Ranime Alameddine

Astrid Wahl

Fuwei Pi

Kaoutar Bouzalmate

Laurent Limozin

Anne Charrier

Kheya Sengupta
email: sengupta@cinam.univ-mrs.fr.

Printing functional protein nano-dots on soft elastomers: from transfer mechanism to cell mechanosensing

Living cells sense the physical and chemical nature of their micro/nano environment with exquisite sensitivity. In this context, there is a growing need to functionalize soft materials with micro/nano-scale bio-chemical patterns for applications in mechanobiology. This however is still an engineering challenge.

Here a new method is proposed, where sub-micronic proteinpatterns are rst formed on glass and are then printed on to an elastomer. The degree of transfer is shown to be governed mainly by hydrophobic interactions and to be inuenced by grafting an appropriate uorophore onto the core protein of interest. The transfer mechanism is probed by measuring the forces of adhesion/cohesion using atomic force microscopy. The transfer of functional arrays of dots with size down to about 400 nm, on elastomers with stiness ranging from 3 kPa to 7 MPa, is demonstrated. Pilot studies on adhesion of T lymphocytes on such soft patterned substrates are reported.

In the last decades more and more experiments have conrmed that living cells are sensitive to the mechanics of their immediate environment. They behave dierently on sti and soft substrates in terms of adhesion, migration, actin organization, force generation, dierentiation and a host of other properties. 15 In separate studies, it was shown that adherent cells respond to the way adhesive ligands are distributed or grouped into micro or nano-patterns. 69 Single molecules of adhesive ligands that are separated by distances larger than a cut-o length scale of about 80 nm fail to support adhesion, spreading, proliferation and growth of connective tissue cells like broblasts. 7,10 Intriguingly, the behavior of lymphocyte-like cells may depend not on ligand spacing but on average ligand density. 9,1113 Directing cell behavior and fate, in a cell type and ligand dependent manner and through physical means, is of major interest for tissue-engineering applications. 14 In the general context of cell biology as well as the specic context of lymphocytes, there is therefore a lot of interest in patterning of soft substrates but even at the micro-scale this is a current engineering challenge. 15,16 At the nanoscale there is, so far, only one available technique that has been applied to cell studies, 17 which is based on patterning soft hydrogels employing block copolymer micelle lithography (BCML) for the production of surfaces patterned with gold nanoparticles on a hard template, then transfer the pattern to a polymeric hydrogel. 18,19 This technique is therefore dependent on the chemistry and optics of gold and is limited to the visco-elastic range of the hydrogel. Another technique with the potential to achieve nano-scale features on a hydrogel is deep UV etching followed by transfer, but so far this has been mainly conned to larger, micron size patterns and is also limited by the visco-elastic range of the hydrogel. 20,21 Moreover, in the view of seminal work dissecting possible dierences between cells on hydrogels and on elastomers, 14 patterning both types of material is important.

A large number of techniques have been devised to nanopattern hard inorganic substrates like glass or silicon, 22,23 including photo or electron-beam lithography, [START_REF] Bucknall | Nanolithography and patterning techniques in microelectronics[END_REF] dip pen nanolithography, [START_REF] Li | [END_REF]26 and nano-imprint technologies. 10,27 An universal technique to transfer such patterns to soft substrates will address a specic need in the community. Micro and nano contact printing, which uses elastomeric stamps with a pattern of relief structures to transfer molecules to glass or gold surface, is now an ubiquitous tool in biology. 6,2835 These techniques, and their oshoots, depend on the high anity of the ink molecules for the target surface which is typically glass or gold. The reverse transfer of protein patterns from glass to elastomer was demonstrated at micron-scale on Polydimethylsiloxane (PDMS) but was limited by a requirement of treatment of PDMS leading to its hardening. 36,37 Similar transfer to soft native PDMS turned out to be possible, 38,39 but only under very special conditions. Here we present the key idea that by tuning molecular anities of the transferred proteins via grafting of selected chemical moieties, the reverse transfer from glass to an elastomer can be systematically controlled. We demonstrate the transfer at sub-micron scale onto very soft PDMS and present pilot experiments with cells. We show that the transfer can be predicted from measured adhesive/cohesive forces between the protein molecules and the glass/PDMS surface. We argue that the degree of hydrophobicity is the major, and the presence of ionic groups is a minor factor that governs the transfer. The pattern and transfer process is presented schematically in Figure 1. In brief, to pattern glass, cover-slides were thoroughly washed and a colloidal bead mask was formed on them as described before. 38 A uorosilane (Trichloro(1H,1H,2H,2H-peruorooctyl)silane, PFOTCS) was deposited from the vapor phase on the glass through the bead-mask. The beads were removed, revealing a layer of hydrophobic uorosilane patterned with holes. The hydrophobic regions were passivated by absorption of a triblock poloxamer (chosen to be Pluronic F68, see SI). The holes exposing bare glass were subsequently back-lled with a protein of choice. The protein of choice here is variously functionalized bovine serum albumin (BSA) which was usu-ally biotinylated (bBSA) and additionally labeled with a uorophore, or neutravidin (NAV) also conjugated to a uorophore. As examples, we chose two well known uorophores : the hydrophobic dye Texas Red (TR) and the hydrophillic dye Atto-488 (Atto). The binding steps are schematically presented in Figure 1, and were checked by imaging with atomic force microscopy (AFM) (Figure SI.1). At this stage, the cover-slide is chemically patterned with protein nano-dots, and is henceforth called the glass-master. A second glass cover-slide was coated with a layer of elastomer precursor solution by spin-coating and was appropriately cured. The elastomers used here include: a polydimethyl based silicone rubber (corresponding to sylgard-184 used with base to cross-linker ratio of 10:1, henceforth called PD), or a silicone gel containing additional methylphenyl groups and phenyl polymers (corresponding to Q-gel 920 used with ratio of 1:2, henceforth called MP), or, for certain experiments, another polydimethyl based silicone rubber (corresponding to CY 52-276, henceforth called PD2). All three types of elastomers were either used as is after curing or were exposed to oxygen plasma (accordingly, the latter are henceforth called pPD, pMP or pPD2). SI table 1 and 2 report the Young modulus and contact angle measurements on the dierent surfaces, showing that a stiness range of about 3 kPa to 7 MPa was covered and that the elastomers can be arranged in order of hydrophobicity as PD2 ≈ MP > PD pMP > pPD2 > pPD. For some experiments, the plasma exposed PDMS was further functionalized with a organo-aminosilane and glutaraldehyde (gluMP and gluPD2), re-rendering them hydrophobic.

The surface of the elastomer layer was brought into physical contact with the glass-master in presence of a drop of water. The presence of water, in agreement with other reports 40 facilitates the transfer. A minimal pressure was applied manually to ensure conformal contact. Since both the surfaces are at and do not appreciably deform on contact, pressure is not a crucial parameter and it was veried that the quality of transfer is not sensitive to the applied pressure. The surfaces were carefully separated the next day to obtain the patterned elastomer. The patterned surface was imaged using epi-uorescence microscopy (Figure 2) and AFM (Fig

- ure SI.2).
Visual inspection of images in Figure 2 reveals that bBSA functionalized with Texas Red dye (bBSA-TR) transferred on PD (a,b) but the same core protein functionalized with Atto-488 dye (hbBSA-Atto) failed to transfer (c,d). bBSA-Atto could however be transferred to pPD (e,f). We note that on one hand, from the molecular structure of Atto and TR we expect TR to be more hydrophobic than Atto, the latter having an isolated primary amine group that is prone to losing an anion. On the other hand, native PD is hydrophobic but plasma treated pPD is hydrophillic. Thus, bBSA could be transferred to native hydrophobic PDMS if conjugated to a hydrophobic dye, and to hydrophillic PDMS if conjugated to a hydrophillic dye. These observations lead us to test a variety of proteins and elastomer surfaces. The table in Figure 2 summarizes the success, or not, of the attempted transfers. As conjectured, proteins (bBSA, BSA or NAV) labeled with hydrophobic dyes transfer well on hydrophobic PDMS: either native or glutaraldehyde treated (PD,MP,PD2,gluMP,gluPD2), whereas bBSA labeled with hydrophilic dyes transfers well on hydrophilic, plasma treated elastomers (pPD,pMP). Cross transfer is possible only in some cases. To put these observations on a quantitative basis, we selected the transfer of bBSA-TR or bBSA-Atto onto PD,MP,pPD and pMP. Transfer ratio of proteins inside the dots (a) and outside the dots (poloxamer covered zones) (b). Calculated from data presented in gure SI.5. Data are for bBSA-Atto or bBSA-TR dots and PDMS type PD, MP,pPD or pMP, as indicated ages for these transfers and Fig. 2 summarizes the dot size and contrast (see SI text for details of data analysis). The parameters reported here are averages calculated from at least 3 samples, with at least 6 elds each. The transfer of the pattern to both PD and MP conserves the size, but transfer to pPD or pMP increases the dot size slightly (Ttest: p<0.001). The contrast is systematically diminished on transfer, implying that the amount of protein transferred from within the dots is not identical to the transfer outside the dots. To characterize this, a transfer ratio was dened as I elastomer max /I glass max . A similar transfer ratio can be separately calculated for intensity outside the dots (I elastomer min /I glass min ) to quantify of the amount of transfer in the polaxamer coated zones. Here, Imax and Imin are the maximum and minimum intensity in the pattern which essentially correspond to the peak intensity in the dots and the background intensity out-side the dots (see Figure ). The transfer ratio inside (Figure 4a) clearly shows that the chemical nature of the grafted uorophore as well as the elastomer surface inuences the success of transfer. For the same uorophore and elastomer, the transfer ratio outside (Figure 4b) roughly similar to the transfer inside, and is often non-negligible. It is clear that to obtain good patterns, the transfer of proteins inside the dots should be maximal. Outside, in the ideal case, while making the glass-master, there should be no protein absorbed on the regions passivated with the poloxamer and even if there is some protein absorbed, it should not be transferred to PDMS. In practice, the amount of protein absorbed and transferred both depend on the quality of the poloxamer layer. In light of the non-negligible transfer in all cases, the strategy here was to minimize protein absorption outside the dots at the glass-master stage. The poloxamer type and concentration used was optimized accordingly (data SI Figure SI.6). In this work, an inside transfer ratio > 0.2 is considered a successful stamping, irrespective of the outside transfer ratio.

To verify the hypothesis that the forces of adhesion, possibly originating from hydrophobic/hydrophilic anities, govern the success of transfer, we quantied the eective force of adhesion of the protein on glass and elastomers using AFM force curves. 41,42 The protein of interest was covalently bound to a glass bead attached to the AFM cantilever (Figure SI.7). The protein covered bead was approached and made to touch a test surface which was either bare clean glass, or bare elastomer, or glass covalently functionalized with the same protein. The retraction curves were analyzed in order to extract the force of adhesion. As control measurements, we also obtained force curves from intermediate steps of functionalization to ensure that the functionalization steps were correctly realized. PD turned out to have a very strong non-specic adhesion with the bead, probably due to van der Wall's interactions and these measurements were not amenable to interpretation. However, forcecurves could be consistently measured and interpreted for pPD (Figure SI.8). For MP, such measurements were difcult even after plasma treatment, probably due to its extreme softness. The force of adhesion for pPD (called FpP D ), glass (called F glass ), and protein coated glass (called Fprt) are summarized in table 1.

Table 1 shows that the force required to separate a protein layer from glass is less than that required to pull apart two layers of protein (F glass < Fprt). Therefore the protein multi-layers expected to be present on the dots on the glass-master are transferred to the elastomer by peeling from glass, in practice, they in fact fracture due to presence of defects. Comparing the adhesion of bBSA-TR and bBSA-Atto, we see that the latter has a stronger interaction with pPD. This is consistent with the higher transfer ratio (about 0.6 for bBSA-Atto and about 0.4 for bBSA-TR) reported for bBSA-Atto in Figure 4a.

Let us now re-examine the transfer table (Figure 1). As discussed above, the hydrophobic bBSA-TR transfers well on both PD and MP, with the transfer being better on the latter (TrRatio about 0.3 and 0.6 respectively). Consistent with this, hydrophilic FITC transfered to pMP. Crosstransfers (hydrophobic on hydrophilic or vice versa), may however either fail as expected or be possible due to additional considerations. As expected, the hydrophillic bBSA-Atto fails to transfer to hydrophobic PD. In fact though the transfer is feeble and cannot be detected with the stan-dard camera settings, it can be detected with higher camera amplication (Figure SI.9). Furthermore, PD is known to be slightly negatively charged in aqueous solution at neutral pH, 43 resulting in a additional electrostatic repulsion towards the negatively charged bBSA-Atto. Consistent with this, hydrophilic FITC conjugated bBSA failed to transfer to untreated PD2. However, bBSA-Atto does transfer to some extent (TrRatio about 0.4) to MP, which is even more hydrophobic as judged by contact angle measurements. We rationalize this observation by noting that bBSA-Atto has many phenyl groups that may chemically interact with the phenyl groups on MP through π-π interactions.

The transfer of bBSA (not conjugated to any uorophore) can not be directly tested since in absence of an attached uorophore, bBSA can not be imaged in uorescence microscopy. It needs to be revealed by functionalization with uorescent neutravidin (NAV) after transfer and therefore transfer ratios can not be reported. To do this, after transfer of the bBSA, the bare elastomer around the dots was passivated with a polaxamer and then the NAV was allowed to bind from solution phase (see Figure SI.10 for example). bBSA by itself fails to transfer to PD2 and has very unreliable transfer to MP (data not shown). We conclude that on the glass master, bBSA mainly exposes hydrophilic groups, thus preventing its transfer to the hydrophobic PDMS surface.

To conrm the general hypothesis that the inclusion of a hydrophobic moiety renders a protein more amenable to transfer on hydrophobic untreated elastomers, we checked that neutravidin conjugated to Texas red dye (NAV-TR) transferred well on all the elastomers studied here. However, probably due to drying, the transferred neutravidin was not functional and failed to bind to a biotinylated protein.

In a related set of experiments we functionalized the plasma treated elastomer surface (pPD2 and pMP) with APTES ((3-Aminopropyl)triethoxysilane) and glutaraldehyde, which is known to render the surface hydrophobic. 44 The hydrophobic NAV-TR, transfered well on this type of surface, but hydrophilic bBSA and BSA-FITC showed a very low transfer, again showing that the transfer is governed by physico-chemical anity.

We also compared the impact of hardness on transfer. To minimize artifacts arising from elastomer surface chemistry, hard and soft versions of the same elastomer were employed (Figure SI.11). It is seen that the width does not significantly change in either case and that the transfer is successful in both cases as judged from the calculated transfer ratios.

We next conrmed that the bBSA-TR transferred to an elastomer could be functionalized with NAV. First, the area around the dots, still exposing the PDMS substrate, was passivated using poloxamers.Using NAV labeled with another uorophore (dylight-650), the specic binding of NAV to bBSA-TR dots can be conrmed (Figure SI.10). The NAV can be further functionalized with another biotinylated protein, here an antibody against the CD3 domain in the TCR-complex in T lymphocytes (α-CD3, multibiotinylated UCHT1).

A soft elastomer, chosen to be MP with Young's modulus 20 kPa, was patterned with α-CD3 dots and passivated with a poloxamer. T cells were allowed to interact with this substrate for 30 minutes and were then xed and labeled with an antibody against TCR or with uorescent phalloidin to label actin (SI for details). Cells are seen to adhere, as judged from reection interference contrast microscopy (Figure 5 a), with an irregular contour but with a rather homogeneous adhesion within the contact zone. The actin is in the form of a ring (Figure 5 b), as has been reported in the case of homogeneous α-CD3 absorbed to glass. 45 TCR clusters are detectable and partially co-localize with the underlying anti-CD3 dots (Figure 5 c andd). The overall behavior on patterned glass is fairly similar (Figure SI.12) . The invariance in actin and TCR organization was unexpected since for other cell types, as reported for adhesion to homogeneously distributed ligands, the cell adhesion is diminished on soft substrates and the cytoskeletal organization is strongly impacted. 4,5 A comparison with homogeneously functionalized elastomer reveals that detectable micro-clusters do not form in this case. This is consistent with the dierence observed between patterned and homogeneously functionalized glass substrates. 9 The absorption of proteins to synthetic surfaces is important for a number of applications. For example, a newly implanted prosthesis is rst coated by proteins from the body uids before cells can interact with it. In a laboratory setting, many biology experiments depend on successful protein absorption as the rst step towards functionalization. Yet, the non-specic absorption of proteins to a surface from a solution phase is dicult to model, partly because of the complex and diverse molecular structure of the proteins. In the context of micro-contact printing, several attempts have been made to quantitatively model the transfer and yet they serve at best as indicative and most laboratories still rely on trial and error to test if their protein of interest can be stamped. In fact, it turns out that most proteins of interest readily transfer from traditional PDMS elastomer stamps to glass. Here we have shown that the reverse transfer of proteins from glass to PDMS is also possible, but depends crucially on the physico-chemical interactions between the protein and the PDMS surface.

One point of great practical signicance is that proteins that can not be reverse transferred in native state, can be made to do so after by grafting an appropriate molecular moiety, here chosen to be dierent uorophores. We have shown that proteins grafted with a hydrophobic moiety always transfer well on PDMS with hydrophobic surfacewhich is the case for most available PDMS in their native state. The reverse transfer of proteins that are intrinsically hydrophillic or those that are grafted with a hydrophillic group, onto hydrophobic PDMS is not reliable. However, these typically transfer well on PDMS rendered hydrophilic by plasma treatment. Interestingly, atomic force microscopy based force measurements conrmed that indeed the degree of reverse transfer depends on the force required to detach a bead, grafted with a layer of protein, from the surface of the PDMS.

We showed that the protein pattern created by reverse printing from at stamps remains functional and can be further functionalized with a more complex protein of choice. We demonstrated the adhesion of T cells to RCP-patterns functionalized with an antibody against the TCR complex. Consistent with previous observations on glass, the TCR can gather to form micro-clusters on patterned but not on homogeneously coated PDMS. The knowledge of mechanisms governing reverse transfer elucidated here should open the way to systematically using such glass based at-stamps to pattern elastomers with any desired protein molecules.

Acknowledgement The authors thank Pierre Dillard for

Figure 1 .

 1 Figure 1. Schematic representation of the fabrication of protein nano-patterns on glass and soft substrates: (a) Deposition of uorosilane from a gas phase through a self-assembled colloidal bead mask on a glass substrate. (b) Removal of the mask and grafting of a poloxamer (pluronic) to passivate the uorosilane covered area. (c) Functionalization of the bare patches with the desired protein. (d) Thin layer of elastomer supported on a glass coverslide. (e) Transfer of the protein from glass to elastomer by reverse contact printing in presence of water. (f) Protein pattern on the elastomer.

Figure 2 .

 2 Figure 2. TOP: Epi-uorescence images of protein dots on glass master before transfer and on elastomer after transfer with same camera and display settings for all images. (a,b) bBSA-TR to PD. (c,d) bBSA-Atto to PD. (e,f) bBSA-Atto to pPD. Insets display Fourier transforms of the corresponding images to emphasize the ordering of the lattice. BOTTOM: Table summarizing the transfer of proteins to elastomer surfaces. Rows correspond to one kind of protein, the core being either BSA or NAV, which are then decorated with various uorophores as indicated. Hydrophilic molecules are depicted in blue and hydrophobic in red. Columns correspond to dierent elastomers, either native (hydrophobic, in red) or plasma treated (hudrophilic, in blue). Successful transfers are indicated with and unsuccessful ones with ×. Note that the intersection between identically colored lines and columns invariably results in a successful transfer (red or blue circled checkmark). The intersection between dierently colored lines and columns results in failed transfer in most cases ( × symbol).

  Fig.SI.3 shows representative im-

Figure 3 .

 3 Figure 3. Quantication of the bBSA nano-dots from the epiuorescence images before and after the transfer from glass to elastomer. Values are medians and error bars are median absolute deviation, both averaged over at least 3 independent samples, each with at least 6 elds each containing hundreds of dots. (a) Dot-size (FWHM of the intensity prole). (b) Contrast of the dots with respect to the background. Data are for bBSA-Atto or bBSA-TR dots and PDMS type PD, MP,pPD or pMP, as indicated

Figure 4 .

 4 Figure 4.

  Figure 5. T-cells adhered on soft elastomer (MP) patterned with nano-dots of α-CD3 antibody, observed after 30 minutes of spreading. a) RICM image showing a at membrane topography in the cell adhesion zone. b) TIRF image showing peripheral actin organization. c) TIRF image of labelled TCR on the cell surface d) epi-uorescence image of the underlying α-CD3 dots. Arrows on c and d point to partial co-localization of the TCR with the α-CD3 dots. Scale bar: 4 µm.

Table 1 .

 1 Adhesion force measurement. Each value is extracted from 100 independent force curves taken at 3 dierent regions for at least 2 dierent samples. (nN) F glass (nN) FpP DM S (nN) BSAb TR 4.8 ± 1.5 2.7±0.8 5.1±0.6 BSAb Atto 488 10.5 ± 3.5 2.3 ± 0.8 12.8±1.7 fruitful discussions regarding cell experiments, and Martine Biarnes for help with cell culture. This work was partially funded by European Research Council via grant no. 307104 FP/2007-2013/ERC-Stg SYNINTER.

Fprt

Supporting Information Available:

The le SI.pdf is available free of charge and contains tables (Young's modulus and contact angles), gures (AFM images, force-curves, untreated optical images, complementary data) and details on materials and methods. This material is available free of charge via the Internet at http://pubs.acs.org/.


Dr Mohammed H Elmahmoudy

Dr Sahika Inal
email: sahika.inal@kaust.edu.sa

Dr Anne Charrier

Prof Ilke G G Uguz

George G Malliaras

Dr Sébastien Sanaur

Malliaras

Tailoring the Electrochemical and Mechanical Properties of PEDOT: PSS Films for Bioelectronics

Keywords: organic bioelectronics, poly(3, 4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT: PSS), organic electrochemical transistor, thin films, cross-linker, Young's modulus

Introduction

Bioelectronics uses electrical signals to interact with biological systems. Sensors that allow for electrical read-out of important disease markers, and implants/stimulators used for the detection and treatment of pathological cellular activity are only a few examples of what this technology can offer [START_REF] Karunakaran | Biosensors and bioelectronics[END_REF][START_REF] Lin | [END_REF] . In the last few decades, due to their intriguing electroactive and mechanical properties, organic electronics or -conjugated materials have been extensively explored regarding their use in bioelectronics applications [3][4][5][6][7][8] . Historically, the interest in organic electronic materials stemmed from their soft and flexible nature which dampens the mechanical properties mismatch with tissue [7] . This less "foreign" surface enhances the signal transfer to/from cells in vitro [9] . It also elicits a small foreign body response when used in vivo, improving the performance as well as the lifetime of the implanted device. The other Revised Manuscript attractive feature of -conjugated materials and more particularly of conducting polymers for bioelectronics is their mixed electronic/ionic conductivity [10][11][12] . Mixed conductivity enables coupling between the electronic charges in the bulk of the organic films with ion fluxes in biological medium. This translates into low electrochemical impedance at the biotic interface and therefore efficient transduction as well as stimulation of biological signals. As a matter of fact, the materials research for bioelectronics strives for soft materials that exhibit low impedance.

The prototypical material of organic bioelectronics is the conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) doped with polystyrene sulfonate (PSS). PEDOT: PSS is commercially available, water-dispersible conjugated polymer complex that can be cast into films of high hole and cation conductivity, good charge storage capacity, biocompatibility, and chemical stability. In PEDOT: PSS films, PEDOT chains accumulate in a pancake-like morphology surrounded by a PSS network [11] . While hole transport is facilitated within/among the PEDOT aggregates, the PSS phase attracts a considerable amount of water, enabling penetration and transport of ions in the film. Ion transport, and consequently electrochemical activity, benefits from such hydrated pathways. This, however, brings together the challenge of maintaining the integrity of films when exposed to an aqueous environment such as the biological tissue for in vivo applications and the cell culture media for in vitro studies.

In order to avoid delamination/disintegration of the films in aqueous environment, PEDOT: PSS dispersions are typically mixed with other chemical compounds. Some studies have reported blending the dispersion with water soluble polymers such as the polyvinyl alcohol (PVA) [13][14][15][16] . The main problem with these blends is the dramatic drop in the conductivity. For instance, when mixed with PVA at a weight ratio of 20 and 60 %, the electrical conductivity of films dropped by 1 and 5 orders of magnitude, respectively [13] . The silane based crosslinking agent, 3-glycidoxypropyltrimethoxysilane (GOPS), on the other hand, was reported to make relatively stable PEDOT: PSS films (with conductivities up to 800 S cm -1 ) for a large variety of bioelectronics devices [17][18][19][20][21][22][23][24] , when used at a particular concentration in the dispersion (0.1 wt%). The epoxy group in GOPS (see the inset of Figure 1 for the chemical structure of GOPS) can react with amines, thiols, and acids, as well as interacting with itself and covalently with SiO2 substrates [25] . Although the interaction mechanism between GOPS and PEDOT: PSS is yet unclear, XPS studies suggested that the cross-linker can polymerize in water to form a multilayered structure [26] . However, depending on its concentration in the film, GOPS might change electronic and ionic transports as well as mechanical properties of PEDOT: PSS, presumably due to alterations on the structure and morphology of films. Only a few reports have touched upon the effect of GOPS concentration on the electrical properties of the resulting films [27] . Nevertheless, it is essential to maintain the electrical performance and softness of PEDOT: PSS while improving the thin film stability in aqueous media using a stabilizer such as GOPS. This is particularly crucial for long term use of bioelectronic devices, a direct example being the organic electrochemical transistors (OECTs) which are chronically implanted into cortex to record neural activities.

Balancing these needs requires a systematic synthetic work or processing related interventions which aim to improve the electrochemical properties while not impeding mechanical properties. Moreover, materials with high performance electronic properties and physical characteristics matched to those of the tissue have great potential to bring forth applications for soft electronics. Here, post-processing can provide alternative modification routes as crystalline materials exhibiting high charge mobility typically have low mechanical resilience.

In this work, we investigate the effect of GOPS content in PEDOT: PSS dispersions on the properties of films spun cast from these formulations. We find out that the concentration of GOPS has a tremendous, yet gradual impact on the electrical, electrochemical, and mechanical properties of the PEDOT: PSS/GOPS films and that there is an optimum concentration which maximizes a particular feature of the film such as its water uptake or elasticity. The benefits of aqueous stability and mechanical strength with GOPS are to be compensated by an increase in the electrochemical impedance. Our findings suggest that a trade-off cross-linker concentration exists, which enables sufficient electrical conductivity with mechanical robustness and stability in aqueous environment.

Results and Discussion

Effect of the cross-linker on electrical properties

To gain insight into the effect of the cross-linker on the electrical properties of dry PEDOT: PSS films, we casted films from dispersions containing a variety of GOPS concentration (0.05, 1, 2.5, 3.5 and 5 wt%) in addition to a constant concentration of the conductivity enhancer, ethylene glycol (EG, 5 vol%) and the surfactant, dodecyl benzene sulfonic acid (DBSA, 0.002 vol%). The films were spin-cast at the same speed on four sets of substrates: Au-coated polyimide films (Au thickness: 100 µm, surface area: 96.7 mm² and 24 mm²) and glass substrates of different geometries (25 x 25 mm², 75 x 25 mm²). Figure 1 shows that GOPS content in the film affects the bulk conductivity significantly: the highest conductivity (ca. 460 S.cm -1 ) is observed for the film containing the least amount of GOPS (0.05 wt%), whereas the conductivity dropped by 4 times (120 S.cm -1 ) with a GOPS concentration of 5 wt%. These results are in agreement with those of Zhang et al who reported a gradual decrease in the conductivity of PEDOT: PSS film (cast from a dispersion with 5 vol% glycerol and 0.5 vol% of DBSA) with an increase in GOPS concentration [27] . The Clevios PH1000 PEDOT: PSS dispersion has a polymer content of 1.15 wt%, with a PEDOT to PSS ratio of 1:2.5 (ca. 0.3 wt% PEDOT and ca. 0.8 wt% PSS). It is intriguing that although PEDOT: PSS comprises only 18.5 wt% in the presence of 5 wt% GOPS, the conductivity of the film exceeds 100 S.cm -1 .

Moreover, the films processed from dispersions that contained more GOPS are thicker than the ones that had less of the cross-linker (ca. 3x difference between 0.05 wt% and 5 wt% of GOPS, Figure S1). We attribute this to an increase in the viscosity of the dispersions in the presence of GOPS. On the other hand, interactions of the cross-linker with the non-volatile additives in the dispersion might lead to an increased material content since the amount of cross-linked network in the film with respect to the PEDOT: PSS increases with GOPS content [27] . This can as well account for the observed decrease in the conductivity as a larger content of non-evaporating and non-conducting species would be present in the film [27] .

However, for our case, i.e., dispersions containing EG, X-ray studies showed no evidence for EG remaining in the films [11] . We therefore attribute the decrease in electrical conductivity to a dilution effect of the conducting phase by the cross-linker. Indeed, hole mobility also decreases ca. 4 times in the range of cross-linker concentrations investigated. The mobilities were estimated by measuring OECTs prepared from PEDOT: PSS formulations of different GOPS concentrations (Figure S2 and experimental section). For instance, while the hole mobility of the 0.05 wt% GOPS-cast film is 6.4 cm 2 V -1 s -1 , this value drops to 1.7 cm 2 V -1 s -1 for the 5 wt% GOPS-cast film. It is likely that GOPS limits the extent of aggregation of PEDOT chains by introducing crosslinks into the system. In an OECT, upon application of a positive gate voltage (VG), cations from the electrolyte are injected into the channel, compensate the sulfonate groups of the PSS and deplete the holes of the PEDOT (See Figure S2 for a schematic of an OECT). This mechanism is measured as a decrease in the drain current (ID). The performance of an OECT is therefore evaluated as its transconductance (gm=

𝜕𝐼 D 𝜕𝑉 G
), i.e., the extent of the modulation of the drain current with a change in gate voltage. As the GOPS content in the channel increases, not only the channel becomes less conductive, we also measure a gradual decrease in the transconductance of OECTs (Figure 2).

Cross-linked PEDOT: PSS properties in aqueous environment

Since applications in bioelectronics necessitate an aqueous environment, it is critical to characterize the properties of the polymer film in aqueous working conditions. Using the quartz crystal microbalance dissipation (QCM-D), we quantify the swelling capability of the PEDOT: PSS/GOPS films cast on quartz crystals when exposed to DI water or an aqueous solution of NaCl. In these measurements, a decrease in the frequency (f) accompanied with an increase in dissipation (D) with the inflow of water indicates an increase in the mass of the film, i.e. swelling due to uptake of molecules. Here, the inflow of NaCl induces a further decrease in f for all samples, attributed to penetration of solvated ions in addition to the trapped water molecules. In order to quantify the swelling of the films casted from different GOPS concentrations, we treated our data both with Sauerbrey model which directly correlates the change in f to coupled mass (more appropriate for rigid films) and with Kelvin-Voigt model which is considered typically for soft films (see Experimental section). The swelling percentages estimated from these two models are summarized in Table S1. The results suggest a reduction in the swelling capacity of films with an increase in GOPS content (Figure 3a). The ability of PEDOT: PSS film to uptake water drops from 397% to 12% when GOPS concentration increases from 0.05 to 5 wt%. Using AFM to estimate the thickness of PEDOT: PSS films before and after exposure to DI water, Duc et al. reported a swelling ratio of a 40 ± 1% for a PEDOT: PSS film that contained 1 wt% of GOPS [28] . This value is well below our estimations (ca. 266%) for the same formulation. These authors also reported 660 ± 90% swelling for a PEDOT: PSS film that does not contain GOPS. Using the same technique, Stavrinidou et al. reported 155 ± 53 % of swelling for the film prepared in the absence of GOPS and 35 ± 4 % in the presence of 1 wt% of GOPS [19] . These variations in the reported values could be due to the characterization techniques, the film/dispersion preparation, and the measurement conditions. The latter is particularly challenging to control since PEDOT: PSS films change their volume rather rapidly due to the humidity in the environment. Our results are rather meant to demonstrate the relative decrease in swelling with changing the GOPS content in PEDOT: PSS/GOPS films. For our measurements, we dried the films under vacuum over night to ensure that the film has minimal water trapped prior to its interactions with water molecules. Notably, we observed that such a dry film requires ca. two hours under constant water flow to reach a steady-state change in frequency, i.e., fully hydrated state.

(Table S1). Interestingly, although the film that contained a higher GOPS content was thicker than the one that had less GOPS, its stabilization time was shorter: 30 min for 5 wt% GOPScast film in comparison to ca. 103 min for 1 wt% GOPS-cast film. Taken together, the trend of stabilization time and GOPS content is consistent throughout the whole formulation series.

It is absolutely mandatory to investigate the impedance characteristics of conducting materials dedicated to bioelectronics applications. Figure 3b shows the electrochemical impedance (Z) of PEDOT:PSS/GOPS films measured at 1 kHz as a function of the cross-linker concentration.

Here, since the films had different thicknesses due to variations in GOPS content (Figure S1), we normalized Z values measured at 1 kHz for a 100nm-thick film. First, the Bode plots (log Z vs log frequency) were fit to an equivalent circuit model (RC) to extract the resistance (R) and capacitance (C) values. Then, the capacitance was estimated for a 100 nm thick film using

𝐶 ′ = 𝐶×100 𝑑
, where C' is the normalized impedance and d (in nm) is the film thickness. Finally, C' was substituted in the impedance formula (|𝑍 ′ | = √𝑅 2 + 1 𝜔 2 𝐶' 2 ) to estimate the normalized impedance (𝑍 ′ ), knowing that the change in R is negligible. Our results show that 1 kHz impedance increases indistinctly as the film contains more GOPS. This is in fact consistent with the trend in swelling. At low GOPS concentrations, the film uptakes more water (Figure 3a), suggesting that the ions of the electrolyte can more readily penetrate and travel inside the polymer film without significant accumulation at the polymer/electrolyte interface, leading to low impedance values. Likewise, less swelling at high GOPS content impedes ionic mobility in the film, resulting in higher impedance.

Finally, we studied the effect of GOPS on the mechanical properties of PEDOT: PSS films in solution via Nano-indentation experiment using the tip of an atomic force microscope (Figure 4a). During the course of the experiment, the tip-sample distance is modulated and the subsequent interaction between the tip and the sample is monitored through the vertical displacement of the cantilever probe. Young's modulus can be extracted from such forcedistance curves using the appropriate model. In this experiment, we used a derived model of the Sneddon contact mechanics assuming a conical tip with a non-negligible radius of curvature at its apex in contact with a flat surface [29] . The applied force (F) and the indentation (h) are related with the Young modulus (E), the Poisson ratio ( ~ 0.5), the radius of curvature of the tip apex (R), and the half opening angle of the tip () as in the following:

𝐹 = 2𝐸 𝜋.(1- 2 ) {2𝑅ℎ[1 -𝑡𝑎𝑛(𝜃)] + ℎ 2 𝑡𝑎𝑛(𝜃)} (1)
For each sample prepared, a series of 50 measurements was performed at different locations of the film. A typical challenge encountered for thin films is that the deformation of the film under the tip goes through continuous change by the presence of the hard substrate underlying the sample. This results in an overestimation of the Young moduli. In our case, the thickness of the films varies from 60 nm for the 0.05 wt% GOPS-cast film to 180 nm for the 5 wt% GOPS. In order to overcome challenges related to thin films, we performed the measurements at varying indentation depths. We found the Young's modulus to drastically increase with the indentation depth, reflecting the substrate effect and that it can therefore be minimized with low indentation depth experiments (Figure S3). Therefore, we limited the indentations to 10 nm. The roughness of the PEDOT:PSS/GOPS films was also estimated from the AFM images taken in water (Figure S4). Samples containing 0.05 and 3.5 wt.% of GOPS exhibited an average surface roughness of 1.46 and 1.43 nm (root mean square = 1.98 and 1.81 nm)

respectively. These roughness values are much smaller than the chosen indentation depth (10nm). Figure 4b shows that the Young modulus of the films increases from 90 MPa for the 0.05 wt% of GOPS to 150 MPa for the 1 wt% of GOPS. Above 2.5 wt%, the cross-linker doesn't seem to influence the elasticity of the films (ca. 350 MPa). Moreover, we observe that the mechanical stiffness of PEDOT: PSS films decreases dramatically (by ca. 25x) in aqueous environment compared to air (Figure S5). The decrease in film stiffness in DI water is directly related to the swelling of the polymer. Our results are in agreement with those reported by GPa) [30][31][32] . These results suggest that GOPS is a versatile additive that can be used not only to improve the aqueous stability of films but also to modify their elasticity.

As par these results, considering the use of PEDOT: PSS based OECTs for long term implantations in the brain [22] , we intended to evaluate the performance of devices over several days. The OECT that was selected for this test had GOPS content that led to films with optimal water uptake, softness and conductivity: 1 wt%. Figure S6a depicts the change in the transconductance of an OECT comprising 1 wt% GOPS in the channel over 21 days, within sub-chronic period, measured in PBS. In order to obtain stable current values at day 0 (prior to the performance evaluation experiments), the devices were incubated in water overnight followed by multiple current-voltage cycles. This enabled the dissolution/diffusion of low molecular components in the film into the solution. In fact, another study reported that when gold electrodes coated with electropolymerized EDOT were soaked in a buffer for several days, the impedance of some of these electrodes raised steadily associated with decreases in charge storage capacity. This decrease was attributed to partial delamination of the PEDOT coating in PBS [33] . Nevertheless, our devices (width = 10 µm, length = 5 µm, thickness = 275∓25 nm) showed stable transconductance values (gm= 7.3 ∓ 0.2 mS) over the course of this study. In a separate study, we found that the devices that contain even higher (3.5 wt%)

GOPS in the channel had similar performance in stability, tested over 5 days in aqueous environment (Figure S6b). Overall, the results obtained from 5 different OECTs suggest that the films cast with 1% wt of GOPS maintain their structural integrity in aqueous media and but also exhibit adequate long-term electrical performance.

Conclusions

In this work we investigated the electrical, swelling, electrochemical, and mechanical properties of PEDOT: PSS films modified with varying amounts of the silane based crosslinker, GOPS. As the cross-linker content increases from 0.05 to 5 wt%, we observed a drop in the bulk conductivity from ca. 530 to 120 S.cm -1 , a decrease in the swelling from ca. 397%

to 12%, and a relative increase in the electrochemical impedance (from ca. 15 to 20 Ohms at 1 kHz for films with thickness of 100 nm with a surface area of 96.7 mm²). The benefits of aqueous stability with GOPS are therefore to be compensated by losses in electronic transport and increase in the electrochemical impedance. Nevertheless, the presence of the cross-linker led to an increase in the mechanical strength of the films when they are hydrated (ca. 90 to 300 MPa in DI water for 0.05 and 5 wt% of GOPS in the dispersion, respectively), as these films uptake significantly less amount of water. We also emphasize the tolerance of PEDOT:

PSS films to a large quantity of the cross-linker (only 18.5 wt% of PEDOT: PSS in the solution can lead to conductivity up to 100 S.cm -1 ). GOPS aids obtaining highly conducting films with excellent mechanical integrity in aqueous media. Moreover, devices that contain 1 wt% GOPS, which is a concentration that leads to film with high electrical conductivity with sufficient mechanical stability, exhibit steady performance over 3 weeks. These results suggest that variations in the concentration of such a dispersion additive like GOPS can enable facile co-optimization of electrical and mechanical properties of a conducting polymer film.

Experimental Section

Sample Preparation: PEDOT: PSS (Clevios PH-1000 from Heraeus Holding GmbH.), dodecyl benzene sulfonic acid (DBSA; 0.002 vol%), ethylene glycol (EG; 5 vol%) and GOPS (ranging from 0.05 to 5 wt%) were mixed, sonicated for 30 minutes at room temperature and then filtered using 1.2 µm hydrophilic syringe filters (Minisart, from Sartorius Stedim Biotech). The substrates were cleaned and exposed to plasma oxygen for 2 minutes at 100

Watts for surface activation and further cleaning. All films were spin cast at 2500 rpm for 40 sec. The films were then baked at 140°C for 1 hour. The thickness of PEDOT: PSS films was determined using a Dektak mechanical profilometer.

Electrical and Electrochemical Characterization:

We measured the sheet resistance (RS in Ω.sq -1 ) of PEDOT:PSS/ GOPS films cast on glass substrates using a four-point probe (Jandel RM3-AR). Given the film thickness (d), we could calculate the resistivity (ρ = RS × d, where ρ is resistivity in Ω.cm) from which the conductivity (1/ρ in S.cm -1 ) was obtained.

Electrochemical impedance spectroscopy (EIS) was performed in NaCl solution (0.1M) via an impedance spectrometer (potentiostat/galvanostat, Metrohm Autolab B.V.) with a threeelectrode configuration, where the polymer-coated substrate is the working electrode, a Pt mesh is the counter electrode, and Ag/AgCl is used as a standard reference electrode. EIS was performed over a range of 10 kHz to 1Hz with an AC 10 mV sine wave, and a DC offset of 0 V. In order to extract capacitance (C), and the resistance, R, the spectra of films were fit to an (RC) equivalent circuit using NOVA software.

OECT fabrication and characterization: OECTs were fabricated using photolithography, as previously described [34] . Briefly, 150 nm thick gold lines were patterned on a glass slide with impedance matching method reported for OECTs [21] .

Swelling Measurements:

The swelling of the thin polymer films was investigated by quartz crystal microbalance with dissipation set-up (QCM-D) (Q-Sense, from Biolin Scientific).

PEDOT: PSS dispersions at a given GOPS concentration were spun-cast on cleaned goldcoated Q-sensors. They were then kept under vacuum overnight to ensure complete drying of the film. Filtered DI water or aqueous NaCl solution (0.1 M) were flown over the samples at 24 ℃ at a flow rate of 50 -100 µL.min -1 controlled by a peristaltic pump. The adsorbed mass (∆𝑚) can be approximately estimated from ∆𝑓 using the Sauerbrey equation:

∆𝑚 = -𝐶 ∆𝑓 𝑛 𝑛 ( 2
)
where 𝐶 is the mass sensitivity constant (17.7 ng.cm -2 Hz at 𝑓 = 5 MHz) and ∆𝑓 𝑛 is the change in resonance frequency at 𝑛 th overtone [35] . We used the 5 th overtone for our calculations. Given the initial thickness of the films, we could estimate the water uptake.

Kelvin-Voigt viscoelastic model was also used (equations 3, 4, and 5) where G* is the complex shear modulus, ρ is the density (kg m -3 ),  is the viscosity (G"/) (kg ms -1 ), µ is the elasticity (G') (Pa), and δ is the thickness (m) [36] .

G* = G' + jG'' = µ + j2πf (3) Δf = f1 (n, f, ρf, µf, δf) (4) ΔD = f2 (n, f, ρf, µf, δf) (5)
Mechanical Characterization: Young's modulus was obtained from the force-curve measurements that were realized by using an NTEGRA AFM system (from NT-MDT). In all experiments AFM tips (NSC35 from Mikromash) were used with typical resonant frequency of 150 kHz, spring constants ranging from 5 to 12 N.m -1 and apex radius of 8 nm as verified by scanning electronic microscopy. For each tip, the spring constant was determined using the thermal noise method after obtaining the deflection sensitivity of the cantilever by pressing the AFM tip against a hard reference silicon surface. The measurements were all performed in water after allowing the samples to hydrate for 2 hours.

  S1813 photoresist, exposed to UV light using a SUSS MBJ4 contact aligner, and developed using MF-26 developer. Upon the deposition, a standard metal lift-off process in acetone was employed and gold interconnects and pads were insulated from the electrolyte by a 1.5 µm parylene C film deposited using a SCS Labcoater 2. A second sacrificial layer of parylene C was coated, patterned with AZ9260 photoresist, developed, and selectively etched by an CF6/O2 plasma using an Oxford 80 plus to define the transistor channel. Finally, PEDOT:PSS dispersion was cast and the sacrificial layer of parylene C was peeled, and the devices were baked at 110 °C for 1 hour.The PEDOT: PSS channel had a width/length (W/L) of 50 μm/50 μm. The transistors were operated in the common source configuration with a Ag/AgCl pellet electrode (Warner Instruments) immersed in NaCl solution (0.1M). The characterization was performed using a National Instruments PXIe-1062Q system. The gate bias was applied and controlled using a NI PXI-6289 modular instrument, and current recorded with either the NI PXI-4145 SMU or a NI PXI-4071 digital multimeter. The recorded signals were saved and analyzed using customized LabVIEW and MATLAB software. Hole mobilities were extracted using

Figure 1 .

 1 Figure 1. Electrical conductivity of PEDOT: PSS films cast from dispersions with GOPS

Figure 2 .Figure 3 .

 23 Figure 2. The transconductance (gm= 𝜕𝐼 D 𝜕𝑉 G ) of OECTs comprising PEDOT:PSS channels of

Figure 4 .

 4 Figure 4. a) Schematic of the AFM tip indenting PEDOT: PSS thin film b) The change in

Figure S2 .

 S2 Figure S2. Hole mobility of PEDOT: PSS films, extracted from working OECTs, as a

Figure S3 .Figure S4 .

 S3S4 Figure S3. Young's modulus diverging with the indentation depth of a PEDOT: PSS film

Figure S5 .

 S5 Figure S5. Mechanical elasticity of PEDOT:PSS (5 wt% GOPS) in air (black) and in DI

Table S1 .

 S1 Swelling ratios of PEDOT: PSS films in DI water and in NaCl solution (0.1 M) with varying GOPS content (using Sauerbrey and Kelvin-Voigt models). Standard deviation is calculated according to the swelling capacity estimated via different models.

	GOPS	Swelling using Sauerbrey model (%)	Swelling using Voigt model (%)		Standard deviation	Time to stabilize
	(wt %)							(min)
		DI	NaCl	DI	NaCl	DI	NaCl
	0.05	283	397	364	480	58	57	66
	1.0	266	386	387	497	78	86	103
	2.5	113	136	224	248	79	78	61
	3.5	10	17	128	130	80	83	38
	5.0	2	12	127	138	89	88	31

Acknowledgements

The work was financially supported by the Agence Nationale de la Recherche (grant number: ANR-14-CE08-0006). The authors would like to also thank David C. Martin for the fruitful discussions.

Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff))

Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2013.

Supporting Information

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

Supporting Information


S D Vergani

J Palmerio

R Salvaterra

J Japelj

F Mannucci

D A Perley

P D'

T Avanzo

M Krühler

S Puech

Boissier

P D'avanzo

T Krühler

M Puech

S Boissier

S Campana

S Covino

L K Hunt

P Petitjean

G Tagliaferri

The chemical enrichment

Keywords: Gamma-ray burst, general -Galaxies, abundances -Galaxies, star formation

published or not. The documents may come

Introduction

It has been established that long gamma-ray bursts (LGRBs) are linked to the explosions of massive stars, both from the studies of their host galaxy formation sites (Fruchter et al. 2006;Svensson et al. 2010) as well as from detections of accompanying supernova emission (GRB-SN; see Cano et al. 2016 for a review). It is still not clear which conditions give rise to LGRBs or what is the relation between the progenitors of LGRBs and those of other explosions resulting from deaths of massive stars (e.g., Metzger et al. 2015).

The progenitors of nearby core-collapse supernovae can be directly identified as resolved stars in archived high-resolution images of their birth places (Smartt 2015). However, LGRBs have a lower occurrence rate (e.g., Berger et al. 2003;Guetta & Della Valle 2007) and are usually observable at cosmological distances, for which their birth places cannot be resolved. Our understanding of LGRB progenitors therefore depends on linking the predictions of different stellar evolution models with the observed properties of LGRB multiwavelength emission (e.g., Schulze et al. 2011;Cano et al. 2016) and their host galaxy environment (see Perley et al. 2016a for a review). In this work, we focus on the latter.

While metallicity is not the only factor that might affect the efficiency of the LGRB production (e.g., van den Heuvel & Portegies Zwart 2013; Kelly et al. 2014;Perley et al. 2016b), it has been one of the most studied in the past as the metal content of the progenitor star is considered to play a major role in the formation of a LGRB explosion. Single-star evolution models predict that the metallicity of LGRB progenitors should be very low (e.g., Hirschi et al. 2005;Yoon & Langer 2005;Woosley & Heger 2006): in this way the progenitor star can expel the outer envelope (hydrogen and helium are not observed spectroscopically) without removing too much angular momentum from the rapidly rotating core. Higher metallicity values are allowed in the case of the models presented by Georgy et al. (2012), also depending on the different prescriptions between the coupling of surface and core angular momentum in the star. Alternatively, the LGRB progenitors could be close interacting binaries, in which case the metallicity is a less constraining factor (e.g., Fryer et al. 2007;van den Heuvel & Yoon 2007). Strong observational constraints are clearly needed to understand which of the evolutionary channels could produce a LGRB.

Different observational works on LGRB host galaxies in the literature have indeed revealed that their metallicities are mostly subsolar [START_REF] Modjaz | [END_REF]Levesque et al. 2010a;Graham & Fruchter 2013;Vergani et al. 2015;Krühler et al. 2015;Perley et al. 2016b;Japelj et al. 2016). The evidence is corroborated by numerical simulations (e.g., Nuza et al. 2007;Campisi et al. 2011;Trenti et al. 2015). In particular, Campisi et al.

Article number, page 1 of 4 arXiv:1701.02312v1 [astro-ph.HE] 9 Jan 2017 (2011) studied LGRB host galaxies in the context of the mass metallicity (e.g. Tremonti et al. 2004) and fundamental metallicity (Mannucci et al. 2010(Mannucci et al. , 2011) ) relations of field star-forming galaxies by combining a high-resolution N-body simulation with a semi-analytic model of galaxy formation. Campisi et al. (2011) find that a very low metallicity cut is not necessary to reproduce the observed relations. However, previous observational works present one or more of the following issues: (i) they are based on incomplete biased samples (e.g., Levesque et al. 2010a); (ii) they are based on stellar masses directly determined from observations, but on metallicities inferred from the mass-metallicity relation (e.g., Perley et al. 2016b); (iii) they use metallicities directly determined from the observations, but do not consider the stellar masses (e.g.: Krühler et al. 2015); and (iv) they are based on samples limited to small redshift ranges (e.g., 0 < z < 1) as in Japelj et al. (2016).

In this paper we study the metallicity of the host galaxies of the complete Swift/BAT6 sample (Salvaterra et al. 2012) of LGRBs at z < 2, visible from the southern hemisphere. Combining the observed properties with simulations, we study their behavior in the stellar mass -metallicity relation (MZ) and fundamental metallicity relation (FMR). After the description of the sample and new data (Section 2), we present the results in Section 3 and discuss them in Section 4.

All errors are reported at 1σ confidence unless stated otherwise. We use a standard cosmology (Planck Collaboration et al. 2014): Ω m = 0.315, Ω Λ = 0.685, and H 0 = 67.3 km s -1 Mpc -1 . The stellar masses and star formation rates (SFR) are determined using the Chabrier initial mass function (Chabrier 2003).

The sample

Our sample is composed of the 27 host galaxies of the Swift/BAT6 complete sample of LGRBs at z < 2 with declination Dec < 30 • . As the spatial distribution of GRB is isotropic, this restriction does not introduce any bias in our results. The choice to select only the LGRBs that are well observable from the southern hemisphere was due to the availability of the Xshooter spectrograph (Vernet et al. 2011) at the ESO VLT (Very Large Telescope) facilities, which, thanks to its wide wavelength coverage, makes possible the detection of the emission lines necessary to determine the SFR and metallicity of the host galaxies at z < 2. In particular, metallicity is available for 81% of the sample (an estimate of the metallicity was not possible for five host galaxies only).

As the original Swift/BAT6 sample is selected essentially only on the basis of the LGRB prompt γ-ray flux, and no other selection criterion is applied when gathering the galaxy sample (except the southern hemisphere visibility), our sample does not suffer of any flux bias. Indeed, no correlation has been found between the prompt γ-ray emission and host galaxy properties (see e.g.: Levesque et al. 2010b;Japelj et al. 2016). Furthermore, dark bursts are correctly represented in the sample (see Melandri et al. 2012). The restriction to the southern hemisphere at z < 2 maintains this condition, with 26% of LGRB of the sample being dark.

For the part of the sample at z < 1, Vergani et al. (2015) and Japelj et al. (2016) report the tables with the objects in the sample and their properties (including stellar masses, SFR and metallicity). The restriction to the Dec < 30 • excludes GRB 080430 and GRB 080319B from the sample used in this work.

The properties (redshift, stellar mass, SFR, and metallicity) of the 1 < z < 2 part of the sample are reported in Table 1. The stellar masses were taken from Perley et al. (2016b), with the Notes. There are 4

LGRBs in the 1 < z < 2 sample for which we could not determine the metallicity of their host galaxies: GRB 050318, GRB 050802, GRB 060908, and GRB 091208B. Indeed, there are no useful spectra to this purpose for the host galaxies of GRB 091208B and GRB 050318. For the host galaxies of GRB 050802 and GRB 060908 we obtained X-shooter spectroscopy (Prog. ID: 097.D-0672; PI: S.D. Vergani), but the spectra do not show sufficient emission lines to allow the metallicity determination. * : from new/unpublished X-shooter observations presented in this paper (see Table 3).

exception of the host galaxies of GRB 071117 and GRB 080602, which are not part of the Perley et al. (2016b) sample, and for which we determined the stellar masses using Spitzer observations and the same prescription as Perley et al. (2016b). The host of GRB 071117 lies very close (∼ 2 �� ) to a red galaxy, and, therefore, the spatial resolution of the Spitzer observations allowed us to obtain only an upper limit on its infrared flux. We therefore also performed a spectral energy distribution fitting using the host galaxy photometry (see Table 2) following the same prescriptions as Vergani et al. (2015), and found log(M � /M � )∼ 9.9. Notes. The g, r, i, z magnitudes have been determined from GROND (Greiner et al. 2008) observations, whereas for the K value we used VLT/HAWKI observations (Prog. ID: 095.D-0560; P.I.: S.D. Vergani).

The SFR values were taken from Krühler et al. (2015) with the exception of the host galaxies of GRB 061007, GRB 061121 and GRB 071117, not included in that work. We obtained the VLT/X-shooter spectroscopy of these three host galaxies (ESO programs 095.D-0560 and 085.A-0795, PI: S.D. Vergani and H. Flores, respectively). We processed the spectra using version 2.6.0 of the X-shooter data reduction pipeline [START_REF] Modigliani | Observatory Operations: Strategies, Processes, and Systems III[END_REF], following the procedures described in Japelj et al. (2015). The measured emission line fluxes are reported in Table 3. We determine the SFR from the Hα fluxes (corrected by the extinction determined through the Balmer ratio), with the same prescriptions as Krühler et al. (2015).

Following the same prescription as in Japelj et al. (2016), we determined the metallicity of the objects in the sample with the Maiolino et al. (2008) method on the strong emission line fluxes reported in the literature (Piranomonte et al. 2015;Krühler et al. 2015) or on those measured by us; in the relevant cases, the results are consistent within errors to those already reported in the literature. Mannucci et al. (2010Mannucci et al. ( , 2011)). The dark blue curve and area correspond to FMR relation and of its quartiles obtained using the simulation of Campisi et al. (2011). The cyan curve and area correspond to the best-fit model results.

In Fig. 1 we plot the host galaxies of our sample in the MZ and FMR spaces. The dearth of high metallicity galaxies is evident as well as the fact that there are more massive galaxies at the higher redshifts (1 < z < 2) than at z < 1.

At low stellar masses (log(M * /M � ) < 9.5) there is some agreement with the MZ relation and FMR found for general starforming galaxy populations (see also Japelj et al. 2016), whereas massive LGRB host galaxies are clearly shifted toward lower metallicities than predicted by the general relations.

While the MZ relation evolves in redshift, the FMR has the advantage that it is redshift independent in the redshift range considered here, hence strengthening the statistics of our results. For the general population of star-forming galaxies with log(M � ) -0.32log(SFR)� 9.2, the FMR is valid up to z ∼ 2.2, has been defined over SFR and stellar mass ranges encompassing those of the host galaxies in our sample, and has a smaller scatter (0.06 dex) than the MZ relation Mannucci et al. (2010Mannucci et al. ( , 2011)).

To verify that our results are independent of the method used to determine the metallicity, we used the Kobulnicky & Kewley (2004) R23 method to determine the metallicities of the 21 host galaxies for which the relevant lines to use this metallicity indicator are available. The resulting MZ plot confirms the avoidance of super-solar metallicity and the shift of high stellar mass host galaxies toward lower metallicity than those found for general star-forming galaxy populations at similar stellar masses and redshifts.

We stress that the five galaxies in the sample for which we could not determine the metallicity (GRB 050318, GRB 050525, GRB 050802, GRB 060908, and GRB 091208B) are all faint galaxies, not hosting dark GRBs, and with stellar masses log(M * /M � )< 9.2 (three of these galaxies have log(M * /M � )< 8.7 ; see Vergani et al. 2015;Perley et al. 2016b). A super-solar metallicity for a large portion of these host galaxies is therefore extremely unlikely. For two of these galaxies (GRB 050525 and GRB 050802) SFR limits are available (Japelj et al. 2016;Palmerio et al. in preparation). Under the conservative hypothesis that they follow the FMR relation, we can derive limits on their metallicities from their SFR and stellar masses of 12 + log(O/H) < 8.1, 8.4, respectively.

We further investigate the implications of our observational results by comparing them with the expectations of a dedicated numerical simulation of the LGRB host galaxy population presented in Campisi et al. (2009Campisi et al. ( , 2011)), coupling high resolution numerical simulation of dark matter with the semi-analytical models of galaxy formation described in De Lucia & Blaizot (2007). Previous work (De Lucia et al. 2004) has shown that the simulated galaxy population provides a good match with the observed local galaxies properties and relations among stellar mass, gas mass and metallicity. Moreover, Campisi et al. (2011) shows that the simulations nicely reproduce the observed FMR of SDSS galaxies and its spread. Following Campisi et al. (2011) we compute the expected number of LGRBs hosted in each simulated galaxy, assumed to be proportional to the number of shortliving massive stars (i.e., star particles less than 5 × 10 7 yr in age), applying different metallicity thresholds (Z th ) for the GRB progenitor, with probability equal to one below Z th and zero otherwise. We construct the FMR of simulated hosts in the redshift range z = 0.3 -2 and we determined the best-fit value of Z th by minimizing the χ 2 against the BAT6 host data in the same redshift interval. The best-fit model (see Fig. 1) is obtained for Z th = 0.73 +0.08 -0.07 Z � (1σ errors). This is consistent with indirect results inferred from the distribution of the LGRB host stellar masses at z < 1 (Vergani et al. 2015) or of the infrared luminosities over a wider redshift range (Perley et al. 2016b).

Discussion and conclusions

In this paper we considered the properties of the host galaxies of the complete Swift/BAT6 sample of LGRBs (Salvaterra et al. 2012) that are visible from the southern hemisphere and at z < 2. We studied them with respect to the MZ and FMR relation of field star-forming galaxies. This is the first study considering at the same time the SFR, metallicity (both directly determined from the host galaxy spectroscopy), and stellar masses for a complete sample of LGRBs and on a large redshift range. Furthermore, we use LGRB host galaxy simulations to interpret our results.

Thanks to the sample extension to z ≈ 2, we could double the sample size compared to Japelj et al. (2016) and show for the first time that LGRB host galaxies do not follow the FMR. We find that LGRBs up to z ≈ 2 tend to explode in a population of galaxies with subsolar metallicity (Z ∼ 0.5-0.8 Z � ). Our results are well reproduced by LGRB host galaxy simulations with a metallicity threshold for the LGRB production of Z th ∼ 0.7 Z � .

A&A proofs: manuscript no. VerganiHGz2LE1c

Table 3. Emission line fluxes (corrected for MW absorption) of the host galaxies of GRB 061007, GRB061121, and GRB071117 in units 10 -17 erg s -1 cm -2 . Upper limits are given at the 3σ confidence level. Notes. (a). Line strongly affected by a sky line. To determine the host galaxy properties we fixed its value to [O ii]λ3729/1.5 (low electron density case; Osterbrock 1989). (b) Lines falling on too noisy regions to determine a significant upper limit. (c). The line is contaminated by a sky line.

The flux has been determined by a Gaussian fit, using the part of the line not contaminated by the sky.

Although strong metallicity gradients (> 0.1 -0.2 dex) are unlikely (on the basis of low-redshift, spatially resolved LGRB host galaxies observations; Christensen et al. 2008;Levesque et al. 2011;Kruhler et al. in preparation), we cannot exclude that they are at play in the couple of galaxies showing evidences of super-solar metallicities (as, e.g., in the case of GRB 060306; see also Niino et al. 2015). The existence of some super-solar hosts may as well indicate, however, that the formation of LGRBs is also possible above the general threshold, although at much lower rate. Applying smoother cutoffs to the metallicity, instead of the step function used here, shifts Z th toward lower values depending on the functional shape used. The present statistics does not allow us to discriminate between different cutoff shapes, therefore we do not go into further detail. We point out however that none of them succeed in reproducing the super-solar metallicity value. It should also be stressed that the GRB 060306 metallicity is very uncertain with pretty large error bars.

The relatively high metallicity threshold found in this work is much higher than required from standard collapsar models (but see Georgy et al. 2012). Binary stars are a possible solution as progenitors, although detailed models studying the role of metallicity on the fates of binary stars are missing. However, it is important to note that the metallicities determined using strong emission lines are not absolute values (see Kewley & Ellison 2008). In our case, they are relative to the Kewley & Dopita (2002) photoionization models on which the Maiolino et al. (2008) method is based. On the one hand, some works seem to indicate that those models may overestimate oxygen abundances by ∼ 0.2-0.5 dex compared to the metallicity derived using the so-called direct T e method (see e.g., Kennicutt et al. 2003;Yin et al. 2007). On the other hand, other works (see e.g., López-Sánchez et al. 2012;Nicholls et al. 2012) found that the oxygen abundances determined using temperatures derived from collisional-excited lines could be underestimated by ∼ 0.2-0.3 dex. In principle, the simulations should be independent of these models and therefore the curves derived in this work from simulations should not be affected by this issue.

The Z th ∼ 0.7 Z � threshold should not be considered, therefore, as an absolute value. Nonetheless, to be in agreement with the metallicities (Z≤ 0.2 Z � ) needed in most LGRB single massive star progenitor models, all the metallicities presented here should be systematically overestimated, most of them by at least ∼ 0.5 dex.

  Fig. 1. Top panel: MZ plot. The dots correspond to the host galaxies of the Swift/BAT6 sample of LGRBs at z < 2, color coded depending on their redshift as shown in the right bar. The lines correspond to the relations found for field galaxies at the redshift indicated next to each line. Bottom panel: The FMR plane. The dots correspond to the host galaxies of the Swift/BAT6 sample of LGRBs at z < 2, color coded depending on their redshift as shown in the right bar. The gray line corresponds to the FMR found byMannucci et al. (2010Mannucci et al. ( , 2011)). The dark blue curve and area correspond to FMR relation and of its quartiles obtained using the simulation ofCampisi et al. (2011). The cyan curve and area correspond to the best-fit model results.

Table 1 .

 1 Swift/BAT6 sample of LGRB host galaxies at 1 < z < 2 with metallicity determination, visible from the southern hemisphere.

	Host galaxy GRB080413B GRB090926B GRB061007 * GRB061121 * GRB071117 * GRB100615A GRB070306 GRB060306 GRB080605 GRB080602 GRB060814	redshift 1.1012 1.2427 1.2623 1.3160 1.3293 1.3979 1.4965 1.5597 1.6408 1.8204 1.9223	Log(M � /M � ) 9.3 10.28 9.22 10.31 < 10.12 9.27 10.53 10.5 10.53 9.99 10.82	SFR [M � yr -1 ] 2.1 +3.1 -1.2 26 +19 -11 5.8 +4.8 -4.8 44.2 +19 -10 > 2.8 8.6 +13.9 -4.4 101 +24 -18 17.6 +83.6 -11 47.0 +17 -12 125.0 +145 -65 54.0 +89 -19	Metallicity 12 + log(O/H) 8.4 +0.2 -0.2 8.44 +0.18 -0.20 8.16 +0.18 -0.13 8.5 +0.09 -0.06 8.4 +0.15 -0.09 8.14 +0.26 -0.22 8.45 +0.08 -0.08 9.12 +0.18 -0.42 8.46 +0.08 -0.08 8.56 +0.2 -0.3 8.38 +0.14 -0.28

Table 2 .

 2 Observed AB magnitudes (corrected by the Milky Way extinction) of GRB 071117 host galaxy.

	Host galaxy GRB 071117	g 24.4 ± 0.1	r 24.7 ± 0.2	i 24.8 ± 0.3	z > 24.4	K 22.9 ± 0.2

Acknowledgements. This work is based in part on observations made with the Spitzer Space Telescope (programs 90062 and 11116), which is operated by the Jet Propulsion Laboratory, California Institute of Technology under a contract with NASA. SDV thanks M. Rodrigues, H. Flores and F. Hammer for useful discussions. JJ acknowledges financial contribution from the grant PRIN MIUR 2012 201278X4FL 002. TK acknowledges support from a Sofja Kovalevskaja Award to Patricia Schady. We thanks G. Cupani for sharing his expertise on Xshooter data reduction.