Katia Pérez

Sébastien Leveneur

Fabrice Burel

Julien Legros

Daniela Vuluga

Anionic synthesis and end-functionalization of polymyrcene in a flow microreactor system

Introduction

In the framework of sustainable development over the last decade, the interest of scientists towards bio-sourced materials has become obvious, and terpene derivatives have thus been widely studied to obtain different types of materials. [1][2][START_REF] Hadjichristidis | Macromolecular Engineering: From Precise Synthesis to Macroscopic Materials and Applications[END_REF][START_REF] Monica | [END_REF][5] Due to the presence of several double bonds in their structure, terpenes can easily be considered as monomer substitutes for polybutadiene, a prominent member of the commodity polymers. Among the most interesting monomers of this family, myrcene (owing a isoprene motif) has been polymerized by various pathways, radical, 6,7 anionic, 8 or coordinative, 9 leading to polymyrcene (PMYR) high molar mass (Mn 20000-60000) with low dispersity (1.3<Đ<2.0). [9][10][11] However, the preparation of materials with interesting architectures and properties is highly depending on the controlled size of the polymers (ie oligomeric) that will be embedded. In addition, it is necessary to introduce functional groups on these oligomers to transform them into reactive entities able to be integrated into the materials. Thus, the quest for a flexible and sustainable method for the controlled polymerization of myrcene into oligomers is a tackling challenge.

However, the sustainability of a chemical reaction/process must be considered as a whole; if most of the concerns focus on the substrate source and the reagents/solvents involved the tools used to perform this transformation and the energy consumed are as important as the other parameters. In this sense, miniaturised reactors working in continuous flow have been shown to be invaluable tools to perform reactions with excellent grip on mass and heat transfer, allowing thus to reach high selectivity without cryogenic conditions, sometimes unavailable under classical batch conditions. [12][13][14] Moreover, such devices offer the possibility to handle and treat large volumes of chemicals -even hazardous-without exposing the operator. [15][16][17][18][19][20] Thus, the handling of pyrophoric alkyl lithiums can be easily done in a flow system and very selective and challenging transformations have been reported with incomparable results with regard to conventional batch conditions. [21][22][23][24][25][26][27][28][29] Such organolithium compounds are very useful promoters of anionic polymerization and flow polymerization of reactive petroleum-based olefins (acrylate, styrenes) has been successfully reported by Yoshida and Nagaki. [30][31][32][33][34][35] Along these lines, we now report that the polymerization of myrcene (a biobased analogue of isoprene) can be finely controlled to afford low molar mass PMYR, with the kinetics supported by a predictive model. As a proof of concept for the functionalization of PMYR in an integrated flow device, we also demonstrate that a carboxylic acid moiety can be easily introduced from CO2 (Figure 1). Telescoped polymerization and functionalization with CO2 under flow conditions. For the functionalization of polymyrcene with CO2 a similar system in continuous flow was used as previously mentioned above. However, in the second micromixer, instead of methanol, CO2 gas is introduced by connecting a gas tank to a mass flow controller (Bronkhorst High-Tech BV). This inlet is connected to a second reactor (Reactor 2: ID = 0.75 mm, L = 30 cm) to generate the PMYR functionalized with a carboxyl group, before collection in a vial containing 2 mL of HCl 2 N. The procedure for collection, extraction and analysis of the sample was performed as above.

Results and discussion

This section describes the anionic polymerization of myrcene under batch and flow conditions, in order to get polymyrcene (PMYR) with finely controlled molar mass, and also to introduce a carboxylic acid moiety on PMYR by direct carbon dioxide incorporation.

Polymerization under batch conditions

First experiments were performed under batch conditions in a simple flask under stirring in a thermostated bath at 10 °C as already performed for anionic polymerization. 36 Whereas s-BuLi is mostly used for this purpose, n-BuLi is also efficient but the latter is much more stable in ethereal solvents in a broader range of temperatures, preventing the competitive decomposition of THF via reverse (3+2) cycloaddition when non-cryogenic conditions are applied. 37,38 The results of the polymerization of myrcene into PMYR with n-BuLi (myrcene/n-BuLi = 17:1, providing a theoretical mass of 2316 g/mol) at various scales (from 110 mg to 17 g) at various reaction times are provided in Table 1. Adding 110 mg of myrcene to a n-BuLi /THF solution, allowed to obtain increasing masses of polymer according to reaction time (entries 1-4). Thus, quenching with methanol after 2 min yielded an oligomer with 4 units (M = 650). Increasing the reaction time allowed to gain mass up to 2400 after 30 min, with intermediate values M = 1050 (10 min), 2200 (20 min). It is therefore possible to finely control the polymerization process to reach precise molar mass (Mn) value for tailored made oligomers with a small amount of starting materials. Increasing the amount of myrcene to 1 g afforded M = 970 after only 1 min (entry 5) and a maximum molar mass M= 2330 was obtained after 8 min (entry 8). A significant scale up was finally performed on 17 g of myrcene. In this case, the effect of the scale was tremendous since a single minute reaction allowed to reach M = 2060 (entry 12). Such an effect of the quantity of starting materials on the reaction rate can only be attributed to an (expected) exothermic behavior of the reaction despite the bath thermostated at 10 °C. Thus, the reaction was reproduced in the presence of a thermal probe inside the reaction medium: as soon as the myrcene was added to the medium the reaction jumped from 10 to 50 °C within 2 minutes. Regarding dispersity (Đ), the values obtained are regular and in the classical range for anionic polymerization (<1.5).

From the experiments reported in Table 1, the following conclusions can be drawn: it is possible to generate custom made oligomers from an anionic process with a precise number of units provided that the reaction is performed on a very small scale (ca. 100 mg). As soon as the scale is increased, an uncontrolled exothermic process occurs leading to a competitive consumption of organolithium species (vide supra), 37,38 with an increase in the monomer/organolithium ratio, provoking thus a rapid and uncontrolled gain in molar mass.

Therefore, the obtaining of small oligomers in high amounts cannot be obtained under batch conditions.

It must be noted that, for the 17 g experiment, lower final molar masses are observed probably because n-BuLi is used directly from the commercial bottle, as provided (conversely to the experiments performed on lower scales (0.11 and 1.0 g) where it was dissolved prior to use), avoiding thus early degradation of the initiator.

Polymerization under microflow conditions

Flow microreactors exhibit unique features that perfectly fit with our purpose: the size of the reactor allows to react extremely small quantity of monomers within a subminute reaction time, [30][31][32][33]35 with expected highly positive consequences on the precise control of the molar mass of the PMYR. Moreover, the thermal transfers are extremely well mastered as well avoiding the process to get out of hand. It is also worth noting that since the reactor is working under continuous flow, the quantity of PMYR produced is related to the operating time of the reactor rather than on the size of the reactor (ie under batch conditions). Thus, we implemented a flow set-up for the anionic polymerization of myrcene, as depicted in Figure 2. Our experiments were thus performed with a flow system equipped with a stainless steel reactor composed of a T-shaped mixer and tubing with ID = 750 µm, the outlet being collected in an aqueous acidic solution (Table 2). The first experiments (entries 1-5, table 2) were performed to determine the influence of the temperature at constant t R = 75 s. At this fast reaction time, a temperature of 10 °C allowed to reach a short Mn = 700 (ie 5 monomer units) whereas 20 °C led to Mn = 1100 (8 units) with a plateau at 30 °C with Mn = 1350 (10 units). Therefore, performing the reaction at a temperature close to room temperature (10 to 20 °C) is energy saving and allows fine control of the Mn of PMYR. Moreover it avoids the competitive decomposition of THF in presence of organolithium bases at higher temperatures. [37][38][39] Regarding the microstructure of PMYR obtained by anionic polymerization, the effect of the solvent has been highlighted in two distinct papers: Avila-Ortega reported that the reaction of myrcene in pure benzene with s-BuLi afforded 1,4 microstructure as major isomer (85%) while Hillmeyer and Hoye described the 3,4 isomer as major product (60%) along with 1,4 (30%) and 1,2 (10%) adducts in THF with s-BuLi (1.4 M in hexanes). 40,41 An NMR comparison of the structures obtained herein under batch and flow conditions were also performed. In batch, it showed that the 3,4 isomer is the major adduct (57%) along with 1,4 (33%) and 1,2 isomers (10%). In flow, the proportion of the 3,4 isomer rises to 66% accompanied with 1,4 (22%) and 1,2 (12%). However, this is hardly assignable to the nature of the reactor, but rather to the proportion of hexanes in the polymerization medium. Whereas, in batch, n-BuLi was used as provided (2.5 M in hexanes), the commercial solution was diluted to 0.3 M for practical reasons in the flow inlet. This results in a significant variation of the V/V percentage of hexanes in the reaction medium (hexanes/THF): 14% (batch) and 29% (flow) with obvious consequences on the reactivity and selectivity. 42 The influence of the residence time t R was further evaluated at both these temperatures from t R = 19 to 600 s by modifying the flow rates in a fixed length reactor (L = 0.5 m; entries 1-6, table 3). As expected, the molar mass increased according to t R as well as to the temperature. For example, the increase goes from Mn = 400 to 800 at 10 °C, and from 450 to 1100 at 20 °C. By working at a precise residence time, either by varying the flow rate or the reactor length, gave the same results. This means that mixing (influenced by the flow rate in the mixing zone) does not seem to be a major parameter in this reaction between n-BuLi (in hexane) and myrcene (in THF). Probably because an instantaneous anionic initiation of myrcene by n-BuLi occurs even at low flow rates in our conditions (THF). Thus, the t R can be tuned either through the flow rate or the reactor size, indifferently. This was also confirmed by results reported in entries 9-11. Therefore, we chose to perform final experiments for the controlled synthesis of low-Mn PMYR at fixed flow rate with various reactors at 10°C (entries 12-19) and 20 °C (entries 20-25). With the flow system it is therefore possible to precisely select the number of monomer units with Mn = 450 (t R = 19 s; entry 4) to 2100 (t R = 600 s; entry 19). It is worth to note that such a fine t R tuning (and therefore Mn of PMYR) is unreachable in a conventional batch reactor.

In order to confirm the living character of the polymerization, it was decided to trap a relevant group instead of a simple hydrogen atom. Indeed, whereas this hydrogen should come from the methanol inlet, competitive processes could also occur (such as deprotonation of THF) and quench the polymerization in an early manner. For this, an inlet of TMSCl was connected to the flow polymerization reactor (Fig. 3). A representative experiment was performed with t R2 = 227 s, and the degree of functionalization was determined by 1 H NMR analyses by comparing the (CH3)3 groups from TMS with the CH3 group from the n-Bu initiator: a 73% degree of functionalization was measured, showing thus the living character of this process. It should be noted that the uncomplete functionalization of the polymer with the TMS group is much likely due to the traces of HCl in the TMSCl (injected neat).

From these results it can be concluded that, in a flow system, an anionic living polymerization of myrcene can be performed in a flow microreactor: the molar mass (Mn) of PMYR can be modulated by adapting the residence time and the temperature within the flow reactor, conversely to batch polymerization which depends on the amount of material used. Thus, a kinetic model can be of interest to better apprehend this type of reaction under flow conditions.

Kinetic modeling of myrcene polymerization in continuous flow

Non-ideal mixing conditions could occur in batch conditions, decreasing the accuracy of kinetic constant evaluation. To overcome such issue, microfluidic conditions was used to estimate kinetic constants for myrcene polymerization.

The study of anionic myrcene polymerization in batch conditions was performed by Gonzalez-Villa et al. in 2019. This study was carried out in cyclohexane initiated by n-butyllithium at different temperatures (55, 63, and 71 °C). For example, at 55 ° C a propagation constant of kp = 1.24 L(1/b)/ (mol^ (1/b)*min) was evaluated. 43 The anionic polymerization myrcene follow the reaction path depicted in Figure 4. The reaction rates of step 1 and 2 were expressed as:

𝑅 1 = 𝑘 1 [𝑛𝐵𝑢𝐿𝑖][𝑚𝑦𝑟𝑐𝑒𝑛𝑒] 𝑅 2 = 𝑘 2 [𝐼𝑛𝑡𝑒𝑟𝑚𝑒𝑑𝑖𝑎𝑡𝑒][𝑚𝑦𝑟𝑐𝑒𝑛𝑒]
We assumed a plug flow model for the continuous reactor. The material balances are expressed as: Thus, the material balance for each compound is:

The kinetic modeling was performed in ModEst software; ODEs were solved by ODESSA solver. 44,45 Myrcene concentration was used as an observable. The objective function was defined as OF= ([M]exp-[M]sim) 2 .

The minimization of the objective function was done by the simplex and Levenberg-Marquardt algorithm.

The estimated kinetic constants: k1 (T), k1 (Tref), k2 (T), k2 (Tref), and a modified Arrhenius equation was expressed as follows:

𝑘 1 (𝑇) = 𝑘 1 (𝑇 𝑟𝑒𝑓 ) * 𝑒 [ -𝐸𝑎1 𝑅 ( 1 𝑇 - 1 𝑇 𝑟𝑒𝑓 )]
Equation 6

𝑘 2 (𝑇) = 𝑘 2 (𝑇 𝑟𝑒𝑓 ) * 𝑒 [ -𝐸𝑎1 𝑅 ( 1 𝑇 - 1 𝑇 𝑟𝑒𝑓 )]
Equation 7

From Table 4, one can notice that the credible interval for k1 is quite large, that could be explained by the fact that we do not measure the intermediate concentration. From the preliminary modelling, it seems that the rate constant k1 was temperature independent, for that reason we put Ea1=0 J/mol.

The credible intervals for the other kinetic constants are narrow, meaning that the parameters are well identified.

The correlation matrix (Table 5) shows that the correlations are negligible in this case. According to Toch et al., 46 two parameters are correlated if their binary correlation coefficient is higher than 0.95. Globally, Figures 5 and6 show that the model can fit the experimental concentration of myrcene.

End-functionalization of PMYR with CO2 in a flow system

Feedstock-derived functional oligomers are attractive as molecular building blocks in replacing oil-based moieties. Among polyterpene derivatives amine ω-terminated β-myrcene polymers have been synthesized as well as hydroxytelechelic PMYR, these last being used as toughening agents. [47][48][49] To our knowledge acid end-groups have never been introduced on PMYR yet. 43,[50][51][52] As stated in the introduction, continuous flow systems exhibit higher mixing between chemical reagents, with major improvements in gas/liquid mass transfer. In this line, Yoshida and Jamison independently reported that the challenging trapping of CO2, which often suffers from unselective consecutive competitive additions (carboxylic acid → ketone → tertiary alcohol) could be performed in a selective fashion in flow reactors. 53,54 Thus, a flow reactor system fed with an organometallic reagent (R-Li or R-MgX) in a first inlet, and a CO2 tank connected to a mass flow controller in a second inlet provided the selective mono-addition product R-CO2H without over-addition products, even without cryogenic conditions. Conversely, consecutive competitive over-addition occurred in batch reactors. 53,54 Based on these reports, we envisioned that the concatenated polymerization/CO2H functionalization in flow synthesis of ketones would be a mean to overcome all the drawbacks associated with the analogous batch synthesis: 1) as shown above the Mn of PMYR can be finely tuned, 2) the higher interfacial contact between gas and liquid phases should enhance the selectivity of carboxylation. Moreover, it is possible to use stoichiometric amounts of CO2 in flow, whereas it is generally much challenging under batch conditions. Thus, CO2 was introduced in the previous flow set up implemented for the controlled anionic polymerization of myrcene was connected a third inlet connected to the CO2 tank and controlled by mean of a mass flow controller (constant flow rate of 1.36 mL/min under 1 bar). The outlet was collected in an aqueous acidic solution (Figure 8). Thus, after polymerization of myrcene in a stainless steel flow microreactor (ID = 0.75 mm, L = 3 m) as described above [Q(myrcene) = 0.25 mL/min, Q(n-BuLi) = 0.1 mL/min, t R = 227 s] and CO2 was delivered in a further inlet (Reactor 2: ID = 0.75 mm, L = 30 cm). Delightfully, this telescoped process afforded the target PMYR terminated by a useful CO2H functional moiety PMYRCO2H (Table 6, entries 3-5). Increasing the mass flow rate -and therefore increasing the number of equivalents of CO2allowed to reach a functionalization degree FD = 95% with 3 equiv. of gas with regard to n-BuLi. It is worth to note that a parallel experiment was conducted under batch conditions (Table 6, entries 1 and 2) which afforded a different result: a ketone connected to two PMYR moieties was obtained as major product as attested by 13 C NMR in which a signal at  = 207.19 ppm, typical of a -C(=O)-moiety appeared (in contrast to (CO2) = 178.48 and 177.89 ppm detected in the flow experiment. This difference of reactivity (confirming thus the previous observations by Yoshida and Jamison with small organometallics) is depicted in Fig. 9, along with the corresponding reaction paths. Thus, in any reactor, PMYR-Li adds onto CO2 to afford the intermediate A. Due to lower liquid/gas mass transfer in the batch reactor, there is, in this case, coexistence between PMYR-Li and A that can undergo a second addition to yield B. This latter, upon elimination of Li2O, provides the ketone (PMYR)2C=O 1. Beside mixing issues, a plausible explanation for this divergence of reactivity lies on a major dichotomy between batch and flow conditions: the composition of the reaction medium according to space and time. In a batch reactor, the chemical composition of the reaction medium evolves with time as the conversion of reagents/substrates to products increases. In contrast, under flow conditions, the conversion increases along the reactor: thus, any point of the flow reactor corresponds to a specific state of the progress of the reaction, with -ideally-a completion at the outlet of the reactor. In our case, this major feature avoids reactions between PMR-Li and the primary product A, and thus undesired competitive products, with high benefits for chemical selectivity toward PMYR-CO2H 2. 55

Conclusions

In conclusion, we have shown that the anionic polymerization of myrcene can be finely controlled under microfluidic conditions to yield custom polymyrcene (PMYR) oligomers. The common issues associated to mixing and temperature control, directly related to the amount of starting material used, are circumvented under these flow conditions. Moreover, a kinetic model has been developed that can perfectly predict the outcome of the polymerization. In addition, thanks to the microfluidic reactor, we have been able to trap CO2, at the end of the polymerization, in a fully selective fashion to avoid the consecutive competitive reactions and introduce a useful carboxyl function. This work opens the door to a new sustainable process to obtain controlled and functionalized oligomers.

Fig. 1 .

 1 Fig. 1. Microflow synthesis of PMYR by anionic polymerization

Fig. 2 .

 2 Fig. 2. Flow polymerization of myrcene.

Fig. 3 .

 3 Fig. 3. Assessment of living polymerization in the flow microreactor.

Fig. 4 .

 4 Fig. 4. Reaction path for the anionic polymerization of myrcene.

Table 4 .

 4 Estimated kinetic constants and statistical data

Fig. 5 .

 5 Fig. 5. Simulation vs experimental trend of concentration vs residence time at 10 ° C.

Fig. 6 .Fig. 7 .

 67 Fig. 6. Simulation vs experimental trend of concentration vs residence time at 20 ° C.

Figure 7 (

 7 Figure 7 (parity plot) shows that the model can predict the experimental concentrations.

Fig. 8 .

 8 Fig. 8. Telescoped anionic polymerization of myrcene/functionalization with CO 2 in a flow system (MFC = mass flow controller).

Fig. 9 .

 9 Fig. 9. End-functionalization of PMYR: reactivity according to batch or flow conditions.

Typical procedures Anionic polymerization under batch conditions. All

	shaped micromixer (stainless steel, 500 µm of internal diameter
	(ID)) connected to a tubular microreactor (stainless steel, 750
	µm ID, 1 m length) at the desired flow rate. This microreactor is
	immersed in a water bath thermostated at 10 o C. The reaction
	mixture then feed a second T-shaped micromixer (500 µm ID)
	to be quenched with methanol (flow rate 0.1 mL/min). The
	quenched mixture is passed through a second tube of 300 mm
	length and 750 µm ID before collection in a vial containing 2 mL
	of HCl 2 N (three dead volumes of reagents were run through
	the system before collection to ensure reactor equilibrium,
	approximately between 2 and 5 mL were collected for analysis).
	The quenched mixture is extracted with dichloromethane (3 x
	10 mL), washed with water (20 mL), dried over magnesium
	sulfate, and concentrated under vacuum to afford a crude
	mixture, analyzed by NMR.
	Polymerization and functionalization with TMS under flow
	conditions. The flow system is composed of a first inlet
	containing a solution of myrcene in THF (2 M) and a second inlet
	with n-BuLi solution (0.3 M in hexanes). The solutions are
	passed through a T-shaped micromixer (stainless steel, 500 µm
	of internal diameter (ID)) connected to a microreactor (stainless
	steel, 750 µm ID, 3 m length) at the desired flow rate. This
	microreactor is immersed in a water bath thermostated at 10
	°C. The resulting solution is introduced in a second T-shaped
	micromixer (500 µm ID) connected to a trimethylsilyl chloride
	(TMSCl) inlet. The mixture is passed through a second tube of
	300 cm length and 750 µm ID before collection in a vial
	containing 2 mL of HCl 2 N. The procedure for collection,
	extraction and analysis of the sample was performed as above.
	5 M in hexanes) is
	introduced under stirring. After 5 minutes, 1.4 mL of myrcene
	(8 mmol) is introduced in one portion. After the time selected
	(1, 2, 8 min, etc.), the reaction is quenched with a
	methanol/water mixture (2:2 mL). The resulting solution is then
	extracted with dichloromethane (310 mL), washed with water
	(20 mL), dried over magnesium sulfate, and concentrated under
	vacuum to afford a crude mixture, analyzed by mean of NMR.
	Anionic polymerization under flow conditions. Stainless steel
	reactors were dried overnight in an oven at 120 °C, before
	cooling in a dessicator over silica gel under vacuum. The flow

experiments were run in oxygen-and moisture-free environment. Glassware was dried under vacuum using a Schlenk line by heating the flask with a heat-gun before cycling dry argon and vacuum. 2.6 mL of anhydrous THF is introduced in a 50 mL-round bottom flask under inert atmosphere (Ar) and then cooled with a 10 °C water bath. Then, 1.4 mL of hexane is introduced and 0.19 mL of n-BuLi (2.system is composed of a first inlet containing a solution of myrcene in THF (2 M) and a second inlet with n-BuLi solution (0.3 M in hexanes). The solutions are passed through a first T-

Table 1 .

 1 Anionic polymerization of myrcene with n-BuLi in batch at various scales. a

	Entry	Quantity of	t (min)	Mn 1 H RMN	Mn GPC	Đ	Yield (%) d	Conv. (%) e	Mn theo. f
		myrcene (g)		(g/mol) b,c	(g/mol) b
	1	0.110	2	650	600	1.23	28	28	648
	2		10	1050	1300	1.26	53	45	1227
	3		20	2200	1850	1.19	71	95	1644
	4		30	2400	1900	1.23	91	104	2107
	5	1	1	970	900	1.24	20	42	463
	6		2	800	950	1.14	32	35	741
	7		4	1700	1600	1.12	61	73	1412
	8		8	2330	2050	1.14	90	101	2084
	9		10	2200	2800	1.24	59	95	1366
	10		30	2700	3250	1.23	68	117	1574
	11		60	2600	3000	1.35	65	112	1505
	12	17	1	2060	2300	1.13	88	89	2038
	13		10	2100	2000	1.17	92	91	2130

a Performed at 10 °C with myrcene/n-BuLi = 17:1. b Average molecular weight. c 1 H NMR yields are calculated by comparison of the relative intensity of CH3 group of the initiator (δ = 0.87 ppm) with that of vinyl CH of PMYR (δ = 4.75 ppm). d based on the mass of PMYR recovered after full removal of volatiles (including myrcene). e based on the Mn ( 1 H NMR)/2316100. f Theoretical Mn = yield2316/100.

Table 2 .

 2 Anionic polymerization of myrcene with n-BuLi under flow conditions at various temperatures and scales. a,b According to set-up depicted in Fig. 2. b Myrcene/n-BuLi flow rate (mL/min) : 0.25/0.1 (68 mg/min for myrcene); t R = 75 s; L(reactor) =1 m; ID(reactor) = 0.7 mm; [myrcene] in THF = 2 M; [n-BuLi] in hexane = 0.3 M. c Average molecular weight. d Calculated by comparison of the relative intensity of CH3 group of the initiator (δ = 0.87 ppm) with that of vinyl CH of PMYR (δ = 4.75 ppm). e based on the mass of PMYR recovered after full removal of volatiles (including myrcene). f based on the Mn ( 1 H NMR)/2316100.

	Entry	T	Mn c,d	Mn c	Đ	Yield	Conv.	Mn
		(°C)	1 H RMN	GPC		(%) e	(%) f	theo. g
			(g/mol)	(g/mol)
	1	10	700	650	1.27	28	30	648
	2	20	1100	1000	1.23	43	47	996
	3	30	1350	1050	1.18	65	58	1505
	4	40	1500	1200	1.19	73	65	1690
	5	50	1350	1200	1.21	58	58	1343

a g Theoretical Mn = yield2316/100.

Table 3 .

 3 Anionic polymerization of myrcene with n-BuLi under flow conditions, different scales at 10 °C and 20 °C. a

	Entry	T (°C)	t R (s)	L (m) b	Mn 1 H RMN	Mn GPC	Đ	Yield (%) e	Conv (%) f	Mn theo. g
					(g/mol) c,d	(g/mol) c
	1	10	19	0.5	400	300	1.18	10	17	231
	2		38		450	400	1.44	13	19	301
	3		75		800	650	1.10	26	35	602
	4	20	19	0.5	450	400	1.25	18	19	416
	5		38		700	600	1.25	26	30	602
	6		75		1100	900	1.30	40	47	926
	7	10	38	8.0	600	350	1.16	29	26	671
	8		75		900	550	1.23	47	39	1088
	9	10	75	0.5	800	650	1.10	26	35	602
	10			1.0	700	650	1.27	28	32	648
	11			8.0	850	550	1.23	47	37	1088
	12	10	38	0.5	450	400	1.44	13	19	301
	13		75	1.0	700	600	1.27	28	30	648
	14		120	1.6	1000	850	1.33	42	43	972
	15		157	2.0	1150	900	1.19	54	50	1250
	16		180	2.4	1300	1050	1.16	57	56	1320
	17		227	3.0	1050	1200	1.43	47	45	1088
	18		302	4.0	1500	1400	1.20	69	65	1598
	19		600	8.0	2100	1700	1.36	54	91	1250
	20	20	38	0.5	900	600	1.25	40	39	926
	21		75	1.0	1100	850	1.26	51	47	1181
	22		120	1.6	1450	1050	1.36	78	63	1806
	23		157	2.0	1650	1450	1.36	73	71	1690
	24		180	2.4	1500	1500	1.34	78	65	1806
	25		302	4.0	1700	1800	1.18	74	73	1713

a ID(reactor) = 0.7 mm; [myrcene] in THF = 2 M; [n-BuLi] in hexane = 0.3 M; flow rates are adjusted according to t R = Vreactor/Q. b Reactor length. c Average molecular weight. d Calculated by comparison of the relative intensity of CH3 group of the initiator (δ = 0.87 ppm) with that of vinyl CH of PMYR (δ = 4.75 ppm). e based on the mass of PMYR recovered after full removal of volatiles (including myrcene). f based on the Mn ( 1 H NMR)/2316100. g Theoretical Mn = yield2316/100.

Table 5 .

 5 Parameter correlation matrix

	Parameters	k1 (L/mol/s)	kp (L/mol/s)	Eap (J/mol)
	k1 (L/mol/s)	1.00	0.00	0.0
	kp (L/mol/s)	-0.84	1.00	0.0
	Eap (J/mol)	-0.49	0.49	1.0

Table 6 .

 6 Experiments for the end-functionalization of PMYR with CO2 at 10 °C. a

	Entry	Conditions	t (s) b	Mass flow rate (mg/min)	Molar flow rate (mmol/min)	Molar ratio n-BuLi/CO 2	Mn (g/mol) 1 H NMR	GPC	ᴆ	Product	FD (%) c
	1	batch	60	-	-	bubbled gas	1750		950	2.50	ketone	-
	2	batch	120	-	-	bubbled gas	1800	1350	2.17	ketone	-
	3	flow	7.6	1.32	0.03	1:1	1000	1200	1.58	acid	30
	4	flow	4.6	2.64	0.06	1:2	1050		850	1.69	acid	64
	5	flow	3.6	3.96	0.09	1:3	1415	1100	1.19	acid	95
	a According to set-up described in Fig. 7. b Entries 1,2: bubbling time of CO2 in the batch reactor; Entries 3-5: residence time in reactor 2 (t R2 ).

c

Functionalization degree (FD) calculated by

1

H NMR by comparing the relative intensity of CH(CO2H) (δ = 3.05 ppm) with the CH3 group of butyl chain initiator of PMYR (δ = 0.86 ppm).

Acknowledgements

The authors thank INSA Rouen Normandy, University of Rouen Normandy, the Centre National de la Recherche Scientifique (CNRS), Labex SynOrg (ANR-11-LABX-0029), Carnot Institute I2C, the graduate school for research XL-Chem (ANR-18-EURE-0020 XL CHEM) and Region Normandie for their support. Catherine Legrand and Denis Danvy (laboratory COBRA) are thanked for the SEC and IR analyses. K.P. is grateful to the Ministerio de Educación Superior, Ciencia y Tecnología of the Dominican Republic for the fellowship (CALIOPE Program).

Notes and references

Conflicts of interest

There are no conflicts to declare.


Pascal Thébault

Mohamad Ammoun

Rym Boudjemaa

Aimeric Ouvrard

Karine Steenkeste

Bernard Bourguignon

Marie-Pierre Fontaine-Aupart

Surface functionalization strategy to enhance the antibacterial effect of nisin Z peptide

Keywords: Antimicrobial peptides, antibacterial surfaces, XPS, FITR-ATR spectroscopy, SHG, confocal microscopy

One of the main challenges when building antibacterial surfaces with antimicrobial peptides (AMPs) is to preserve their antimicrobial activity after stable immobilization of the peptides. Among all parameters, order/conformation of self-assembled monolayers, used as spacer, is one the most important. Herein we report the covalent immobilization of the nisin Z peptide on a gold surface functionalized with a self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) alone or mixed with 6-mercaptohexanol, used as a spacer. The MUA acid is activated by treatment with carbodiimide/N-hydroxysuccinimidine and then reacts with nisin Z to form amide bonds via the N terminal part of the peptide. We have characterized each step of the surface modification using X-ray photoelectron spectroscopy, FTIR-ATR spectroscopy and contact angle measurements. The combined results show the success of each functionalization step. Additionally, SFG brings information on the orientation and conformational ordering of the self-assembled monolayers. Indeed, a better order of MUA25 layers compared to MUA was observed due to the spacing of carboxylic acid groups. The antibacterial activity of the immobilized AMPs against Staphylococcus aureus is evaluated using confocal microscopy and bacterial counting: it increases with a better order of the SAMs rather than a greater peptide concentration. This study provides fundamental insights on how to engineer AMPs and substrate to produce efficient biocidal surfaces.

INTRODUCTION

The colonization of abiotic surfaces by bacteria (microbial contamination) is a real plague for public health (nosocomial infections, chronic wounds, rejection of implants ...), food industry (alterations and poisoning of foodstuffs), cosmetics (product degradation causing skin problems for the consumer) or industrial sector (biocorrosion phenomena, occlusions of cooling circuits ...) [START_REF] Alvarez-Ordóñez | Biofilms in Food Processing Environments: Challenges and Opportunities[END_REF].

It is now accepted that for any microorganism, life in a fixed form (called sessile) is favored over a suspended lifestyle (called planktonic). In most cases, this results in the formation of a biofilm that allows the bacteria to persist and even to resist in an unfavorable environment [START_REF] Gupta | Biofilm, pathogenesis and prevention-a journey to break the wall: a review[END_REF]. This "way of life" (sessile state) has been shown to make bacteria up to 1000 times less sensitive to antibiotic or other bactericidal treatments than their planktonic counterparts [START_REF] Levin-Reisman | Antibiotic tolerance facilitates the evolution of resistance[END_REF], making the fight against bacterial contamination of surfaces very problematic. In this context, prevention seems preferable to any treatment. The best approach may rely on new surface coatings that resist or prevent adhesion of bacteria and/or their growth to form biofilms.

Bacterial adhesion on surfaces strongly depends on the physico-chemical properties of both the microorganisms and the substrate. The surface of bacteria are amphiphilic while they are globally negatively charged. Therefore a common approach relies on the development of highly hydrophobic or negatively charged surfaces to preclude the adhesion of bacteria and /or their growth [START_REF] Zhang | Superhydrophobic surfaces for the reduction of bacterial adhesion[END_REF][START_REF] Roy | Antibacterial hydrogels of amino acid-based cationic amphiphiles[END_REF][START_REF] Morán | Superhydrophobic polypyrene films to prevent Staphylococcus aureus and Pseudomonas aeruginosa biofilm adhesion on surfaces: high efficiency deciphered by fluorescence microscopy[END_REF]. However, microorganisms will inevitably adhere to some extent to these coatings due in particular to the micro-heterogeneity of positive charges on the bacteria surface [START_REF] Chappaz | Imagerie moléculaire corrélative de nanosondes fonctionnalisées pour l'étude de l'hétérogénéité physico-chimique spatiale de microorganismes associés aux surfaces[END_REF] and surprisingly, despite a slower initial adhesion, surface growth of the adhering bacteria may be higher [START_REF] Gottenbos | Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria[END_REF]. Another strategy is the immobilization of active biocides (quaternary ammoniums, antibiotics, metal salts, metallic nanoparticles…) on the surfaces to promote bacterial killing [START_REF] Tischer | Quaternary Ammonium Salts and Their Antimicrobial Potential: Targets or Nonspecific Interactions?[END_REF][START_REF] Alves | Mini-review: Antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces[END_REF]. This approach also presents some drawbacks, especially because antibacterial agents with high potential toxicity can be released and contribute to environmental pollution and to the emergence of multi-resistant bacterial strains. Such a situation has required to consider new biocidal candidates displaying anti-adhesive and/or antibacterial properties with low toxicity as well as surface coating strategies for long term biocidal stability.

In this sense, developing surfaces using a covalent binding process of antimicrobial peptides (AMPs) appears very attractive [START_REF] Onaizi | Tethering antimicrobial peptides: Current status and potential challenges[END_REF][START_REF] Costa | Covalent immobilization of antimicrobial peptides (AMPs) onto biomaterial surfaces[END_REF][START_REF] Kamra | Covalent immobilization of molecularly imprinted polymer nanoparticles on a gold surface using carbodiimide coupling for chemical sensing[END_REF][START_REF] Vreuls | Prevention of bacterial biofilms by covalent immobilization of peptides onto plasma polymer functionalized substrates[END_REF][START_REF] Mularski | Atomic Force Microscopy Reveals the Mechanobiology of Lytic Peptide Action on Bacteria[END_REF][START_REF] Paris | Elaboration of antibacterial plastic surfaces by a combination of antiadhesive and biocidal coatings of natural products[END_REF][START_REF] Héquet | Optimized grafting of antimicrobial peptides on stainless steel surface and biofilm resistance tests[END_REF]. Compared to antibiotics, some AMPs offer the advantages of having a broad spectrum of activity, of not promoting bacterial resistance and of being active at very low concentrations [START_REF] Alves | Mini-review: Antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces[END_REF][START_REF] Yala | Elaboration of antibiofilm materials by chemical grafting of an antimicrobial peptide[END_REF]. Otherwise, covalent immobilization of AMPs is reported to limit their release, which avoids the drawbacks mentioned above.

We are interested in developing such biocidal surfaces against Staphylococcus aureus, one of the most frequent agents of biofilm-related infections in medical and food environments and one of the most critical multidrug-resistant bacteria [START_REF] Archer | Staphylococcus aureus biofilms: Properties, regulation, and roles in human disease[END_REF]. In this context, the covalent immobilization of the AMP nisin Z onto surfaces may be a promising strategy to create materials with friendly environmental and health impact. nisin Z, produced by Lactic acid bacteria, is the only one peptide recognized as safe by the U.S. Food and Drug Administration [START_REF] Guiotto | PEGylation of the antimicrobial peptide nisin A: problems and perspectives[END_REF]. Its antimicrobial activity in the nanomolar-range, its lack of toxicity for humans and its resistance to thermal treatments lead to its massive use in food industry as a preservative against Gram-positive bacteria including S. aureus [START_REF] Alves | Mini-review: Antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces[END_REF][START_REF] Joshi | Synthesis and antibacterial activity of nisin-containing block copolymers[END_REF].

Reported studies have highlighted that the antimicrobial activity of covalently immobilized AMPs onto surfaces is generally lower than that of their soluble counterparts [START_REF] Alves | Mini-review: Antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces[END_REF]. It can be due, to the impossibility for immobilized AMPs to cover all the surface of the target bacteria as in solution, but only one side of the cell, limiting the multiple interactions with the bacteria cell wall surface. The peptide mode of action of the AMP must also be considered. Typically nisin Z can bind lipid II with high affinity to form pores in the cytoplasmic membrane of target cells and also block peptidoglycan biosynthesis, thus inhibiting cell-wall formation [START_REF] Zendo | Lactococcal membrane-permeabilizing antimicrobial peptides[END_REF]. According to this mode of action, one could expect that the immobilized nisin Z peptides would lose their antibacterial activity against Gram positive bacteria having peptidoglycane cell wall above the cytoplasmic membrane. Indeed, the length of the immobilized peptide standing up on the surface is ~ 40-50 Å (33 peptidic bonds), ten times too short to allow the peptide to penetrate the cell wall of S. aureus bacteria (of thickness ~40 nm) [START_REF] Boudjemaa | Impact of Bacterial Membrane Fatty Acid Composition on the Failure of Daptomycin To Kill Staphylococcus aureus[END_REF]. The activity of the immobilized peptides also strongly depends on several parameters of the functionalized surfaces (peptide surface density, length and flexibility of the spacer that binds the AMP to the surface, etc..), all these requiring to be mastered for a better control of surface chemistry and topography.

In this study, we are interested to decipher the role of self-assembled monolayers orientation used as spacer on the activity of immobilized nisin Z. This point has not been systematically addressed because it requires nanoscopic specific method such as sum frequency generation (SFG) vibrational spectroscopy, a nonlinear optical technique that is interfaced specific. The method was used to characterize two different self-assembled monolayers (SAMs) of 11mercaptoundecanoic acid (MUA) alone or mixed with 6-mercaptohexanol, to covalently immobilize the nisin Z peptide on a gold model surface. Then the antibacterial efficiency of these AMP surfaces against S. aureus was evaluated through fluorescence confocal microscopy and bacteria counts.

Experimental section

Materials

Mercaptoundecanoic acid (MUA, C11H22O2S), 6-mercaptohexanol (C6OH), Nhydrosuccinimide (NHS), absolute ethanol, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Hydrochloric acid (HCl) and Phosphate buffer saline (PBS) tablets were purchased from Sigma-Aldrich. Nisin Z was purchased from Anhui mimetals development (China). It was chosen because it is the only AMP authorized by the FDA and the WHO. nisin Z is a cyclic antibiotic of 34 amino acids [START_REF] Guiotto | PEGylation of the antimicrobial peptide nisin A: problems and perspectives[END_REF][START_REF] Schneider | Analysis of nisin A, nisin Z and their degradation products by LCMS/MS[END_REF][START_REF] Van Den Hooven | NMR and circular dichroism studies of the lantibiotic nisin in non-aqueous environments[END_REF][START_REF] Rink | Dissection and Modulation of the Four Distinct Activities of nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation[END_REF], largely randomly coiled in aqueous solution [START_REF] Dykes | Structural Variations in nisin Associated with Different Membrane Mimicking and pH Environments[END_REF]. It differs from nisin A by the replacement of a histidine by an asparagine residue at position 27. Because nisin Z contains only one histidine residue, it has a higher solubility at physiological pH (the pKa of His being ~6.5 in water). The stock solutions were prepared at 10 mg/ml in HCl 0,0001N. The gold surfaces (11x11 mm size, purchased from Arrandee (Germany)) are borosilicate glass substrates coated with layers of chromium and gold of thicknesses 2.5 ± 1.5 nm and 250 ± 50 nm respectively. The surfaces were annealed in a butane flame to ensure a good crystallinity of the topmost layers, rinsed in a bath of absolute ethanol during 10 min and then dried under a flow of dry nitrogen before use.

Surface preparation and characterization

Cleaned gold surfaces were immersed in absolute ethanol containing MUA (10 mM) or a binary mixture (25/75) of MUA (2.5 mM) and 6-mercaptohexanol (HS(CH2)6 OH) (7.5 mM). These substrates named Au-MUA and Au-MUA25 respectively were then (i) stirred on an orbital shaker during 3 h in order to ensure optimal homogeneity of the adlayer and (ii) thoroughly rinsed successively in an absolute ethanol and milliQ water baths on an orbital shaker during 10 min before (iii) drying under a flow of dry nitrogen.

The functionalized surfaces were then immersed in a solution of EDC/NHS (10/20 mM) in milliQ water for 90 min at room temperature (Au-MUAact and Au-MUA25 act surfaces). The resulting succinimidyl ester-terminated surfaces were copiously rinsed in milliQ water baths on an orbital shaker during 10 min and dried under a flow of dry nitrogen. Finally, a 150 µl drop of nisin Z (1 mg/ml in PBS, pH 7.4) was deposited on the activated substrates at room temperature during 2h. This concentration is large enough to ensure the grafting of at least one peptide by activated MUA chain. The final Au-MUA-Nis and Au-MUA25-Nis substrates were successively rinsed in PBS and milliQ water baths on an orbital shaker during 10 min and dried under a flow of dry nitrogen.

Each step of the process was characterized by a combination of XPS, FTIR-ATR and watercontact angle methods as reported in Supplementary Information.

SFG vibrational spectroscopy

Our home-made "BB"-SFG setup (for Broad Band) was described in details previously [START_REF] Bulard | Non-Invasive Vibrational SFG Spectroscopy Reveals That Bacterial Adhesion Can Alter the Conformation of Grafted "Brush" Chains on SAM[END_REF]. It uses tunable IR pulses (4 µJ/pulse, 120 fs pulse duration, and 150 cm -1 bandwidth) and "visible" pulses (806 nm, 2 µJ, adjustable duration and bandwidth 1-6 ps  15-2.5 cm -1 , the spectral resolution being determined by the visible laser bandwidth). The two laser beams were superimposed onto the substrates in a collinear co-propagating configuration at the incident angle of ~66° in p polarization. The laser spot size on the sample surface was ~100 µm. After thorough rejection of light from the visible beam by dichroic filters, the beam emitted at the sum frequency was focused on the entrance slit of a spectrometer of resolution 0.4 cm -1 centered at ≈650 nm or ≈714 nm for detection of the CH and amide bands respectively, equipped with a cooled CCD camera. The SFG spectra were collected during 30 to 150 seconds to obtain an acceptable signal to noise ratio. SFG vibrational spectra were collected for all substrates in the air. Attempts to obtain SFG signal using s polarization of the visible and IR beams failed. SFG vibrational spectra were recorded in two different spectral ranges: the 2800-3000 cm -1 range for analyzing CH3 and CH2 vibrational bands (in this case the IR pulse frequency was set at 2900 cm -1 ) and the range 1500-1700 cm -1 for analyzing amide bands (in this case the IR pulse was set at ≈ 1600 cm -1 ). In this 1500-1700 cm -1 range, absorption by gas-phase water molecules is so strong that the IR laser is almost completely absorbed at the frequencies of the strongest absorption lines. Therefore, the set-up (IR laser cavity and all the optical path to the sample) was purged by dry air one night in advance and the key optical mounts were motorized for last minute adjustments. In addition, an airlock was used for sample introduction allowing to install samples with minimal water vapor contamination. The residual small imprint of the water spectrum on the spectral profile of the IR laser was then easily removed before deconvolution of the vibrational bands against the non-resonant background of the SFG spectrum. The rest of spectral analysis was as described in earlier papers [START_REF] Bulard | Non-Invasive Vibrational SFG Spectroscopy Reveals That Bacterial Adhesion Can Alter the Conformation of Grafted "Brush" Chains on SAM[END_REF].

It must be recalled that there are two major specificities of SFG vibrational spectroscopy with respect to FTIR-ATR spectroscopy ( 29): (i) the vibrational bands of molecules adsorbed on gold interfere with a non-resonant (NR) background. In BB-SFG configuration, this background has the spectral shape of the IR femtosecond laser (SFG reference spectra in Figure 2). It provides a reference for comparison of intensities from one sample to the other (while absolute intensities of different spectra are difficult to compare because they are strongly dependent on optical alignment). (ii) Due to the coherent nature of SFG emission, the amplitudes of identical chemical groups sum up instead of intensities, with two possible consequences (both arising in this study): (a) if the orientation distribution of the transition moments of the contributing chemical groups is isotropic, then the intensity vanishes. In such a case, a low intensity means "disorder" in the sense of an isotropic distribution of the different chemical groups. (b) in the case where the sample is highly ordered with a centrosymmetric arrangement of the contributing chemical groups, then the SFG intensity also vanishes. This is typically the case of CH2 bands of all-trans alkyl chains in SAMs, for which zero intensity means "order" [START_REF] Bulard | Non-Invasive Vibrational SFG Spectroscopy Reveals That Bacterial Adhesion Can Alter the Conformation of Grafted "Brush" Chains on SAM[END_REF].

Antibacterial effect of nisin Z coated surfaces

Staphylococcus aureus ATCC 27217 strain (BioMérieux, France) was used in the present study.

After two subcultures in TSB at 37°C (one 8-h followed by an overnight culture of a stock strain culture cryotube kept at -20°C in TSB), cells were harvested by centrifugation, washed twice in sterile NaCl (150 mM) and suspended in TBS. Optical density was measured at 600 nm to control the initial bacterial concentration (~10 8 UFC/ml). The cleaned substrates (Au substrates, Au-MUA and Au-MUA25-without and with nisine Z) were incubated with 500 µl of the bacterial suspension in a static mode over 1h30 at 37°C (in this condition, the S.aureus cells could adhere but not grow on the surface). It must be noted that only the functionalized side of the gold-coated surface was accessible for the bacteria to adhere. Before observation, the samples were carefully rinsed to eliminate non-adherent cells (which can be dead cells during time of adhesion) and further recovered with sterile NaCl (150 mM).

The visualization of bacterial adhesion and subsequent cell development on nisin Z functionalized substrates was performed using a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, France) at the Centre de Photonique pour la Biologie et les Matériaux (CPBM) (Orsay, France). Bacteria were stained with 2.5 µM Syto9® (Life Technologies, France), a green cell permeant nucleic acid dye, and 30 µM propidium iodide (PI) (Life Technologies, France), a red nucleic acid dye that can easily penetrate cells with compromised plasma membranes. Syto9® and PI were sequentially excited at 488 nm and 543 nm, respectively, and their fluorescence emissions were collected between 500 and 600 nm for Syto9® and between 640 and 750 nm for PI. Images were acquired using a 63x oil immersion objective with a 1.4 numerical aperture. The size of the confocal images was 512 by 512 pixels corresponding to image sizes of 246 x 246 µm 2 for zoom 1 and 82 x 82 µm 2 for zoom 3. For each sample, 5 different areas were analyzed. The reproducibility of the results was checked by using three different sets of functionalized substrates and bacterial culture batches. Each fluorescence intensity image corresponds to an average of 4 frames.

A total of 15-20 images per zoom were analyzed. On each image, the bacterial counting was performed using ImageJ image processing software. Due to the satisfying correspondence between cell counting on images obtained using zoom 1 and zoom 3 the data are pooled together. Analyses of variance (ANOVA) were performed using Statgraphics software (Manugistic, Rockville, MD). Evaluated factors were considered statistically significant when P values associated with the Fischer test were below 0.05.

Results and discussion

Surfaces preparation and characterization

A schematic representation of the three steps to construct the AMP coated surfaces is depicted in Figure 1. This process is specific for the AMP immobilization via an amine function, which is more efficient than via a carboxylic acid function [START_REF] Paris | Elaboration of antibacterial plastic surfaces by a combination of antiadhesive and biocidal coatings of natural products[END_REF].

The first step is the functionalization of the gold surfaces by self-assembled monolayers composed of (i) mercapoundecanoic acid or (ii) a mixture (25/75) of MUA and 6- mercaptohexanol. The latter is expected to insert between the MUA chains, in order to limit (i) the repulsive interactions between carboxylate functions which could impact the monolayers conformation and/or (ii) residual unreacted carboxylic acid functions (with AMP) which could promote bacterial adhesion by hydrogen bonds.

The second step is the conversion of the MUA carboxylic acid functions into NHS esters by the carbodiimide chemistry allowing the AMP attachment through an amide function. The EDC and NHS concentrations were chosen in order to prevent the formation of byproducts [START_REF] Sam | Semiquantitative Study of the EDC/NHS Activation of Acid Terminal Groups at Modified Porous Silicon Surfaces[END_REF].

Finally, the third step is the covalent immobilization of nisin Z on activated surfaces which occurred to preferentially select an AMP linkage with its N-terminal amide function. For this purpose, the pH was maintained at 7.4 to prevent the unwanted deprotonation of the -amine groups of the three lysine residues (pKa 12.2) [START_REF] Joshi | Synthesis and antibacterial activity of nisin-containing block copolymers[END_REF]. To maintain the nisin Z antibacterial effect, it is of importance that the lysine residues do not bind to the surface and remain available to interact electrostatically with bacteria. XPS, FTIR-ATR and surface wettability were used to attest to the surface functionalization and further to the immobilization of the nisin Z peptides. All results are detailed in the supplementary information. However, XPS analysis seems to demonstrate a higher peptide immobilization on Au-MUA compared to Au-MUA25 (if we consider that all the NHS ester groups are converted). But, we never observe a four times larger effect for Au-MUA compared to Au-MUA25 substrates, suggesting that the nisin peptides could dimerize on the surface as reported [START_REF] Casford | The Structure of Lipid Bilayers Adsorbed on Activated Carboxy-Terminated Monolayers Investigated by Sum Frequency Generation Spectroscopy[END_REF].

SFG characterization

The SFG spectra of the different surfaces were measured both in the C-H stretching region (2700-3100 cm -1 ) (Figure 2A) and in the amide band region (1400-1700 cm -1 ) (Figure 2B). In this latter domain, no SFG signal was expected before peptide immobilization. It was the case for Au-MUA and Au-MUA25 samples but upon surface activation, a weak signal arised. It was ascribed to the side-reaction product N-acyl urea (identified from FTIR spectroscopy) (Supplementary information), which contains amide bonds. In the C-H stretching region, the SFG spectra of the control Au-MUA and Au-MUA25 surfaces exhibit three vibrational bands centred at 2929 cm -1 , 2907 cm -1 and 2856 cm -1 (Figure 2A). These frequencies are in good agreement with previous reported data [START_REF] Chen | Probing α-Helical and β-Sheet Structures of Peptides at Solid/Liquid Interfaces with SFG[END_REF]. The 2856 and 2929 cm -1 bands are assigned to the symmetric and asymmetric methylene stretching modes of MUA, respectively. Several assignments of the band at 2907 cm -1 were reported. It may correspond to a Fermi resonance of the CH2 groups. Another assignment was the symmetric stretching mode of the CH2 groups at the bottom (S-CH2 still observed for octadecanethiol on Au ( 27)) or the top (CH2-C=O) of the MUA chain. Their specific local environments may result in a different frequency than the other CH2 groups. However, considering the SFG spectrum after activation of the surface by EDC/NHS (Figure 2A) which leads to the transformation of the acid group into the ester group without modifying S-CH2, this band at 2907 cm -1 was significantly weakened. This highlighted that the contribution of CH2-C=O at the top of the alkyl chain is larger than that of S-CH2. It must be noted that this band at 2907 cm -1 is too weak to be observed in FTIR-ATR spectra, where the strong bands of the numerous CH2 groups dominate the spectrum (Supplementary information).

The observation of these different bands confirms what has been noticed from FTIR-ATR measurements (Supplementary information), that alkyl chains of Au-MUA and Au-MUA25 surfaces are not as expected perfectly ordered both before and after activation. In such case, as mentioned above, the contribution of the CH2 groups to the SFG spectra should vanish. This disorder of the alkyl chains may be due to steric hindrance and/or to the presence of H-bonds among terminal acid groups but also to electrostatic repulsion between these terminal groups in the case of elimination of protons during sample preparation. All are expected to distort alkyl chain conformation from all-trans. Interestingly, the dilution of MUA into mercaptohexanol results in a clear weakening of CH2 bands, both from Au-MUA to Au-MUA25 and from Au-MUAact to Au-MUA25act. This highlights the success of the strategy to increase the order of the MUA layer by the distance of the COOH/COO -groups.

The attachment of the succinimidyl ester termination also results in an intensity decrease of the symmetric and asymmetric CH2 bands (from Au-MUA to Au-MUAact and from Au-MUA25 to Au-MUA25act), which can be ascribed to a conformation of the alkyl chains closer to all trans thanks to the elimination of the interactions between COOH or COO -groups.

: SFG spectra of Au-MUA and Au-MUA25 surfaces in (A) the C-H stretching region and (B) the amide bands region. Experimental (black lines), fitted (red lines) SFG spectra, and deconvoluted vibrational bands (green lines). The SFG reference spectrum (blue dashed line)

is also presented.

The binding of nisin Z was ascertain from XPS, FTIR-ATR and water-contact angle measurements as reported in Supplementary Information. It results also in three major changes in the SFG spectra: (i) the appearance of the bands corresponding to the methyl groups (19 methyl groups are present in nisin Z) at 2876 cm -1 and 2930 cm -1 corresponding the symmetric and asymmetric stretching modes, respectively. What is striking is their weak intensity relative to their large number, but it is not unexpected considering their presumably large orientation distribution in such a complex molecule (25): if it was completely isotropic, the bands would vanish completely. (ii) The reversed intensities of symmetric and asymmetric CH2 bands with respect to the activated surfaces. Since, on metallic surfaces, dipole moments parallel to the surface plane do not contribute to SFG, and since the CH2 asymmetric dipole moment is along the H H direction, the intensity of the asymmetric mode can be larger than the symmetric one if the CH2 plane is not parallel to the surface and the H H direction is normal to the surface.

This configuration hardly occurs in the case of an alkyl chain in a well ordered SAM. cm -1 , corresponding to amide I and amide II vibrational modes, respectively. It ascertains qualitatively the presence of amide groups on the surface. However, in this case also, quantitative analysis is impossible: nisin Z has a polycyclic structure [START_REF] Rink | Dissection and Modulation of the Four Distinct Activities of nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation[END_REF] and contains 33 amide groups, so the situation is comparable with that of CH3 modes: if the orientation distribution of the amide groups is close to being isotropic, then their SFG signature is expected to be very weak, which is the case. Additionally, the formation of nisin Z dimers on the surface, as suggested from XPS data (supplementary information), may also be responsible of this low SFG signal. Interestingly, we observe the presence of both amide I and amide II bands. Previous studies [START_REF] Ye | Orientation Difference of Chemically Immobilized and Physically Adsorbed Biological Molecules on Polymers Detected at the Solid/Liquid Interfaces in Situ[END_REF] have investigated by SFG the orientation of the R-helical peptide cecropin P1 (containing 31 amino acids) deposited on polymer surfaces. The selective presence of the amide I band was explained by the fact that the peptide structure contains only α helices. The selective presence of amide I was also observed for peptides with a β-sheet structure [START_REF] Chen | Probing α-Helical and β-Sheet Structures of Peptides at Solid/Liquid Interfaces with SFG[END_REF]. By contrast, nisin Z has a polycyclic structure [START_REF] Rink | Dissection and Modulation of the Four Distinct Activities of nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation[END_REF] and contains no helix. Therefore, our results suggest that the presence of both amide I and II bands is related to the polycyclic structure of nisin Z.

Antibacterial effect of nisin Z coated surfaces

Adhesion of S. aureus bacteria on Au-MUA and Au-MUA 25 surfaces was imaged and quantified using fluorescence confocal microscopy allowing to control the homogeneity of the surfaces.

As observed on Figure 3a, (zoom 1 and 3), the adhered cells appeared homogeneously distributed, isolated or in some groupings (a well-known behavior for S.aureus cells). It was previously reported that the surface topography of Au deposited on glass substrates presents platens of 1-2µm 2 separated by grooves of 20-30 nm depth [START_REF] Bulard | Non-Invasive Vibrational SFG Spectroscopy Reveals That Bacterial Adhesion Can Alter the Conformation of Grafted "Brush" Chains on SAM[END_REF]. The S.aureus cells seem to adhere on these platens and not on the grooves due to their small thickness with respect to size and form of bacteria (coccoid structure of ~1µm).

If we now consider the quantification of the adhered S. aureus bacteria, no statistical valid difference in surface coverage was obtained for Au, Au-MUA and Au-MUA25 (Figure 3b).

Neither the higher hydrophilicity of the surfaces grafted with MUA chains by comparison with a gold surface (Table 3), nor the higher charge of the Au-MUA than the Au-MUA25 surfaces via the carboxylic groups of the MUA chains have significant effect on the cell adhesion. Thus, the interactions between bacteria and the surface are rather short-distance interactions in agreement with the low number of adhered bacteria (~5x10 6 bacteria/ cm 2 ) (Figure 3b).

Assuming an average bacterium section of 1 µm², this corresponds to ~ 5.0 % of the bacterial surface coverage (Figure 3b). It is also interesting to note the low coverage of cells with damaged membrane and /or dead cells (in red on Figure 3a) on Au, Au-MUA and Au-MUA25 surfaces. Their quantification (~10%, Figure 3d) is comparable to what is commonly encountered in the process of bacterial adhesion. This reveals that neither the adlayer of MUA chains nor the alcohol function of 6-mercaptohexanol chains have any damaging effect on the cell membrane.

The covalent attachment of nisin Z to the Au-MUA and Au-MUA25 surfaces leads to a decrease in the number of adhered S. aureus after 1h30 (~ 70% for Au-MUA-Nis substrates and 95% for Au-MUA25-Nis substrates) by comparison to the corresponding surfaces without peptides (Figure 3 b,c). Furthermore, the percentages of dead or damaged cells on the surfaces with nisin Z are higher (~20%) by comparison with Au-MUA and Au-UA25 substrates (~10%) (Figure 3d).

Figure 3 (a) Representative confocal fluorescence images with zooms 1 and 3, highlighting both live (green) and dead or damaged (red) S.aureus cells immobilized on different surfaces for 1h30. Bacteria were stained with Syto9/IP LIVE/DEAD staining dyes. (b) Average concentrations of adhered bacteria on the different surfaces and the corresponding average percentages of coverage (%) of the surfaces. The presented data are the average of measurements in three spots of the same surface (c) inhibition of adhesion of S.aureus on Au-MUA-Nis and Au-MUA25-Nis surfaces as compared to Au-MUA and Au-MUA25 substrates respectively. (d) Average percentages of dead or damaged cells on each surfaces

Undoubtedly, the nisin Z peptide although immobilized on a surface has a biocidal activity against S. aureus bacteria. As already mentioned, the mode of action of immobilized nisin Z to kill bacteria cannot be through the "pore model" created on the cytoplasmic cell membrane, the peptide length being too short. The electrostatic interaction of nisin Z with the outer surface of bacteria is sufficient for its lethal activity. A detailed study of antimicrobial effects of positively charged surfaces on adhered Gram positive bacteria similar to S. aureus has shown that while bacteria are strongly attracted by the surface charges, they cannot grow [START_REF] Gottenbos | Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria[END_REF]. The interpretation is that the cell division cannot occur because the required cell elongation is prevented by the strong electrostatic interactions. Such a process can interpret our results. It is consistent with the increase of dead and/or damaged bacteria in the presence of nisin Z (Figure 3d). It tends to show that the bacteria first adhere to the surface but are then permeabilized by the grafted peptide, modifying their cell wall surface and allowing the entry of PI as observed for Magainin I on Gram positive bacteria [START_REF] Humblot | The antibacterial activity of Magainin I immobilized onto mixed thiols Self-Assembled Monolayers[END_REF]. Thus on nisin Z grafted surfaces, the bacteria die "naturally" being unable to divide on the surface. Our results are also in agreement with the better efficiency when the covalent grafting of nisin Z is via its N terminal part, leaving available the lysine residues for electrostatic interaction and in particular its terminal C part, identified as the one that allows to permeabilize the target membrane [START_REF] Rink | Dissection and Modulation of the Four Distinct Activities of nisin by Mutagenesis of Rings A and B and by C-Terminal Truncation[END_REF].

The most important result proves in this study concerns the role of the order of the surface; the better order of the Au-MUA25-Nis substrate, as revealed by SFG analysis, results in a higher antibacterial effect even if higher amount of nisin Z were immobilized on Au-MUA.

Conclusion

We have developed a model of AMP surfaces by the covalent immobilization of the nisin Z peptides via its N terminal part on carboxyl functionalized gold substrate using carbodiimide coupling, the active C terminal part of the peptide being available to interact with the bacteria.

Using XPS, FTIR-ATR and SFG spectroscopy and water contact angle measurements, we have verified successful surface modification after each reaction steps. Interestingly, SFG measurements allow to highlight that the conformational ordering of the Au-MUA25 substrates is significantly improved by comparison to Au-MUA substrates. Consequently, the antibacterial activity of the so-immobilized nisin Z surfaces can achieve (95%) against S.

aureus, depending on the surface order of the attachment centers of the peptides and on the peptide orientation. These findings provide essential information for the development of antibacterial surfaces based on covalently immobilized peptides.

The whole procedure for constructing such AMP surfaces is simple, reproducible and efficient.

Furthermore, the safety of antimicrobial peptides for human health has been demonstrated.

XPS measurements of gold surface after MUA and MUA/(CH2)6 OH immobilization and after nisin Z binding are illustrated in Figure S1 and summarized in Table S1. The XPS spectra of unmodified gold surfaces show C1s and O1s peaks, as expected when Au surface are not prepared in ultra-high vacuum conditions [START_REF] Kamra | Covalent immobilization of molecularly imprinted polymer nanoparticles on a gold surface using carbodiimide coupling for chemical sensing[END_REF]. They are mainly due to hydrocarbon impurities (C1s peak) and to -OH from water molecules (O1s peak). Comparison of the XPS data for the gold surfaces before and after MUA functionalization, revealed the appearance of a sulfur peak and the decrease of Au4f intensity both on Au-MUA and Au-MUA25 surfaces (Table S1), confirming immobilization of thiols on the gold surfaces (13,35,S1). The attenuation of the Au4f peak by the adlayer was more important for Au-MUA corresponding to a higher thickness of MUA layer compared to MUA25 (11 carbons for MUA chain vs 6 carbons for mercaptohexanol). The high resolution analysis of the S2p peaks (Table S2, Figure S1) reveals the presence of two sulfur components, one at 162.1 eV (S2p3/2) and 163.4 eV (S2p1/2) attributed to sulfur atoms bound to the gold surface (in red on Figure S1) and the second one at 163.7 eV (S2p3/2) and 164.9 eV (S2p1/2) corresponding to free thiol functions (in blue on Figure S1) (S2).

The free thiols content was larger for Au-MUA (45%) compared to Au-MUA 25 (30%). The presence of free sulfur is probably due to the formation of a partial bilayer through hydrogen bonds between the carboxylic terminal groups of thiols bonded to the surface and free thiols from the solution (in this case, the S atom outwards from the surface). This was further confirmed by IR spectra analyses (see FTIR-ATR section). The high resolution XPS spectra of the C1s region for Au-MUA and Au-MUA25 substrates (Figure S1, Table S2), were best fitted S2). A lower C-S/C-O component at 286.5 eV and higher C-C/C-H

and COOH contributions at 285eV and 289.5 eV, were observed for the MUA layer. These differences can be explained by the longer alkyl chain average length for MUA and the lower carboxyl group concentration for Au-MUA25.

The success of nisin Z grafting on the two types of surfaces was confirmed by (i) the significant decrease of Au4f peak intensity, (ii) the appearance of a nitrogen peak (N1s) centered at 400.2 (Table S1, Figure S1) assigned to the amide functions of the peptide [START_REF] Humblot | The antibacterial activity of Magainin I immobilized onto mixed thiols Self-Assembled Monolayers[END_REF] and (iii) an approximatively twice larger free sulfur peak after nisin Z immobilization (Figure S1) due to the presence of sulfur atoms from thioether functions in the peptide [START_REF] Paris | Elaboration of antibacterial plastic surfaces by a combination of antiadhesive and biocidal coatings of natural products[END_REF][START_REF] Héquet | Optimized grafting of antimicrobial peptides on stainless steel surface and biofilm resistance tests[END_REF]. These effects were more pronounced for Au-MUA-Nis surface by comparison to Au-MUA25-Nis. Infrared spectroscopy measurements further confirm the chemical composition of the surfaces at each step. The FTIR-ATR spectra for Au-MUA and Au-MUA25 showed similar peak positions in the C-H stretching region, corresponding to asymmetric (2928 cm -1 ) and symmetric (2856 cm -1 ) stretching modes of the CH2 alkyl chains of MUA and 6-mercaptohexanol (Figure S2). The unexpected shoulder at 2960 cm -1 corresponds to the asymmetric band of the methyl group. It was due to impurities related to MUA conditioning as revealed by the spectrum of a KBr pellet of MUA, where it is present (S1). The corresponding symmetric band of this methyl group is expected at ≈2880 cm -1 . It is not clearly visible, presumably because it is weaker than its asymmetric counterpart and is hidden in the wings of other bands. The frequency of the asymmetric methylene stretching is reported to be sensitive to conformational ordering: the more ordered the alkyl chain, the lower the frequency (2918 cm -1 for a well-ordered alkyl chain) (S3). Therefore, the position of the asymmetric band of Au-MUA and Au-MUA25 before and after activation at 2928 cm -1 suggests a certain disorder of the thiol layers. This was supported by the IR spectra measured in the frequency range 1800 cm -1 -1500 cm -1 . Figure S2). The two bands at 1742 cm -1 and 1714 cm -1 were attributed to the CO stretching mode of non-hydrogenbonded and hydrogen-bonded carboxylic acid groups, respectively [START_REF] Casford | The Structure of Lipid Bilayers Adsorbed on Activated Carboxy-Terminated Monolayers Investigated by Sum Frequency Generation Spectroscopy[END_REF][START_REF] Humblot | The antibacterial activity of Magainin I immobilized onto mixed thiols Self-Assembled Monolayers[END_REF]. The band at 1714 cm -1 is consistent with the ability of carboxylic acids to undergo strong intermolecular hydrogen bounding interaction. This can occur from head to head MUA chain dimers forming a partial bilayer of MUA chains, as already suggested from XPS data. Nevertheless, dimerization of carboxylic groups of neighboring MUA chains may also exist, which would imply disorder within the layer. Elsewhere, the large absorption signals in the range 1570-1540 cm -1 and the band at 1463 cm -1 are ascribed to the asymmetric and symmetric stretching modes of COO - respectively. It reveals that some deprotonated COO -groups are present. They are able to induce electrostatic repulsion and presumably a relative disorder of the surface layer (S4). SFG measurements further support this disorder of the Au-MUA and Au-MUA25 layers. the persistence of the prominent peak at 1744 cm -1 corresponding to the transformation of the acid terminal group into ester, (ii) the appearance of two weak bands at 1788 cm -1 and 1820 cm - of the 1714 cm -1 absorption band highlights that the activation leads to the disruption of intermolecular hydrogen bonds between carboxylic groups. Smaller broad bands observed in some experiments at 1656 cm -1 and ~1550 cm -1 (not visible in the spectra of Figure S2), evocative of amide I and amide II bands, respectively, can be ascribed to the limited formation of the stable N-acyl urea byproduct due to defects in the activated layer [START_REF] Sam | Semiquantitative Study of the EDC/NHS Activation of Acid Terminal Groups at Modified Porous Silicon Surfaces[END_REF]. Finally, the broad band extending between 1500 cm -1 and 1400 cm -1 corresponds to bending modes of the different CH bonds. It cannot be exploited because it shows no structure.

Although the nisin Z peptide has an appreciable number of CH, CH2 and CH3 groups in various positions with respect to amide groups, the CH stretching spectrum does not show any significant change (Figure S2). However, the nisin Z grafting is ascertained by the appearance of the two strong bands at 1673 cm -1 and 1542 cm -1 that can be ascribed to the amide I and amide II bands of the peptidic backbone. In addition, a band centered at ~1739 cm -1 is still present. It may correspond to the free carboxylic group of the C-terminated part of Nisin and/or from remaining non-activated carboxylic groups.

Water contact angle measurements

Static water contact angle measurements of all samples were performed using a sessile drop technique with deionized water as a probe liquid. The instrument used was DSA 30S Krüss contact angle system goniometer. To check surface uniformity and reproducibility, six measurements were acquired on each of two different samples by depositing six droplets (2.5 µl milliQ water) at different points of the surface (11mm x 11mm).

Table 3 summarizes the changes in the water contact angles when moving from Au to functionalized substrates. In each case, the water contact angle values were constant over the entire sample surface demonstrating that the successive functionalization of the gold surfaces were homogeneous. When the gold surface was modified by MUA, an increase of the surface hydrophilicity is observed due to the introduction of polar head groups (COOH) and the hydrophilicity of the mixed OH/COOH terminated surface (Au-MUA25) is higher than that of a COOH terminated surface (Au-MUA), which is consistent with the literature (12,S2). Upon EDC/NHS activation, the water contact angle increased by ~7-10°, indicating lower hydrophobicity of the surface due to the formation of the succinimide ester intermediate (13,S5). The nisin Z immobilization rendered the surfaces slightly less hydrophilic, as expected considering the amphiphilic nature of the peptide. No difference between Au-MUA and Au-MUA25 substrates was observed.

Figure 1 :

 1 Figure 1: Schematic representation of steps involved for covalent nisin Z immobilization on gold surface. Step 1: MUA and HS(CH2)6 OH adsorption on gold surface named Au-MUA and Au-MUA25 surfaces; step 2: esterification of the COOH functionalities by NHS/EDC; step3: covalent binding of nisin Z, named Au-MUA-Nis and Au-MUA25-Nis surfaces.

Figure 2

 2 Figure 2: SFG spectra of Au-MUA and Au-MUA25 surfaces in (A) the C-H stretching region

  It must be related to a specific CH2 group inside nizin Z, or to a specific modification of the alkyl chain conformation imposed by the bonding of the AMP. It can be also noted that despite the presence of additional CH2 groups in nisin Z, no significant change of the intensity of the CH2 bands is observed, indicating again an isotropic distribution of the dipole moments. Interpreting in more details the relative intensities of the CH bands would require in-depth density functional theory (DFT) calculations of SAM-Nis conformation on the surface, which is beyond reach of the present study. (iii) The appearance of two bands with weak intensities at ~1650 cm -1 , and ~1550

  with four components corresponding to C-C/C-H bonds at 285 eV, carbon in α position of heteroatom (S and O atoms) at 286.2 eV, carboxylate functions at 288 eV and carboxylic acid functions at 289.5 eV (35,S2). Percentages of the four components were different between MUA and MUA-25 (Table

Figure S2 :

 S2 Figure S2: FTTR-ATR spectra of the three consecutive steps leading to the nisin Z immobilization: (a) Au-MUA or Au-MUA25, (b) Au-MUAact or Au-MUA25act by NHS/EDC, (c) covalent binding of Nisin Z, Au-MUA-Nis or Au-MUA25-Nis.

Table S1 :

 S1 Surface atomic composition of gold and modified gold surfaces determined by XPSThe proportion of component composition from peak areas were performed using the CasaXPS software system and applying a Gaussian/Lorentzian ratio (70/30).

	S2p Figure S1: High resolution XPS spectra of S2p, C1s and N1s regions before and after nisin Z
	Au-MUA-Nis immobilization. The spectra were acquired with a Thermo Electron K-Alpha Spectrometer Au-MUA25-Nis
	200 CPS using a monochromatic Al-K-Alpha X-ray source (1486.6 eV) with a spot size of 400 µm, a pass
	energy of 50 eV and through a range of -10 to 1350 eV with pass energy of 200 eV. The binding Atomic composition (%) Surfaces Au 4f C 1s O 1s S 2p energy was referenced by setting the maximum C 1s peak at 285.0 eV. The deconvolution of N 1s Gold 60.4 29.4 10.2 -peaks from the spectra was performed using the CasaXPS software system and applying a -Au-MUA 46.8 38.1 13 2.1 Au-MUA -Au-MUA25 Gaussian/Lorentzian ratio (70/30)
	Au-MUA25 Au-MUA-Nis FTIR-ATR measurements Au-MUA25-Nis FTIR-ATR measurements were obtained using a Brüker Vertex 70 FTIR spectrometer, equipped 57.8 27.3 12.3 2.6 -19.8 54.2 14.3 2.2 9.5 32.2 41.8 15.5 2.5 8 168 166 164 162 160 158 168 166 164 162 160 158 with an attenuated total reflection (ATR) accessory (PIKE MIRacle crystal plate diamond/
	Binding energy (eV)						Binding energy (eV)
	C1s ZnSe) and a mercury cadmium telluride (MCT) detector cooled down by liquid nitrogen. 100
	Au-MUA-Nis spectra were accumulated over the spectral region 3100 to 1400 cm -1 with a spectral resolution Au-MUA25-Nis Table S2: C1s and S2p chemical composition (%) of modified surfaces 5000 CPS of 4 cm -1 . The background was recorded (average of 100 scans) on the ATR unit without any
	Carbon components (%) substrate pressed against the crystal.					Sulfur components (%)
		C-C/C-				COO -/
	Surfaces	H (285		C-S/C-O (286.5 eV)	N-C=O (288	COOH (289.5 eV)	S-Au (162.1 eV)	Free S (163.7 eV)
	Au-MUA	eV)				eV)	Au-MUA25
	Au-MUA	72.7		13.4		6.1		7.8			55.2	44.8
	Au-MUA25	64.7		22.8		6.4		6.1			69.6	30.4
	AU-MUA-Nis	59.1		23.7		15.6		1.6			28	72
	Au-MUA25-Nis 56.5 Binding energy (eV) 292 290 288 286	284	26.7 282		15.3 292	290	1.5 288	286	45 284	282	55
								Binding	energy	(eV)
		N1s
				Au-MUA-Nis
			CPS
			1000
				Au-MUA
		406		404	402	400	398	396	394
					Binding energy (eV)

Table S3 :

 S3 contact angle measurements

	Surfaces	Water contact angle (±1.5°)
	Gold	86
	Au-MUA	52
	Au-MUA25	45
	Au-MUAact	61
	Au-MUA25act	51
	Au-MUA-Nis	62
	Au-MUA25-Nis	60

, the signature of the ester groups of the imide ring (30,25,S5) (FigureS2). The disappearance

ACKNOWLEDGMENT

We thank Farah Savina (ISMO, Université Paris Saclay), Ariane Deniset (ICP, Université Paris Saclay) and Julien Deschamp () for their helpful technical advice during contact angle, FTIR-ATR measurements and bacterial preparation respectively. M.A.is grateful for the financial support by the Lebanese Association for Scientific Research (LASER).

Therefore, such biocidal surfaces present great potential in packaging and industrial production surfaces (cosmetic, food, medical devices...) to avoid product contamination.

AUTHOR INFORMATION

Corresponding Author * E-mail address: pascal.thebault@univ-rouen.fr

Graphical Abstract

Supplementary Information

Surface characterization XPS measurements.