domain: chim spi phys.cond.cm-scm

text:
Dr Rémi Merindol
email: remi.merindol@umontpellier.fr

Dr Nicolas Martin

Dr Thomas Beneyton

Jean-Christophe Baret

Serge Ravaine

Prof. J C Baret

Prof. S Ravaine

Fast and ample light controlled actuation of monodisperse all-DNA microgels

The assembly of adaptive hierarchical soft-materials that resemble living tissues requires responsive building blocks of controlled dimensions. While DNA self-assembly provides an exceptional tool for nanoscale architectural control, responsive DNA microstructures remain scarce. Here, we address two challenges controlling the size of DNA microstructures and embedding them with fast and ample structural response. For size-control, we combine arrested phase separation and microfluidic confinement to produce monodisperse all-DNA particles. For responsiveness we implement a light controlled coil-globule transition of the microgel DNA network powered by an azobenzene cationic surfactant. The photoinduced trans-cis isomerization of the azobenzene moiety reduces its affinity for DNA which results in fast, large amplitudes microgel swelling. Finally we demonstrate the assembly of light responsive periodic microgel lattices as proof-of-concept hierarchical all-DNA materials.

Introduction

Biological materials consist of hierarchical assemblies of biomolecules that constantly sense and interact with their environment. [1,[START_REF] Buehler | Materiomics: Multiscale Mechanics of Biological Materials and Structures[END_REF] Reproducing such an adaptive multi-scale organization is attracting growing interest to build life-inspired materials. [START_REF] Merindol | [END_REF] Hydrogels and microgels that dynamically respond to external cues across length scales are particularly crucial for cell studies and tissue engineering. [4][5][6] The bottom-up construction of such dynamic hierarchical materials requires the design of building blocks with the highest number of orthogonal control over their geometry, interactions, functionalization sites and responsive properties. DNA is the best example of such versatile building blocks at the molecular scale. [7] DNA selfassembly provides artificial materials with molecular structural control (e.g. DNA origami…), [8] easy functionalization with multiple DNA labelled probes [9] and programmable dynamic behaviour with enzymatic [10,[START_REF] Padirac | Proc. Natl. Acad. Sci[END_REF] and strand displacement reactions. [START_REF] Zhang | [END_REF] DNA-based materials also naturally respond to various stimuli, including heat, [13] pH, [14,15] metal ions [16] or biomolecules. [17] This set of orthogonal control offers a robust and modular platform to develop personalized medicine, [18] study cell behaviour, [5] and mimic biological non-equilibrium processes. [19] Recent works on DNA self-assembly target a structural control at always larger length scales towards micrometric objects [20,21] and macroscale materials. [22] Yet, sequence-driven selfassembly of individual DNA molecules into defined superstructures becomes slow and difficult at the micron scale as the number of accessible microstates increases exponentially. Mesoscale building blocks offer a convenient way to speed up the assembly of hierarchically structured materials. [6] Phase transitions in polyelectrolyte solutions provides means to spontaneously prepare such mesoscale structures. [23,24] When combined to microfluidics, the structures are highly controlled as exemplified with DNA solutions producing liquid micro-compartments [25] or with the preparation of crystalline particles of clonal DNA sequences. [26,27] We recently combined DNA liquid-liquid phase separation and supramolecular hybridization to explore new assembly landscapes for the pathway controlled formation of mesoscale all-DNA microgels. [28] However, the arrested phase separation at the origin of these DNA colloids yielded highly polydisperse particles, which prevented their assembly into well-defined superstructures. [29,30] Developing monodisperse all-DNA microgels represents a crucial step to assemble exotic photonic superstructures [29,30] and embed them with responsive properties, [31] or produce hierarchical soft materials that can adapt and process signals. [32,33] Beyond structural control, there is a growing interest to design responsive DNA. Owing to its great spatiotemporal capability, light is the most promising trigger for such a purpose. For instance, azobenzene photoswitches grafted onto DNA chains were used to promote volume changes of hybrid DNA-polymer hydrogels in response to light due to photoreversible DNA hybridization. [34] Yet, as opposed to fully synthetic polymer microgels whose radii easily double or triple upon irradiation due to coil-globule transition, [35] hybrid polymer-DNA hydrogels display small volume changes because the azobenzene groups only impact the DNA crosslinks and not the full hydrogel network. [15,34] Other example of responsive DNA microgels either burst open upon photothermal trigger, [28] dissolve due to enzymatic degradation, [36] swell due to metaloenzyme catalysed polymerization, [32] or grow irreversibly with the insertion of DNA hairpins. [37,38] As opposed to the rich diversity of responsive synthetic microgels driven by coilglobule transition, [39] we lack DNA microstructures with fast and ample structural response.

Here, we report the formation of monodisperse all-DNA microgels of controllable size via droplet-based microfluidics, and show that a cationic azobenzene photoswitch (azobenzenetrimethyl-ammonium bromide, azoTAB) promotes fast, large and reversible volume changes of the microgels upon light irradiation. This light-actuated swelling and compaction process arises from trans-cis photoisomerisation of azoTAB that impacts its binding affinity to DNA chains and induces globule-coil transition of the backbone conformation. [40][41][START_REF] Estevez-Torres | Proc. Natl. Acad. Sci[END_REF] We further report self-assembly of the monodisperse photoresponsive DNA microgels into regular superstructures as a step towards light adaptive hierarchical DNA materials that can undergo large scale structural reconfigurations. Our results describe an efficient approach to design size-controlled photoresponsive DNA microstructures as versatile building block for the assembly of responsive biomaterials.

Assembly of monodisperse all-DNA microgels

All-DNA microgels were produced by arrested phase separation of long single-stranded DNA (ssDNA), obtained by rolling circle amplification (RCA) of a circularized template with 29 polymerase, in the presence of divalent counterions, as reported previously. [28] (Figure S1) We here used a template consisting of (i) a 20 bases long homo-adenine run that promotes phase separation at high temperature in the presence of magnesium or calcium counterions, (ii) a selfcomplementary (palindromic) domain that forms duplexes upon cooling, therefore stabilizing the DNA-rich phase into soft microgels, and (iii) a recognition (bar-code) domain that remains single stranded and serves as an addressable anchor that can be labelled using fluorescent oligonucleotides (Figure S2). In bulk, the arrested phase separation produced polydisperse microgels (Figure S2) due to uncontrolled coalescence and ripening of DNA-rich droplets formed upon heating, as observed previously. We therefore developed a procedure to form monodisperse all-DNA microgels using droplet-based microfluidics (Figure 1a). Surfactantstabilized monodiperse water-in-oil drops of diameter DDrop = 35 μm encapsulating a homogeneous solution of long ssDNA and calcium ions (50 mM CaCl2) were first produced using a flow-focusing microfluidic device (Figure 1b and Figure S3). Liquid-liquid DNA phase separation within the microfluidic emulsion drops was then induced by thermal treatment at 85°C for 2 h. During this incubation time, we observed the nucleation of small DNA-rich droplets and their fusion into a larger single micro-droplet (Figure S4 for short incubation time).

Cooling down to room temperature induced gelling of the phase-separated DNA droplets to produce a single microgel per droplet (Figure 1c). We can estimate the fluctuation of concentrations as the square-root of the number of DNA molecules per drop, in our system these variations fall between 7 % at 3 mg.L -1 and 1 % at 100 mg.L -1 (taking 200 kg.mol -1 as average molecular weight). Since each droplet initially contains similar amount of DNA, phase separation results in microgels with similar size. The final step consisted in destabilizing the microfluidic emulsion to remove the oil phase and collect the microgels by centrifugation. The collected microgels were monodisperse (Figure 1d), as expected from the narrow distribution of the number of DNA per droplet, and could be re-dispersed into the desired buffer for downstream applications. We systematically investigated the relation between the initial DNA concentration in the homogeneous solution and the diameter of the DNA microgels both in the calcium solution (50 mM CaCl2) and at lower ionic strength in Tris-EDTA (TE) buffer (10 mM Tris, 1 mM EDTA) (Figure 1e-h). As expected, the microgel diameter increased with increasing initial DNA concentration. The DNA microgels also exhibited a larger diameter at lower ionic strength, up to 3.2 times the initial diameter corresponding to a 33-fold volume increase, consistent with swelling due to enhanced electrostatic repulsion between DNA strands. We further observed that the microgel diameter grew linearly with the cubic root of the initial DNA concentration (Figure 1f). If we assume that all the DNA strands initially present in one microfluidic drop end up in the final microgel, we can fit the data using Equation 1, which gives the diameter of the DNA microgel (Dmicrogel) as function of the initial microfluidic drop diameter (DDrop), initial DNA concentration (Ci) and the DNA concentration in the microgel (DNA). Our microfluidic drops were 35 µm in diameter and we measured 0.63 ± 0.03 µmLmg -1 and 2.0 ± 0.1 µmLmg - 1 for the slope of the linear fit of particles respectively in the calcium solution and in TE buffer.

The DNA content in the microgels was therefore calculated to be 17 ± 2 wt% in Ca 2+ and 0.53 ± 0.8 wt% in TE buffer, respectively. Although we considered the possibility that some DNA remained in solution after thermal treatment, the linear fit in Figure 1g crosses the abscissa very close to the origin (at a concentration below 0.01 mg.L -1 ), which suggests that the remaining DNA in solution is negligible in this case. We were yet unable to observe the formation of DNA microgels below 3 mg.L -1 of total DNA concentration. 1)

𝑫 𝒎𝒊𝒄𝒓𝒐𝒈𝒆𝒍 = 𝑫 𝑫𝒓𝒐𝒑 * √ 𝑪 𝒊 𝝆 𝑫𝑵𝑨 𝟑 (Equation

Light controlled actuation

The above observations exemplify the high volume change capability of the monodisperse all-DNA microgels. Here we demonstrate that we can use a cationic azoTAB photoswitch (Figure 2a) to control this process. AzoTAB has previously been exploited for the reversible photocontrol of DNA coil-globule transition and coacervation in solution. [40][41][START_REF] Estevez-Torres | Proc. Natl. Acad. Sci[END_REF][START_REF] Martin | [END_REF] Addition of trans-azoTAB at concentrations above 1 mM to monodisperse all-DNA microgels prepared at 100 mg.mL -1 and suspended in TE buffer induced a significant microgel compaction (Figure 2b and Figure S5), with a maximum ~2.6-fold decrease in the microgel diameter, corresponding to a ~17-fold volume decrease. In comparison, UV-adapted cis-azoTAB did not impact significantly the microgel size on the whole range of concentrations tested (Figure 2b), so that irradiation of the dark-adapted microgels with UV light in the presence of 3 mM trans-azoTAB resulted in significant microgel swelling due to trans-cis azoTAB photoisomerisation (Figure 2a,c and Figure S5). Reversibly, blue light irradiation favoured the compaction of the UVadapted microgels due to cis-trans azoTAB photoisomerisation (Figure 2a,c). This lightactuated swelling/compaction process could be repeated over multiple UV/blue-light irradiation cycles without any apparent fatigue (Figure 2c). Addition of magnesium ions decreased the light-driven microgel swelling capability (Figure 2c,d), as expected from screening of inter-DNA electrostatic repulsion at higher ionic strength (Figure S5), but more regular collapsed microgels were produced (Figure 2e) compared to low ionic strength conditions (Figure S5), consistent with higher reorganisation dynamics in the presence of competing counterions. [START_REF] Martin | [END_REF] In the following experiments, we thus chose to fix the concentration of magnesium ions to 3 mM to maintain a regular collapsed state while retaining a sufficiently large swelling ratio. In such conditions, optical microscopy revealed a ~2-fold change of the microgel diameter upon light irradiation, corresponding to a ~8-fold volume change (Figure 2d-f). The swelling process was typically completed within a few seconds (Movie S1), and the kinetics of the process could be modulated by varying the light intensity (Figure S6). Confocal fluorescence microscopy images of DNA microgels functionalized with fluorescently labelled complementary oligonucleotides confirmed that the light-actuated compaction and swelling process occurred in both xy and zy directions (Figure 2g,h and Movie S2). Significantly, the variation of fluorescence intensity within the microgels showed that the fluorophores followed the DNA network during compaction (higher fluorescence, Figure 2g) and swelling (lower fluorescence, Figure 2h), which indicated that supramolecular DNA hybridization was unaffected by the surfactant conformation, unlike what has been reported for azobenzene-grafted DNA strands. [34]

Responsive superstructures

We last investigated the assembly of all-DNA monodisperse microgels, to form photoresponsive superstructures. Although sequence controlled interaction between DNAdecorated colloids provides a powerful platform to elaborate complex materials, the narrow crystallisation range makes its implementation difficult. [29,44] Therefore, we relied here on a simpler strategy. We compacted the repulsive DNA colloids at low ionic strength by slow drying of a microgel suspension in TE buffer at room temperature. Slow water evaporation gradually concentrated the colloids, eventually leading to a compact periodic layer of microgels that was either immobilized onto the underlying glass coverslip surface (Figure 3a-c) or remained as a free-standing film (Figure 3d-i) depending on the rehydration protocol (See Methods). Similarly to dispersed microgels, in presence of TE buffer containing magnesium ions (3 mM) and trans-azoTAB (3 mM) these microgel assembly are light responsive. We observed that the 5.4 ± 0.5 μm period of the compacted microgels immobilized onto the coverslip surface did not change during UV light irradiation (Figure 3a-c), as expected from the fixed position of the microgels. As a result, light-actuation swelling/deswelling of the immobilized microgel layer produced a reversible switch in the surface coverage from 55% (dark, collapsed microgels) to 85% (UV, swollen microgels)(Movie S3). In comparison, the lattice period of free standing microgel films could be reversibly photoswitched between ~3.6 μm (dark or blue light, Figure 3e) and ~5.2 μm (UV light, Figure 3f) due to light-actuated microgel swelling/deswelling. The period photoswitch was achieved in a few seconds, and could be repeated several times (Movie S4). Significantly, while we observed mostly homothetic transformations in small microgel assemblies (Figure 3e,f and Movie S4), larger pieces of self-assembled microgels tended to fold and twist, which resulted in large amplitude reconfigurations under UV light (Figure 3g-i and Movie S5). Although this goes beyond the scope of this communication, localized swelling could provide a general strategy to control non trivial motion mechanisms [45] (e.g. between light-responsive DNA microgels and passive DNA coated silica particles). assembly whose period varies under light induced swelling. e,f). g-i) CLSM showing the large amplitude reorganisation of a large particle assemblies. All images are taken in TE with 3 mM MgCl2 and 3 mM of AzoTAB, using Microgels prepared at 100 mg.L -1 . Scale bars for a-c and g-i, 20 µm and for d,e 10 µm.

Conclusions

These results show that all-DNA materials present fast and wide light-triggered swelling/compaction properties in the presence of a cationic azobenzene photoswitch. Such swelling, based on coil-globule transition in the hydrogel network, surpasses what has been reported in hybrid DNA-polymer hydrogels. [34] We demonstrated this effect using monodisperse all-DNA microgels and showed its transfer to the next structural level with the actuation of microgels superstructures. Since azoTAB has been used to control DNA transcription, [START_REF] Estevez-Torres | Proc. Natl. Acad. Sci[END_REF] our results also provide new routes for light-controlled cell-free gene expression in DNA microgels. [46] Our microfluidic production process also improves on the previous microgel preparation protocols and enables the assembly of size-controlled DNA microgels with diameters that are one order of magnitude smaller than the microfluidic emulsion drops, thereby decreasing the size accessible by simple gelling of the whole drops' content. [47,48] We show that the improved size control of all-DNA microgels enables their assembly into periodic structures, which was impossible with the previously reported process. [28] Overall, our results offer new tools for the design of responsive DNA materials, opening perspectives for drug-delivery, nanomechanical devices and tissue engineering applications.

Experimental Section

Materials

ssDNA oligomers were purchased from EurogenTech as summarized in Table S1. The enzymes T4 ligase (2.5 WU.µL -1 ), Exonuclease I (20 U.μl -1 ), Exonuclease III (200 U.μL -1 ) as well as Deoxynucleotide triphosphate (dATP, dTTP, dGTP and dCTP) 100 μM were purchased from Jena bioscience. Φ29 polymerase (10 U.μL -1 ) was purchased from Lucigen. Inorganic pyrophosphatase (0.1 U.µL -1 ) was bought from Thermofisher. Magnesium AzoTAB was synthesized using a three-step reaction described previously by azocoupling p-ethoxyaniline with phenol, followed by alkylation with dibromoethane then quaternisation with trimethylamine.

DNA synthesis

The RCA process, schematized (Figure S1a), follows three main steps, the circularization of linear templates (I->II), the removal of unligated products(II->III) and the template amplification (IV). For circularization the linear template (100 µM in TE) and its corresponding ligation strands (100 µM in TE) are mixed to reach a final concentration of 5 μM in TE buffer containing additionally 100 mM NaCl (total 20 μL). The solution is heated up to 85°C and cooled down to 25°C at 0.5°C.min -1 . After hybridization 4 μL of 10 X commercial ligase buffer (Jena Bioscienc: 500 mM TRIS-HCI pH 7.8 at 25 °C, 100 mM MgCl2, 100 mM dithiothreitol, 10 mM ATP and 25 mg.ml -1 BSA), 14 μL of water and 2 μL of T4 Ligase (2.5 WU.μL -1 ) are introduced in the tube containing the template strand, gently mixed and left to react for 3 hours at room temperature. The enzyme is deactivated by heating the mixture for 20 min at 70°C. Unligated products are removed using exonucleases that digest linear products from their extremities. The absence of "ends" in circularized DNA prevents their digestion. To this end, 2 μL of Exonuclease I (20 U.μL -1 ) and 2 μL Exonuclease III (200 U.μL -1 ) are introduced and the Table 1. Sequences used, with their name the purification grade and modifications. a These sequences are converted into polymers using RCA. Hence, the complementary nucleobase base structure is found in the multiblock copolymers.

mixture is left overnight at 37°C. The enzymes are then deactivated by heating at 80°C for 40 min. The templates are purified by filtration over Amicon Ultracentrifugal filters with a 10 kDa cut-off (Merck Millipore) and rinced 3 times using TE buffer in the same filter. The ssDNA concentrations are measured using a Nanodrop 2000C (Fisher scientific) and the solutions are diluted to 1 μM using TE buffer. A control experiments which followed exactly the same step as discussed above, except that the T4 DNA ligase was replaced by 2 µL of water, shows Figure S1b, that the enzyme can digest all the non-ligated DNA strands.

For RCA, 10 μL circularized template (1 μM) are mixed with 150 μL of ultrapure water, 2 μL of RCA primer (10 μM in TE buffer), 20 μL of commercial 10 X polymerase buffer (Lucigen: 500 mM TRIS-HCl, 100 mM (NH4)2SO4, 40 mM Dithiothreitol, 100 mM MgCl2), 4 μL of Φ29

Polymerase (10 U.μL -1 ), 0.5 μL of pyrophosphatase (2 U.μL -1 ). The mixture is left for 1 hour at room temperature in order to pre form the enzyme DNA complexes before addition of 10 μL of an adjusted dNTP mix (total dNTP concentration of 100 mM the proportion of each bases correspond to the expected product composition) to start the amplification reaction. The samples are kept for 48 hours at 30°C before inactivation for 10 min at 80°C. The product is then concentrated by filtration over Amicon Ultracentrifugal filters with a 30 kDa cut-off (Merck Millipore) and rinsed 3 times using 400 μL of TE buffer. The RCA product is rediluted into 50 µL of TE buffer, homogeneized at 95 °C for 10 min, the amount and purity of the ssDNA concentration are then measured using a Nanodrop 2000C (Fisher scientific) spectrophotometer using 33 μg.OD260 -1 as standard absorbance coefficient for single stranded DNA.

Mechanism of the arrested phase separation

We used template A, through the manuscript to optimize the assembly conditions and control the mean particle diameter. The sequence B was used for microgel superstructure formation and confocal imaging because it can be labelled using an Atto565 fluorophore which, unlike SYBR gold, does not interact with the wavelength used for azobenzene actuation. Both sequences behave similarly in all conditions tested. Sequence A is identical to sequence p(A20-i-XL) from our previous publications and sequence B functions identically. [28] The role of each domain of the DNA sequence as well as the schematic representation of the DNA microgel formation is given Figure S2a. The sequence of the RCA product contains homoadenine runs that drives aggregation, a bare code domain for fluorescent labelling and a self-hybridizing sequences that forms supramolecular crosslink. Upon heating in presence of Magnesium (+II)

or Clacium (+II) ions in solution the DNA strands form liquid coacervate due to the presence of the homoadenine runs. Upon cooling the self-hybridizing sequence forms supramolecular crosslinks which stabilize the DNA-rich phase against dissolution. As shown in Figure S2b, without compartmentalization the process yields polydisperse particles. A detailed protocol has already been published previously. The sequence B formed through the exact same mechanism since they only differ in terms of bare code domain.

Preparation and self-assembly of monodisperse microgels

Preparation of the microfluidic emulsion: First an homogeneous solution of RCA products was obtained by diluting the RCA product at 1.5 times the target concentration into 200 µL of into TE buffer and heating the solution up to 95 °C for 15 min for homogenization. Then then 30 µL of CaCl2 solution at 500 mM and 70 µL of TE buffer are introduced in the mixture. The solution was used immediately for the preparation of the microfluidic emulsion. We used fluorinated Oil (Novec7500, 3M) supplemented with 2 % of a perfluoropolyetherpolyethyleneglycol block-copolymer(PFPE-PEG-PFPE) as continuous phase. The preparation of the microfluidic devices made of poly-(dimethylsiloxane) (PDMS, Sylgard 184) and the synthesis of the block-copolymer surfactant were performed as previously reported. [50,51] The device consists of a flow-focusing junction with a 30x30x20 µm nozzle (Figure S3), where the aqueous and oil phase are respectively injected into the central and side channels. The solutions are injected using 1 mL syringes (Terumo) and PTFE tubing (Fisher Scientific) with an inner diameter of 0.3 mm and an outer diameter of 0.76 mm. The flow was controlled using a Nemesys syringe pumps (Cetoni). We typically used volumetric flows of 400 µL.h -1 for the DNA solution and 600 µL.h -1 for the fluorinated oil solution. The resulting emulsion contains 35 µm droplets which are directly gathered into a 1.5 mL microtube while the oil excess is removed continuously.

Arrested phase-separation in confinement: After production we covered the microfluidic emulsion using few drops of mineral oil to avoid evaporation. The tube was placed inside a temperature controlled heater (Thermomixer, Eppendorf) without agitation and heated for 2 h at 85 °C. After cooling down to room temperature, the microgels were either gathered via destabilisation of the microfluidic emulsion or imaged using fluorescence microscopy. For confocal imaging, we introduced 2 µL of SYBR gold (diluted at 100X into TE buffer) into 45 µL of emulsion and left the mixture overnight in the fridge for the dye diffuse across the organic phase.

Note that the assembly time is significantly longer than what was previously used. Indeed if when providing insufficient time for the phase separated compartment forming inside a single droplet to merge we obtained polydisperse DNA microgels. (Figure S4) Rehydrating the assemblies using a TE buffer containing 3 mM of magnesium yields periodic lattices of particles immobilized on the glass slides. On contrary, a treatment at 60 °C for 5 min followed by a rehydration using a TE buffer containing 0.1 % of Triton X100 yields free standing microgel films floating in solution. The AzoTAB, and if needed magnesium, are then introduced in solution for light driven actuation. The diameter of the microgels was measured using ImageJ on >20 microgels in the same field of view, and the average value and standard deviation reported.

Gathering
Light-mediated swelling/compaction of the microgels was monitored on samples containing 3 mM azoTAB in TE buffer or in TE supplemented with 3 mM MgCl2 solution. UV or blue light irradiation of the microgels was performed using external LEDs operating at 365 nm or 450 nm, respectively. On/off actuation was performed by alternatively turning on/off the UV and blue light sources. The diameter of the microgels was measured using ImageJ on >20 microgels in the same field of view, and the average value and standard deviation reported. Movies of microgels' swelling and compaction were acquired during irradiation using MicroManager and processed with ImageJ.

Light

Figure 1 .

 1 Figure 1. Preparation of monodisperse DNA microgels via arrested phase-separation in microfluidic confinement. a) Schematic representation of the confined phase separation yielding monodisperse DNA microgels. b-d) Confocal laser scanning micrographs (CLSM) at different stages of the process. b) Homogeneous DNA solution in the microfluidic emulsion before heating. c) Individual microgels in the emulsion after phase separation. d) Monodisperse microgels gathered after destabilization of the emulsion. e) Schematic representation of the confined microgels and purified objects dispersed in low and high ionic strength solution. f) Graph presenting the diameter of the final microgels as function of the initial DNA concentration. Open circle correspond to the swollen diameter measured in TE and the filled dot correspond to the microgels in 50 mM CaCl2. Note that the data are plotted with a cubicroot scales as x-axis. g) Superposition of CLSM and transmission optical micrographs showing the microgels as prepared from different initial DNA concentrations in their microfluidic emulsion. h) CLSM showing the purified microgels in TE. We used "Fire" look-up-table from ImageJ to show fluorescence through the manuscript. Error bars on f) represent standard deviation from the average value measured on at least 20 microgels. Scale bars, b-d) 20 µm and g,h) 10 µm.

Figure 2 .

 2 Figure 2. Photoresponsive DNA microgels using azobenzene surfactants. a) Molecular structure of the two conformation of azobenzenetrimethylammonium bromide (azoTAB) and the corresponding state of the DNA microgels. b) Graph presenting the diameter of DNA microgels prepared at 100 mg.L -1 in TE as function of the concentration of trans-(dark) or cis-(purple) azoTAB. c) Variation of the diameter of microgels in presence of 3 mM of AzoTAB, either with or without 3 mM Mg 2+ , under 5 cycles of UV and blue light irradiation. d-f) optical micrographs and g,h) confocal laser scanning micrograph, showing swelling and deswelling of the DNA microgels (3mM AzoTAB, 4 mM MgCl2) under UV (e,h) and blue light irradiation (d,f,g). The left panel of micrographs (g,h) present a ZY cut, and the right panel a XY cut. The position of the cut on each image is represented by a red arrow at the bottom. Error bars on b,c) represent standard deviation from the average value measured on 20 microgels from the same field of view. Scale bars d-f) 10 µm, and g,h) 5 µm.

Figure 3 .

 3 Figure 3. Photoresponsive microgels assemblies. a-c) Shematic representation a) and corresponding CLSM showing the photoinduced change of surface covered by an array of DNA microgels b,c). d-e) Shematic representation d) and corresponding CLSM of a small microgel

[ 43 , 49 ]

 4349 PCB-mounted LEDs operating at 365 ± 4.5 nm (model M365D2) or 450 ± 9 nm (model M450D3) were purchased from Thorlabs, Inc. and adapted on a custom-made aluminium heat sink. The LEDs were controlled by a T-Cube LED driver that was purchased from Thorlabs, Inc. Thermal ramps were performed on a thermocycler (T100, Bio-Rad), DNA concentrations were measured using a NanoDrop 2000 C (Fisher Scientific) spectrophotometer with a standard value of 33 μg.OD260 -1 . Confocal laser scanning microscopy (CLSM) was performed on Leica TCS SP2 microscope.

  AA T TTT TTT TTT TTT TTT TTT T CG GAT GCG CAT CCG GAA CCC GTA CGA TCC TCT AAA A HPLC 5' Atto 565 (NHS ester)

  the monodisperse DNA microgels: To destabilize 100 µL of microfluidic emulsion we introduced 20 µL of 0.2 M EDTA solution, 10 µL of 1 % Triton X-100 solution, and 10 µL of perfluorooctanol. The mixture was then shaken vigourously and left 10 min on the bench top until the two phases become separated by a clear interface. The aqueous supernatant was then gathered in a separated tube. The particles were directly imaged by adding 5 µL of SYBR gold diluted 10 times in TE buffer to 50 µL of particle suspension. For light driven actuation and superstructure assembly the DNA particles were washed three times by centrifugation at 2000 g for 10 min and redispession in fresh TE buffer. The concentration of the suspension obtained by redissolving 2 µL of DNA suspension diluted in 8 µL of TE (by heating at 95°C for 2 min) was measured by UV-vis spectroscopy. Finally we adjusted the stock microgel concentration to 200 mg.L -1 using TE buffer. For confocal microscopy experiments the DNA microgels were functionalized using 1 µL of 100 µM fluorescent oligomer (Atto565-B) for 10 µL of particle suspension at 200 mg.L -1 . The microgels were washed and redispersed in fresh TE a last time by centrifugation before use. Superstructure assembly: Periodic microgel assemblies were obtained by drying a concentrated microgel suspension. Typically 20 µL of fluorescently labelled microgels suspension at 200 mg.L -1 was concentrated to about 1 g.L -1 by centrifugation. A drop of suspension was cast onto a glass slide and left to evaporate slowly at room temperature. Marangoni flow concentrate the microgels on the edge of the drop during evaporation and yield compact microgel assembly.

4. 5 .

 5 photodetector (model 918D-UV-OD3R, Newport Corporation, USA) along the emission axis

  -actuated reversible swelling of DNA microgels: Light-responsive microgels were prepared by adding 3 mM trans-azoTAB to fluorescently labelled microgels. The suspension of trans-azoTAB-doped fluorescent microgels was then loaded into an observation chamber and the microgels were left to settle for 5 minutes. The microgels were imaged by confocal fluorescence microscopy on a Leica SPE confocal laser scanning microscope attached to a Leica DMIRE2 inverted microscope and equipped with a ×63 oil immersion lens (HCX PL APO, 1.4 NA). Excitation (and emission) wavelengths were set to 561 nm (emission: 575 -675 nm) to monitor Atto564 fluorescence. UV light irradiation was performed using an external LEDs operating at 365 nm positioned at ~2 cm from the microscope slide. In comparison, blue light irradiation was performed using the in situ 488 nm wavelength of the confocal laser.On/off actuation was performed by alternatively turning on/off the UV and blue light sources. Z-stacks of the microgels were acquired using the built-in Leica Application Suite software and zy and xy projections obtained using ImageJ. Movies of microgels' swelling and compaction were acquired during irradiation using the built-in Leica Application Suite software and processed with ImageJ. The DNA superstructures were imaged by confocal microscopy during light-actuation using the same set-up as for the individual microgels (see above). Movie S5. Confocal fluorescence microscopy video showing multiple swelling/compaction of a larger free-standing DNA microgel film under alternating UV and blue light irradiation, showing large scale reconfigurations. False colouring using the Fire LUT is used. Movie is shown at 10-time speed at 3.5 frames per second. Total duration of recording was 6 min in real time. Scale bar = 20 μm.

Figure S5. a )Figure 6 .

 a6 Figure S5. a) Graph presenting the evolution of the swelling ratio of the microgels under UV

Acknowledgements

This work was supported by LabEx AMADEus (ANR-10-LABX-42) in the framework of IdEx Bordeaux (ANR-10-445IDEX-03-02), France. JCB acknowledges the additional financial support of the Région Nouvelle Aquitaine. RM also thanks A. Walther and A. Samanta for providing a sample of DNA to start the project.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

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Supporting Information


H Mhamdi

R Benabderrahmane Zaghouani

T Fiorido

J.-L Lazzari

M Bendahan

W Dimassi

Rabia Benabderrahmane
email: rabia.benabderrahmane@gmail.com

Study of n-WO3/p-porous silicon structures for gas sensing applications

Keywords: Tungsten oxide, Porous silicon, NO2 detection response time, recovery time

In this work, we report on the gas sensing properties of n-WO3/p-porous silicon (PS) structures. Nanostructured WO3 films are deposited by simple chemical method via dip-coating of PS in tungsten hexachloride solution.

The morphological and structural properties of the elaborated structures were investigated using Atomic Force Microscope (AFM), Raman and Fourier Transform Infrared (FTIR) Spectrometers. The NO2-sensing performances of the sensor were measured at different operating temperatures ranging from room temperature to 250 °C, different NO2 concentrations and different time exposure. The sensor showed a p-type semiconducting behavior which probably owning to the strong effect of the hetero-junction between the n-WO3 nanoparticles and p-porous silicon substrate. The obtained results confirm the suitability of these structures to detect low NO2 concentrations up to 1 ppm at moderate temperatures. These sensors exhibit fast response and recovery times.

Introduction

During last decades, there has been an increasing demand for high-performance gas detection systems to be used in various fields such as environment monitoring, automotive and pharmaceutical industries. There is a permanent quest for reduced sizes, high-throughput and cost-effectiveness sensors. In particular, sensors based on metal-oxide semiconductors (MOX) have been extensively studied offering low cost, high stability, and compatibility with microelectronic technology. The most studied MOX are titanium dioxide (TiO2) [START_REF] Sertel | Development of MgO:TiO2 thin films for gas sensor applications[END_REF], zinc oxide (ZnO) [START_REF] Oliveira | Enhancement of acetylene gas sensing properties for ZnObased gas sensor produced by plasma immersion ion implantation and deposition[END_REF], Tin oxide (SnO2) [START_REF] Barbosa | Investigation of electronic and chemical sensitization effects promoted by Pt and Pd nanoparticles on Single-crystalline SnO nanobelt-based gas sensors[END_REF], molybdenum oxide (MoO3) [START_REF] Fu | Ultra-fast and highly selective room-temperature formaldehyde gas sensing of Pt-decorated MoO3 nanobelts[END_REF], and tungsten oxide (WO3) [START_REF] Yin | Synthesis of Ag quantum dots sensitized WO3 nanosheets and their enhanced acetone sensing properties[END_REF]. These materials, with interesting physical and chemical properties, exhibit a resistive response to different gases. In fact, they present naturally non-stoichiometric structures [START_REF] Kaur | Effect of indium-doped SnO2 nanoparticles on NO2 gas sensing properties[END_REF] attributed to oxygen vacancies leading to a semiconductor behavior [START_REF] Shewale | H2S gas sensing properties of nanocrystalline Cu-doped ZnO thin films prepared by advanced spray pyrolysis[END_REF]. The operating principle of a MOX sensor is based on the adsorption of oxidizing or reducing molecules on the surface of the sensitive layer leading to its conductivity increase or reduction respectively. Currently, in the most developed sensors, the sensitive layers are generally deposited on an insulator layer with a transducer permitting their heating at an optimal temperature and the resistance measurement. Many solutions are proposed to improve the MOX sensors performance such as structuring the sensitive layer, which considerably increases the number of adsorption sites. In this work, a way of improvement could be reached by increasing the porosity and the active surface of the oxide, which can be obtained by using a porous silicon substrate. Porous silicon, with its large specific surface area, can form a new generation of gas sensors with semiconductor oxides. Initially developed in the "Silicon On Insulator (SOI)" technology, porous silicon (PS) is pushed back to the research scene as 'the miracle material' after the rediscovery of its photoluminescence at room temperature by Canham in 1990 [START_REF] Canham | Silicon quantum wire array fabrication by electrochemical and chemical dissolution of wafers[END_REF]. In addition to its photoluminescence, porous silicon can act as an antireflection (ARC) layer with low reflectivity in the visible spectrum by trapping light in its pores. Its optoelectronic properties could be modulated by porosity and thickness [START_REF] Zaghouani | Study of WO3-decorated porous silicon and Al2O3-ALD encapsulation[END_REF].

In particular, tungsten oxide may form with porous silicon an interesting gas sensor. It is considered as n-type semiconductor with a band gap around 3 eV. This multifunctional material combines many features: good chemical stability in aqueous solutions, outstanding electrochromic, photochromic and photocatalytic properties [START_REF] Blackman | Atmospheric Pressure Chemical Vapor Deposition of Crystalline Monoclinic WO3 and WO3-x Thin Films from Reaction of WCl6 with O-Containing Solvents and Their Photochromic and Electrochromic Properties[END_REF][START_REF] Gomez-Solis | Facile synthesis of m-WO3 powders via precipitation in ethanol solution and evaluation of their photocatalytic activities[END_REF][START_REF] Liao | Electrochromic properties of nanocomposite WO3 films[END_REF]. WO3 could be elaborated by different techniques such as: chemical vapor deposition [START_REF] Blackman | Atmospheric Pressure Chemical Vapor Deposition of Crystalline Monoclinic WO3 and WO3-x Thin Films from Reaction of WCl6 with O-Containing Solvents and Their Photochromic and Electrochromic Properties[END_REF][START_REF] Gordona | Atmospheric pressure chemical vapor deposition of electrochromic tungsten oxide films[END_REF], spray pyrolysis [START_REF] Li | Electrochromic films produced by ultrasonic spray deposition of tungsten oxide nanoparticles[END_REF], sol gel [START_REF] Sharma | FTIR and absorption edge studies on tungsten oxide based precursor materials synthesized by sol-gel technique[END_REF], electrodeposition [START_REF] More | Electrodeposition of nano-granular tungsten oxide thin films for smart window application[END_REF] and atomic layer deposition (ALD) [START_REF] Malm | Atomic layer deposition of WO3 thin films using W(CO)6 and O3 precursors[END_REF]. In the literature, WO3 used in gas sensors has been reported in different forms as nanowires [START_REF] Yan | Preparation and room temperature NO2sensing performances of n-porous silicon/n-WO3 nanowires heterojunction[END_REF][START_REF] Wang | Room temperature NO2 gas sensing of Au-loaded tungsten oxide nanowires/porous silicon hybrid structure[END_REF], nanoparticles [START_REF] Yan | Room temperature NO2-sensing properties of WO3 nanoparticles/porous silicon[END_REF], thin films [START_REF] Lee | Effect of substrate on NO2-sensing properties of WO3 thin film gas sensors[END_REF] permitting the detection of different gases: nitrogen dioxide (NO2) [START_REF] Yan | Room temperature NO2-sensing properties of WO3 nanoparticles/porous silicon[END_REF][START_REF] Lee | Effect of substrate on NO2-sensing properties of WO3 thin film gas sensors[END_REF], hydrogen sulfide (H2S) [START_REF] Urasinska-Wojcik | H2S sensing properties of WO3 based gas sensor[END_REF], ozone (O3) [START_REF] Gillet | Grain size effect in sputtered tungsten trioxide thin films on the sensitivity to ozone[END_REF], hydrogen [START_REF] Ahsan | Low Temperature Response of Nanostructured Tungsten Oxide Thin Films towards Hydrogen and Ethanol[END_REF], ethanol [START_REF] Ahsan | Low Temperature Response of Nanostructured Tungsten Oxide Thin Films towards Hydrogen and Ethanol[END_REF]. The WO3-based sensor response at an operating temperature is depending strongly on the substrate [START_REF] Lee | Effect of substrate on NO2-sensing properties of WO3 thin film gas sensors[END_REF] and the microstructure features [START_REF] Li | NO2-sensing properties based on the nanocomposite of n-WO3-x/nporous silicon at room temperature[END_REF].

In this work, we propose the use of porous silicon as a substrate for WO3 deposition. This work highlights an original study with a focus on the NO2 detection performance of n-WO3/p-porous silicon hetero-junction obtained by simple chemical methods.

II. Experimental details :

1. Samples preparation :

Porous silicon samples were elaborated by stain etching method in a (HF, HNO3, H2O)= (1, 2, 3) in volumetric ratio solution for 2 min using solar grade boron-doped single crystalline silicon substrates (100) with a resistivity of ̴ 1-3 Ω.cm and a thickness of 200 µm. Silicon substrates were successively dipped in boiling acetone for 10 min, in ethanol for 5 min and deionized water in order to eliminate organic greases and metallic surface contaminants. Substrates were then immersed in 10 % acid hydrofluoric for 1 min to eliminate the native oxide, rinsed in deionized water and dried with nitrogen.

After formation of PS layers, substrates were dip-coated for 10 s in a solution containing tungsten hexachloride (WCl6) to deposit WO3 layers. The WO3 solution with a concentration of 0.035 mol/L was prepared using WCl6 powder, 50 % ethanol and 50 % water under stirring for 3 h at ambient temperature. Finally, the solution was stored in air during 7 days before being used [START_REF] Alaya | Enhancement of physical properties of stain-etched porous silicon by integration of WO3 nanoparticles[END_REF][START_REF] Badilescu | Study of sol-gel prepared nanostructured WO3 thin films and composites for electrochromic applications[END_REF].

Samples characterization:

The WO3 deposition was analyzed using a Fourier Transform Infrared (FTIR) Nicolet MAGNA-IR 560 Spectrometer and a Raman "LabRAM HR JOBIN YVO Technology HORIBA Scientific" spectrometer at room temperature under the excitation of a 632 nm He-Ne laser. The samples morphology was interpreted by a digital instrument Nanoscope 3100 Atomic Force Microscopy (AFM). The surface reflectivity was measured by a PerkinElmer Lambda 950 UV/VIS/NIR spectrometer equipped with an integrating sphere. Photoluminescence (PL) analysis was done by home-made equipment with a 447 nm laser.

Gas sensing measurement:

To perform electrical measurements, aluminum (Al) parallel electrodes, with a thickness of about 100 nm, were deposited by thermal evaporation system on samples surface. The gas-sensing performances of the n-WO3/p-PS sensors were evaluated in a static gas sensing testing system, shown in Fig. 1 (a). The sensor was placed on a heating plate connected to a temperature controller used to adjust the operating temperature. The sensor dynamic response was obtained by measuring the sensor resistance change under gas. Fig. 1 (b-c) present a schematic illustration of the proposed sensor composed of Al/WO3/PS structure and its photography showing the Al electrodes and the different dimensions.

In this work, the gas sensor response is defined by the following equation (Eq. 1): 100 .

a g a R R R S   (Eq. 1)
Where Ra and Rg are respectively the sensor resistance measured in air and under gas.

The sensor performance is also described by the response and recovery times. The response time is defined as the time required for the resistance to reach 90 % of the total value change after gas exposure. Accordingly, recovery time is the time necessary for the resistance to return to 10 % above initial resistance after the gas removal.

III.

Results and discussion:

1. Porous silicon characterization:

The porous silicon layer is formed by etching the silicon substrate in an acidic solution. The stain-etching process is composed of two chemical reactions as proposed by Robbins and Schwartz [START_REF] Robbins | Chemical Etching of Silicon[END_REF]. The first reaction leads to the surface oxidation by the nitric acid as described by the equation (Eq. 2):

(Eq.2)

In a second step, the formed silicon dioxide is etched by the HF (Eq. 3):

O H SiF H HF SiO 2 6 2 2 2 6    (Eq. 3) O H NO SiO HNO Si 2 2 3 2 4 3 4 3    
The overall reaction is described by (Eq. 4): 3Si + 4 HNO3 + 18 HF → 3 H2SiF6 + 4NO + 8H2O (Eq. 4) This etching technique leads to the formation of porous silicon layer.

The porous silicon FTIR absorption spectrum is presented in Fig. 2 (a). We distinguish the principal recorded vibration bands: SiHx stretching mode at 2112 cm -1 , Si-O-Si stretching mode at 1092 cm -1 , SiH2 scissors mode at 907 cm -1 , Si-Si stretching mode at 628 cm -1 , SiHn wagging mode at 672.7 cm -1 and O-SiHx wagging mode at 861 cm -1 [START_REF] Alaya | Enhancement of physical properties of stain-etched porous silicon by integration of WO3 nanoparticles[END_REF].

In Fig. 2 (b), porous silicon and silicon substrate reflectivities are compared. A large decrease in the reflectivity of PS (about 5% in the visible) is obtained. Indeed, PS is known for its ability to trap light in the pores thus decreasing the reflections and increasing the light absorption probability within the active region.

Due to its indirect band gap, the silicon emits weak infrared photoluminescence however the porous silicon exhibits a high visible photoluminescence at ambient temperature [START_REF] Canham | Silicon quantum wire array fabrication by electrochemical and chemical dissolution of wafers[END_REF]. Fig. 2(c) shows the PS sample photoluminescence spectrum performed in the wavelength range of (480 nm-750 nm) at room temperature attributed to the nanometric size crystalline structures.

WO3 deposition on porous silicon surface:

Porous silicon, with its high specific surface, will play the role of a template for the WO3 deposition. In order to study the deposition, a comparison of the FTIR spectra of PS and WO3/PS samples was done (Fig. 3 (a)). The two spectra show almost the same bands with a clearly reduction of SiHx and Si-O-Si peaks intensities for the WO3/PS sample suggesting the replacement of H atoms by WO3 molecules [START_REF] Alaya | Enhancement of physical properties of stain-etched porous silicon by integration of WO3 nanoparticles[END_REF]. After zooming different zones (A, B, C) in the spectral region (400 cm -1 -1000 cm -1 ) and the deconvolution of different peaks, the W-O bands are depicted.

The deconvolution of the peak around 906 cm -1 in the zone A (Fig. 3 (b)) shows peaks at 927.7 cm -1 and 898.9 cm -1 corresponding to W═O and W-O-W bridging modes respectively [START_REF] Diaz-Reyes | Obtaining of films of tungsten trioxide (WO3) by resistive heating of a tungsten filament[END_REF][START_REF] Deepa | Electrodeposited tungsten oxide films: annealing effects on structure and elechtrochromic performance[END_REF]. The peaks deconvolution in the zone B (Fig. 3 (c)) shows a band at 782.9 cm -1 attributed to W-Ointra-W bridging stretch mode [START_REF] Mendoza-Aguero | Optical and structural characterization of tungsten oxide electrodeposited on nanostructured porous silicon: effect of annealing atmosphere and temperature[END_REF]. In the zone C (Fig. 3 (d)), the peak at 466 cm -1 is describing the W-Ointra deformational mode.

Although some peaks could be ascribed to W-O-W and W=O bridging modes, FTIR does not fully evidence the layers chemical composition. That's why Raman analysis was performed. The Raman spectrum of WO3/PS sample is presented in Fig. 4. A principal Raman peak observed at 520 cm -1 is attributed to the scattering of the first order optical phonon of c-Si. As reported in the literature, two Raman bands characterizing WOx, are located in (200-500 cm -1 ) and (600-1000 cm -1 ) spectral regions associated to O-W-O bending vibration modes and O-W-O stretching vibration modes respectively. In the WO3/PS sample Raman spectrum, we notice the presence of two weak broad bands centered at 335 cm -1 and 900 cm -1 characterizing the amorphous nature and assigned to bending and stretching vibration modes of the monoclinic WO3 [START_REF] Ahsan | Low Temperature Response of Nanostructured Tungsten Oxide Thin Films towards Hydrogen and Ethanol[END_REF].

The PS surface modification by the WO3 deposition was also studied by AFM. The 3D AFM topographies of the PS and WO3/PS samples are presented in Fig. 5 (a-b). Comparing the two AFM images, we notice a slight modification of PS surface morphology after WO3 treatment. The untreated PS layer containing pores is considered as an adhesive surface for the WO3 after immersing wafers into the WCl6 solution. The WO3 deposition is noticed by the roughness changes which varied from 132 nm to about 220 nm for PS and WO3/PS sample respectively.

Gas sensing properties:

Fig. 6 depicts the WO3/SP structure resistance variation under dry air at different temperatures. As noticed, the resistance is decreasing with temperature increase. The resistance evolution is thermally activated presenting two different slopes attributed to the existence of two different mechanisms. As reported for metal oxides [START_REF] Madhi | Sensing characteristics of zinc oxide thin films deposited by spray pyrolysis onto tubular Pyrex substrate[END_REF], these mechanisms are essentially the thermal excitation of electrons and oxygen adsorption.

The WO3/PS sensor is exposed to 4 ppm of NO2, with humidity of 0%, at different temperatures ranging from room temperature to 250 °C in order to determine the optimal operating temperature. Fig. 7 shows the gas sensor responses as a function of the operating temperature. For ambient temperature, no response is obtained. As is commonly known, the WO3-based sensors are working at high temperatures exceeding 300 °C. The WO3/PS sensor proposed in this work exhibits a response which initially increased from 50 °C to 100 °C then it decreased. The best response of 40% is obtained for an operating temperature of 100 ° C. In addition, the response and recovery times are measured for the different operating temperatures as displayed in Table 1. The obtained values are low and comparable to those reported in the literature [START_REF] Urasinska-Wojcik | ultrasensitive WO3 gas sensors for NO2 detection in air and low oxygen environment[END_REF]. The recovery time is enhanced with the temperature increase as the gas molecules desorption is promoted at higher temperature. The best response time is obtained at the optimal operating temperature (100 °C). The time-dependent resistance evolution at 100 °C for two consecutive cycles is shown in Fig. 8. It is noticed that this sensor exhibits a good repeatability as a function of time.

Further measurements were performed for lower NO2 concentrations (1 to 3 ppm) during an exposure time of 1 min (Fig. 9 (a)) at the operating temperature of 100 °C. As we can notice, a sensor response is obtained up to 1 ppm of NO2. The response increases from 10 % to 20 % for gas concentration varying from 1 to 3 ppm respectively because the sensor is exposed to more gas. The sensor was also exposed to 4 ppm of NO2 during different exposure times (1 to 3 min). As shown in Fig. 9 (b), the response increases with a total recovery of the resistance in each measurement.

The resistance evolution shows also that the sensor resistance decreases when injecting the gas which is contrary to the typical tungsten oxide sensors. In fact, WO3 behaving as n-type semiconductor shows an increase of its electrical resistance when exposed to an oxidizing gas such as NO2. However, the obtained p-type behavior may be attributed to the porous silicon substrate. In order to determine exactly where the conductivity occurs when injecting the gas, porous silicon sensor without WO3 deposition was tested under 4 ppm of NO2. Fig. 10 shows the PS resistance response exposed to 4 ppm of NO2 at 30 °C which is the optimal operating temperature for this sensor. This result already confirms that the porous silicon presence justifies the optimal operating temperature decrease in the WO3/PS sensor. It can also be seen from the time-dependent resistance evolution that PS sensor displays good signal repeatability in addition to a good return to the initial resistance value showing good reversibility. The crucial remark is that the observed p-type behavior is attributed to the porous silicon.

According to previous works [START_REF] Zaghouani | Study of WO3-decorated porous silicon and Al2O3-ALD encapsulation[END_REF], we have shown that the substrate surface is not completely covered by tungsten oxide deposited by the proposed chemical method. So, the injected gas interacts with the PS surface as well as with the WO3 nanostructures. These nanostructures form a hetero-junction with porous silicon in which appears a potential barrier created by the work functions difference of the two materials. Moreover, it has been shown that, in addition to oxygen adsorption, the interface density of states at the porous layer surface play an important role in the NO2 detection. In fact, the porous silicon has H-terminated surface which is chemically unstable due to its formation mechanism leading to the appearance of dangling bonds in ambient conditions. Indeed, the reduction or the oxidation of the surface influences the interface density of states and thus the carrier concentration in the PS surface.

The gas selectivity is an important key parameter to evaluate the gas sensor performance. The WO3/PS sensor is exposed to reducing and oxidizing gases at 100 °C during 1 min: 100 ppm NH3, 4 ppm NO2, 200 ppb O3, and 20 ppm CO. The sensor response S is defined as:

a g R R S 
for reducing gases (Eq. 5)

And as

g a R R S 
for oxidizing gases (Eq. 6)

Where Rg and Ra are the sensor resistance under gas and in the air, respectively.

The responses are presented in Fig. 11 (a). As noticed, the elaborated WO3/PS sensor detects all the exposed gases except the CO. In the literature, there are still problems to detect some gases such as CO, CO2, SO2 and more research is needed to establish a better selectivity. The obtained responses are comparable. In order to determine the best gas to detect, the response and recovery times are measured. As shown in Fig 11 (b), the recovery times are very high in the case of NH3 and O3 (45 and 9 min, respectively). However, for NO2, the sensor exhibits the lowest response and recovery times. Therefore, we can consider that the presented sensor is more adapted to detect NO2.

IV. Conclusions:

In summary, the n-WO3/p-porous silicon structure was prepared for NO2 sensing. A simple and low-cost chemical method was proposed to deposit WO3 nanostructures on porous silicon substrate. It was found that the prepared sensors are promising for NO2 sensing with low concentrations. Fourier transform infrared spectroscopy analysis shows that WO3/PS sample presents peaks ascribed to O-W-O and W=O bridging bonds.

Raman spectrum of the WO3/PS sample presents two weak broad bands centered at 335 cm -1 and 900 cm -1 characterizing the amorphous nature and assigned to bending and stretching vibration modes of the monoclinic WO3. AFM analysis shows changes in the surface roughness which varied from 132 nm for PS sample to about 220 nm for the WO3/PS sample. These sensors present fast response and recovery times at a moderate temperature (100 °C). Despite these interesting results, a further amelioration of these sensors is necessary. In particular, a better control of the oxide deposition by the amelioration of the porous silicon surface could be reached by using electrochemical etching method. Tables Table 1: The recovery and response times of the WO3/PS sensor exposed to 4 ppm of NO2 at different operating temperatures.

Figure 1 :

 1 Figure 1: (a) The schematic diagram of the static gas sensing test system (b) Schematic illustration of the sensor (c) Photography of the sensor showing the Al electrodes.

Figure 2 :

 2 Figure 2: (a) FTIR absorption spectrum of PS sample (b) Reflectivity spectra of silicon and PS samples (c) PL spectrum of PS sample.

Figure 3 :

 3 Figure 3: FTIR absorption spectra of: (a) PS and WO3/PS samples, amplification of (b) zone A (c) zone B (d) zone C for the WO3/PS sample.

Figure 4 :

 4 Figure 4: Raman spectrum of the WO3/PS sample.

Figure 5 :

 5 Figure 5: AFM topographies (a) untreated PS surface (b) WO3/PS sample.

Figure 6 :

 6 Figure 6: Sensor resistance variation as a function of the operating temperature in ambient air.

Figure 7 :

 7 Figure 7: Dynamic response of the WO3/PS sensor as a function of the operating temperature.

Figure 8 :

 8 Figure 8: Resistance response of the WO3/PS sensor exposed to 4 ppm of NO2 at 100 °C.

Figure 9 :

 9 Figure 9: (a) WO3/PS sensor response to different NO2 concentrations during 1 min at 100 °C (b) Resistance response of the WO3/PS sensor at 100 °C exposed to 4 ppm of NO2 during different durations.

Figure 10 :

 10 Figure 10: Resistance response of the PS sensor exposed to 4 ppm of NO2 at 30 °C.

Figure 11 :

 11 Figure 11: (a) WO3/PS sensor response to 4 ppm of NO2, 100 ppm NH3, 200 ppb O3 at 100 °C (b) The recovery and response times of the WO3/PS sensor exposed to different gases.

  Figure 2

  Figure 4

3 Figure 11

 311 Figure 7

Table 1

 1

	Operating	50	100	150	200	250
	temperature (°C)
	Response time (s)	35	25	39	41	45
	Recovery time (s)	38	23	19	12	8

Acknowledgements:

This work was supported by the Tunisian Ministry of Higher Education and Scientific Research.

Conflict of Interest Statement:

The authors declare that they have no conflict of interest


Alexandre Merlen
email: merlen@univ-tln.fr

Cédric Pardanaud
email: cedric.pardanaud@univ-amu.fr

Katharina Gratzer

Stéphane Coussan

Denis Machon

Alexis Forestier

David Bergé-Lefranc

Trang N T Phan

Virginie Hornebecq

Spectral fluctuation in SERS spectra of benzodiazepin molecules: The case of oxazepam

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

Drug abuse remains a serious public health problem all around the world and the rapid and efficient detection of drugs in blood or other body fluids remains a challenge. Among all the techniques in development, Surface Enhanced Raman Spectroscopy (SERS) appears as highly promising with respect to selectivity and sensitivity [1][2][3] . Indeed, standard Raman Spectroscopy has already proven to be efficient for drugs identification [4] but it suffers a major limitation: its very low cross section. In this way, this technic can be used for the analysis of bulk samples but in blood or saliva, the concentration of drugs is usually too low for an efficient and direct analysis based on classical Raman spectroscopy. As an alternative, SERS combines the chemical selectivity of Raman spectroscopy associated to a very high sensitivity, as single molecule detection has already been proven with this method [5] . It is based on the gigantic electromagnetic enhancement obtained with plasmonic nano-antennas [START_REF] Etchegoin | Principles of surface-enhanced Raman spectroscopy and related plasmonic effects[END_REF] . This enhancement occurs in the near field of the nano-antenna, a few nanometers above its surface. In most SERS studies, those nano-antennas are either gold or silver nanoparticles. Usually gold is privileged due to its better chemical stability and lower toxicity compared to silver, even if this last point remains subject of debate [START_REF] Johnston | [END_REF] . The experimental protocol for performing SERS is straightforward: the solution is deposited on a SERS substrate, usually made of gold nanoparticles or nanostructures, and the spectrum is directly recorded via a standard Raman spectrometer. In some cases, the measurement can be directly performed in a solution containing the nano-antennas. Thus, except the elaboration of nano-antennas, SERS does not require any specific experimental device and can be easily performed. Since its development in the 70's, many methods have been developed for the preparation of efficient SERS substrates, with a large variety of shape for the nano-antennas [8] : spheres, triangles, pyramids, stars etc…..Nevertheless the preparation of efficient nano-antennas is not the only key point in SERS. Another important point is the nature of the chemical interaction between the metallic nanostructures and the probe molecules, this feature is generally called the "chemical enhancement" of SERS [9] . It has led to an extensive debate in the SERS community but it is generally admitted that it is related to a charge transfer mechanism between the metal and the molecule [10] . Its relative contribution to the final enhancement of the Raman signal remains unclear as it varies depending on the combination of sample and antenna, however, either way, this feature underlines that the interaction between the nano-antenna and the molecules must be taken into account for the interpretation of SERS spectra.

In the specific case of benzodiazepine, SERS has already proven to be efficient [11][12][13] . Those pharmacological molecules are used as tranquilizers and sleep-inducing agents in particular. Doctors can prescribe a benzodiazepine for legitimate medical conditions but any abuse can create addiction and have severe health consequences. The detection of those molecules is a challenge and SERS is a promising alternative to other established techniques. The basic principle of benzodiazepine detection is based on a spectral analysis by identification of characteristic vibrational modes. The main problem with this approach is that previous SERS studies of benzodiazepine have reported differences between the powder Raman spectrum of those molecules and the observed SERS spectra. The authors have suggested that those differences should be a sign of the interaction of the molecule with the metallic surface without giving too much detail. In order to address reproducibility and biases introduced by measurements, it is necessary to understand the origin of those spectral differences. Any changes in the spectral fingerprint of a molecule should be clearly identified and understood for the further development of SERS as a reliable detection technique. Here, comparing SERS measurements and DFT calculations, an explanation for the spectral fluctuations of SERS spectra of oxazepam, a benzodiazepine molecule, is proposed.

Experimental and methods

SERS samples are based on nanoporous silica matrix containing gold nanoparticles in its pores (dpore is close to 8-9 nm) [14] . They were prepared following the procedure described in [15] . Transmission Electron Microscopy observations and low wavenumber Raman measurement (below 10 cm -1 ) confirmed the presence of gold nanoparticles in the silica matrix with typical sizes close to 5 nm (see supplementary information). With such small nanoparticles, the crystallinity of gold is very poor and their surfaces should exhibit a large variety of various adsorption sites for oxazepam molecules. Oxazepam was adsorbed on the material surface using a classical procedure. A known mass of solid is put in contact with an oxazepam solution of known concentration. After reaching equilibrium (12 hours), the supernatant is recovered and the equilibrium oxazepam concentration is determined. In this way, it is possible to calculate the adsorbed amount of oxazepam per gram of solid. In this study, this value is close to 2 mol/g . All the results obtained in this study were obtained with dried samples consisting of a red powder. This color is due to the gold nanoparticles. SERS measurements were performed in backscattering geometry on grains (zones of analysis were selected to be as flat as possible to optimize signal intensity) using 633 nm laser excitation with a LabRam HR800 spectrometer from the Horiba Jobin Yvon Company. It must be mentioned that we have also used a 514.5 nm laser but we have not observed any Raman signature with this excitation wavelength. To avoid any cross-contamination among different samples via the objective we have used a ×50 long working distance objective (NA: 0.5). The theoretical spatial resolution with this configuration is ≈ 0.8 μm. Power on the sample can be tuned from 10 mW on a 1.8 μm 2 surface down to 0.1% of this value using optical filters. Mapping mode was performed on flat zones with steps in x and y directions equal to 1 μm. We also used a furnace to perform in-situ Raman measurements from room temperature to 250°C. Pressure of Ar was kept at 1.5 bar to limit oxidation effect.

Numerical calculations, and in particular DFT, are essential for the interpretation of SERS spectra [16,17] . Those calculations have been carried out at the B3PW91/6-31G* level of theory using the GAUSSIAN09 [START_REF] Frisch | Gaussian[END_REF] suit of programs. We have calculated the structure of the isolated oxazepam molecule together with its set of harmonic frequencies.

Results

Fig 1:

Raman spectra of oxazepam powder (blue, inverted for clarity), oxazepam in silica matrix with gold nanoparticles (SERS, red), silica matrix with gold nanoparticles (black) and silica matrix (green). Excitation wavelength: 633 nm Typical Raman spectra of the different samples are shown in figure 1. For all of them, the characteristic Raman modes of the silica matrix are clearly observed between 400 and 550 cm -1 . Some other weaker modes at around 600, 800 and 1000 cm -1 are also associated to the silica matrix [START_REF] Biswas | [END_REF] . When gold nanoparticles are incorporated in the matrix, these modes are still visible, without any significant increase of their intensity, suggesting that there is no electromagnetic enhancement between gold and silica. Broad modes at around 1600 cm -1 are visible, they can be associated to carbon impurities formed during the calcination process. When oxazepam molecules are added, the Raman spectrum is strongly modified. It exhibits now the typical features of the SERS modes of this molecule in addition to the modes from the silica matrix. A mode at 270 cm -1 is sometimes observed (see supplementary information). It can be attributed to the Au-N stretching mode [13] but we do not consider it as a clue for the absorption of oxazepam on gold through its nitrogen atoms as this mode is not systematically observed and it is also present without oxazepam. Its origin remains unclear; we suggest that it could be attributed to the adsorption of nitrogen present in the air.

It has already been reported that the SERS spectrum of oxazepam is not identical to its powder signal, and we clearly observed these differences. This result suggests that we are in experimental conditions where a SERS effect occurs for this molecule with this substrate. To confirm this hypothesis we performed similar measurements with oxazepam adsorbed on the silica matrix without gold nanoparticles and we have not observed any signature from the molecule. This proves the crucial role of gold in the detection of oxazepam.

Gold nanoparticles play two distinct roles in the detection of oxazepam:

-They promote the adsorption of oxazepam molecules in the silica matrix.

-As nano-antenna they enhance the Raman response of the molecules, the so-called Surface Enhanced Raman response.

These two contributions are necessary to get a high sensitivity in the detection of oxazepam molecules, even at low concentration. Usually in SERS, a low concentration induces a low signal to noise ratio. To get a measurable signal, the basic idea is to increase the laser intensity. In figure 2 we report the evolution of the SERS signal with laser power for a fixed concentration. Evolution of the Raman spectrum with laser power (left) and temperature (right). The two peaks marked with a star are coming from the furnace. A typical spectrum of amorphous carbon is also shown for comparison [20] . The vertical offset between spectra is only to improve comparability.

We clearly observe an evolution of the SERS spectrum of oxazepam. At low power excitation, all modes are visible and the associated peaks are sharp. They progressively broaden with the increase of laser power and some modes are not visible anymore with laser power higher than 25 %. In particular, we note the disappearance of the phenyl band around 1000 cm -1 . This first result indicates that laser power plays a role in SERS measurement, it should be maintained to a rather low value (close to 0.5 mW/μm 2 ) as otherwise the recorded spectrum might be modified. For Raman spectroscopy, it is generally admitted that high laser intensity increases the sample temperature and its spectral effect should be similar to high temperature measurement. Thus, in figure 2 we have also reported high temperature measurements up to 250 °C. At first sight, we observe the same features for high temperatures and high laser power: peaks broadening and disappearance of some modes. Nevertheless, if we look carefully at the spectra, some subtle differences are visible. The most obvious is the broadening: it is much more important with temperature increase compared to laser power increase. We can reasonably assume that high temperature degrades the oxazepam molecule. Therefore, the spectral signature of this molecule is progressively transformed into broad D and G bands, characteristic of amorphous carbon. At the maximum tested temperature (250 °C), the SERS spectrum is identical to amorphous carbon confirming the molecule degradation. Such feature has already been observed for C60 [21] for instance. This is clearly not the case with laser power: even if the G band becomes very broad, the D band keeps a low intensity and the final spectrum, at maximum laser power, is different from amorphous carbon. This result suggests that temperature and laser power do not play the same role. Another interesting point in our measurements is the spatial inhomogeneity of the SERS signal. In figure 3, a Raman map of the 1000 cm -1 mode of the oxazepam molecule is reported. We focused our attention on this mode for different reasons: it is sharp (easily identifiable), typical of the oxazepam molecule (associated to a benzene group vibration as can be seen in figure 3), far from the silica and carbon impurities modes. In addition, high temperature and high laser power measurements have suggested that this mode is sensitive to oxazepam degradation. Thus, it is a clear fingerprint of the oxazepam molecules in the matrix. The mapping exhibits a broad distribution of the mode intensity. Most of pixels exhibit a rather low signal but in some specific places the intensity is greatly enhanced. We can reasonably assume that this result is not related to an inhomogeneous distribution of oxazepam molecules in the silica matrix, even if a slight contribution of this feature cannot be totally excluded. TEM images of the sample (see supplementary information) exhibit a rather homogenous matrix and it is hardly believable that the adsorption of oxazepam molecules should occur in specific places, at least for a micrometer resolution. We rather suggest that this distribution is related to the nano-antenna efficiency. As can be seen in FDTD calculations of the supplementary information, the electromagnetic enhancement depends on the confinement of the gold nanoantenna in the silica pores. We believe that the distribution of Raman intensity shown in figure 3 is related to the distribution of this enhancement. It is clear that a very efficient nano-antenna for the 633 nm excitation is localized at the pixel in the center of the image giving the highest Raman intensity. This is also probably the reason why we have not observed any signal with the 514.5 nm excitation, gold nanoantenna being far less efficient tor this excitation wavelength. These features prove once again that we are performing SERS measurements of the oxazepam molecules.

In the map shown in figure 3, we also have an inhomogeneity of the modes distribution. Some typical spectra are visible in figure 4 a). We have focused our attention on two different spectral regions: the first one around 1000 cm -1 and the second at 1600 cm -1 . It is confirmed here that the SERS spectrum of oxazepam is different from its powder Raman spectrum. We also note that SERS spectra are all slightly different from one location to the other. At 1000 cm -1 , the powder Raman spectrum exhibit one intense mode whereas in the SERS spectra a doublet is sometimes observed. In the 1600 cm -1 region, the powder Raman spectrum exhibit two sharp modes whereas SERS spectra show much broader peaks associated to a larger number of modes which cannot be attributed to carbon impurities. Such a feature has already been reported by Trachta et al. [12] : they have noticed these differences between SERS and powder Raman spectrum but not the fluctuation in the SERS signal. They attributed those differences to molecular orientation and concluded that the molecule should be adsorbed with the molecular plane orthogonal to the metal surface. As mentioned in the introduction, the role of chemical interaction between the molecule and the metallic surface in essential in SERS [22] . For molecules with a strong affinity for gold, such as thiol molecules [23] , this question can be easily addressed. This is not the case here: oxazepam has no specific chemical function with a very high affinity for gold. The question of the adsorption mechanism of this molecule remains unclear. This molecule has the possibility adsorb on the gold surface through different chemical interactions. To check this hypothesis we performed DFT calculations of the molecule in interaction with a few gold atoms. We have not performed the calculation with a complete gold surface as the crystallinity of our gold nanoparticles is certainly poor. The calculated Raman spectra are shown in fig. 4 b). Independently from any molecular orientation effect, it appears clearly that the calculated Raman spectra exhibit spectral differences depending on the position of the gold atoms. When gold atoms are close to the aromatic ring, a doublet appears in the 1000 cm -1 region and 3 bands are present in the 1600 cm -1 region. This result is in good accordance with our measurements. When the gold atoms are located close to the N atoms, 2 modes are also present in the 1600 cm -1 region, one being shifted down to almost 1500 cm -1 . In addition, the calculated spectra do not show modes at 1000 cm -1 anymore. This result suggests that the absence in the SERS spectrum of this mode does not automatically mean that the oxazepam molecule has been degraded. This point is particularly interesting if we recall the influence of laser power in the evolution of the SERS spectrum of oxazepam. We have previously discussed the fact that high temperature transforms the molecule into amorphous carbon but laser power has a different influence: it causes the broadening of the 1600 cm -1 modes, without the appearance of a D band and the 1000 cm -1 mode is not visible anymore (see fig. 2). This is very similar to what is observed in the calculated spectra of oxazepam with gold atoms close to nitrogen, if we take into account the general broadening of Raman modes induced by the inevitable laser heating. Thus we suggest that the evolution of the SERS spectrum of oxazepam is related to changes in adsorption sites of the molecule on the gold surface and not to its degradation. We also propose the same explanation for the spectral fluctuation in the SERS spectrum as shown in fig. 4. Those fluctuations are the signature of different absorption sites on the gold surface. This hypothesis is corroborated by the comparison between SERS spectra and the powder spectrum. In all SERS spectra, the 1600 cm -1 modes are broader. DFT calculations have demonstrated that many Raman modes are present in the range 1500-1600 cm - 1 as soon as the molecule is in interaction with gold atoms. The distribution of various absorption sites on the surface of gold nanoparticles will lead to a general broadening of the modes in the same spectral range, and indeed this is exactly what is experimentally observed. In the 1000 cm -1 spectral range, comparison between measured and calculated spectra leads to the same conclusion: a single intense mode is observed for the pristine molecule, in accordance with powder Raman spectrum, whereas doublets or no modes at all are calculated if the molecule is in interaction with gold, which corresponds exactly to the different measured SERS spectra. We have excellent agreement between calculated and measured spectra considering our hypothesis on the existence of various adsorption sites.

Conclusion

Based on our DFT calculations and SERS measurements, we can conclude that the differences between powder and SERS spectra of oxazepam are not related to any orientation effect but to specific interactions between the molecule and the gold surface, temperature-dependent adsorption sites to be specific. Any change in the absorption site will cause the observed fluctuations in the SERS spectrum. Our findings also suggest that this mechanism can be activated through laser illumination. This study gives important conclusions for the further development of SERS in drugs detection: first, the SERS spectrum can be rather different compared to the powder spectrum and second, the precise control of laser power is essential.

Fig 2 :

 2 Fig 2:Evolution of the Raman spectrum with laser power (left) and temperature (right). The two peaks marked with a star are coming from the furnace. A typical spectrum of amorphous carbon is also shown for comparison[20] . The vertical offset between spectra is only to improve comparability.

Fig 3 :

 3 Fig 3: Raman map of the 1000 cm -1 mode of the oxazepam molecule. The size of each pixel is 1m ×1 m. Excitation wavelength: 633 nm

Fig 4 :

 4 Fig 4: a) Typical experimental SERS spectra of oxazepam extracted from the mapping shown in figure3, in comparison with the powder spectrum. b) DFT calculations of free oxazepam and in interaction with Gold atoms.

Acknowledgments: this work was funded by the French research national agency (ANR) through the project DOMTOM (16-CE09-0021-05).


Marc M Barzman

Pierre Ricci

Emilie Labussière

How research can contribute

come

Local development of territoires >The Community-conscious Farmer

Farmers contribute to the economic attractiveness of territoires. Local stakeholders recognise the multiple services they provide

General context

• Europe is no longer a major food exporter To face the rapid decrease in chemical options, very significant efforts are urgently needed to increase the range of effective and affordable solutions.

This requires a coordinated plan to:

> encourage public and private research on new crop protection technologies and facilitate the regulatory conditions for their availability on the market, > support multidisciplinary research on whole systems -an emerging field -as a way to design truly innovative IPM strategies, > develop information, education and recognition of these integrated strategies for the benefit of farmers, advisers and other actors of the food chain, including the general public, > maintain a momentum at the European level to create synergies from national efforts.

ENDURE stresses the fact that the strict stand taken by the EU on pesticide legislation calls for a parallel, sustained and equally determined action to promote the design and implementation of new solutions in order to develop IPM schemes that contribute to sustainable development while preserving the competitiveness of European

Creating an enabling policy environment

> The European Parliament has approved new European Union pesticides legislation introducing new regulations on the production and licensing of plant production products and introducing new rules on their use.

> ENDURE, the European Network for the Durable Exploitation of Crop Protection Strategies, has conducted a foresight study that seeks to provide crop protection stakeholders with the tools they need to tackle proactively future challenges. The process has enabled ENDURE to draw up five very different scenarios for European crop protection heading towards the year 2030.

> From these scenarios we intend to discuss what scientific knowledge already exists to satisfy their varied demands, which research areas will need to be strengthened further and which additional measures will be needed to accompany ongoing changes.

Economy and public decision

Sociology of innovation

Coupling economic and environmental models

Valuation of ecosystem services

European response to global challenges

	Challenges for research
	> Valuation of ecosystem services and design of a re-
	muneration process
	> Apply ecological theory to cultivated ecosystems
	> Sociology and economics of local stakeholding
	Challenges for policy makers
	> Recognised ecosystem services > Informed citizens regarding crop protection issues

• Foreign income from non-agricultural sectors • Territoires are EU instruments for economic growth European agriculture • Farming essential to making territoires attractive • Diversified according to local demands • Provides multiple services: food, landscape, biodiversity, natural resources, recreation Crop protection • Local negotiations determine choice of plant protection strategies

• Natural processes as alternatives to less-acceptable control methods

• Diversity is a tool to manage pest communities

> The Energy-saving Producer Farmers produce food for local consumption in cities as well as the countryside General context

European response to global challenges > The Sustainable Food Provider Farmers ensure food self-sufficiency for Europe. They manage reliable and stable production systems. General context

		• Urban: fruit and vegetable production in micro-
		farms and industrial units. Vertical agriculture, hy-
		droponic, intensive organic methods
		• Rural: arable and bioenergy crops in large farms
		with livestock integration. Limited use of machinery,
		no-till practices, reliance on nitrogen fixation
		Crop protection
		• Urban: zero-pesticide, crop diversity to spread the
		risk, healthy material and sanitation, resistant varie-
		ties, biological control by augmentation
	Challenges for research > Harnessing ecological processes to stabilise agro-ecosystems > Modelling and assessing redesigned cropping sys-	• Rural: targeted use of plant protection products. Rotation, polyculture, relay crops and living mulch against weeds. Resistant varieties against new pests of bioenergy crops
	tems > Breeding approach: adapt cultivars to systems > Predicting and limiting evolution of pests	Challenges for research > Whole-systems approach for an optimal trade-off between minimising energy and reducing pest risk
		> Urban: ecological engineering and high-tech
	Challenges for policy makers > Stabilisation policy to promote long-term strategies > Targeted incentives to support farmers in transition	> Rural: development of green pesticides Challenges for policy makers > Access to biological control agents

• Fear of food crises • Agriculture for domestic consumption European agriculture • Europe-wide planned food production • Crop allocation, minimising pest incidence • Sustainable management of the resources key to future production Crop protection • Systems inherently less vulnerable to pests • Ecological engineering, exploitation of biodiversity • Exploitation of synergies between complementary control methods phase Globalised

free market >The Specialised High-tech Grower Farmers are part of a successful web of economic activities. They concentrate on spe- cialised high-quality export products. General context

	Crop protection
	• Sophisticated strategies • Resistant and sanitised plant material • Controlled conditions • New technologies for pesticide application
	Challenges for research
	> Cross-sectoral and multi-disciplinary innovation > New technologies applied to crop protection
	> Ecological engineering
	Challenges for policy makers
	> Support access to alternatives to pesticides
	> Encourage SMEs developing controlled and confined
	high-tech agriculture

• Growing world demand for food • A globalised and free market

European agriculture

• Contributes to the knowledge-based bioeconomy • Diverse: unique food products, green chemistry, top grade ornamentals, pharmaceuticals...

• Vertical integration: from genotypes to market

Globalised free market > The Commodity Market Player Farmers are important actors in the European economy. They compete on commodity markets. General context

	European agriculture
	• Basic crops for export
	• Large farms, homogeneous cropping systems • Partition of land use: intensive agriculture / conser-
	vation and recreation areas
	Crop protection
	• Best cost/benefit ratio: chemical control • Ability to detect, monitor and trace residues and to
	assess impact
	• Legally accountable stakeholders
	Challenges for research
	> Basic knowledge to develop green chemicals
	> Sustained efficacy of chemicals > Efficient indicators of impact > Dynamics of controversies on health and the envi-
	ronment
	Challenges for policy makers
	> Fast-track registration for green chemicals > Environmental objectives fully integrated into agri-
	cultural policy

• Growing world demand for food • A globalised and free market ENDURE sees IPM as a continuously improving process in which innovative solutions are integrated and locally adapted as they emerge and contribute to reducing reliance on pesticides in agricultural systems.

study European Crop Protection in 2030

	Food Quality and Safety

This foresight exercise was carried out by researchers from: AU, University of Aarhus > Denmark INRA, Institut National de la Recherche Agronomique > France WUR, Wageningen University > The Netherlands RRES, Rothamsted Research > United Kingdom with the contribution of all other ENDURE partners.

ENDURE Foresight

> The European network of excellence ENDURE brings together more than 300 researchers from 18 institutions in 10 European countries to contribute to the development of farming systems less reliant on pesticide use.

> ENDURE aims at:

• Assembling a permanent research community

policy European Network for the Durable Exploitation of Crop Protection Strategies Contact ENDURE

  Coordinator > Pierre RICCI Assistant Coordinator > Marco BARZMAN endure.coord@sophia.inra.fr Centre de recherches INRA / 400, route de Chappes / BP 167

•

Providing scientific support to

Biological diversity Innovative technologies

	Plant	Green
	genetics	chemicals
	Soil
	Ecological processes	Newly recruited technologies
	Landscape	System
		-Integration
		-Management
		-Modelling tools
	Transition and new path	Social and economic processes
		Risk
		management
	Local governance
	and extension
	services

Information technologies