free height of the channel is larger. The free height of the channel and consequently the thickness of the hydrogel in the channel can be deduced from the fluorescence intensity. The calibration of the intensity of the fluorescence signal in grayscale was achieved using a stairway channel with five different heights. The fluorescence intensity was measured for each stair for temperatures ranging from 20 to 50°C. It was found to be the same for each stair (with a maximum deviation of 5% from the mean value) confirming no dependence with temperature. The normalized fluorescence intensity (to the maximum value measured for the highest stair channel) increases linearly with the channel height as expected; the calibration of fluorescence intensity is displayed on the microscopy image of the stairway channel in Figure 10. The thickness of the hydrogel stair pattern (and consequently the swelling ratio) in the channel at various temperatures can be then determined from the measure of the fluorescence intensity. For temperatures below the LCST (< 32°C), the PNIPAM hydrogel is supposed to be totally swollen. The thickness of the star pattern in water is found to be about four times the dry thickness measured in air before the integration into the microfluidic channel, which is in good agreement with the value determined by ellipsometry and shown in Figure 4. For temperatures above the LCST (> 32°C) with the PNIPAM hydrogel supposed to be collapsed, the swelling ratio found is around 1.5, again in good agreement with ellipsometry measurements.

The hydrogel swelling is homogeneous on the whole star pattern. The star pattern has exactly the same shape in air and in water with no lateral swelling or edges deformation. Actually, as the hydrogel pattern is synthesized in dry state in air and then exposed to water, the hydrogel buckling would arise from inhomogeneous compressive strains due to surface attachment. These buckling instabilities were observed for surface-attached hydrogel patterns for which the thickness and the width are the same order of magnitude. [START_REF] Dupont | Swelling-Induced Instabilities in Microscale, Surface-Confined Poly(N-Isopropylacrylamide) Hydrogels[END_REF] In our case, the surface-attached hydrogel pattern is much wider than thick (the thickness is a few micrometers while the star size is close to millimeter) so that the swelling of hydrogel is unidirectional (perpendicular to the substrate) without lateral deformation.

Figure 1 .Figure 2 .

 12 Figure 1. (Left) Dry thickness of PNIPAM hydrogel films as function of polymer concentration

Figure 3 .

 3 Figure 3. Final dry thickness of PNIPAM network film as function of activation time. The effect

Figure 5 .

 5 Figure 5. FTIR-ATR spectra of PNIPAM films. The absorption spectra are obtained with

Figure 5

 5 Figure5shows FTIR-ATR spectra of PNIPAM films. Five absorption spectra are compared: the

Figure 6 .

 6 Figure 6. Optical microscopy images of PNIPAM hydrogel patterns. The patterns are obtained

CFigure 7 .

 7 Figure 7. (A) Optical profilometer image of patterned PNIPAM hydrogel squares on glass

Figure 8

 8 Figure 8 displays square enclosures patterns of hydrogels grafted on glass substrate using 20 m-

Figure 8 .

 8 Figure 8. (A) Top view and 3D view of patterned PNIPAM hydrogel squares on glass substrate

Figure 9

 9 Figure 9 shows top view pictures of hydrogel pyramidal structure of millimeter size grafted on

Figure 9 .

 9 Figure 9. (A) Top view picture of hydrogel patterns with pyramidal multilayers structure

Figure 10

 10 Figure 10 shows fluorescence microscopy images of microfluidic channels filled with

Figure 10 .

 10 Figure 10. Fluorescence microscopy images in a microfluidic channel. (A) and (B) Pictures of a

Figure 4. (A) and (B) Swelling ratio of PNIPAM hydrogel films as function of activation time. The swelling ratio h w /h a is the ratio between the hydrogel thickness in water and the dry thickness in air. The swelling ratio is measured at 25°C below the LCST (blue) and at 40°C above the LCST (red). The effect of UV irradiation (square markers of Figure B) and thermal


Benjamin Chollet

Mengxing Li

Ekkachai Martwong

Bruno Bresson

Christian Fretigny

Patrick Tabeling

Yvette Tran
email: yvette.tran@espci.fr

Multiscale Surface-Attached Hydrogel Thin Films with Tailored Architecture

Keywords: hydrogel, thin film, responsive, surface-attached, multiple-networks

A facile route for the fabrication of surface-attached hydrogel thin films with well-controlled chemistry and tailored architecture on wide range of thickness from nanometers to micrometers is reported. The synthesis, which consists in crosslinking and grafting the preformed and enereactive polymer chains through thiol-ene click chemistry, has the main advantage of being wellcontrolled without addition of initiators. As thiol-ene click reaction can be selectively activated by UV-irradiation (in addition to thermal heating), micro-patterned hydrogel films are easily synthesized. The versatility of our approach is illustrated by the possibility to fabricate various chemical polymer networks, like stimuli-responsive hydrogels, on various plane solid substrates such as silicon wafers, glass and gold surfaces. Another attractive feature is the development of new complex hydrogel films with targeted architecture. The fabrication of various architectures for polymer films is demonstrated: multilayer hydrogel films in which single-networks are stacked one onto the other, interpenetrating networks films with mixture of two networks in the same layer, nanocomposite hydrogel films where nanoparticles are stably trapped inside the mesh of the network. Thanks to its simplicity and its versatility this novel approach to surfaceattached hydrogel films should have strong impact in polymer coatings area.

Introduction

Hydrophilic polymer coatings that provide specific properties such as wettability, permeability, adhesion or friction properties have drawn considerable attention in various fields, including biomedicine or chemical engineering. [START_REF] Knoll | Functional Polymer Films[END_REF] The surface properties can be controlled and improved with suitable polymer materials and in particular stimuli-responsive polymers. [START_REF] Stuart | Emerging Applications of Stimuli-Responsive Polymer Materials[END_REF][START_REF] Döring | Responsive Hydrogels -Structurally and Dimensionally Optimized Smart Frameworks for Applications in Catalysis, Micro-System Technology and Material Science[END_REF] Layer-by-layer polymer assemblies and polymer brushes are so far the most usual polymer coatings. Layer-bylayer assemblies where polymer layers are assembled by physical bonds (hydrogen bond, electrostatic or hydrophobic interactions) have become a universal approach for the facile fabrication of multicomponent films on solid supports. Polymer brushes for which polymer chains are chemically grafted to surface to ensure the durability of the coating are extensively employed in the past two decades. However, layer-by-layer assemblies and brushes as coatings can have some major inconvenience. For polymer brushes, the thickness of polymer brushes which is ruled by the polymer chain length is restricted to low submicrometer. As layer-by-layer assemblies require many cyclic steps, high thickness could be the limitation.

Hydrogel films go beyond and are a real alternative to layer-by-layer assemblies and polymer brushes as stable and durable polymer coatings. [START_REF] Tokarev | Stimuli-Responsive Hydrogel Thin Films[END_REF][START_REF] Kuckling | Responsive Hydrogel Layers -From Synthesis to Applications[END_REF] Hydrogel thin films associate both advantages of hydrogels and films. The architecture of hydrogel films can be inspired from that of macroscopic hydrogels. In recent years, the design of hydrogels has been a hot topic in the research to tailor the structures for well-controlled properties over a large range. [START_REF] Richtering | Gel Architectures and Their Complexity[END_REF] For example, the poor mechanical properties of hydrogels materials can be remarkably improved with suitable architectures, such as interpenetrating networks [START_REF] Sun | Highly Stretchable and Tough Hydrogels[END_REF] and especially double-networks [START_REF] Gong | Double-Network Hydrogels with Extremely High Mechanical Strength[END_REF][START_REF] Gong | Why Are Double Network Hydrogels so Tough?[END_REF] and also hybrid gel materials in which solid nanoparticles are embedded in the polymer network. [START_REF] Haraguchi | Effects of Clay Content on the Properties of Nanocomposite Hydrogels Composed of Poly(N-isopropylacrylamide) and Clay[END_REF][START_REF] Lin | Effect of Polymer-Particle Interaction on the Fracture Toughness of Silica Filled Hydrogels[END_REF][START_REF] Rose | Nanoparticle Solutions as Adhesives for Gels and Biological Tissues[END_REF] Moreover, the decrease of the feature size in thin films is an appropriate way to create stimuli-responsive hydrogels with fast response. Actually, the response time is roughly expressed as L 2 /D, where L is the thickness of the hydrogel film and D is the collective diffusion constant of the gel network. It indicates that small size of hydrogels (by downsizing to hydrogel films) would give faster response. For example, the response time is millisecond for micron-thick gel films. The properties of these active materials can be interestingly explored for the fabrication of miniaturized devices with short response time.

Despite their huge potential, there is a lack of clear and universal strategy for the controlled synthesis of surface-attached hydrogel films. We propose a novel, simple and versatile approach to fabricate surface-attached hydrogel films with well-controlled chemistry on wide range of thickness from nanometers to micrometers. [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF] This approach must allow easily the adjustment of chemical properties (for example, responsiveness) and physical properties (e.g. size and structure) of the films. Here, we demonstrate the facile route for the fabrication of tailored hydrogel films with responsive properties, the responsiveness providing additional freedom to polymer films. However, the strategy can be extended to more general polymer networks films.

Hydrogel films have responsive properties if the polymer chains used for crosslinking are responsive. If mechanical properties are the purpose, it is possible to vary the properties of the polymer network from glassy to rubber.

The strategy chosen for the synthesis of surface-attached chemical hydrogels consists in crosslinking preformed and functionalized polymers by thiol-ene click chemistry. It is preferred to the approach starting from monomers which are then simultaneously polymerized and crosslinked by radical polymerization. In this latter approach, the reaction mixture containing monomers, crosslinkers and initiators is generally confined between two planar substrates (one is functionalized to attach the hydrogel film and the other is non-sticky to detach the film) separated by spacers 14 so that very thin films (submicrometer size) are unfeasible. An additional difficulty is the constraint of controlled atmosphere to avoid oxygen which inhibits radical polymerization. The fabrication of hydrogel films is delicate given that synthesis at surface is very sensitive due to a high surface to volume ratio. To overcome this difficulty, our strategy is to preform polymer chains first and then crosslink and attach them to the surface by click chemistry. Here, thiol-ene click chemistry [START_REF] Hoyle | Thiol-enes: Chemistry of the Past with Promise for the Future[END_REF][START_REF] Kade | The Power of Thiol-ene Chemistry[END_REF] is chosen since the thiol-ene reaction is advantageously performed without any initiator addition and can be activated by either thermal heating or UV-irradiation, resulting in facile patterning of hydrogel films. Many thiol molecules are commercially available to suit the crosslinking reaction and the surface modification: bifunctional thiol (or dithiol) as crosslinkers or gold surfaces modifiers, mercaptosilane to functionalize silicon and glass substrates. The strategy based on thiol-ene click chemistry is likely more straightforward and versatile than the approach using photo-crosslinkable polymers developed by Kuckling et al. with dimethylmaleimide groups [START_REF] Kuckling | Photo-Cross-Linkable PNIPAAm Copolymers. 1. Synthesis and Characterization of Constrained Temperature-Responsive Hydrogel Layers[END_REF][START_REF] Harmon | Photo-Crosslinkable PNIPAAm Copolymers. 2. Effects of Constraint on Temperature and pH-Responsive Hydrogel Layers[END_REF][START_REF] Harmon | Photo-Crosslinkable PNIPAAm Copolymers. 4. Effects of Copolymerization in Cross-Linking on the Volume-Phase Transition in Constrained Hydrogel Layers[END_REF][START_REF] Harmon | Photo-Crosslinkable PNIPAAm Copolymers. 5. Mechanical Properties of Hydrogel Layers[END_REF][START_REF] Kuckling | Bilayer Hydrogel Assembly[END_REF] and Toomey et al. with benzophenone groups. In this article, we report the facile route for the fabrication of hydrogel films with responsive properties and tailored architectures. The potential of our approach is demonstrated with different kinds of plane solid substrates, silicon wafers, glass substrates and gold surfaces, and various stimuli-responsive polymers, for example poly(N-isopropylacrylamide) for its responsiveness to temperature and poly(acrylic acid) for its pH-sensitivity. We show the synthesis of surface-attached hydrogel films on wide range of thickness from nanometers to micrometers. Spin-coating is chosen as coating technique, but other coating methods can be envisaged, for example dip-coating if thicker films are desired. As discussed, the strategy also allows the creation of patterns if thiol-ene reaction is UV-activated instead of thermally annealed. Another attractive feature of the approach is the development of new complex hydrogel films with various targeted architectures. We show the ability to build multilayer hydrogel films inspired from layer-by-layer assemblies, interpenetrating networks films and nanocomposite hydrogel films which are inspired from the architecture of macroscopic hydrogels. The purpose of this article is to show the facile and universal route for the fabrication of surface-attached hydrogels on external and flat surfaces. The wide possibilities of architecture of the hydrogels are demonstrated: micrometer-size patterns, multilayers, interpenetrating networks, nanocomposite hydrogels. This article is the starting point of our group for many studies on both fundamental aspects and promising applications of surface-attached hydrogels using this synthesis strategy.

Experimental Section

Thiol-modification of substrates. Thiol-modification of silicon wafers using 3mercaptopropyltrimethoxysilane is described in detail in a previous article. [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF] Briefly, silicon wafers are cleaned in a freshly prepared "piranha" solution before being immersed in a solution of anhydrous toluene with 3 vol% of mercaptopropyltrimethoxysilane under nitrogen for 3 hours.

Gold surfaces are obtained by evaporation of a gold film (with thickness around 100 nm) on microscope glass slides. A chromium adhesion layer ( 3 nm) is first deposited to improve adhesion of the gold coating. Gold surfaces are functionalized with thiol self-assembled monolayer using a solution of dithioerythritol at 1 mM in chloroform for 2 hours. The thiolmodified surfaces are rinsed in solvent (toluene or chloroform) in ultrasonic bath to remove unreacted thiol.

Synthesis of hydrogel films. Ene-functionalized polymers are coated on thiol-modified substrates by spin-coating. A solution containing ene-functionalized copolymers (at various concentration and molecular weight) and dithioerythritol crosslinkers is dropped onto thiol-modified solid substrates (the ratio of dithioerythritol to ene-functionalized copolymer units is 15 times, corresponding to a molar excess of bifunctional dithioerythritol of 30). The conditions of spincoating are fixed with the final angular velocity of 3000 rpm and the spinning time of 30 seconds. The polymer films are annealed at 120°C for 16 hours under vacuum to activate thiolene reaction. For deep UV-irradiation ( = 254 nm) instead of thermal activation, either a common 8 Watt fluorescent lamp with photomasks or a 240 Watt DILASE laser is used. The bilayers films are fabricated using thermal process. The bilayers architecture is made of two stacked polymer network layers which are successively obtained after 16 hours annealing at 120°C. The dry thickness of each layer is equal to 300 nm (obtained using a 254 kg/mol PNIPAM at concentration of 4 wt% in the solution for spin-coating). The total thickness of the bilayers is 600 nm. For the synthesis of interpenetrating networks films, the first network (with dry thickness equal to 300 nm) is synthesized in the same way as single-network films. The formation of the second network (300 nm) inside the first network is obtained by favoring the interdiffusion of chains under saturated atmosphere before crosslinking them. The sample is left under methanol/butanol saturated atmosphere at ambient temperature during 48 hours. It is then put at 120°C during 16 hours to achieve the crosslinking reaction for the formation of the second network. All hydrogel films are rinsed in methanol and sonicated before being dried under nitrogen to remove unreacted polymers so that remained polymers are chemically cross-linked and covalently grafted to the substrate. Silica-PNIPAM hybrid hydrogel films are prepared by crosslinking polymer chains in the presence of silica nanoparticles dispersed inside. Silica Ludox suspensions (Dupont) which are known for their weak polydispersity were used for the fabrication of hybrid gel films. Ludox-TM50 silica particles diameter of 30 nm (characterized by scanning electron microscopy, dynamic light scattering and small angle neutron scattering) was chosen. Ludox-TM50 is initially purified by dialysis against water during two weeks to remove Na + stabilizing counter ions (this step is necessary for the addition of the polymer organic solution). After dialysis, a stable suspension is obtained, with the silica concentration around 20 wt%. Ene-functionalized PNIPAM is first dissolved in the mixture of methanol and pure water at a certain concentration, the solution stirred for one night. Then the dialyzed Ludox-TM50 suspension is introduced into the solution whose amount is decided by the ratio between polymer chains and silica particles(with respect to the ratio between methanol and water as V/V = 4/1). Dithioerythritol crosslinkers (with molar excess of 30) are added just before spin-coating. After spin-coating, the samples are immediately put at 120°C under vacuum for 16 hours. The samples are then rinsed in methanol with ultrasonic bath to remove the unreacted polymers and dried with N 2 flow.

Patterns of surface-attached hydrogels. Patterns are obtained by performing the UV-light irradiation with common 8 Watt fluorescent lamp through a photomask. The photomask is placed over the spin-coated polymer film and the sample is irradiated for 2 hours. Photomasks are fabricated by patterning a 100 nm-thick layer of chromium on quartz plates. Quartz is transparent to UV radiation unlike the 100 nm-thick layer of chromium. To etch the mask design on the chromium layer, a photoresist masking material (Microposit S1813 G2 Photoresist from DOW Chemical) is first deposited according to the design using photolithography. The chromium layer is then chemically wet-etched and the masking material was finally removed. Another way to obtain patterns is to achieve the UV-activation of the thiol-ene reaction with a laser spot which is focused on the surface of the polymer film. The laser spot is shifted over the surface to draw the patterns with very high resolution. This laser lithography is performed using a KLOE DILASE 650 device equipped with a 266 nm laser light source.

Ellipsometry. The thickness of hydrogel films is measured with spectroscopic ellipsometer

Nanofilm EP3 (Accurion GmbH, Germany). For the measures in air, we use the model with two layers between two semi-infinite media, the silicon substrate (n = 3.87) and the ambient air, the first layer comprising silica and silane (n = 1.46) which thickness was determined before grafting the hydrogel film, the second layer being the hydrogel film which thickness is measured and refractive index is 1.50 (taking in account that hydrogel films contain less than 10% of water in air with humidity ratio between 20% and 60%). In situ measurements in water are performed using a temperature-controllable liquid cell with thin glass walls fixed perpendicularly to the light path and the angle of incidence is fixed at 60°. The polymer hydrogel film is modeled as a single layer with the thickness and a constant refractive index between that of water (1.33) and of the polymer (1.52). Since the hydrogel film is covalently attached to the substrate, the polymer amount should keep the same when immersed in water with and with the volume fraction of polymer in water. The swollen thickness , the refractive index of the film in water and the dry thickness are deduced from the fitting of the experimental data. The two equations should provide the same value of to ensure that the fitting is reliable. Both hydrogel dry films and hydrated films are uniform over a large area (e. g., several centimeters) of the substrates. Each thickness data correspond to the mean measure of at least three samples. For dry films, the standard deviation from the mean value is 7% at the maximum. [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF] The refractive index of the polymer film in water is chosen to be approximated with a linear model instead of Lorentz-Lorenz equation. It is slightly overvalued by the linear model, the divergence being at the maximum 0.1% (for the weakest value of equal to 0.2), which is well below the accuracy of the refractive index of pure polymer and also much lower than the tolerance accepted for the fitting of  and  experimental data (1%). Moreover, as the refractive index and the thickness are measured independently by ellipsometry, the value of the thickness is not impacted by this slight divergence of the refractive index.

For hybrid hydrogels, there are three components in the film: polymer, silica and water. So the refractive index in water should be written as for components i. Considering the refractive index of silica equal to 1.46, we have the following system:

The refractive index of the film in water and the thicknesses of the film in air and in water are deduced from the fitting of the experimental data. The ratio is fixed by the synthesis and corresponds to the silica/polymer ratio shown in the Supporting Information. The equations should provide consistent values of the volume fraction of polymer and silica in the hybrid gel film, and , so that the fitting is reliable.

Atomic Force Microscopy. All AFM images are acquired with a Bruker ICON microscope controlled by a Nanoscope V. Height and phase images are obtained in tapping mode using a tip with a spring constant of 40 N/m and a resonance frequency close to 300 kHz. The working conditions are the following: free amplitude of about 20 nm, amplitude reduction equal to 0.95, scan frequency of 0.5 Hz and images acquired with 512 x 512 pixels. We check that all the images are acquired in repulsive mode. The phase difference between the cantilever excitation at its resonance and the response of this cantilever are measured by the internal ICON lock-in amplifier. Both height images and phase images are displayed after baseline subtraction so that the height and phase values are relative values. The mean roughness is estimated from the half width of the height histogram of each image.

Results and Discussion

Strategy for the synthesis of hydrogel thin films

Surface-attached hydrogel films are synthesized by simultaneously crosslinking and grafting responsive polymers by thiol-ene click reaction. The preformed and ene-reactive polymer is coated on thiol-modified substrate with dithiol crosslinkers (Figure 1). The preformed polymers provide responsive properties of hydrogel films. The synthesis of ene-functionalized polymers can be carried out in two steps for ene-functionalized PNIPAM (one step for PAA) as described in detail in a previous paper.13

First, P(NIPAM-co-AA) copolymers are synthesized by free radical polymerization (this step is not necessary for PAA homopolymer which can be easily and cheaply purchased). Since there is no need to control the polydispersity index of polymer chains which are aimed to be postcrosslinked, free radical polymerization is the best option, as it is much straightforward compared to other polymerization methods. The initiator used for radical polymerization in water is ammonium persulfate /sodium metabisulfite redox couple. By varying the concentration of the reducing agent, polymer chains can be obtained with different molecular weight, as shown by Bokias et al. [START_REF] Bokias | Molar Mass Control of Poly(N-isopropylacrylamide) and Poly(acrylic acid) in Aqueous Polymerizations Initiated by Redox Initiators Based on Persulfates[END_REF] Second, the copolymer chains are randomly functionalized with ene-groups by peptide reaction. Because there are carboxyl groups (-COOH) in the polymer chains and allylamine has both amino (-NH 2 ) and ene-group, the peptide reaction is exploited to graft allylamine onto polymer chains in the presence of 1-(3-dimethyl-aminopropyl)-3ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). EDC is used as the dehydration agent and NHS as the addition agent to increase yields and decrease side reactions. [START_REF] Roy | Hydrophobically Modified Xanthan: an Amphiphilic but Not Associative Polymer[END_REF] Both radical polymerization and peptide reactions are performed in water for a facile recovery of the ene-functionalized polymer after purification by dialysis against water and freeze-drying. However, if the purification is necessary for the characterization of the enefunctionalized polymer (SEC, NMR), it is not required for the synthesis of chemical hydrogel film grafted to solid substrate. The washing of surface-attached film is enough to remove all undesired species such as residual monomers, initiators, reactive products and byproducts from synthesis of ene-functionalized polymer. homogeneous (melt) polymer films from polymer solutions such as dip-coating, spray-coating or spin-coating. We chose the spin-coating technique to synthesize hydrogel films on wide range of thickness from nanometers to micrometers. The spin-coating technique has also the advantage to necessitate a little polymer. For example, as only 0.2 ml of solution is needed to cover a surface of 1 cm 2 , a polymer concentration of 1 wt% requires only 2 mg of polymer. The solvent blend selected to prepare the polymer solution must solubilize both ene-functionalized polymer and dithiol crosslinkers and also allow the spreading of the polymer film on thiol-modified substrates by spin-coating. The mixture of butanol and methanol (V/V = 1/1) is preferred as the solvent blend for PNIPAM and the mixture of methanol and formic acid (V/V = 7/3) for PAA.

The film thickness is strongly affected by many factors such as the volatility of the solvent, the final angular velocity and the viscosity of the polymer solution. [START_REF] Norrman | Studies of Spin-Coated Polymer Films[END_REF] Here, films with different thickness are obtained by changing the viscosity with the variation of polymer molecular weight and polymer concentration. Figure 2 demonstrates that surface-attached hydrogel films can be synthesized on wide range of thickness from nanometers to micrometers. As expected, the film thickness increases with the viscosity of polymer solution, and so with polymer concentration or molecular weight. For molecular weight of 66 kg/mol, submicrometer thicknesses can be obtained with high precision (below 5% deviation) by varying the concentration below 10 wt%.

For higher molecular weight, the films have a bigger deviation of the thickness but the advantage is the large range with the formation of micrometric films. The results are shown here for PNIPAM layers with various molecular weight but the same are obtained for PAA films. In particular, we observed that the film thickness is similar for PNIPAM and PAA using spinning polymer solutions with same concentration, same molecular weight and suitable solvent (see Supporting Information). The dependence of the film thickness on the viscosity of the polymer solution is clearly demonstrated in the insert of Figure 2. In the spin-coating process, the film thinning can be modeled by hydrodynamic equations describing radial out-flow and solvent evaporation. [START_REF] Meyerhofer | Characteristics of Resist Films Produced by Spinning[END_REF] The thickness of ultrathin polymer films can be expressed by the scaling: h   -1/2  1/2 C p where h is the final film thickness,  is the spin speed,  is the initial solution viscosity and C p the polymer concentration. The power law dependence of the viscosity with the exponent of 1/2 [START_REF] Extrand | Spin Coating of Very Thin Polymer Films[END_REF] is preferred to the value of 1/3 [START_REF] Hall | Spin Coating of Thin and Ultrathin Polymer Films[END_REF] assuming that all the solvent flashes off at a certain time. This assumption is in agreement with our spin-coating conditions with the use of very volatile solvent such as methanol. As the viscosity of polymer solution can be expressed as function of the concentration and the molecular weight as C p M  (Mark-Houwink parameter  is between 0.5 for theta solvents and 0.8 for good solvents). Finally, the thickness of polymer films is given by the expression:

h   -1/2 M /2 C p 3/2 .
The film thickness is plotted as M /2 C p 3/2 using  = 0.8.

Experimental data are satisfactorily aligned on the same master curve on a large range of concentration (from 1 to 10 wt%) and large range of thickness from nanometer to micrometers.

Note that in our spin-coating experiments, the spin speed was fixed at 3000 rpm, but the variation of this parameter could be as valuable as that of polymer concentration and molecular weight to reach the desired thickness.

The formation of surface-attached hydrogel films can be achieved by either UV-irradiation or thermal activation. Deep selective UV-irradiation allows the activation of thiol-ene reaction without any initiator to simultaneously crosslink polymer chains and graft them on thiolmodified substrate. Figure 3 shows PNIPAM film thickness as function of the irradiation time.

The experiments were performed using a commercial 8 Watt fluorescent lamp at a wavelength of 254 nm. The film thickness increases with the exposure time and reach a plateau after 2 hours.

The maximum thickness reached for each polymer concentration (in the solution for spincoating) is the same than that obtained by heating at 120°C for 16 hours. The variation of the film thickness with the exposure time during the first two hours is probably due to partial crosslinking of polymer chains, the uncrosslinked chains being removed by washing. The same kinetics is observed for PAA surface-attached hydrogel films, as shown in Supporting

Information. In addition to time saving, the advantage of the UV-activation is the ability to create patterns of surface-attached hydrogels. The patterning of hydrogels can be obtained using common 8 Watt fluorescent lamp with photomasks. This patterning process corresponds to a negative photolithography. It can also be achieved without photomask by using 240 Watt laser technology such as DILASE650 device. As a simple illustration, Figure 3 shows a clear patterning of PNIPAM hydrogels on silicon surface with 6 microns-wide lines using the fluorescent lamp through a photomask. Images of the patterns were obtained with a reflected light microscope.

The reflective silicon surface appears bright and the hydrogel patterns appear in black tones. instabilities resulting from the hydrogel buckling when hydrogel patterns synthesized in dry state are exposed to water. These buckling instabilities are due to inhomogeneous compressive strains that arise from the geometric constraints. [START_REF] Dupont | Swelling-Induced Instabilities in Microscale, Surface-Confined Poly(N-isopropylacrylamide) Hydrogels[END_REF] Actually, our surface-attached hydrogel patterns are rather wide than thick (the thickness is in general ten times lower than the width) so that the swelling of hydrogel is unidirectional without out-of-plane deformation. Hydrogel patterns will be the subject of a forthcoming paper and in particular the fabrication and characterization of patterns with multi-responsive properties will be focused.

Stimuli-responsive properties of hydrogel films

Temperature-responsive properties of PNIPAM hydrogel films are demonstrated with the measure of the thickness of hydrogel films in water at different temperature (Figure 4). The swelling ratio is defined as the ratio between the thickness of the hydrogel film in water (swollen thickness) and in air (dry thickness). Indeed, from the measure of water content in PNIPAM hydrogel film with controlled humidity ratio (data not shown here), PNIPAM hydrogel films are shown to contain less than 10% of water in air (for humidity ratio roughly varying from 20% to 60%). For simplicity, the thickness of PNIPAM hydrogel film in air is assumed to be the dry thickness. The swelling ratio is independent of the thickness in the whole range from 100 nm to a few micrometers. The data were obtained with hydrogel films of supposedly same crosslinks density. This assumption seems consistent since the synthesis of the hydrogel films is performed with the same (2%) reactive copolymer and same (30 times the ene-reactive groups) excess of dithioerythritol crosslinkers. As experimental conditions used the same except the thickness, hydrogel films can be supposed to be chemically the same. The crosslinking is likely uniform without any gradient in the direction normal to the surface as ene-functionalized polymers and dithiol crosslinkers are homogeneously mixed before spin-coating (both are totally soluble in the solvent used). Actually, it was shown that surface-attached hydrogel films have abrupt density profile with weak interface width. Experimental data from ellipsometry and neutron reflectivity could be satisfactorily fitted with a model of one unique and uniform layer for PNIPAM hydrogel films. [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF] Unfortunately, spectroscopic methods probing surfaces are unable to provide information on the crosslinks density. For example, XPS technique only probes nanometric layers (the standard penetration depth of the technique is about 5 nm) and cannot give average measure of the whole hydrogel films. Infrared spectroscopy in ATR (Attenuated Total Reflection with micrometric penetration depth) is more suitable to probe the whole films. However, the spectra analysis is problematic for wave number range below 1600 cm -1 due to the high absorbance of silicon substrates used as (infrared) waveguides. Also, the ratio of thiol-ene reaction cannot be quantified as the absorption peak which is characteristics of S-C bond (around 700 cm -1 ) is in this range of low wave number. Moreover, the thiol-ene ratio is weak with a maximum of 2%, the ratio of ene groups measured by 1 H NMR being 2% (see in Supporting Information).

Swelling ratio

Temperature (°C)

The measure of the thickness in the swollen state is a way to control the synthesis of hydrogel films since it provides (indirectly) the crosslinks density. The weaker the crosslinks density, the more the hydrogel films swell. Differently from macroscopic hydrogels, surface-attached polymer networks are expected to swell linearly, i.e. only in the direction normal to the substrate and not in the direction parallel (the dimension of the surface is infinite compared to the perpendicular direction). The Flory-Rehner theory [START_REF] Flory | Statistical Mechanics of Cross-Linked Polymer Networks. II. Swelling[END_REF] for the swelling of hydrogels extended to one-dimension swelling is appropriate, as shown by Toomey et al. [START_REF] Toomey | Swelling Behavior of Thin, Surface-Attached Polymer Networks[END_REF] They determined the swelling ratio (or linear degree of swelling) as function of the proportion of photo-crosslinkable units from 0.5% to 14.3% for 100 nm-thick films. Our swelling ratio of 4 found with 2% enereactive polymer is in agreement with their results.

If the swelling ratio is 4 for temperature below the LCST, the value of 1.5 is found for temperatures above the LCST. It means that collapsed PNIPAM hydrogel films are not free of water but indeed contains roughly 30% of water. It is in good agreement with the results from Kuckling's group [START_REF] Kuckling | Photo-Cross-Linkable PNIPAAm Copolymers. 1. Synthesis and Characterization of Constrained Temperature-Responsive Hydrogel Layers[END_REF][START_REF] Harmon | Photo-Crosslinkable PNIPAAm Copolymers. 2. Effects of Constraint on Temperature and pH-Responsive Hydrogel Layers[END_REF] and Toomey et al. [START_REF] Vidyasagar | Temperature Induced Volume-Phase Transitions in Surface-Tethered Poly(N-isoprylacrylamide) Networks[END_REF] They also found that water is not entirely expulsed from collapsed PNIPAM hydrogels no matter the crosslinks density. The swelling-collapse phase transition of responsive hydrogel films with swelling ratio between 4 and 1.5 is quite high amplitude (almost 3), which is interesting for applications exploiting temperature-responsive properties. This amplitude of swelling-collapse transition was also found by Toomey et al. [START_REF] Vidyasagar | Temperature Induced Volume-Phase Transitions in Surface-Tethered Poly(N-isoprylacrylamide) Networks[END_REF] The amplitude could be increased with weakly cross-linked hydrogels. However, weakly cross-linked hydrogels (with high water content) are not mechanically stable and can irreversibly be damaged under constraints due to the swelling. Polymer chains are then removed during the washing step.

Another point which could be valuable for applications is the sharp transition within a temperature range of 3°C (here from 33°C to 36°C). The identical sharp transition with high amplitude is found for surface-attached PNIPAM films in the whole range of the thickness from 100 nm to 1 m. In a previous publication, we have also demonstrated that the swelling ratio which only depends on the cross-links density is the same for a few micrometers-thick films. [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF] Mulilayers hydrogels and interpenetrating networks The dry thickness of the hydrogel films (or thickness measured in air) increases linearly with the number of deposited layers (Figure 5). The linear increase is demonstrated for two different thickness of the elementary layer (15 nm and 65 nm) decided by the concentration of the spincoating solution as shown previously in Figure 2. The hydrogel can be any polymer PNIPAM or PAA. It provides evidence the simplicity of the method to fabricate stable and durable multiresponsive polymer films. The potential of multilayers hydrogel films is the chance to vary the thickness at will in comparison to layer-by-layer polymer assemblies in which the elementary adsorbed layer is a few nanometers-thick due to the obligatory physical interaction between two adjacent layers. For interpenetrating networks, the mixture of two networks is targeted. The strategy is accordingly different from that used for multilayers films. After the formation of the first network, the polymer chains coated for the second network need to diffuse inside the first network before being crosslinked. It is not possible with the dry thickness of films to differentiate the architecture of bilayers BL and interpenetrating networks IPN as the dry thickness (measured in air) corresponds to the amount of polymer. However, a bilayers film and an IPN film made from the same single-network (with same polymer amount and same crosslinking ratio) are expected to swell differently. The swelling ratio at 25°C of a bilayers film nm) is synthesized in the same way as single-network film. The polymer chains for the formation of the second network are then coated with dithiol crosslinkers on top of the gel film (the dry thickness of the second film is also equal to 300 nm). The total interdiffusion of chains inside the first network is favored by the swelling or stretching of the first network under vapor saturated atmosphere (methanol/butanol for PNIPAM film). Finally, the thermal process allows the crosslinking reaction for the formation of the second network. At 25°C below the LCST of PNIPAM, the swelling ratio of bilayers film is the same as that of single-network film, as expected. The swelling ratio of 4 is also the value found in Figure 4. It clearly proves architecture of stacked layers with very little interdiffusion of chains between the layers. The stability of bilayers is probably ensured by entanglements between peripheral chains and thiolene bonds between the chains of the first layer and those of the second layer. The swelling ratio of IPN film is half the swelling ratio of single-network film and bilayers film since the whole IPN film can be considered as twice crosslinked due to the complete interdiffusion of the two networks. At 40°C above the LCST of PNIPAM, in the collapsed state, the swelling ratio is around 1.5 for all films whatever the architecture. Collapsed PNIPAM gel films are not free of water while containing approximately 30% of water independently of the architecture and the thickness of the gel films, as discussed previously.

Here, we show a way to fabricate interpenetrating networks film by successively coating reactive polymers of the two (or more) networks. It should be noted that the other approach to interpenetrating networks films could be the use of two (or more) distinctive and selective chemical reactions to simultaneously crosslink the two (or more) networks. [START_REF] Tillet | Chemical Reactions of Polymer Crosslinking and Post-Crosslinking at Room and Medium Temperature[END_REF] The advantage is the simultaneous coating of the two polymers for the formation of interpenetrating networks.

Surface-attached hybrid hydrogel films

Another example of architecture of hydrogel films with great potential is the hybrid gel films in which nanoparticles are incorporated inside the network. An illustration of nanocomposite hydrogel films is given with silica solid particles stably trapped inside PNIPAM hydrogel. The nanocomposite networks are achieved by simply adding the silica nanoparticles to the solution of polymer for coating. The silica particles used in this study are commercially available (Ludox aqueous suspensions). The particles have a spherical shape with a mean diameter of 30 nm, which is much higher than the mesh size of the polymer network. The synthesis of silica-polymer hybrid hydrogel films is very similar to that of single-network gel films, except that silica nanoparticles are added to the polymer solution before spin-coating. As for single-network, multilayers and interpenetrating networks films, thiol-ene click reaction allows the covalent crosslinking of the polymer network and covalent grafting to the surface, the hybrid hydrogel network being obtained with silica particles trapped inside. The thickness of silica-polymer hybrid hydrogel film can be easily controlled by adjusting the molecular weight and concentration of the polymer and the silica/polymer ratio (see Supporting Information). As expected, the dry thickness of the hybrid gel film (measured in air) increases with the silica content. For example, for a given molecular weight of polymer (66 kg/mol) and concentration of polymer in the solution for spin-coating (5%), the thickness increases from 200 nm to 455 nm for a volume fraction of silica particles from 0 to 27%. Silica-PNIPAM hybrid gel films can be synthesized on a wide range of volume fraction of particles. It should be highlighted that the volume fraction of particles can reach values above 60%, the value of 64% corresponding to the random close-packing of monodisperse spherical objects. In the hybrid network films containing high volume fraction of solid particles, the empty space is probably filled by polymer chains.

AFM images of the free surface of hybrid hydrogel films are shown in Figure 7. Two samples with different volume fraction of silica particles are probed in air, one with low concentration of silica at 2.1% and the other with very high concentration at 63.4% which is near the closepacking. AFM images provide information on the dispersion of silica nanoparticles at the free surface of the gel film. The height and phase images of the hybrid gel film containing 2.1% of silica give evidence the good dispersion of the nanoparticles. The dispersion is obvious on the phase image because of the high contrast between the PNIPAM matrix and the silica particles. In the height image of the hybrid gel containing 63.4% of silica, nanoparticles with a typical size of about 30 nm can be observed, which is in good agreement with the diameter of the silica spheres characterized by scanning electron microscope. Actually, due to their high volume fraction in the hybrid gel film, the solid particles distribute on the surface in very high density corresponding to a nearly close-packing distribution with almost no polymer network showing up. As expected in such configuration, the phase image of the hybrid gel with 63.4% of silica is quite uniform:

indeed, almost all the surface is covered by silica particles giving the same phase value. The roughness of both hybrid gel films are a few nanometers. Even if the roughness is a little higher than that of single-network films, it remains much lower than the thickness of the film, so that the hybrid gel film can be considered as flat.

Swelling ratio

Volume fraction of particles

Figure 8 shows the swelling ratio of the hybrid gel films in water at 25°C and 40°C. At 25°C, the obvious trend is a clear decrease of the swelling ratio as the silica particles content increases, from a swelling ratio of 4 (for the pure polymer matrix without silica) to 1.2-1.5 (for the highest particles content of 63.4%). The presence of silica in the hybrid gel film gives rise to topological constraints which restrict the swelling ability of the polymer network. In addition to the geometrical constraints, attractive interactions at the interface through hydrogen bonds between silica particles and PNIPAM chains should be taken into account. The swelling restriction in these hybrid gel films is comparable to the swelling restriction shown for macroscopic silicafilled elastomers [START_REF] Berriot | Reinforcement of Model Filled Elatomers: Experimental and Theoretical Approach of Swelling Properties[END_REF] and hybrid hydrogels containing silica nanoparticles. [START_REF] Carlsson | Nano-Hybrid Self-Crosslinked PDMA/Silica Hydrogels[END_REF] At 40°C, the swelling ratio has no obvious variation with the volume fraction of silica, the values fluctuating between 1.2 and 1.5. There is unsurprisingly no effect of the silica particles in the collapsed state of the hydrogel. The gap of the swelling ratio between 25°C and 40°C is smaller when there are more silica particles in the hybrid hydrogel. For very high volume fraction of silica particles near the close-packing, the swelling ratio of the hybrid gel is the same in the swollen and collapsed states,

showing no responsive property.

Conclusion

A simple and versatile approach to surface-attached hydrogel thin films with well-controlled chemistry and targeted architecture was demonstrated. Our strategy to fabricate chemical hydrogel films is to preform ene-functionalized polymers first and then crosslink and attach the chains to the surface by thiol-ene click chemistry. A powerful consequence of this approach is the facile patterning of hydrogel films as thiol-ene reaction can be selectively activated by UVirradiation (in addition to thermal heating). A very attractive feature of the hydrogel films is the development of new complex hydrogel films with various architectures. We demonstrated the fabrication of multilayer hydrogel films inspired from layer-by-layer assemblies, interpenetrating networks and nanocomposite hydrogel films which are inspired from the architectures of macroscopic hydrogels. Multilayer hydrogel films are very promising as simple way to achieve structured and multifunctional films with long-term stability. The potential of multilayers hydrogel films is the chance to vary the chemical and physical properties of each elementary layer at will, for example responsiveness and thickness. Along with that, the interpenetrating networks films are the alternative architecture to bilayer films as they are the mixture of two crosslinked structures inside the same layer. The IPN architecture can be interestingly exploited to generate double-networks films by analogy with double-networks materials. Meanwhile, nanocomposite hydrogel films are also expected to have improved mechanical properties similarly to hybrid gel materials. Actually, hydrogel coatings with targeted architectures offer great potential. The versatility of the approach is a powerful toolbox to tune physical properties of these hydrogel coatings, such as optical properties with multilayers for Bragg dielectric mirrors [START_REF] Hiller | Reversibly Erasable Nanoporous Anti-Reflection Coatings from Polyelectrolyte Multilayers[END_REF][START_REF] Wang | Metallodielectric Photonic Structures Based on Polyelectrolyte Multilayers[END_REF][START_REF] Nolte | Creating Effective Refractive Index Gradients within Polyleectrolyte Multilayer Films: Molecularly Assembled Rugate Fillers[END_REF] or mechanical properties with double-networks and hybrid gels for reinforcement or healing. [START_REF] Gong | Double-Network Hydrogels with Extremely High Mechanical Strength[END_REF][START_REF] Gong | Why Are Double Network Hydrogels so Tough?[END_REF][START_REF] Haraguchi | Effects of Clay Content on the Properties of Nanocomposite Hydrogels Composed of Poly(N-isopropylacrylamide) and Clay[END_REF][START_REF] Lin | Effect of Polymer-Particle Interaction on the Fracture Toughness of Silica Filled Hydrogels[END_REF][START_REF] Rose | Nanoparticle Solutions as Adhesives for Gels and Biological Tissues[END_REF]

Figure 2 .

 2 Figure 2. Dry thickness of PNIPAM hydrogel films as function of polymer concentration in the

Figure 3 .

 3 Figure 3. (Left) Dry thickness of PNIPAM hydrogel films as function of UV-irradiation time for

Figure 4

 4 Figure 4 also displays squared patterns achieved from direct writing with the DILASE laser

Figure 4 .

 4 Figure 4. Swelling ratio of PNIPAM hydrogel films as function of temperature for various dry

Figure 5 .

 5 Figure 5. Dry thickness of PNIPAM hydrogel multilayers film as function of the number of

Figure 6 .

 6 Figure 6. (Left) Swelling ratio of PNIPAM hydrogel films for various architectures: SN for

  be the same as the ratio of single-network film and twice the ratio of IPN film because the whole IPN film can be considered as twice crosslinked due to the interdiffusion of the two networks.

Figure 6

 6 Figure 6 displays the swelling ratio of PNIPAM hydrogel films in water at 25°C (below the

Figure 7 .

 7 Figure 7. Height (top) and phase (bottom) tapping AFM images of surface-attached silica-

Figure 8 (

 8 Figure 8 (Top) Swelling ratio of silica-PNIPAM nanocomposite hydrogel films in water at 25°C

Acknowlegments. We gratefully thank the French National Research Agency (ANR), the China Scholarship Council (CSC) and the Ministry of Science and Technology of Thailand (MOST) for their financial support.