Caroline Pereira

Jean-Sébastien Baumann

Vincent Humbot

Céline Falentin-Daudré
email: falentin-daudre@univ-paris13.fr

Biological properties of direct grafting by ultraviolet irradiation of vinyl benzyl phosphonic acid onto titanium surfaces

Keywords: Titanium, Poly(vinyl benzyl phosphonic acid) grafting, Osseointegration, bioactive, UV

Titanium (Ti) and its alloys are the most effective metals for structural implantable device applications. However, Ti-based materials are passively integrated into the bone, resulting in a purely mechanical attachment. Consequently, the loss of osseointegration often leads to implant failure. Therefore, enhancing bone formation surrounding the implant is primordial. In previous investigations conducted in our laboratory, grafting bioactive polymers with sulfonate groups, such as poly(sodium styrene sulfonate) (polyNaSS), was demonstrated to increase the adherence and differentiation of osteoblast cells. In this context, this contribution proposes to functionalize Ti with a phosphonic acid-based polymer, poly(vinyl benzyl phosphonic acid) (poly(VBP)). A two-step UV-initiated grafting polymerization was developed to covalently graft VBP into Ti surfaces. The surfaces were characterized using colorimetry, Fourier-transformed infrared spectra recorded in an attenuated total reflection (FTIR-ATR), X-ray photoelectron spectroscopy (XPS) techniques, and water contact angle (WCA) measurements. The Ti substrates were evaluated for cell viability, spreading, alkaline phosphatase activity, and calcium formation using MC3T3-E1 osteoblast cells. The interaction of Ti grafted samples with osteoblast cells, was significantly improved as well as the cell/surface interaction. Together, these findings demonstrated that poly(VBP) grafted on Ti surfaces improved osteoblasts' early cell adhesion and spreading activities, crucial for osseointegration applications.

Introduction

Titanium (Ti) and its alloys, employed in the biomedical field since the early 1950s [START_REF] Brånemark | Intra-Osseous Anchorage of Dental Prostheses:I[END_REF], have demonstrated the success of their mechanically-compatible properties as biomedical devices. However, despite their biocompatibility, titanium implants are not optimally osseointegrated, and an inflammatory process resulting in fibrous capsule development can ensue. As a result, implants are encased by fibrous tissue, isolating them from the bone tissue and causing implant loosening [START_REF] Esposito | Biological factors contributing to failures of osseointegrated oral implants[END_REF][START_REF] Roos-Jansaker | Treatment of peri-implant infections: a literature review[END_REF].

The success of implant surgery is primarily based on the complex osseointegration mechanisms involving both mechanical and biochemical aspects occurring at the contact between the bone tissue and the implant surface. The effectiveness of biomaterial placement is closely linked to the osseointegration phenomenon that occurs when the bone tissue adjusts to the presence of the implant through the bone remodeling mechanisms. Therefore, an exemplary bone/implant interface would consist of bone merged with the implant surface without a fibrous connective tissue layer in between.

To improve the bone uptake of the implant, researchers have developed different strategies. Modifications of the morphological surface properties (roughness, porosity) using various methods, essentially mechanical treatments, allow an essential range of roughness and textures that promote bone bonding [START_REF] Ellingsen | Surface configurations of dental implants[END_REF]. Other approaches have been used to improve osseointegration, including physical modifications (thermal treatment, plasma treatment) [START_REF] Park | Surface characteristics and bioactivity of oxide film on titanium metal formed by thermal oxidation[END_REF][START_REF] Saldaña | Thermal oxidation enhances early interactions between human osteoblasts and alumina blasted Ti6Al4V alloy[END_REF][START_REF] Carradó | Structural, Microstructural, and Residual Stress Investigations of Plasma-Sprayed Hydroxyapatite on Ti-6Al-4 V[END_REF], chemical modifications (silane treatments, protein coating) [START_REF] Liu | Biomimetic coprecipitation of calcium phosphate and bovine serum albumin on titanium alloy[END_REF], and electrochemical treatments (anodic oxidation) [START_REF] Sul | Characteristics of the surface oxides on turned and electrochemically oxidized pure titanium implants up to dielectric breakdown: J o u r n a l P r e -p r o o f the oxide thickness, micropore configurations, surface roughness, crystal structure and chemical composition[END_REF][START_REF] Le Guéhennec | Surface treatments of titanium dental implants for rapid osseointegration[END_REF]. Moreover, to improve osteoblast responses and bone-implant integration, chemical modifications were made to directly functionalize Ti surfaces with various chemical groups, like hydroxyl [START_REF] Park | Surface characteristics and bioactivity of oxide film on titanium metal formed by thermal oxidation[END_REF], carboxyl [START_REF] Michiardi | Bioactive polymer grafting onto titanium alloy surfaces[END_REF], sulfonic groups [START_REF] Chouirfa | Grafting of architecture controlled poly(styrene sodium sulfonate) onto titanium surfaces using bio-adhesive molecules: Surface characterization and biological properties[END_REF], and phosphate groups [START_REF] Adden | Phosphonic Acid Monolayers for Binding of Bioactive Molecules to Titanium Surfaces[END_REF]. As a result of our prior research, polymers bearing ionic chemical functions have been found to modulate cell attachment and spreading [START_REF] Khadali | Modulating Fibroblast Cell Proliferation with Functionalized Poly(methyl methacrylate) Based Copolymers: Chemical Composition and Monomer Distribution Effect[END_REF][START_REF] Anagnostou | Osteoblast functions on functionalized PMMA-based polymers exhibiting Staphylococcus aureus adhesion inhibition[END_REF]. The arrangement of these ionic groups throughout the polymer chains affords effective sites that operate with extracellular proteins vital in cell response, such as fibronectin [START_REF] Felgueiras | Contribution of fibronectin and vitronectin to the adhesion and morphology of MC3T3-E1 osteoblastic cells to poly(NaSS) grafted Ti6Al4V[END_REF]. Despite the effectiveness of the approaches described above, numerous limitations remain, such as poor coating-to-substrate adhesion and sophisticated and time-consuming chemical processes. Grafting polymerization induced by ultraviolet (UV) light is an easy, quick, and low-cost approach for introducing desired functional groups to the surfaces of materials without changing their bulk characteristics. Our laboratory has developed a direct UV grafting process of a bioactive monomer such as sodium styrene sulfonate (NaSS) [START_REF] Chouirfa | Grafting bioactive polymers onto titanium implants by UV irradiation[END_REF]. By activating the Ti surface with hydroxyl, it is possible to generate active radicals under UV irradiation. The polymerization of functional monomers is induced by radical species, resulting in bioactive groups on the surface of Ti.

One interesting molecule to graft is a phosphonic acid-based monomer. Studies have shown that titanium and polymers functionalized with phosphate groups (-PO 3 H 2 ) exhibit enhanced bioactivity due to their bone-like apatite-induced ability [START_REF] Datta | Electrospun nanofibers of a phosphorylated polymer-A bioinspired approach for bone graft applications[END_REF][START_REF] Viornery | Surface Modification of Titanium with Phosphonic Acid To Improve Bone Bonding: Characterization by XPS and ToF-SIMS[END_REF][START_REF] Viornery | Osteoblast culture on polished titanium disks modified with phosphonic acids: Osteoblast Culture on Polished Titanium[END_REF]. In recent years, vinyl phosphonic acid was used as a coating to manufacture bone graft replacements [START_REF] Dey | Poly(vinylphosphonic acid-co -acrylic acid) hydrogels: The effect of copolymer composition on osteoblast adhesion and proliferation: PVPA-co -AA HYDROGELS[END_REF][START_REF] Dey | Synthesis and Characterization of Poly(vinylphosphonic acid-co -acrylic acid) Copolymers for Application in Bone Tissue Scaffolds[END_REF] and functionalize bone scaffolds [START_REF] López-Pérez | Plasma-induced polymerization as a tool for surface functionalization of polymer scaffolds for bone tissue engineering: An in vitro study[END_REF] to stimulate regenerating bones. These findings suggested the beneficial influence of phosphonate groups on implant osseointegration.

The current work described a method for improving bone-implant integration by functionalizing Ti with a phosphonic-acid-based monomer: vinyl benzyl phosphonate (VBP). This study used a two-step UV-induced graft polymerization of VBP to functionalize Ti surfaces with phosphonate groups. Ti oxidation produces peroxides on the surface, which generate radicals when exposed to UV light, initiating the monomer VBP polymerization. Various factors for optimizing poly(VBP) grafting on titanium substrates were investigated. The surface grafting effect was chemically characterized. Furthermore, in vitro investigations on MC3T3-E1 osteoblast cells were assessed to evaluate how the grafted Ti surfaces influenced cell adherence, spreading, and differentiation.

Materials and Methods

Materials

Grade 2, commercially pure Ti disks of 10 mm diameter and ≈ 1 mm thickness purchased from Goodfellow (supplier set in Lille, France) were used as raw surfaces. Both sides of the Ti were polished with 500 and 1200 grit SiC papers.

(4-Vinylbenzyl)phosphonic acid (VBP) was supplied by Specific Polymers (supplier set in Montpellier France) and stored at 4°C. The monomer was used without further purification, as it is provided without polymerization inhibitor.

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Recrystallization in a combination of ethanol/water (Carlo Erba) (10: 90 v/v) overnight at 70°C was used to purify sodium styrene sulfonate (NaSS, Sigma). The product was then dried overnight at 50°C under air pressure and kept at 4°C as previously described [START_REF] Chouirfa | Grafting bioactive polymers onto titanium implants by UV irradiation[END_REF][START_REF] Helary | A new approach to graft bioactive polymer on titanium implants: Improvement of MG 63 cell differentiation onto this coating[END_REF]26].

2,2'-azobis(2-methylpropionitrile) (AIBN) was refined by the same recrystallization described above for 1h at 30°C. The purified AIBN was vacuum-dried at 30°C and stored at 4°C.

The solvents used for the cleaning treatment, activation procedure and functionalization of titanium surfaces were used without further purification.

Methods

Cleaning treatment of titanium substrates

The surfaces were ultrasonically cleaned for 15 minutes following the polishing procedure with different solvents: acetone, cyclohexane, isopropanol, and distilled water (dH 2 O). After that, Ti surfaces were then stripped for 1 min with stirring in Kroll's reagent (2% hydrofluoric acid (Sigma) ; 10% HNO 3 (Acros organics) and 88% dH 2 O), followed by 15 min of ultrasonic washing in five successive distilled water baths. Ti substrates were then dried in an oven at 50°C.

Activation procedure of titanium substrates

Briefly, Ti substrates were chemically oxidized by immersion with an acidic solution (H 2 SO 4 /H 2 O 2 50:50 v/v, Sigma) to activate the surfaces with hydroxide and peroxide groups. Then, the Ti surfaces were rinsed with dH 2 O to remove any traces of the Piranha solution.

Grafting procedure of VBP onto titanium surfaces

A 1 M VBP solution in dimethyl sulfoxide (DMSO) was poured into a flask with a circular bottom that contains 2% of 2,2'-azobis(2-methylpropionitrile) (AIBN). After degassing the solution with argon for 30 minutes, the oxidized Ti substrates were quickly added to the solution. The Ti surfaces immersed in the VBP/DMSO solution containing 2% AIBN were then irradiated with UV radiation (365 nm, 160 mW/cm 2 with a power of 38%) at ambient temperature for 2h while stirring (Fig. 1). The grafted surfaces were then washed with a mixture of DMSO/methanol (50:50 v/v, Sigma). Then the surfaces were washed with methanol for 48h to remove nonreacted monomers and non-grafted homopolymers. Finally, the poly(VBP) grafted Ti surfaces were dried overnight at 37°C.

Under normal temperature and pressure conditions, UV sources for the grafting technique come from an LED lamp (Omnicure) with a produced power ranging from 200 W to 500 W at 365 nm.

Surface characterization

Fourier-transformed infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), water contact angle measurements (WCA), and the toluidine blue (TB) colorimetric technique were used to assess the existence of poly(VBP) on grafted Ti surfaces.

In attenuated total reflectance (ATR) mode, FTIR spectra were collected between 4000 and 600 cm -1 . Ti disks were pushed equally against a diamond crystal (4000-500 cm -1 ) (512 scans). After that, each spectrum was fitted and examined.

Under ultra-high vacuum circumstances (≤10-10 Torr), photoelectrons emission was studied at a 90° take-off angle. The chemical contents of the surface were analyzed using XPS (from the Omicron Argus spectrometer in Taunusstein, Germany) with a monochromated Alkα radiation source (hv = 1486.6 eV) and a 300 W electron beam. The survey spectrum was captured with a pass energy of 100 eV and the high-resolution

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areas with 20 eV. The C1s binding energy of aliphatic carbon atoms, 284.8 eV, was used to calibrate the binding energies. Casa XPS v.2.5.15 software (Casa Software Ldt, UK) was used to profile high-resolution spectra, and the resultant peaks regions were utilized to compute the elemental composition.

The wettability of the samples was measured after each surface treatment phase by measuring WCA with one drop (2 µl) of dH2O using a Kruss DAS10 goniometer at ambient temperature. Six samples from each group were examined, with three measurements made on each to determine the average contact angle 20 seconds after the water drop made contact with the surface. The contact angle was evaluated using the DSA drop shape analysis tool after photos of the droplet were obtained.

The TB colorimetric technique was used to evaluate the quantity of poly(VBP) grafted on Ti surfaces, following the methodology established by Helary et al [START_REF] Helary | A new approach to graft bioactive polymer on titanium implants: Improvement of MG 63 cell differentiation onto this coating[END_REF]. To facilitate the complexation of TB with the PO 3 2groups from the polymer, Ti surfaces were submerged in a TB aqueous solution (5 x 10 -4 M, pH = 10) at 30°C for 6 hours. To remove the non-complexed dye, the surfaces were washed with 5 x 10 -3 M sodium hydroxide in dH2O. A combination of acetic acid/dH2O (50/50 v/V Sigma) was used to de-complex the disks for 24 hours. Finally, the concentration of de-complexed TB was determined using a Perkin-Elmer Lambda 25 spectrometer using visible spectroscopy at 633 nm. Three Ti samples of each condition (non-grafted, oxidized, poly(NaSS) grafted, and poly(VBP) grafted) were used per analysis.

Preparation of titanium disks for biological assays

All substrates (ungrafted, oxidized, poly(NaSS), and poly(VBP) grafted) were cleaned and sterilized before interaction with the osteoblast cells. Ti disks were washed three times for three hours in 1.5 M sodium chloride (NaCl, Fisher), 0.15 M sodium chloride, pure water, and phosphate-buffered saline solution, respectively (PBS, Gibco). For 15 min, each side of the Ti substrates was sterilized using an ultraviolet germicidal lamp (UV, 30 W, 254 nm). The samples were kept overnight on well-plates with Gibco's Dulbecco's Modified Eagle's Medium (DMEM). Finally, the samples were put in wells with enriched DMEM medium (1% penicillin, 1% glutamine, 10% fetal bovine serum) and kept until the experiments began.

Osteoblastic cell culture

All tests were carried out using MC3T3-E1 cells procured from the American Type Culture Collection (ATCC). The cells were cultivation was based on the protocol established by Felgueiras et al [START_REF] Felgueiras | Contribution of fibronectin and vitronectin to the adhesion and morphology of MC3T3-E1 osteoblastic cells to poly(NaSS) grafted Ti6Al4V[END_REF]. The cell culture media were replaced twice a week. All investigations employed just early passage (P2-P6) cells. To separate the cells, trypsin-EDTA (Gibco) was utilized. All studies used a cell loading dose of 1 x 10 5 cells/ml, with 1 ml of cell suspension introduced onto each Ti surface kept in a 24-well tissue culture polystyrene plate. Analysis of variances (ANOVA) was used to analyze quantitative data.

Cell viability

A MTT (3-(4,5-dimethyl thiazolyl-2) -2,5-diphenyltetrazolium bromide) test was used to determine the vitality of the cells. The Ti substrates were first seeded for 24 hours. The samples were then transferred to a fresh 24-well plate and washed twice with 1 ml of PBS per well after the culture. The wells were then filled with 50 µl of 5 mg/ml MTT in a DMEM medium without phenol red. The samples were incubated at 37°C with 5% CO 2 for 4 hours. After incubation, all of the solutions were withdrawn from the wells, and the formazan crystals were dissolved in 1 ml of DMSO for 10 min during orbital stirring. A UV-Vis reader was used to measure the solution's absorbance (PerkinElmer lambda 25).

Cellular spreading and morphology

Environmental scanning electron microscopy pictures (ESEM -Hitachi TM3000) were used to analyze the cellular morphology on ungrafted, oxidized, poly(NaSS), and poly(VBP) grafted surfaces 1h, 4h, and 24h after incubation.

The Ti surfaces were first cleaned in PBS after being withdrawn from the cultures. The cells were then fixed for 30 minutes at 4°C with 4% formaldehyde (Sigma) in PBS.

Differentiation: Alkaline phosphatase activity

ALP is a precursor to the development of osteoblasts. After 7 and 14 days of culture, its activity was measured after p-nitrophenyl phosphate was converted to p-nitrophenol at 37°C. The enzyme was isolated from cell's membrane for 1 hour under agitation using Triton x 100. The cellular suspension was coupled with 20 mM J o u r n a l P r e -p r o o f p-nitrophenyl phosphate substrate in a 2-amino-2-methyl-propanol buffer in the same quantities and kept at 37°C for 30 minutes. A UV-vis spectrometer set to 405 nm was used to measure the amount of p-nitrophenol generated (Perkin-Elmer). The enzyme activity was normalized to the protein mass and expressed in nmol of pnitrophenol generated per minute (expressed in mg). A commercially available colorimetric assay was used to determine the amount of ALP in the sample (Bio-Rad protein kit assay).

Extracellular matrix mineralization: Calcium production

After 3 and 4 weeks of culture, the mineralization of calcium deposits was assessed. The Ti surfaces and culture medium were removed, washed two times in PBS, and transferred to a fresh well containing 1 ml of 15% (w/v) trichloroacetic acid solution to dissolve calcium-phosphate crystals. To test the calcium concentration, 10 µl of the supernatant was added to 1 ml of arsenazo-III (0.2 mM in PBS) containing calcium reagent after 1 h of continuous gentle shaking. The calcium concentration was estimated by comparing absorbance at 650 nm to a standard linear curve of CaCl 2 in trichloroacetic acid (15% w/v) from 50 to 1000 µg.ml -1 .

Statistical analysis

Each experiment was carried out three times and yielded the same findings. The data were provided as mean standard deviation with n = 9. The standard deviations are indicated by the error bars in the graphs. A one-way analysis of variance (ANOVA) was used to compare grafted and non-grafted samples, followed by a multiple comparison correction using the Tuckey test. A p-value < 0.05 was deemed significant.

Results and Discussion

Functionalization of titanium surfaces with bioactive polymers

Free radical polymerization of vinyl benzyl phosphonic acid, initiated by the formation of radicals generated by titanium peroxide, was used to covalently graft phosphonate groups to titanium surfaces. The yields of the grafting procedure were computed after the grafting density of poly(VBP) was assessed using the TB colorimetric technique. Controls were titanium oxidized (Ti-OH) and UV-exposed samples. Control samples showed no blue staining, indicating that the coloring was attributable only to the dye being de-complexed from the bioactive polymer's monomer units and, as a result, the proof of poly(VBP) grafted on the titanium surface. However, toluidine blue complexation of the VBP monomer or poly(VBP) adsorbed on the titanium surface might generate the staining. To reject this hypothesis, poly(VBP) and VBP monomers at a concentration of 1 M were introduced with Ti-OH samples for 1 day. The polymerization of VBP monomers was induced by UV irradiation of the VBP aqueous solution containing titanium-oxidized samples, resulting in the formation of radicals of peroxide groups (Fig. 1). There was no interaction with the dye beyond this period, confirming that the staining is only due to covalent bonding between the Ti surface and the polymer (results are shown in Fig. 3). To identify the best grafting conditions, variables including polymerization time (UV irradiation exposure duration) and monomer concentration, were studied.

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The quantity of grafted polymers assessed as a function of UV irradiation exposure duration (Fig. 2) for a monomer concentration of 1 M revealed that the greatest value was attained after 2 hours of UV irradiation exposure (160 mW/cm 2 38% power). Hence, this period was chosen for the investigation of additional characteristics. Furthermore, a concentration of 1 M of VBP monomer allows 14.5±2 µg.cm -2 of grafted polymer, whereas a concentration of 0.5 M of VBP monomer produces lower yields (Fig. 3). To achieve high grafting degree yields, all polymerizations were carried out in an inert gas environment with 2 h of UV irradiation and a monomer concentration of 1 M. This study aimed to investigate the potential benefit of a poly(NaSS)-mimicking polymer with a phosphonic acid moiety for improving osseointegration. For this purpose, it was necessary to find the optimal grafting conditions to obtain a good yield.

The exposure time to UV irradiation and the concentration of the monomer were tested to find the best grafting conditions. The colorimetric characterization technique revealed that the grafting of poly(VBP) on titanium surfaces could reach a grafting density of 14.5 µg.cm -2 , a high value compared to the one found in the literature for the grafting of bioactive molecules [START_REF] Porté-Durrieu | Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V: physical characterization and interest towards human osteoprogenitor cells adhesion[END_REF][START_REF] Müller | Influence of surface pretreatment of titanium-and cobalt-based biomaterials on covalent immobilization of fibrillar collagen[END_REF][START_REF] Chollet | RGD peptides grafting onto poly(ethylene terephthalate) with well controlled densities[END_REF]. Therefore, the method used in this study is appealing for industrial application since only 2 stages (surface activation then UV irradiation) are required to achieve a high yield of covalent grafting on titanium implants.

Chemical characterization of grafted titanium surfaces with poly(VBP)

FTIR, XPS, and WCA were used to verify the presence of poly(VBP) on grafted surfaces. First, FTIR-ATR spectra of grafted titanium revealed the existence of phosphonate groups' distinctive bands: the P=O stretching vibration mode at 1264 cm -1 , P-O-C stretching at 985 cm -1 , all related to the -PO(CH 2 ) group, and C=C stretching at 1509 cm -1 of the phenyl group (Fig. 4). The absorption at 3050 cm -1 and 2915 cm -1 were attributed to C-H streatching in CH 2 . The vibrational studies of organic and phosphonate compounds were used to assign these bands [START_REF] Nakanishi | Infrared absorption spectroscopy[END_REF][START_REF] Danilich | Infrared study of perfluorovinylphosphonic acid, perfluoroallylphosphonic acid, and pentafluoroallyldiethylphosphonate[END_REF][START_REF] Popa | Synthesis and Characterization of Phosphonate Ester/Phosphonic Acid Grafted Styrene-Divinylbenzene Copolymer Microbeads and Their Utility in Adsorption of Divalent Metal Ions in Aqueous Solutions[END_REF][START_REF] Yamabe | Novel phosphonated perfluorocarbon polymers[END_REF].

The elimination of the signal owing to the C=C stretching vibration mode of the vinyl group of the monomer was the most convincing proof of polymer formation (Fig. 4). No specific poly(VBP) peaks were detected on non-grafted Ti surfaces and Ti oxidized samples. The peak observed on non-grafted Ti at 1100 cm -1 is attributed to the presence of a thin oxygen layer on the Ti surface. This observation is in agreement with XPS measurements, as shown in Figure 5 and Table 1. XPS studies were performed on materials under various settings: non grafted Ti (Ti NG), oxidized Ti (Ti-OH) and poly(VBP) grafted Ti (Ti-poly(VBP)). Table 1 summarizes the atomic composition determined by XPS measurements. A thin oxide layer of TiO 2 naturally occurs on the non-treated Ti surface, as shown in Figure 5 by the presence of oxygen (O1s) at 530 eV; in addition, adventitious carbonaceous contamination is also observed by the presence of C1s signal at 285 eV. As a result, Ti2p (458 eV), O1s (530 eV), C1s (285 eV) are components classically seen on titanium surfaces [START_REF] Viornery | Surface Modification of Titanium with Phosphonic Acid To Improve Bone Bonding: Characterization by XPS and ToF-SIMS[END_REF].

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After activation, less carbon contamination was observed at 289.3 eV, Figure 6 (a) on the activated titanium surfaces with a more intense contribution at 285.4 eV. In addition, on the oxygen region, Figure 6 (b), a very intense peak observed at 532.3 eV, assigned to hydroxyls and oxygen radicals, confirmed the activation of the titanium surface.

The titanium surface grafted with poly(VBP) causes a rise in carbon (by 2.17) and an oxygen reduction. The polymer has a high carbon and low oxygen content, which accounts for the observed fluctuation. The primary marker of poly(VBP) is the appearance of the P2p peak at 132 eV, clearly confirming the successful grafting of poly(VBP) at the titanium surface. Moreover, after the poly(VBP) grafting, the titanium substrate is hardly distinguishable, suggesting a polymer equivalent thickness around 10 nm. This conclusion was made by observing the results of the XPS measurements in Table 1. The Ti2p peak (458 eV) decreases with grafting of poly(VBP) (18.7 at. % on ungrafted Ti samples and 0.6 at. % on grafted Ti samples), the titanium substrate becomes hardly visible. To investigate the surface's wettability, WCA was measured on polished Ti, stripped Ti, oxidized Ti, and poly(VBP) grafted Ti (Fig. 7). The hydrophilic nature of the surface is enhanced by the production of Ti-OH (contact angle around 25°) in the first step and the presence of poly(VBP) in the second phase (contact angle around 42°). After the grafting procedure, the Ti surface becomes more hydrophilic compared to a stripped Ti surface with a contact angle of around 66°. After validating the grafting protocol, the effective presence of the polymer was verified by FTIR-ATR, XPS, and WCA analysis. XPS analysis revealed that the poly(VBP) grafted titanium surfaces have a significant atomic percentage of phosphorus (6.2 at. %). Furthermore, compared to the non-functionalized titanium, the grafted surface becomes more hydrophilic (with a contact angle value that decreases by 20°). This value is consistent with the literature's water contact angle measurements results obtained for phosphonic acid [START_REF] Danahy | Self-assembled Monolayers of α,ω-Diphosphonic Acids on Ti Enable Complete or Spatially Controlled Surface Derivatization[END_REF]. The hydrophilic/hydrophobic balance of a surface influences cellular spreading at the cells/implant contact, with hydrophilic surfaces having a beneficial influence on cell adherence [START_REF] Feng | Characterization of surface oxide films on titanium and bioactivity[END_REF][START_REF] Zhao | High surface energy enhances cell response to titanium substrate microstructure[END_REF].

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Cell viability

The percentage of viable cells was determined by MTT assay after 1 day of MC3T3-E1 cells culture (Fig. 8). All Ti surfaces revealed at least 70% of viable cells during the entire assay and were therefore considered non-cytotoxic. These findings demonstrate that functionalized Ti surfaces do not affect the viable cell rate of MC3T3-E1. Moreover, the value found for poly(VBP) grafted Ti surfaces was substantially (p=0.01) greater than the non-grafted Ti and the oxidized Ti surfaces, indicating a favorable outcome of poly(VBP) on the osteoblast cell response. Furthermore, when comparing poly(NaSS) grafted surfaces (112±6% viable osteoblast cells) and poly(VBP) grafted surfaces (118±4.2% viable osteoblast cells), it is interesting to note a slightly better cellular response for the surfaces grafted with the phospho nic acidbased polymer J o u r n a l P r e -p r o o f Fig. 8. Percentage of viable osteoblast cells over non-grafted titanium, oxidized, poly(VBP), and poly(NaSS) grafted titanium after 24 h incubation.

Cellular spreading and morphology

Cell spreading and morphology analyses were performed by environmental scanning electron microscopy (SEM) to take a broad look at the morphology of osteoblast cells. MC3T3-E1 osteoblast cells were cultivated on non-grafted Ti, oxidized Ti (Ti-OH), poly(NaSS), and poly(VBP) grafted Ti surfaces during 1 h, 4 h, and 24 h (Fig. 9).

After 1 h of culture, SEM images have shown a considerable variation of the MC3T3-E1 osteoblast cells spreading between Ti surfaces. Osteoblast cells were more confluent on grafted surfaces than on non-grafted surfaces. For the same magnification, the nucleus of the cells was more prominent on the grafted surfaces.

After 4 h of culture, osteoblast cells were connecting with a flattened morphology with lamellipodia extensions on oxidized and grafted surfaces. However, on ungrafted surfaces, the cell expansion was very restrained, with most cells exhibiting a round shape, and no evidence of interconnection between the cells was observed. Osteoblasts cultivated on poly(VBP) grafted surfaces elongate their cytoplasmic across the whole material surface, revealing a solid adhesion capacity of MC3T3-E1 osteoblast to the presence of poly(VBP) onto the surfaces.

After 1 day of culture, osteoblast cells' morphology onto non-grafted surfaces has a more extended and flattened form than the polygonal shape observed on oxidized and grafted surfaces. In addition, it was immediately apparent that the amount of cells was considerably higher on the poly(VBP) grafted surfaces. This observation is consistent with the cell viability experiment result depicted in Fig. 8. ImageJ software was used to calculate the surface area covered by the cells after 24 h of culture in each condition. This quantification was done on three separate zones of each sample, and it was discovered that the J o u r n a l P r e -p r o o f grafted samples were considerably (p=0.01) more covered than the non-grafted (26 ± 9% surface covered) and oxidized surfaces (29 ± 5%). Furthermore, when compared to surfaces grafted with poly(NaSS) (44 ± 6%), the proportion of osteoblast cells covered on poly(VBP) grafted surfaces (66 ± 4.5%) was more significant (Fig. 10). As a result, adherent cell populations on poly(VBP) grafted surfaces are strikingly improved compared to nongrafted surfaces. The poly(VBP) grafted surfaces promote osteoblast adhesion and spreading, resulting in favorable conditions for future cell growth. The primary goal of this research was to illustrate the impact of poly(VBP) grafted Ti surfaces on in vitro osteogenic properties. In this context, the bioactivity of the chemically modified Ti surfaces grafted with phosphonic acid-based compounds was studied. For this purpose, cell viability, osteoblast cells' morphology, and spreading through SEM and MTT were performed on non-treated titanium and functionalized titanium surfaces. Furthermore, to account for the bioactivity of both bioactive polymers, the grafting of poly(VBP) was compared to the grafting of poly(NaSS). The UV "grafting from" method published by Chouirfa et al [START_REF] Chouirfa | Grafting bioactive polymers onto titanium implants by UV irradiation[END_REF]26] was used to graft poly(NaSS) onto Ti surfaces. The high values of viable cell densities, improvement of cell morphology, and spreading on poly(VBP) grafted surfaces suggested no cellular toxicity and an enhancement in osteoblast cells' growth. Moreover, after 1 h of culture, SEM images revealed the formation of microcellular extensions called filopodia and adherence of the cells to each other in poly(VBP) grafted Ti substrates. The improved cell spreading could be related to the enhanced hydrophilicity of the surface due to the surface-grafting of phosphonate groups.

Differentiation and mineralization

ALP is a primary indicator of osteoblast differentiation [START_REF] Helary | A new approach to graft bioactive polymer on titanium implants: Improvement of MG 63 cell differentiation onto this coating[END_REF]. The effect of untreated and functionalized Ti surfaces on osteoblast cell development was initially investigated using ALP activity measurements from 7 to 14 days of culture. Results (Fig. 11) showed that ALP concentration was much improved by the grafting of poly(NaSS) and poly(VBP), with maximum concentration found at day 14, which corresponded to the beginning of the extracellular matrix (ECM) development. Before day 14, ALP production was lower because proliferation was dominant. ALP reduced in result of cell maturation's ECM mineralization phase with the apparition of calcium nodules in the second stage of differentiation [START_REF] Martin | Proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63) cultured on previously used titanium surfaces[END_REF]. For the poly(VBP) grafted surfaces, the ALP concentration started to decrease from day 14, showing the beginning of the mineralization phase. Ti surfaces were therefore evaluated for calcium content after 21 and 28 days of culture (Fig. 11). In accord with ALP results, surfaces grafted with poly(NaSS) and poly(VBP) presented statistically superior mineralization than the non-grafted and oxidized surfaces. The difference was even more pronounced after 4 weeks, indicating that the osteoblasts had matured significantly on the poly(VBP) grafted Ti surfaces. These findings revealed that grafting a titanium sample by poly(VBP) promotes osteoblastic differentiation of MC3T3-E1 cells.

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Cell differentiation ability in contact with the implant is crucial for bone regeneration. ALP activity and ECM mineralization were assessed quantitatively to examine osteogenic differentiation of MC3T3-E1 in vitro. Poly(VBP) grafting has been demonstrated to promote osteoblast differentiation. Together, these outcomes showed that phosphonate groups grafted on Ti surfaces enhanced osteoblasts' early cell adhesion and spreading processes. In previous investigations, phosphate groups have been shown to promote osteoblast adhesion, spreading, proliferation, and osteogenic differentiation [START_REF] Zheng | Immobilization of alendronate on titanium via its different functional groups and the subsequent effects on cell functions[END_REF][START_REF] Dadsetan | Incorporation of phosphate group modulates bone cell attachment and differentiation on oligo(polyethylene glycol) fumarate hydrogel[END_REF][START_REF] Nakaoka | Effects of surface chemistry prepared by self-assembled monolayers on osteoblast behavior[END_REF]. One explanation is that phosphate groups are deprotonated under physiological circumstances, which improves calcium ion chelation, a desirable property for transducing osteogenic promptings [START_REF] Wagner | Osteogenic differentiation capacity of human mesenchymal stromal cells in response to extracellular calcium with special regard to connexin 43[END_REF][START_REF] Mellor | Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source[END_REF]. As a result, phosphonic acid's strong affinity for calcium ions has prompted biological uses [START_REF] Sevrain | Phosphonic acid: preparation and applications[END_REF].

Furthermore, osteoblast attachment and spreading are affected by adsorbed fibronectin (Fn), and vitronectin (Vn) on biomaterial surfaces [START_REF] Felgueiras | Contribution of fibronectin and vitronectin to the adhesion and morphology of MC3T3-E1 osteoblastic cells to poly(NaSS) grafted Ti6Al4V[END_REF]. Fn and Vn become more stable in the presence of poly(NaSS), facilitating cell adhesion and growth [START_REF] Mosher | Fibronectin: Review of its Structure and Possible Functions[END_REF][START_REF] Potts | Structure and function of fibronectin modules[END_REF]. Although the hypothesis mentioned above partially explains why osteoblast responses are improved on phosphonic acid-based surfaces, follow-up research should be conducted to understand further the protein cells' interaction with poly(VBP).

Conclusion

A UV-initiated grafting polymerization technique in a two-step process was successfully developed to graft phosphonate polymers. FTIR-ATR, WCA and XPS data showed that chemically introducing phosphonic acid groups to Ti surfaces was a success. The method developed is easily adaptable to functionalize Ti implants for medical purposes. Moreover, it is possible to extend this process to graft poly(VBP) onto titanium alloys, which are extensively encountered in biomedical equipment. Cell survival, ALP activity, and calcification of MC3T3-E1 osteoblast cells cultured onto the poly(VBP) grafted titanium surfaces revealed increased differentiation of these cells, implying the effect of improved grafted implant osseointegration response. The Ti-metal surface's covalently bonded with phosphonic acid molecules act as a scaffold for new bone growth, allowing the implant to attach to the host tissue. These results indicate that poly(VBP) grafted Ti implants could be used in biological applications.

The next stage of our research will further understand the mechanisms at the atomic and molecular levels involved in cell-protein interaction on grafted titanium and perform in vivo study in a rabbit model.

CRediT authorship contribution statement

Fig. 1 .

 1 Fig. 1. Grafting mechanism of poly(VBP) on titanium surface

Fig. 2 .

 2 Fig. 2. Quantity of grafted polymers measured as a function of UV irradiation exposure time at a concentration of monomer [VBP] =1 M.

Fig. 3 .

 3 Fig. 3. Amount of grafted polymers measured at two different monomer concentrations.

Fig. 4 .

 4 Fig. 4. FTIR/ATR spectra of VBP monomer, poly(VBP) polymer, non grafted titanium, and poly(VBP) grafted on titanium surface.

Figure 5 :

 5 Figure 5: XPS survey spectra of titanium surfaces: non grafted (Ti-NG), oxidized (Ti-OH), and grafted with poly(VBP) (Tipoly(VBP)).

Figure 6 :

 6 Figure 6: High-Resolution XPS data for (a) C1s and (b) O1s.

Fig. 7 .

 7 Fig. 7. Water contact angle measurements on polished Ti, stripped Ti, oxidized and poly(VBP) grafted Ti.

Fig. 9 .

 9 Fig. 9. After 1 h, 4 h, and 24 h incubation, SEM images of osteoblast cells onto the non-grafted, oxidized, poly(NaSS), and poly(VBP) grafted Ti surfaces.

Fig. 10 .

 10 Fig. 10. Mean percentage of non-grafted, oxidized (Ti-OH), poly(VBP) and poly(NaSS) grafted surfaces covered by osteoblast cells after 24 h incubation.

Fig. 11 .

 11 Fig.11. ALP activity (A) and ECM mineralization assay (B) of MC3T3-E1 osteoblast cells onto different titanium surfaces (ungrafted, oxidized, poly(NaSS) and poly(VBP) grafted) after respectively 7 and 14, 21, and 28 days of culture.

  Pereira: Methodology, Investigation, Resources, Validation, Data curation, Formal analysis, Visualization, Writingoriginal draft, Writingreview & editing. Jean-Sébastien Baumann: Resources. Vincent Humblot: Resources, Data curation, Formal analysis, Validation, Writingreview & editing. Céline Falentin-Daudré: Supervision, Conceptualization, Methodology, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Validation, Writingreview & editing.

Table 1 .

 1 Surface composition (at. %) of the various titanium samples as revealed by XPS analysis.

	Element Position (eV) Ti-NG (atomic %) Ti-OH (atomic %) Ti-poly(VBP) (atomic %)
	Ti 2p	458	18.7	12.7	0.6
	C 1s	285	33.5	42.2	72.7
	O 1s	530	47.8	45.1	20.5
	P 2p	132	/	/	6.2

J o u r n a l P r e -p r o o f

Acknowledgments

This research was supported by the French Ministry of National Education, Higher Education, and Research. The authors are grateful to University Sorbonne Paris Nord. CRediT authorship contribution statement Caroline Pereira: Methodology, Investigation, Resources, Validation, Data curation, Formal analysis, Visualization, Writingoriginal draft, Writingreview & editing. Jean-Sébastien Baumann: Resources. Vincent Humblot: Resources, Data curation, Formal analysis, Validation, Writingreview & editing. Céline Falentin-Daudré: Supervision, Conceptualization, Methodology, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Validation, Writingreview & editing.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

J o u r n a l P r e -p r o o f

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Biological effect of direct grafting by ultraviolet irradiation of vinyl benzyl phosphonic acid onto titanium surfaces

Caroline Pereira 1 , Jean-Sébastien Baumann 1 , Vincent Humbot 2 , Céline Falentin-Daudré *Corresponding author. E-mail address: falentin-daudre@univ-paris13.fr (C. Falentin-Daudre).

Highlights:

 An ultraviolet-initiated grafting polymerization of phosphonic acid is developed.

 Surface and biological properties of functionalized titanium are assessed.

 Improvement of osteoblast cells adhesion and spreading processes are presented.

 Enhanced functionalization of titanium surfaces for biomedical applications.

J o u r n a l P r e -p r o o f


David Rabus
email: david.rabus@femto-st.fr

Fanny Lotthammer
email: fanny.lotthammer@femto-st.fr

Joscelyn Degret
email: joscelyn.degret@edu.univ-fcomte.fr

Lilia Arapan
email: lilia.arapan@femto-st.fr

Frank Palmino
email: frank.palmino@univ-fcomte.fr

Jean-Michel Friedt
email: jmfriedt@femto-st.fr

Frédéric Cherioux
email: frederic.cherioux@femto-st.fr

Degradation of Sub-Micrometer Sensitive Polymer Layers of Acoustic Sensors Exposed to Chlorpyrifos Water-Solution

Keywords: SAW, pesticide, sensing layer, aqueous stability

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Introduction

Monitoring the increasing number of pollutants in the water table is an ongoing concern for water treatment systems and water resource managers [START_REF]Sustainable development goals[END_REF]. Pesticides, heavy metals, personal care products, natural toxins, and a host of other organic and inorganic chemical pollutants and their products can increase toxicity in water. Among all in the class of water pollutants, pesticides based on organo-phosphates are one of the main hazardous compounds due to their long-term toxicity. The development of innovative sensors that can identify the presence of chemical pollutants in various types of water without preparation for continuous monitoring, as opposed to sampling as needed for current laboratory methods, is still challenging. [START_REF] Tajik | Recent developpments in polymer nanocomposite-based electrochemical sensors for detecting environmental pollutants[END_REF][START_REF] Chapman | Combining chemometrics and sensors: Toward new applications in monitoring and environmental analysis[END_REF] In that sense, direct detection sensors relying on the measurement of a physical quantity upon reaction of an analyte, such as an acoustic velocity in surface acoustic wave (SAW) sensors, have been developed. [START_REF] Adhikari | Polymer-plasticizer coatings for shear horizontal-surface acoustic wave sensors for long-term monitoring of BTEX analytes in liquid-phase[END_REF][START_REF] Adhikari | Investigation of polymer-plasticizer blends as SH-SAW sensor coatings for detection of benzene in water with high sensitivity and long-term stability[END_REF][START_REF] Rabus | Subsurface H2S detection by a surface acoustic wave passive wireless sensor interrogated with a ground penetrating radar[END_REF][START_REF] Ballantine | Surface acoustic wave devices for chemical analysis[END_REF][START_REF] Lange | Surface acoustic wave biosensors: A review[END_REF][START_REF] Gizeli | Biomolecular Sensors[END_REF][START_REF] Devkota | SAW sensors for chemical vapors and gases[END_REF][START_REF] Go | Surface acoustic wave devices for chemical sensing and microfluidics: A review and perspective[END_REF][START_REF] Caliendo | Advances in SAW-based gas sensors[END_REF] However, in all cases, the sensing physical principle is not selective, and the sensor selectivity is brought by the chemical functionalization of the surface. Polymers offer a variety of sensing layers and can be directly integrated in collective processes of fabrication. [START_REF] Mcquade | Conjugated Polymer-Based Chemical Sensors[END_REF][START_REF] Leibl | Molecularly Imprinted Polymers for Chemical Sensing: A Tutorial Review[END_REF] For instance, polymers such as polyepichlorohydrin (PECH), polyisobutene (PIB), and poly (butyl methacrylate) (PBMA) have been widely used for the development of chemical SAW sensors. [START_REF] Liron | The polymer-coated SAW sensor as a gravimetric sensor[END_REF][START_REF] Pan | Effects of temperature and humidity on the performance of a PECH polymer coated SAW sensor[END_REF][START_REF] Sayago | Surface acoustic wave gas sensors based on polyisobutylene and carbon nanotube composites[END_REF] However, polymers also being chemical compounds, they could react with pollutants, which could affect their stability and finally could modify their sensing ability: despite having been used for BTEX detection, using such sensors in water tables contaminated with pesticides might alter their detection capability as will be discussed here. We investigate the stability of these polymers widely used as sensitive layers for the direct detection of an organophosphate pesticide in water-solution, which is representative of the environmental conditions. Within this objective, we choose chlorpyrifos as model of organophosphate pesticide. [START_REF] Rahman | A comprehensive review on chlorpyrifos toxicity with special reference to endocrine disruption: Evidence of mechanism, exposures and mitigation strategies[END_REF] The detection of chlorpyrifos has already been investigated in gas [START_REF] Xu | Development of a Wireless and Passive SAW-Based Chemical Sensor for Organophosphorous Compound Detection[END_REF][START_REF] Skotadis | A sensing approach for automated and real-time pesticide detection in the scope of smart-farming[END_REF] or aqueous [START_REF] Mensah-Brown | Analysis of the absorption kinetics for the detection of parathion using hybrid organic/inorganic coating on SH-SAW devices in aqueous solution[END_REF] phase by using SAW sensors under controlled conditions.

In this work, we monitor the acoustic response of SAW devices functionalized with PECH, PIB and PBMA thin films exposed to chlorpyrifos water-solution in environmental conditions. Then, we assess the instability of the measurements by considering analytical characterization of the thin films prior and after exposure to chlorpyrifos using Fourier-Transform InfraRed Spectroscopy (FT-IR) and Atomic Force Microscopy (AFM). We demonstrate the irreversible degradation of the thin polymer films exposed to chlorpyrifos in water-solution. This instability of sensitive layers seems to be a strong limitation to the popularization of this kind of acoustic sensors in environmental challenge and water resource management in practical deployment scenarios.

Materials and Methods

Design of Sensors and Active Sensitive Layers

Design of Sensors

Surface Acoustic Wave (SAW) reflective delay lines were manufactured by depositing 200 nm thick aluminum interdigitated electrodes acting as transducer (IDT in Figure 1) and mirrors (M1-4 in Figure 1) on YXl/36° lithium tantalate substrate. The split finger transducer and mirrors generate a shear leaky SAW confined to the surface by metallizing the areas between IDTs with a wavelength of 41 µm with a metallization ratio of 50% and an acoustic aperture of 1600 µm, leading to a center frequency around 100 MHz. Both the IDT and mirrors are apodized, with the mirrors designed for reemission of the incoming wave rather than mechanical reflection: mirrors M1-4 act in a regenerative mode where the incoming wave polarizes each mirror, regenerating a SAW towards the transducer (IDT in Figure 1). The four mirrors were included to define four areas free of transducers acting as sensing areas coated with the same metal to confine the pseudo-shear wave to the surface and coated with polymer for sensing capability. The distance from IDT to M1 is 1700 µm, from IDT to M2 is 3400 µm, from IDT to M3 is 4700 µm, and from IDT to M4 is 6200 µm, leading to echoes typically delayed by about 800 ns from each other depending on the acoustic velocity, which itself depends on the polymer layer thickness, starting at 4200 m/s when no polymer is present.

The four areas are selected with two areas patterned with the same sensing polymer to cancel the impact of the probing electronic unit to sensor range (electromagnetic time of flight cancellation by differential measurement); a third area patterned with a polymer insensitive to the compound to be detected for temperature compensation; and a fourth area used here to assess reproducibility, but which could be used to pattern a second sensing polymer to improve selectivity by inducing a different response to the same compound. Thin polymer films are spin-coated to reach thicknesses between 500 and 1200 nm aimed at confining acoustic energy in a Love mode guided in the polymer (see below). The IDT is wired to an Agilent E5071B network analyzer for probing the sensor acoustic response once every 30s by recording the S11 transfer function on a 20 MHz bandwidtha 20% relative bandwidth selected as greater than the sensor transfer function width determined by the electromechanical coupling coefficient-in order to post-process the spectral domain response to reach the time domain response by inverse Fourier-transform. Echoes are identified as local maxima of the magnitude, and the phase is recorded at the associated time delay found, by design of the reflective SAW delay line, between 0.8 and 2 microseconds (Figure 2).

Active Sensitive Layers

All commercially available starting materials and solvents were purchased from Sig-maAldrich, Fisher and used without further purification. All polymer deposits were performed in a Class-100 clean-room on the Karl Suss RC-8 CT-62 spin-coater. All surface activations were performed on PVA/Tepla Giga BATCH 360 M. The thickness of polymer layers was performed with a stylus profilometer Dektak xt (C9). All layers of polymers were characterized by Fourier-transform Infrared-Spectroscopy (FT-IR) by using a spectrum two FT-IR spectrometer from Perkin Elmer. AFM measurements were performed with a Bruker Icon AFM connected to a Nanoscope V controller. The AFM was employed in peak-force tapping mode using Scanasyst-Air-HPI Bruker probes of nominal stiffness of 0.4 N m -1 and a nominal tip radius of 2 nm.

Deposition of Polymers

Polymers Films for SAW Devices

PBMA (90 mg) was dissolved in toluene (1000 mg) and stirred for 30 min at room temperature. The chip sensor surface (aluminum and lithium tantalate) was activated by plasma (O2, 300 W, 10 min). A volume of 250 µL of polymer solution was then deposited on clean surface by using a plastic pipette. PBMA solution was spread at a speed of 2500 rpm with an acceleration of 800 rpm/sec for 30 s. The polymer layers covering contact pads were removed with a cotton-bud dipped in acetone. Then, an annealing at 100 °C for 2 min give a clear, translucent, and homogenous polymer film with a thickness of 980 nm. PBMA (42 mg) was dissolved in toluene (1000 mg) and stirred for 30 min at room temperature. Silicon wafers (size: 3′′, thickness: 380 µm) were cut into 1.5 cm × 2 cm pieces. Then, they were cleaned in piranha solution for 2 min and rinsed with clean water. Silicon surface was activated by plasma (O2, 300 W, 10 min). A volume of 250 µL of polymer solution was then deposited on clean surface by using a plastic pipette. PBMA solution was spread at a speed of 2500 rpm with an acceleration of 800 rpm/sec for 30 s. Then, an annealing at 100 °C for 2 min give a clear, translucent, and homogenous polymer film with a thickness of 200 nm. We used a thinner silicon substrate and a thinner layer of polymer for FT-IR analysis than for AFM or SAW devices in order to decrease the light absorption.

PBMA Films for FT-IR Analysis

PBMA Films for AFM Analysis

A similar procedure used for AFM sample preparation was then used for FT-IR analysis. Polymer solutions were deposited on silicon wafer parts (1000 µm, 1.5 cm × 1.5 cm). The thickness of resulting polymer layers was 500 nm.

Thickness Measurements

Dektak Thickness measurements were collected by using this set of parameters, with range of 6.5µm, hills and valleys as profile, 0.5 mg for stylus force, and a resolution close to 0.1µm/pt.

Thin scratches were applied to the corner and to the center of the PBMA-coated pieces.

Solution of chlorpyrifos

An amount of 40 mg of chlorpyrifos was dissolved into 40 mL of acetonitrile. The resulting solution was then diluted in 960 mL of pure water.

Results

Investigation of Sensing Capabilities

The sensor was designed for wireless sensing of an analyte in liquid phase: such conditions were simulated in a controlled laboratory environment by flowing solutions in an open-well liquid cell with fluids continuously injected and sucked by a peristaltic pump (Peripro-4HS from WPI) running at 5 mL/min. In order to obtain a homogenous aqueous phase, we used a water solution containing 4% of acetonitrile which strongly promotes the dissolution of chlorpyrifos. This solution is named 'blank' in the following parts. All measurements were simultaneously recorded on three parallel sensors for reproducibility assessment, and each cycle of blank-analyte-blank was repeated to assess the stability of the polymer layer. Phase differences between second and first echo on one hand, and between third and first echo on the other hand, aim at cancelling correlated noise such as fluctuations in the level of liquid in the flow cell. Each cycle was completed when an asymptotic behavior was reached, with kinetics lasting multiple hours as shown in Figure 3. Each exposure to chlorpyrifos exhibits a phase decrease indictive of mass loading usually associated with adsorption or absorption of a molecule in or on the polymer layer. The reaction is observed to be reversible since phase rises upon exposure to the blank.

We observe that the phase variations, decreasing being usually associated with mass loading when absorbing analyte molecules in the polymer matrix and increasing being associated with layer stiffening or desorption, are not consistent with injection steps of the pesticide or blank solutions. Indeed, the phase starts dropping upon exposure to the pesticide prior to rising again after 10 h exposure (Figure 3a). On the other hand, exposure to the blank solution around 20 h does not induce a stabilization of the signal or a rise that would be associated with desorption. The same effect is observed on all three parallel channels (Figures 3a and4a) and consistently by differential analysis of echo 1 with echo 2 (Figure 3b) or echo 1 with echo 3 (Figure 4b), eliminating the risk of experimental setup instability. Magnitude of the insertion losses of echo 2 is shown in Figure 4a to emphasize the degradation of the polymer as visible with the increasing losses after each exposure and cleaning steps. The insertion loss increases of several dB are much larger than those usually observed upon polymer, globular protein, or DNA adsorption, hinting at the in-depth degradation process scattering the acoustic wave. [START_REF] Tsortos | Quantitative Determination of Size and Shape of Surface-Bound DNA Using an Acoustic Wave Sensor[END_REF][START_REF] Samarentsis | Hybrid Sensor Device for Simultaneous Surface Plasmon Resonance and Surface Acoustic Wave Measurements[END_REF][START_REF] Oh | Wireless and Simultaneous Detections of Multiple Bio-Molecules in a Single Sensor Using Love Wave Biosensor[END_REF] The same evolution for phase and amplitude is observed for PECH and PIB polymers (see Figures S1-S2). The lack of reproducibility on the quantitative evolution of the phase from one cycle to another leads to the investigation on the stability of the various polymers upon exposure to chlorpyrifos using analytical chemistry techniques.

Physico-Chemical Analysis of Sensitive Layers

In order to understand the variation and the non-reproducible phase variation of SAW devices, we investigated the morphology of the sensitive polymer layers by AFM. The three polymers were deposited by spin-coating onto a silicon substrate as a model of atomically flat surface. We performed three different AFM mappings on (i) the raw In the case of PBMA layer, the roughness is below 2 nm for a length of 30 microns (RMS roughness: 0.3 nm, Figure 5a). This polymer layer was immersed for 12 h in a wateracetonitrile (96:4). The surface is rougher than before the immersion, with a peak-to-valley depth of 30-40 nm (RMS roughness: 11.3 nm, Figure 5b). After immersion in a water-acetonitrile solution of chlorpyrifos for 12 h, the polymer layer was rinsed for 1 h with an acetonitrile-water solution (96:4). The overall surface is significantly different with respect to the previous observations prior to rinsing: holes are observed on the surface, with depths close to 500 nm, equal to the thickness of the polymer layer, and a width varying from 1 to 10 microns (RMS roughness: 321.0 nm, Figure 5c).

The same characterizations were performed on PIB and PECH layers. AFM topography scans and height profiles show the same appearance of crossing holes after the rinsing with water-acetonitrile solution while the layers are only slightly affected with the three first steps (Figures S3 and S4 in ESI).

FT-IR Spectra

Each polymer layers deposited onto a silicon substrate were investigated by FT-IR at different steps of the previous procedure described for AFM investigations.

The spectrum of PBMA layer (blue line in Figure 6) shows three strong bands centered at 2960, 2874 and 1729 cm -1 , respectively. In the spectra recorded after exposition to water solution of chlorpyrifos (red line) and after rinsing with water-acetonitrile solution (yellow line), the three bands are still observed, and some new bands centered at 1576, 1542 1436, 1412, 1023 and 846 cm -1 , respectively, are clearly identified (Figure 6b). In the case of PIB and PECH layers, similar behaviors are observed. New extra-bands are observed in FT-IR spectra, centered at 1164 and 1025 cm -1 and at 1022 and 967 cm -1 for PIB and PECH respectively, after immersion to chlorpyrifos water solution and rinsing with a water-acetonitrile solution (Figures S5 and S6 in ESI).

Discussion

The phase and amplitude variations of the acoustic sensors upon exposure to a wateracetonitrile solution of chlorpyrifos are not reproductible (See Figures 3 and4). AFM experiments demonstrate that chlorpyrifos is involved in this irreproducibility of acoustic response (Figures 3 and4). Strong fluctuations which cannot be explained by the reversible adsorption of chlorpyrifos on the surface of PBMA, PIB or PECH polymers are observed. This effect is striking when comparing our curves to those obtained in the gas phase, which exhibit excellent reproducibility. [START_REF] Skotadis | A sensing approach for automated and real-time pesticide detection in the scope of smart-farming[END_REF] In addition, other work has been carried out in an aqueous medium but with other polymers and under other conditions. [START_REF] Mensah-Brown | Analysis of the absorption kinetics for the detection of parathion using hybrid organic/inorganic coating on SH-SAW devices in aqueous solution[END_REF] Indeed, these authors used buffer solutions (with phosphate buffer solution (PBS)) at a fixed pH of 6.20 while this work uses an aqueous solution without PBS, but whose pH is varying from 6 to 7, depending on the experimental conditions, representative of water table monitoring conditions. In order to confirm the impact of the buffer in the polymer degradation prevention, we investigated by AFM the evolution of a layer of PBMA exposed to 100 mL of a PBS buffered water solution at pH = 6.2 containing chlorpyrifos (Figure 7). In this case, no hole is observed. The layer before and after exposure to the buffered solution exhibit a similar roughness, on the order of a few nanometers as opposed to the holes penetrating through the polymer layer in the absence of PBS (Figure 5d).

Consequently, we suggest that the degradation of polymer layers could be attributed to local variation of pH due to the presence of chlorpyrifos. Indeed, in aqueous media, chlorpyrifos is hydrolyzed which leads to the formation of 3,4,6-trichloro-2-pyridinol and O,O-diethyl hydrogen thiophosphate. These two molecules are weak acids, which can lead to the formation of H + in water (Figure 8). As the chlorpyrifos is hydrophobic, this compound is poorly soluble in the water-acetonitrile (96:4) solution. Therefore, chlorpyrifos can be mainly adsorbed onto the PBMA thin layer, because PBMA contains alkyl chains promoting van der Waals interaction with the aromatic core of chlorpyrifos. Finally, as chlorpyrifos is mainly located at the water-polymer interface, the formation of H + , due to the hydrolysis of chlorpyrifos, is enhanced at this interface, which leads to the enhancement of acidic degradation of the PBMA layer as observed in the AFM images. In addition, pH can be very different at solid-liquid interface from the solution as it has been reported in the case of polymer-water interface [START_REF] Kasuya | Evaluation of Interfacial pH Using Surface Forces Apparatus Fluorescence Spectroscopy[END_REF]. Figure 9 emphasizes the role of acetonitrile in dissolving the products coming from the acidic hydrolysis of PBMA polymer strands. Indeed, exposition of the PBMA layer to the same pH without acetonitrile leads to surface degradation but shallower holes (a peak-to-valley depth of 20 nm with RMS roughness of 4.8 nm, Figure 9a; a peak-to-valley depth of 40 nm with RMS roughness of 10.3 nm, Figure 9b). Similarly, Figure S7 confirms this conclusion by replacing constant pH solution with constant chlorphyrifos concentration and varying acetonitrile concentration: halving the latter significantly reduces the degraded polymer solubilization capability and hence smaller and shallower holes in the polymer layer are observed. Finally, the degradation of polymer thin layers can be explained by a synergistic effect of a local decrease of pH due to the hydrolysis of chlorpyrifos and the presence of acetonitrile which promotes the dissolution of deteriorated polymer layer.

Conclusions

In the context of developing surface acoustic wave transducers acting as passive, wireless cooperative targets for direct detection water table pollutant sensors, we have identified degradation processes when exposing PBMA, PIB and PECH to chlorpyrifos pesticide dissolved in water with concentration determined by the addition of acetonitrile. The degradation is explained following atomic force microscopy and Fourier-transform InfraRed characterization, and is the cause of unstable sensor response over timespans of several hours of exposure of the sensing area to the pesticide. Our work highlights the important role of deep investigations to determine the stability of polymers in their environmental conditions before they are used as a sensitive layer: the results presented here can be generalized to organophosphate direct detection by thin polymer coated sensors whose detection mechanism is more complex than surface absorption of molecule leading to mass loading as found in the literature. The development of new inert polymers for direct detection water table pollutant sensors is currently investigated.

Figure 1 .

 1 Figure 1. SAW sensor layout. The interdigitated transducer (IDT) is located between four mirrors (M1, M2, M3 and M4) positioned at different distances from the transducer in order to introduce different delays. The contact pads are used for the electrical connection of IDT. M1 acts for the reference measurement. The active areas between IDT and mirrors M1 or M3 and between mirrors M3 and M4 on the one hand, M1 and M2 on the other hand, are used for the deposition of the sensitive layer.

Figure 2 .

 2 Figure 2. Frequency domain (top) and time domain (bottom) characterization of the reflective acoustic delay line used in the experiment depicted in this article. While the frequency domain transfer function indicates the spectral characteristics of the probed device, the four echoes as best viewed in the time domain magnitude (blue, bottom, at 0.8, 1.6, 2.4, and 3.2 µs). Fine acoustic velocity is measured through the phase variation (red) at the delay of maximum magnitude of each echo.

  arg(S11) (rad) |S11| (rad) time (us) frequency (MHz) |S11| (rad)

Figure 3 .

 3 Figure 3. Evolution of (a) the insertion loss (IL) of echo 1 and (b) the phase difference between echo 1 and echo 2 separated by 800 ns. The measurement steps are indicated on the bottom for each chart since all steps were timestamped, with water-acetonitrile mixture (named blank) or water-acetonitrile containing chlorpyrifos solution (named pesticide) with PBMA as sensing layer.

Figure 4 .

 4 Figure 4. Evolution of (a) the insertion loss (IL) of echo 2 and (b) the phase difference between echo 2 and echo 3 separated by 1600 ns. The measurement steps are indicated on the bottom for each chart since all steps were timestamped, with water-acetonitrile mixture (named blank) or wateracetonitrile containing chlorpyrifos solution (named pesticide) with PBMA as sensing layer.

Figure 5 .

 5 Figure 5. AFM topography scans (left) and corresponding height profile along the white line (right) of a silicon surface covered with a layer of PBMA (thickness: 500 nm). (a) Flat surface after deposition of PBMA by spin-coating. (b) The surface is still quite flat (RMS roughness: 11.3 nm) after immersion in water-acetonitrile (96:4) solution for 12 h despite some roughness increase with respect to a). (c) After immersion in a water-acetonitrile solution of chlorpyrifos for 12 h then rinsed for 1 h with an acetonitrile-water solution (96:4), the surface of PBMA layer shows deep (200 nm) and crossing (500 nm) holes.

Figure 6 .

 6 Figure 6. FT-IR spectra of a PBMA layer (thickness: 200 nm) deposited on a silicon substrate. In blue, spectrum of PBMA layer after deposition onto silicon substrate; in red, this layer immerged overnight in a water-acetonitrile solution containing 40 mg/L of chlorpyrifos; and in yellow, this layer washed with water-acetonitrile solution during 1.5 h (a) full extended spectrum; (b) zoom corresponding to 400-1800 cm -1 range of spectrum shown in a).

Figure 7 .

 7 Figure 7. AFM topographies (left) and corresponding height profile along the white line (right) of a silicon surface covered with a spin-coated layer of PBMA (thickness: 500 nm). (a) Flat surface after immersion in aqueous PBS solution (pH = 6.2) for 12 h. (b) The surface roughness after immersion in aqueous PBS solution with chlorpyrifos solution for 12 h. The PBMA layer is not modified by the presence of chlorpyrifos.

Figure 8 .

 8 Figure 8. Hydrolysis of chlorpyrifos leading to 3,4,5-trichloro-2-pyrinol and O,O-diethyl hydrogen thiophosphate. These two molecules are weak acids which can lead to the formation of their corresponding base in water.

Figure 9 .

 9 Figure 9. AFM topographies (left) and corresponding height profile along the white line (right) of a silicon surface covered with a spin-coated layer of PBMA (thickness: 500 nm) after immersion (a) in aqueous solution at pH = 5.0 for 12 h and (b) in water-acetonitrile (98:2) solution at pH = 5.0 for 12 h. The PBMA layer exhibits some holes with a depth of 20 nm and 50 nm in (a,b), respectively.

Funding: This research was funded by French National Research Agency through the project UN-DERGROUND, grant number ANR-17-CE24-0037. We also acknowledge partial support of the French RENATECH network and its FEMTO-ST technological facility.

Data Availability Statement:

The data are available upon reasonable request to the authors

Conflicts of Interest:

The authors declare no conflict of interest