Mylan Lam

Vivien Moris

Vincent Humblot

Céline Veronique Migonney

Véronique Migonney1

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

A simple way to graft a bioactive polymer -Polystyrene sodium sulfonate on silicone surfaces

Keywords: silicone, implant, bioactive polymer, grafting Graphical

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Poly(dimethylsiloxane) (PDMS) more commonly known as silicone is an elastomer widely used in a large range of applications such as the fabrication of microfluidic devices [1] or as raw materials for biomedical implants for various surgery applications [2]. The material is known for its inertness, flexibility, low cost, and ease of fabrication. Silicone-based breast implants are used since the 1960s for reconstructive or plastic surgery. However, after long term implantations, several issues appeared such as ruptures and fibrosis. This leads to discuss and re-consider the PDMS non-invasive aspect and biocompatibility. Complications due to breast prosthesis implantations include hematoma, infections, inflations, silicone gel perspirations or the apparition of capsular contractures [3], in the long term.

In the past years, many cases of large cell anaplastic lymphoma (a form of cancer that most of the time requires a prosthesis replacement) have also been reported post-surgery [4] on women with breast implants after 8 -10 years. According to the American Society of plastic surgeons, 694 cases of BIA-ALCL have been identified worldwide since February 2019 (https://www.plasticsurgery.org/). In France, the equivalent association (ANSM) has reported 59 cases including 5 deaths in the country, thus raising the safety issues of such implants. In this study, we have been focused on smooth silicone breast implants as textured implants are being slowly removed from the international market and banned in France since February 2019 (https://www.ansm.sante.fr/). One of the main reasons for all the occurring problems is bacterial contamination during the first implantation surgery. Indeed, investigations of biofilms from explanted implants with capsular contracture showed the presence of Staphylococcus epidermidis. Researchers have proven that the severity of the contracture was directly correlated to the number of colonizing bacteria [5]. The bacterial contamination is supposed to be due to the hydrophobic behavior of silicone surfaces that promotes the adsorption of plasma proteins leading to the adhesion and proliferation of bacteria. In the long term, a deformation around the outer shell of the prosthesis is visible, which may be due to an inflammatory response. This non-controlled host response results in the generation of a fibrous shell around the implant which later leads to complications such as the calcification of the breast implant that is responsible for pains and skin deformations [6].

To overcome this problem, surface modification is needed. A lot of work has been done in the microfluidic field to improve silicone material surface wettability by grafting hydrophilic polymers [1] expecting a better flow of polar liquid through PDMS channels. However, before any modification, silicone surfaces usually need to be oxidized first to generate polar groups on its surface which is really difficult to achieve due to the low reactivity of the silicone. Mostly, PDMS surfaces are activated using UV/ozone [7][8][9] in dry environments or by oxygen plasma treatment to generate oxygen reactive species (-OH;-OOH) [10][11][12]. However, it has been shown that created oxygen species are not stable in time and thus, a quick recovery of PDMS surface hydrophobicity was observed [27].

A silicone-based quarter polymer has been developed previously in the laboratory [13]. It resulted from the UV-combination of two copolymers carrying bioactive groups: on one hand silicone-methacrylate moieties and sulfonate-carboxylate groups on the other hand. This quarter polymer has significantly reduced the bacterial adhesion (S.Aureus & MRSA 88244) by 80% in vitro and by more than a log of difference in vivo (1.5 to 2 log) between a non-grafted (control) sample and a grafted sample [13]. However, the synthesis required several steps which made it difficult to scale up for industrial applications. Another method to improve the biocompatibility consists of the covalent grafting of peptide molecules. But despite the comparable antibacterial activity, the system has a short lifetime and arise high cost [14].

Here we have elaborated a simple method to graft PDMS sample with a bioactive polymer, and we have studied the influence of different synthesis parameters such as surface activation conditions, monomer concentration, and sample's size. We have characterized the grafting by different techniques such as Fourier Transform Infrared Spectroscopy in the ATR mode, ATR-FTIR, water contact angle measurements (WCA), X-ray Photoemission Spectroscopy (XPS), electronic microscopy with chemical analysis (SEM-EDS) or colorimetric measurements.

In the past years, our team has successfully developed functionalization methods of bioactive polymers on metallic surfaces (titanium and its alloys [15][START_REF] Falentin-Daudré | Procédé de greffage de polymères bioactifs sur des matériaux métalliques[END_REF][17]) and polymers surfaces (PET [18], PCL [19][20][21]). The grafting of one specific polymer,the polystyrene sodium sulfonate (polyNaSS) on these surfaces was carried out under heating [15,18,19] or UV irradiation [START_REF] Falentin-Daudré | Procédé de greffage de polymères bioactifs sur des matériaux métalliques[END_REF]17,20,21]. Both techniques allow the creation of radical species permitting the initiation of the polymerization. The grafting of such polymer increases the wettability of the surfaces making them more appropriated for the biological environment. Most importantly, grafted bioactive polymers have also reduced bacterial adhesion [13,22,23]. The mechanism of action can be described as follow: polyNaSS helps to modify the adsorbed plasmatic proteins conformation on the implants making them non-identifiable by the bacteria receptors. This prevents bacterial adhesion and the risk of infection [13,23]. With the described context and inspired by previous work, we aim to develop a simple way to graft polyNaSS on silicone surfaces through a radical polymerization of sodium styrene sulfonate.

In this work, we will be focused on the grafting of the bioactive polymer on the outer shell of smooth silicone breast implants, i.e. the side that is in direct contact with the biological environment. This type of implants is composed of silicone in different forms: the silicone elastomer composing the outer shell, the silicone gel filling the prosthesis and silicone liquid in the mixture of the gel.

Materials and Methods

Material

Silicone samples were extracted from the outer shells of smooth breast implants. The prosthesis was cut in half and silicone gel was rid. Gel residues were removed with two washings in tetrahydrofuran (THF, FISHER Chemical) under ultra-sonication two times, five minutes each. Then, the samples were rinsed in distilled water for five minutes three times. Finally, samples were left dried in a 37°C oven overnight and cut into 1 x 1 cm square-shaped samples before experiments. Sodium styrene sulfonate (NaSS, Sigma), the monomer used for the grafting process is purified by a recrystallization process in a mixture of water/ethanol (Carlo Erba) (10 : 90 v/v) overnight at 70°C. A first Büchner filtration is realized and the filtrate was kept at 4°C for 48 hours before the second filtration. The purified NaSS was then dried under atmospheric pressure at 50°C overnight and then stored at 4°C [15,17] .

UV source used comes from a Lot Quantum Design UV lamp delivering a power at 365 nm under normal conditions of temperature and pressure.

Methods: grafting process

Surface activation: Silicone sample was placed in a round-bottom flask containing distilled water previously degassed and was left under UV irradiation at 365 nm for one hour at 160 mW/cm 2 .

Grafting procedure (cf. Method C): After the surface activation, the sample was transferred to another round-bottom flask containing a degassed aqueous solution of NaSS and left under UV irradiation in the same conditions as described above. The surface was then rinsed in distilled water for 24 hours and dried overnight at 37°C before analyses.

Surfaces characterizations:

After each surface modification step, silicone samples were characterized by Fourier-transform infrared spectra recorded in attenuated total reflection mode (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), colorimetry, water contact angle measurement (WCA). Changing in terms of surface compositions, physical properties or surface textures can be highlighted.

Fourier-transform infrared spectroscopy (FTIR):

Analyses were performed on a Perkin Elmer Spectrum Two Spectrometer in the 600 to 4000 cm -1 spectral range with a resolution of 4 cm -1 . Silicone samples were directly pressed against the diamond crystal for acquirements (128 scans) and obtained spectra were then fitted and analyzed.

X-ray photoelectron spectroscopy (XPS):

To examine possible change in terms of the surface elementary composition after the functionalization, we aim to target the presence of sulfur atoms attributed to polyNaSS grafting silicon samples were analyzed by XPS. These analyses were performed using an Omicron Argus spectrometer (Taunusstein, Germany) equipped with a monochromated AlKα radiation source (hν = 1486.6 eV) working at an electron beam power of 300 W. Photoelectrons emission was analyzed at a takeoff angle of 90°; the analyses were carried out under ultra-high vacuum conditions (≤10 -10 Torr) after introduction via a load-lock into the main chamber. Spectra were obtained by setting up a 100 eV pass energy for the survey spectrum and a pass energy of 20 eV was chosen for the high resolutions regions. Binding energies were calibrated against the C1s binding energy of aliphatic carbon atoms at 284.8 eV. Element peak intensities were corrected by Scofiel factors [26] Casa XPS v.2.3.15 software (Casa Software Ldt, UK) was utilized to fit the spectra and Gaussian/Lorentzian ration was applied (G/L ration = 70/30).

Scanning Electronic Microscopy -Energy Dispersive Spectroscopy (SEM-EDS):

Images were obtained with a HITACHI TM3000 with Oxford energy dispersive spectroscopy (EDS). This technic is mainly used to characterize surface topography with high-resolution image. Coupled with EDS, the device could be used for a qualitative analysis of the elementary composition on the sample's surface.

Colorimetry: Toluidine Blue (TB) is used as a colored indicator to quantify the concentration of polymer grafted on silicone samples. The samples, once grafted and washed were immersed in 5mL of TB (pH=10) for complexation for 6 hours at 30°C. After that, the excess of TB was removed with 3 washings in a solution of sodium hydroxide (NaOH-10 -3 M), five minutes each. Then, complexed TB on sample surfaces was de-complexed by putting the samples in 5 mL of acetic acid (50/50 v/v) for 24hours. TB assays was carried out onto six samples for each conditions. The method is based on the presence of a chromophore group on the TB molecule absorbing in the visible domain at around 633 nm. The molecule has a cationic group N + (CH3)2 allowing it to complex with negatively charged species such as sulfonate SO3 -. According to Ikada et al. [24] work, stipulating that one mole of TB molecule complexes with one mole of carboxylate (-COO -). We assumed that it works for sulfonate groups (-SO3 -) as well. Then we are able to determine the concentration (=grafting rate) of grafted polymers on silicone surface using UV-vis spectroscopy after decomplexation.

Water Contact Angle measurements (WCA): Hydrophobic or hydrophilic behavior of the surface was evaluated by measuring the contact angles on the surface. This is a qualitative measurement of the wettability of the material. In our study, distilled water was used as the solvent and measurements were taken using a DSA10 contact angle measuring system from KRUSS GmbH. Once the drop (2 μL) of distilled water is in contact with the surface, the measure was taken after 20 seconds with the Drop Shape Analysis (DSA) program that gives the value of the contact angle. At least three measures were taken and averaged.

Results and discussion

Bare silicone

The first part of the study was the analysis of bare silicone shell samples. In terms of the surface's aspect, SEM pictures (Figure 1) showed that the shell exhibits two different surface textures. The inner side, ca. the one that was initially in contact with silicone gel, has a textured aspect (roughness visible in Figure 1(a)). On the contrary, the outer side, which is in direct contact with the biological environment once the prosthesis is implanted, is very smooth as seen in Figure 1(b).

The observed difference may be attributed to the way the shell has been manufactured. It is supposed that the outside was in contact with the mold while the inner side was exposed to ambient air. In this work, we will be mainly focussed on grafting bioactive polymer on the outside part of the prosthesis showing almost no surface roughness. PDMS material exhibits methyl groups (-CH3) at its surface which makes the surface very hydrophobic. Water contact measurements on bare silicone samples have confirmed the hydrophobic behavior with high contact angle values of around 105.00° ± 1.00.

The covalent grafting of polyNaSS on such surfaces aims to increase the wettability. First, the surface needs somehow to be activated. In other words, polar groups like hydroxyl groups (-OH), carboxyl groups (-COOH) or hydroperoxide (-O-O-) have to be generated through the methyl groups present on silicone surface to allow the initiation of the NaSS monomer radical polymerization.

The grafting process is based on the "grafting from" method. Active species such as freeradicals are created on the surface allowing then initiation of the radical polymerization.This step is followed by the propagation step where initiated radicals will attack another monomer molecule that will, in turn, generates a radical capable of attacking another monomer. Finally, the reaction ends up with the termination step. In our case, polar groups need to be generated through an activation step to allow the fixation of polyNaSS on the surfaces [17,20,21,23].

Grafting

Standart conditions of grafting -Method A

Several methods that have been developed in the laboratory to activate metallic [15-17] and polymers [18- 21] surfaces were first tested on silicone surfaces. Among which acid activation and ozone activation were tested, detailed protocols are described in Appendix A (method A). Macroscopic degradation was observed in the case of the acid activation. No significant polymer grafting was observed for both conditions according to colorimetric measurements and WCA measurements. Results are all resumed in figure A1.

Direct grafting without activation -Method B

Inspired by previous work realized in the laboratory [20,21] , where polyNaSS was successfully grafted on polymer surfaces without any activation step thanks to the presence of oxygen groups (-OH, -COOH) on the surface at the native state. Under UV irradiation, oxygen radical species are created meanwhile NaSS monomers polymerized on the surface. In the case of silicone surfaces, it was expected to maybe create carbon radicals on silicone methyl groups in the meantime of the polymerization without any previous oxidation step. Experimentally, the silicone sample was placed under UV light at 160 mW/cm 2 in a stirred degassed monomers solution for two hours.

Grafting rates did not show any improvement compared to the UV-control. The values remained practically the same compared: 8.70 x 10 -8 versus 1.05 x 10 -7 mol/g (Figure 2) and are relatively low for a significant grafting rate. Nevertheless, interestingly FTIR spectra (Figure 3) has revealed a feature at 3667 cm -1 which can be attributed to free hydroxyl groups after UV treatment. Moreover, the contact angle measurements showed a slight decrease going from 105° before method-B grafting to 99.74° (Figure 2). Even if the surface remained hydrophobic.

These observations suggested that UV irradiation allows the creation of oxygen groups on silicone surfaces and can be used as the source of energy to effectively activate silicone surfaces.

Grafting with UV activation -Method C

Considering the previous results where UV light efficiency promotes the formation of polar groups on silicone surfaces. It is assumed for the following experiments:

• Activation: 1 hour -UV irradiation (160 mW/cm 2 )

• Polymerization: 1 hour -UV irradiation (160 mW/cm 2 ) in a degassed monomer solution (0.7M) With this method, the obtained results are promising. First, the grafting rates (Figure 4) were much higher compared to the UV-control. More especially they are higher than the grafted samples following the previous grafting process (Method B): 2.22 x 10 -7 mol/g versus 8.78 x 10 -8 mol/g. Statistically, the difference is significant with a p-value equals 0.05(*). XPS analysis also showed a difference in terms of composition : on the grafted sample, sulfur (S 2p) atoms were detected at 174 eV with a rate of 1.94% whereas it was practically undetectable on bare or UV-activated samples (Table 1 Contact angle measurements confirmed the observation (Figure 4). The surface went from really hydrophobic (≃105°) to hydrophilic surface (≃74°). The high standard deviations observed were supposed to be due to the non-homogeneity of grafting on the surface.

We can conclude that polyNaSS has been successfully grafted on the silicone surface with the developed two-step grafting protocol (method C) using UV irradiation. The wettability has clearly been increased. Grafting rates and XPS measurements have confirmed the presence of polyNaSS on silicone surfaces with this method. The mechanism is resumed in Figure 5.

Importance of the grafting under UV irradiation versus under heating (thermal grafting)

Thermal grafting was the initial method developed in the laboratory [15,17,18,19,20,25] to graft polyNaSS on different types of surfaces after the activation step (ozonation or acid treatment). Silicone samples were first activated under UV irradiation, placed into a degassed monomer solution and left in a thermal oil bath for 3 hours at 50°C. The results showed that compared to the previous method, thermal grafting showed any significant improvement in terms of grafting rates. It remained the same value as only UV-activated (control) samples: 8.70 x 10 -8 mol/g (Figure A2-(3)). Contact angle results also confirmed the non-efficacy of the thermal grafting method, the grafted surfaces remained hydrophobic: 108.13° ± 2.44. The explanation could be that thermal energy was not strong enough to break O-H bond (464 kJ/mol) created by UV light during the activation step. As no oxygen radical species were created, the radical polymerization cannot occur. Ozonation or acid treatment activation usually allows the formation of hydroperoxide (O-O) which required less energy (142 kJ/mol) to be broke than O-H bond to create oxygen radical species. As UV only allowed the generation of hydroxyl groups on silicone surface thermal grafting cannot be used for the radical polymerization reaction particularly on silicone surfaces.

Improvement of the grafting homogeneity

Size of the sample

When contact angle measurements were taken, we noticed that the lowest values were concentrated in the same area on the sample. Around this area, the values were slightly higher. It was proposed that it was due to the UV beam wideness which was not wide enough to equally cover the sample's surface. So, to improve the homogeneity of the grafting, the sample dimensions were reduced from 2x2 cm to 1x1 cm samples for the next experiments keeping the same grafting conditions.

On Figure 6-(2), results showed that grafting rates were slightly increased compared to 2x2 cm samples : 2.56 x 10 -7 mol/g versus 2.22 x 10 -7 mol/g. More importantly, wettability was even higher with angles around 40° (*p=0.05) with a lower standard deviation (Figure 7-(4)). The grafting homogeneity was improved.

By this two-step protocol using UV to graft polyNaSS on silicone surface, we have optimal results for 1x1 cm squared-samples.

Monomer concentration

On titanium surfaces, it has been shown that when the monomer concentration was reduced by half : 0.7M to 0.35M, grafting rates were much higher [17] . This allows both the increase of the grafting rates and economic save by using half of the quantity of monomer. In this study, a UVactivated silicone sample was immersed into a round-bottom flask containing a degassed monomer solution concentrated at 0.35M and left under UV irradiation for 1 hour at 160 mW/cm 2 .

Results on Figure 6-(3) showed that with a monomer concentration of 0.35M, the grafting rates increased by four : 8.45 x 10 -7 mol/g versus 2.56 x 10 -7 mol/g.The variation is significant (**p=0.005). The hypothesis suggested to explain such observation, is that the high density of molecules promotes the reaction of homopolymerization of monomers in solution which competes with the radical polymerization occurring on the surface of the sample. A lower density (0.35M) decreases interactions between monomers so it decreases homopolymerization reactions. Accessibility to the surface is then better and thus, it may allow better polymerization rates on silicone surface. In terms of wettability, the contact angles remained practically the same as in the conditions of 0.7M, around 45°.

Reducing activation time

For all previous experiments, the UV-activation time was set at 1 hour. In the next experiments, the duration was reduced by half: 30 minutes. It was expected for the grafting rates to remain at least the same. Results are shown in Figure 8 where it is clear that for 60 minutes or 30 minutes of UV activation, grafting rates remained equivalent. Added to that, WCA also showed a hydrophilic surface with angles around 40°. The polymer grafting process duration time could be reduced to 30 minutes. Furthermore, FTIR spectra shown on Figure 9 also confirm that only after 30 minutes (orange spectra) of UV-exposure hydroxyl groups are already formed with the band at 3670 cm -1 . Obviously, after 45 or 60 minutes, these groups remain present.

All along with the study, we have first successfully grafted the bioactive polymer on silicone surface with an adapted activation step. Following that, we successfully refined the grafting parameters to get the highest rates of grafted polymer and also the most hydrophilic surface with optimum parameters (duration, concentration, sample's dimensions).

Physisorption

Physisorption tests were realized to confirm that polyNaSS has been covalently bond to the surface and not only physisorbed. First, silicone samples were activated at different time duration: 0, 15, 30, 45, 60 minutes under UV irradiation at 160 mW/cm 2 before they were put in an aqueous solution of polyNaSS for 24 hours under stirring. Afterward, samples were rinsed several times with distilled water for 24 hours and dried at 37°. Whatever the duration time of activation, surfaces remained hydrophobic with a contact angle varying around 105° for the physisorbed samples (Figure 10). The grafted sample clearly exhibit a hydrophilic behavior with a contact angle of 40°. On the other hand, grafting rates results also confirm the previous observation. Grafted samples exhibited a higher grafting rates compared to the physisorbed samples. Statistical analysis showed a significant difference between phisisorbed and grafted samples, particularly at the concentration of 0.35M (*p=0.05/**p=0.005/***p=0.001). By these results, we confirm the covalent bond of polyNaSS on silicone surface following the grafting conditions we have developped. The developed "grafting from" method permit a covalent attachment between the polymer and the surface.

Conclusions

We have successfully developed a new approach to covalently graft polyNaSS on silicone breast implant outer shell's surface. Based on the work realized in the laboratory, several methods were tested to graft polyNaSS on silicone surface. Different techniques of activation were tested in vain: piranha solution (surface strong degradation) and ozonation. As well, different grafting methods were tested: UV grafting without activation and thermal grafting also showed really low grafting rates comparable to non-grafted samples. Experiments of UV grafted samples without activation step have did not show any improvement in terms of grafting rates compared to non-grafted sample, but FTIR spectra showed the apparition of hydroxyl groups after UV exposure. This led us to use UV irradiation to activate silicone surfaces: UV-activated samples clearly present a hydroxyl band at 3670 cm -1 and a slight decrease of the contact angle (105° to 95°). Following the activation, polyNaSS was grafted onto the surface under UV irradiation at 160 mW/cm 2 as well.

To conclude, we have fulfilled the main objective which was to find a suitable method to activate silicone surfaces by generating oxygen groups on it thanks to the use of UV irradiation delivering a power of 160 mW/cm 2 . We were then able to graft the bioactive polymer through a radical polymerization under UV. Once the grafting protocol was set up, the experiment parameters were refined to optimize the grafting rates: sample size, monomer concentration, and activation duration.

The developed two-steps UV grafting process adapted for silicone breast implant shell is a low-cost process and most importantly is easy to realize which makes it industrially scalable.

We supposed that piranha solution has created an oxide layer on the sample and meantime has degraded the surface. That is why, this oxidation method cannot be used to activate this silicone material to create polar groups on it.

Figure 1 :

 1 Figure 1 : SEM analysis silicone shell's inner side (a) and silicone shell's outer side (b)

Figure 2 :

 2 Figure 2 : Grafting rates of Non-grafted silicone & Method B-grafted silicone

Figure 4 :

 4 Figure 4 : Grafting rates of UV-activated silicone & Method C-grafted silicone

Figure 5 :

 5 Figure 5 : Developed two-steps mechanism of grafting polyNaSS on silicone surface

Figure 7 :Figure 6 :Figure 8 :

 768 Figure 7 : Grafting rates results of different grafting conditions

Figure 9 :Figure 10 :

 910 Figure 9 : FTIR spectrum silicone samples activated at different time (black:bare silicone/green:15min-UV/orange:30min-UV/blue:45min-UV/red:60min-UV

Table A 1 :

 1 EDS elemental analysis (atomic%) of elements on silicone surfaceFor ozonation activation, ozone was generated using an ozone generator BMT 802 N (ACW) with a pressure of 0.5 bars and an oxygen flow rate of 0.6 L.min -1 . The samples were placed in distilled water where an ozone flow goes through for 20 minutes. Once activated, the sample was put under UV irradiation for 1 hour at 160 mW/cm 2 for the polymerization of NaSS monomers on the surface. In terms of grafting rates, no difference between bare silicone and ozone-treated-grafted silicon was observed. The surface remained hydrophobic with high contact angles around 105°. This led us to rethink the grafting process, especially the adapted activation protocol for silicone breast implant materials (FigureA2).

Figure A 2 :

 2 Figure A 2 : Grafting rates of the different conditions of grafting

Table 1 : elementary composition in atomic percentage obtained from XPS analysis of several silicone treated surfaces

 1 ).

	Element	Position (eV)	Bare silicone (%atomic)	UV-activated silicone	Grafted silicone(%actomic)
				(%atomic)
	O 1s	531	18.4	19.2	18.9
	C 1s	283	50.9	49.2	52.3
	S 2p	174	0.4	0.3	1.9
	Si 2p	101	30.3	31.3	26.8

Acknowledgments: This research was supported by the French Ministry of National Education, Higher Education and Research. Thanks also to the University of Paris 13.

Appendix A

Standard conditions of grafting -Method A

Different activation methods that have been demonstrated efficient on metallic [15,16,17] or polymers [18- 21] surfaces were tested to generate polar groups on silicone surface. Acidic oxidation and ozonation were separately tested to activate the silicone samples in a first step. For the acidic oxidation, silicone sample was immerged into a piranha solution (H2SO4/H2O2 50/50 v/v). It was first soaked in sulfuric acid for 1 minute before hydrogen peroxide was added. The reaction was left for 3 minutes. An exothermic reaction occurred instantly and the surface was immediately attacked in the first seconds after the hydrogen peroxide was added. The sample was then quickly washed in distilled water three times and left dried at 37°. Macroscopically the surface was clearly degraded in the acidic mixture (Figure A 1). The solution strongly attacked the silicone sample with a detachment of a cracking white layer. Images of this newly formed layer were recorded with the SEM-EDS and a difference in terms of elementary composition compared to bare silicone was observed. On the piranha oxidized silicone surfaces the amount of oxygen was clearly increased while the quantity of carbon decreased by 10% (Table A 1).


Ahlem Laribi
email: ahlem.laribi@etu.univ-st-etienne.fr

Séverine Allegra

Mina Souiri

Ridha Mzoughi

Ali Othmane

Françoise Girardot

Legionella pneumophila sg1-sensing signal enhancement using a novel electrochemical immunosensor in dynamic detection mode

Keywords: Legionella pneumophila, Electrochemical Detection, Microfluidic Device, Immunosensor

This work presents a comparison between static and dynamic modes of biosensing using a novel microfluidic assay for continuous and quantitative detection of Legionella pneumophila sg1 in artificial water samples. A self-assembled monolayer of 16-amino-1-hexadecanethiol (16-AHT) was covalently linked to a gold substrate, and the resulting modified surface was used to immobilize an anti-Legionella pneumophila monoclonal antibody (mAb). The modified surfaces formed during the biosensor functionalization steps were characterized using electrochemical measurements and microscopic imaging techniques. Under static conditions, the biosensor exhibited a wide linear response range from 10 to 10 8 CFU/mL and a detection limit of 10 CFU/mL. Using a microfluidic system, the biosensor responses exhibited a linear relationship for low bacterial concentrations ranging from 10 to 10 3 CFU/mL under dynamic conditions and an enhancement of sensing signals by a factor of 4.5 compared to the sensing signals obtained under static conditions with the same biosensor for the detection of Legionella cells in artificially contaminated samples.

Introduction

Legionella pneumophila is the main vector of Legionnaires' disease, a type of pneumonia that can be fatal in humans. Indeed, inhalation of contaminated aerosols can cause bilateral pneumonia [START_REF] Sharma | Atypical Pneumonia[END_REF]. It has been reported that 9% of domestic cases are fatal [START_REF] Dooling | Active Bacterial Core Surveillance for Legionellosis -United States, 2011-2013[END_REF], reaching between 25 to 48% for nosocomial cases in the USA [START_REF] Soda | Vital Signs: Health Care-Associated Legionnaires' Disease Surveillance Data from 20 States and a Large Metropolitan Area -United States, 2015[END_REF]. In France, there was a 75% increase in the number of recorded incidences in 2018 compared to the previous year (Campese, 2018).

Legionella bacteria are found in natural aquatic environments and wet soils [START_REF] Borella | Water ecology of Legionella and protozoan: environmental and public health perspectives[END_REF]. They are known to colonize artificial human-made aquatic ecosystems such as hot water distribution systems, air conditioning systems, and air-cooling towers, thereby giving rise to severe outbreaks of pneumonia [START_REF] Beer | Surveillance for Waterborne Disease Outbreaks Associated with Drinking Water -United States, 2011-2012[END_REF]Mobed et al., 2019a). Approximately 28 sources of sporadic cases involving potable water, hot springs, humidifiers, water fountains, and medical respiratory equipment have been identified [START_REF] Orkis | Environmental sources of community-acquired legionnaires' disease: A review[END_REF].

Therefore, any water system that generates aerosols from water should be considered at-risk [START_REF] Baldovin | May car washing represent a risk for Legionella infection?[END_REF][START_REF] Walker | The influence of climate change on waterborne disease and Legionella : a review[END_REF]. For this reason, effective monitoring of water systems is critical. However, current standardized detection and enumeration methods (ISO 11731 and AFNOR T90 431 for cultures and ISO 12869 and AFNOR T90 471 for quantitative PCR) suffer from limitations that hinder proper and timely diagnosis (Mobed et al., 2019a).

Analysis by culture takes up to 10 days and the recovery of Legionella is not optimal due to the acid treatment and concentration stage [START_REF] Delgado-Viscogliosi | Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp[END_REF]. Viable but nonculturable cells (VBNC), which are compromised cells characterized by a loss of culturability, are not detected using cultures [START_REF] Kirschner | Determination of viable legionellae in engineered water systems: Do we find what we are looking for?[END_REF], whereas it has been demonstrated that they are nonetheless still able to infect amoebae and human macrophages [START_REF] Alleron | VBNC Legionella pneumophila cells are still able to produce virulence proteins[END_REF][START_REF] Dietersdorfer | Starved viable but nonculturable (VBNC) Legionella strains can infect and replicate in amoebae and human macrophages[END_REF]. Several studies have demonstrated that VBNC forms are systematically present in potable water sources due to the presence of disinfection products and their low levels of nutrients [START_REF] Chang | Effects of chlorination and heat disinfection on long-term starved Legionella pneumophila in warm water[END_REF][START_REF] Dietersdorfer | Starved viable but nonculturable (VBNC) Legionella strains can infect and replicate in amoebae and human macrophages[END_REF].

Quantitative PCR (qPCR) is fast and easy to implement. However, it is limited by its inability to discriminate between dead and live cells, not to mention the need for specialized knowledge and experience to operate the equipment.

Furthermore, both techniques require harvesting of water samples and further lab-based analyses.

Contaminated water systems are mainly disinfected using chlorine or heat treatment by exposure to high temperatures (70 °C for 30 min). It has been shown that up to 25% of Legionella bacteria can survive after such a heat treatment, and they are able to regrow after a few months [START_REF] Allegra | Longitudinal Evaluation of the Efficacy of Heat Treatment Procedures against Legionella spp. in Hospital Water Systems by Using a Flow Cytometric Assay[END_REF]. In light of the reasons outlined above, there is hence a clear need to develop fast, sensitive, portable, and easy-to-use devices to achieve continuous monitoring of Legionella that can be applied directly to the water system.

Biosensors are considered to be a valuable alternative in this field. However, despite their remarkable sensitivity and biological compatibility, only a small number of practical studies have investigated the use of immunosensors to address this issue.

In this context, an optical fibre sensor based on surface plasmon resonance has been reported to provide a limit of detection of approximately 10 CFU/mL [START_REF] Lin | Development and application of side-polished fiber immunosensor based on surface plasmon resonance for the detection of Legionella pneumophila with halogens light and 850nm-LED[END_REF]. A similar sensitivity can be obtained using an electrochemical impedance spectroscopy immunosensor [START_REF] Sboui | Characterisation of electrochemical immunosensor for detection of viable not-culturable forms of Legionellla pneumophila in water samples[END_REF], and this LOD is considered to be the lowest detected concentration. For instance, Li et al. [START_REF] Li | Disposable Immunochips for the Detection of Legionella pneumophila Using Electrochemical Impedance Spectroscopy[END_REF] and [START_REF] Lei | Microelectrode array biosensor for the detection of Legionella pneumophila[END_REF] reached LOD values of 10² Cells/mL and 10 5 CFU/mL, respectively, using an impedimetric immunosensor to detect Legionella pneumophila. Using an amperometric magnetic immunoassay assay for Legionella pneumophila detection, Martini et al. reported a LOD of 10 4 CFU/mL, with the possibility of detecting Legionella cells at 10 CFU/mL after a preconcentration step [START_REF] Martín | Rapid Legionella pneumophila determination based on a disposable core-shell Fe 3 O 4 @poly(dopamine) magnetic nanoparticles immunoplatform[END_REF].

Using surface plasmon resonance technology for Legionella pneumophila detection, Enrico et al. [START_REF] Enrico | SPR based immunosensor for detection of Legionella pneumophila in water samples[END_REF] and Foudeh et al [START_REF] Foudeh | Rapid and multiplex detection of Legionella's RNA using digital microfluidics[END_REF] reported LOD values of ~ 10 4 CFU/mL. More recently, Aziziyan et al. have used the negative zeta potential, enhanced by decorating Legionella pneumophila with negatively charged sodium dodecyl sulphate molecules, to achieve a detection limit of 10 3 CFU/mL [START_REF] Aziziyan | Sodium dodecyl sulfate decorated Legionella pneumophila for enhanced detection with a GaAs/AlGaAs nanoheterostructure biosensor[END_REF]. Despite their high sensitivities, these methods require specific equipment, as well as the necessary skills and expertise to properly interpret the sensing data. By using genosensors, which allow the detection of a specific DNA sequence, several authors have been able to achieve very low quantification limits of 10 -21 M (Mobed et al., 2019b) and 10 -14 M [START_REF] Rai | Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp[END_REF].

Nevertheless, despite their high sensitivities, genosensors cannot be used to detect and quantify live bacterial cells in water samples.

In light of these various reasons, in this study, we investigated a new version of a microfluidic immunosensor based on a combination of biosensor technology for Legionella detection and microfluidic systems.

Experimental

2.1.Chemicals and reagents

Phosphate-buffered saline (PBS) (0.01 M, pH 7.4), sulphuric acid (95-97%), bovine serum albumin (BSA, ≥ 96%), hydrogen peroxide solution (H2O2, 30%), acetone, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), and 16-Amino-1-hexadecanethiol hydrochloride (16-AHT) were purchased from Sigma-Aldrich.

BupH™ MES Buffered Saline Packs (MES) were purchased from Thermo Fisher Scientific and ethanol (99.8%) was purchased from Fluka.

Monoclonal mouse anti-Legionella antibody Dp-Lp1-O2 provided by Microbiodetection SARL (Commercy, France) was stored frozen, and three standard solutions were prepared daily at 0.1, 0.05, and 0.01 mg/mL in HEPES-buffered saline (HBS) composed of 10 mM HEPES and 150 mM NaCl.

All chemicals were analytical grade and were used without further purification.

Bacterial strain

A Legionella pneumophila sg1-GFP strain (Lp1-008-GFP) previously validated [START_REF] Mustapha | Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry[END_REF] for the expression of green fluorescent protein (GFP) was cultured on buffered charcoal yeast extract agar (BCYEα medium) containing chloramphenicol and incubated at 37 • C for 72 h to obtain exponential phase forms. All of the bacterial samples were freshly prepared by resuspension of colonies in an appropriate volume of PBS (Dulbecco's Phosphate-Buffered Solution, Sigma, France). An optical density (OD) of 0.2 at 600 nm (Biomate TM3; Avantec, Illkirch, France) was taken as a reference for the initial suspension containing 10 8 CFU/mL. Ten-fold serial dilutions from 10 to 10 8 CFU/ml were prepared and used to study the biosensing performance in both static and dynamic detection mode. The concentration of each independent initial suspension was determined by twice plating 100 µL aliquots of 10 2 and 10 3 dilutions on BCYE agar medium. The Legionella colonies were counted using a Scan 1200 enumerating system-Interscience, after 72 h at 37 °C.

Apparatus

All of the electrochemical measurements were performed at pH 7.4 in a 0.01 M PBS solution containing 0.1 M KCl and 10 mM [Fe(CN)6] 3-/4-(the reaction is controlled by diffusion of the ferri/ferrocyanide system as a redox probe) using a portable bipotentiostat (Metrohm, France SAS). Cyclic voltammograms (CV) were recorded at a scan range from -0.4 to + 0.6 V with a scan rate of 0.05 V/s. Square-wave voltammograms (SWV) were recorded from -0.3 to + 0.5 V using the following parameters: a frequency of 5 Hz, a pulse amplitude of 0.002 V, and a scan rate of 0.05 V.s -1 . To guarantee reproducibility, each experiment was carried out independently at least three times using three different SPEs. The Drop View 8400 software plots the results as SWV and CV curves. The experiments were performed using a threeelectrode system with Au as the working electrode, Pt as the auxiliary electrode, and Ag as the reference electrode.

Validation of functionalization was carried out by electrochemical measurements after each functionalization step and by visualization of the morphological changes using scanning electron microscopy (SEM, Nova Nanosem FEI 200, accelerating voltage 20 kV), and atomic force microscopy (AFM, Agilent 5500 LS instrument, Les Ulis, France). Before being observed by AFM, the SPEs were rinsed using ultrapure water and dried under nitrogen (N2) to prevent the formation of salt crystals. The images were processed using GWIDDION software (version 2.52). The root mean square (RMS) was used to characterize the surface roughness in terms of the irregularity and height distribution. Confocal laser scanning microscopy (CLSM) images were obtained using a TCS-SP2 inverted confocal scanning laser microscope (LEICA Microsystems, Nanterre, France), with argon as an excitation source (488 nm).

Fabrication of the biosensor

Screen-printed electrodes (SPEs), were purchased from Metrohm, France. The SPEs were first rinsed with an acetone and ethanol solution to remove physically adsorbed contaminants.

The working area (4-mm diameter) was then immersed for 1 min in a fresh piranha solution of 3:1 (v/v) 98% H2SO4/30% H2O2 using all the necessary safety procedures. The SPEs were then rinsed extensively with ultrapure water several times and finally dried in air.

Once cleaned, the SPEs were immersed in a solution of 2 mM 16-amino-1-hexadecanethiol 16-AHT in 99.8% ethanol for 24 hours in the dark at 4 °C to form self-assembled monolayers.

The carboxyl groups located on the crystallizable fragment of the heavy chains of the anti-L. pneumophila monoclonal antibody (mAb) were activated using EDC (dissolved in MES) as described by [START_REF] Vashist | Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay[END_REF][START_REF] Vashist | One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics[END_REF] Despite its importance for the biosensing performance [START_REF] Vashist | Comparison of 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide Based Strategies to Crosslink Antibodies on Amine-Functionalized Platforms for Immunodiagnostic Applications[END_REF][START_REF] Vashist | Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay[END_REF][START_REF] Vashist | One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics[END_REF], it is still difficult to ensure the orientation of the antibody during the activation process.

According to the literature, the use of EDC [START_REF] Ismail | Application of APTES-Anti-E-cadherin film for early cancer monitoring[END_REF][START_REF] Vashist | One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics[END_REF] or EDC/N-hydroxysulfosuccinimide (NHS) [START_REF] Chou | Ultra-low fouling and high antibody loading zwitterionic hydrogel coatings for sensing and detection in complex media[END_REF][START_REF] Raghav | Immobilization strategy for enhancing sensitivity of immunosensors: L -Asparagine-AuNPs as a promising alternative of EDC-NHS activated citrate-AuNPs for antibody immobilization[END_REF] or EDC with Sulfo-NHS [START_REF] Dixit | Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays[END_REF] enables chemical activation of the carboxyl groups.

Thus, the antibody linkage occurs through a peptide bond that should, theoretically, not affect its bioactivity.

However, the biosensor efficiency was found to be enhanced when EDC alone was used [START_REF] Vashist | Comparison of 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide Based Strategies to Crosslink Antibodies on Amine-Functionalized Platforms for Immunodiagnostic Applications[END_REF]. We compared the use of EDC alone and the use of EDC along with NHS (data not shown). The results corroborated the previous idea, which is a reason why we adopted this method.

A 50 µL aliquot of the EDC-activated mAb antibodies was then incubated overnight at 4 °C.

A final step consisting of the addition of PBS containing 1% bovine serum albumin (BSA) was carried out in order to block the non-specific binding sites on the sensing area.

2.5.Optimization of the antibody concentration under static conditions

In order to be able to minimize the final cost of the biosensor, we tested the anti-Legionella antibody at 3 different concentrations: 0.01, 0.05, and 0.1 mg/mL. These three concentrations were tested to detect Legionella in static mode as follows: 10 µL of the dilution containing Legionella at 10 CFUs/mL were deposited on the pretreated area of the SPE and then left for 3 h of incubation at room temperature and finally rinsed with PBS solution and characterized electrochemically using CV and SWV. On the same biosensor, 10 µL of Legionella at 10 2 CFUs/mL was added and the same procedure was performed, and so on, each time adding a 10-fold higher concentration of bacteria until Legionella at 10 8 CFUs/mL was reached. This procedure was used to obtain a more reliable indication of the occurrence of sensor saturation.

As in our previous work regarding the detection of Legionella under static conditions [START_REF] Souiri | AFM, CLSM and EIS characterization of the immobilization of antibodies on indium-tin oxide electrode and their capture of Legionella pneumophila[END_REF], the sensor was incubated for 3 hours [START_REF] Ismail | Application of APTES-Anti-E-cadherin film for early cancer monitoring[END_REF] so that the bacterial cell capturing time could be considered to be long enough to assure a comparative results with the dynamic mode of detection. The sensor was kept in a sealed empty Petri dish to avoid surface dryness during the incubation.

Dynamic detection

The Legionella detection experiments under dynamic conditions were carried out in a glove box. The SPE was inserted into a microfluidic cell (volume of the cell: 8 µL; Input Diameter: 1 mm -Metrohm, France) linked to a cell inlet and connected to the tube containing the bacterial suspension (2 mL). Circulation was achieved using a peristaltic pump (Watson-Marlow, type 101 U/R) and the bacterial suspensions were recirculated 5 times at a maximum speed of 100 µL.min -1 . The SPE electrode was then characterized electrochemically. The dilutions were tested using the same cumulative process described for the static mode.

Biosensing performance under dynamic conditions

In order to evaluate the efficiency of this mode of detection, 2 mL aliquots of the precirculated and the post-circulated contaminated samples were evaluated by culture: 500 µL of 10, 10 2 , and 10 3 CFUs/mL samples were plated on four BCYE plates (eight plates for each concentration). For each bacterial concentration, the initial suspension and the recirculated suspension (total volume of 2 mL for each one) were plated on BCYE agar supplemented with chloramphenicol and incubated at 37 °C for 72 h. All of the BCYE plates were observed under UV light at 366 nm to enumerate GFP-L. pneumophila colonies. Flow cytometry assays (FCA) (Cube6, Sysmex) were also carried out to assess the physiological states of the bacteria: dead, alive, and 'viable but nonculturable' (VBNC) before and after fluid circulation.

The quantification protocol was as described in our previous study [START_REF] Allegra | Use of Flow Cytometry To Monitor Legionella Viability[END_REF].

Thus, 2 mL of 10 5 CFU/mL was processed for quantification using this technique.

Results and Discussion

3.1.Validation of the surface modification: layer formation

The subsequent functionalization steps carried out on the bare gold electrode successively resulted in three modified surfaces: 16-AHT/Gold, mAb/16-AHT/Gold, and mAb-BSA/16-AHT/Gold. Each of these three modified surfaces was characterized by microscopic imaging techniques and by electrochemical measurements taking into account the responses obtained with the bare gold surface.

SEM and AFM characterization a) SEM characterization

SEM imaging was carried out on the bare gold surface (Fig. 1a) and on each of the three subsequent modified surfaces (Fig. 1b,1c,1d). As can be seen in Fig. 1a, the surface topography of the typical gold electrode exhibited a high level of definition and homogeneity of the gold film. Compared to the SEM images of the bare gold surface, those obtained from the functionalized surfaces revealed various new 'geometric shapes' that clearly indicated effective immobilization of the corresponding molecules (16-AHT). In terms of the first functionalization step (Fig. 1b), the corresponding geometric shapes appeared to be quite smooth and the shapes had a narrow size range. Furthermore, this range did not change over the entire surface. Such forms could be the result of organized self-assembled monolayers of 16-AHT (Fig. 1b). A similar result was previously reported by Heimel et al. [START_REF] Heimel | Interface Energetics and Level Alignment at Covalent Metal-Molecule Junctions: π-Conjugated Thiols on Gold[END_REF] using the simulated adsorption geometry for 4'-substituted-4-mercaptobiphenyls. These results suggest that the self-assembled molecules (SAM) can undergo a similar formation process. The SEM image of the antibody-modified surface (0.1 mg/mL) revealed grainy forms distributed homogeneously over the entire surface (Fig. 1c), suggesting successful mAb linkage to the 16-AHT modified surface. As can be seen in Fig. 1d, the SEM image of the BSA-treated surface (mAb-BSA/16-AHT/Gold) revealed the presence of more irregular shapes that were between 20 and 50 nm in size. This could be attributed to the adsorption of BSA on non-specific mAb binding areas. Our results, and those reported by others [START_REF] Mathebe | Electrochemistry and scanning electron microscopy of polyaniline/peroxidase-based biosensor[END_REF][START_REF] Zhang | Ultrasensitive electrochemical biosensing for DNA using quantum dots combined with restriction endonuclease[END_REF], confirm the successful use of SEM imaging to characterize modified surfaces during biosensor fabrication. The topography of the bare gold surface and each of the three modified surfaces was analysed using atomic force microscopy (AFM). The roughness parameter was evaluated as the average root mean square roughness (RMS) based on five measurements performed at five different locations (2 x 2 µm 2 ) on the surface. The roughness of the bare gold surface differed from that obtained for the modified gold surface, suggesting the formation of layers of film on the gold surface. Depending on the functionalization step, the analysed surface exhibited a reduced RMS roughness with a substantial standard deviation. These results could be explained by both the surface heterogeneity induced by the chemical modification and the manufacturing process of the screen-printing technology of gold particles suspended in an ink on a ceramic substrate [START_REF] Butterworth | SAM Composition and Electrode Roughness Affect Performance of a DNA Biosensor for Antibiotic Resistance[END_REF]). Yet, ultra-flat surfaces could be obtained using the pressure forming template stripping fabrication technique (0.35 ± 0.05 nm RMS roughness), as previously reported [START_REF] Rubio-Lara | Ultraflat Gold QCM Electrodes Fabricated with Pressure-Forming Template Stripping for Protein Studies at the Nanoscale[END_REF]. Overall, the generation of rough surfaces is one of the most convenient and effective ways to improve sensing signals [START_REF] Gao | On-chip suspended gold nanowire electrode with a rough surface: Fabrication and electrochemical properties[END_REF]. Following the first step of modification (16-AHT) of the gold substrate, the standard deviation decreased, suggesting good coverage of the modified surface. According to Love et al. [START_REF] Love | Self-Assembled Monolayers of Thiolates on Metals as a Form of Nanotechnology[END_REF], SAMs can be fabricated into patterns in the plane of a surface with dimensions ranging from 10 to 100 nm, thus resulting in the formation of a geometric shape through the formation of an assembled monolayer. For the antibody modified surface, the slight decrease in the standard deviation could be the result of the immobilization of the antibody molecules. The addition of BSA appears to indicate better coverage of the surface, which could be due to the adsorption of BSA molecules at different angles, as previously reported [START_REF] Rubio-Lara | Ultraflat Gold QCM Electrodes Fabricated with Pressure-Forming Template Stripping for Protein Studies at the Nanoscale[END_REF].

Electrochemical characterization (SWV and CV)

Electrochemical measurements remain the most effective method for surface analysis during the fabrication process and the performance of the biosensor. In our study, the electrochemical behaviour of the modified surface was investigated using square-wave voltammetry (SWV)

and cyclic voltammetry (CV) of the bare gold electrode and the three subsequent modifications with 16-AHT, mAb, and BSA, respectively.

As can be seen in Fig. 2, the peak current in SWV of the bare gold electrode was lower for each of the subsequent modified surfaces, suggesting immobilization of probe molecules. The highest current response occurred for the gold electrode (red curve). The peak current decreased substantially after treatment of the gold surface with aminothiol molecules (blue curve). The subsequent modification of the 16-AHT/Gold surface with antibody molecules was associated with a further decrease in the corresponding peak current of SWV (green curve), whereas no peak current change was observed after BSA treatment.

The observed decrease of the peak potentials by SWV after each of the two subsequent functionalization steps of the bare gold electrode could be related to a restricted mass transfer of redox species. This could be due to the molecular length of the covalently linked aminothiol and mAb molecules [START_REF] Chen | Effect of Anchoring Groups on Single-Molecule Conductance: Comparative Study of Thiol-, Amine-, and Carboxylic-Acid-Terminated Molecules[END_REF][START_REF] Zhang | Direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor[END_REF].

Furthermore, the reduction of the amplitudes of the current peaks recorded after the functionalization steps was associated with a slight change in the peak potential location. The peak current of the gold electrode was located at + 0.15 V whereas the peak current of the aminothiol-treated electrode was shifted to + 0.13 V. This slight potential shift of the peak current towards a lower voltage for the 16-AHT-modified surface could be related to the presence of a positive electrostatic charge around the H2N-terminated thiol-linked molecules.

Indeed, at the pH that was used (a pH of 7.4), and taking into account a pKa of 10.6 for the NH3+/NH2 couple [START_REF] Kromann | Prediction of pKa values using the PM6 semiempirical method[END_REF], the protonated form of the base predominated (NH3+). Moreover, the current peak for the treated surface with mAbs underwent a positive shift relative to that obtained following the first functionalization step. The corresponding peak appeared at + 0.18 V. This additional shift could be the result of the negative charge of the mAbs carboxyl groups at pH = 7.4. The negative charges introduced after mAb treatment would neutralize the positive charges of the amino functional groups. This could explain the shift of the current peak to a higher voltage following the second step of the functionalization of the biosensor. Similar results have been reported based on electrochemical impedance spectroscopy [START_REF] Sboui | Characterisation of electrochemical immunosensor for detection of viable not-culturable forms of Legionellla pneumophila in water samples[END_REF]. The changes noted with SWV were confirmed by cyclic voltammetry. The same variation was found in the characterized signals as discussed for the SWV results. Of note, there was a greater decrease of the peak current with SWV compared to CV. This could be due to the sensitivity of the SWV technique. Indeed, SWV measures both the forward and the reverse current, while only the forward current is measured in CV [START_REF] Brahman | Highly sensitive stripping voltammetric determination of a biomolecule, pyruvic acid in solubilized system and biological fluids[END_REF].

All of these microscopic and electrochemical measurements suggest that the functionalization steps during the biosensor fabrication were successful. These data are in agreement with the results of the immunofluorescence experiment, which was carried out to make sure that the bioreceptor recognition sites were still accessible after the addition of BSA (using an Alexa

Fluor 555 antibody, data not shown).

Antibody concentration optimization under static conditions

The aim of this part of the study was to estimate the lowest antibody concentration that would still ensure a linear electrochemical response, under static conditions, for bacterial suspensions ranging from 10 to 10 8 CFU/mL (as described in Section 2.4). Thus, three antibody concentrations of 0.01 mg/mL, 0.05 mg/mL, and 0.1 mg/mL were successively investigated.

The biosensor current signals needed for this study were, I mAb-BSA and I detection, which were the recorded signals without bacteria and the bacterial concentrations, respectively. These values could be attributed to the immobilization of bacterial cells, which acted as a barrier to electron transfer reactions on the biosensing surface. Such an impediment of the electron transfer kinetics on the electrode surface has been shown to occur during the formation of antibody/antigen complexes [START_REF] Yun | A nanotube array immunosensor for direct electrochemical detection of antigen-antibody binding[END_REF][START_REF] Zhang | Direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor[END_REF]. In order to confirm the immobilization of captured GFP-L. pneumophila, CLSM was performed. As shown in Fig. 3d, the images obtained indicate a homogeneous distribution of the captured bacterial cells at an antibody concentration of 0.1 mg/mL.

As can be seen in Fig. 3a and Fig. 3b, the ∆I/I values corresponding to antibody concentrations of 0.01 and 0.05 mg/mL did not increase in a linear manner with a Legionella concentration range from 10 to 10 8 CFU/mL. A saturation phenomenon was observed at these two antibody concentrations as the number of Legionella increased. By contrast, at an antibody concentration of 0.1 mg/mL (Fig. 3c), a linear increase in ΔI/I values was observed over a bacterial concentration range from 10 to 10 8 CFU/mL (R 2 = 0.975).

Therefore, under static conditions, the electrochemical biosensor functionalized with an antibody concentration of 0.1 mg/mL exhibited a wide linear response that ranged from 10 to 10 8 CFU/mL.

At an antibody concentration of 0.1 mg/mL, the limit of detection (LOD) was calculated to be 3 x SD (SD: standard deviation, n = 3) obtained from three measurements taken in the absence of bacteria. Three peak currents were recorded in the absence of Legionella, with values of 7.22 (I1), 7.02 (I2), and 5.9 µA (I3), with a mean value of 6.72 µA (I0). Each of these three values (I1, I2, and I3) was then used to obtain the corresponding immunosensor response in the absence of bacteria evaluated as |I0 -I|/I0. The values for the three responses obtained were: 0.075, 0.044, and 0.122, with a mean value of 0.08 and an SD value of 0.04. Therefore, the LOD was calculated to be 3 x SD, thus yielding a value of 0.12 with regard to the immunoresponse. The corresponding LOD value expressed in CFU/mL was then obtained from the calibration curve (Fig. 3c), yielding a value of ~ 10 CFU/mL. This LOD value was expected since the immunosensor responses varied linearly with the logarithmic value of the bacterial concentration over eight orders of magnitude (Fig. 3c), which included 10 CFU/mL. This LOD value is at least one order of magnitude lower than the values reported to date for the detection of Legionella pneumophila using various other biosensing devices. Compared to the previously published works discussed in the Introduction, our immunosensor appears to be the best device to detect Legionella pneumophila at 10 CFU/mL. Moreover, our method has the advantage of a relatively rapid analysis time (180 min), in addition to being portable, easy to operate, and relatively inexpensive compared to the other available technologies. Despite the excellent LOD value that we obtained and these advantages, this detection limit remains quite high compared to the reference levels for cooling towers (< 100 CFU/L) [START_REF] Miranda-Castro | PCR-coupled electrochemical sensing of Legionella pneumophila[END_REF] and thus needs to be improved. Another hurdle to be overcome is that this LOD is subject to the weakness of the corresponding response. As described above, the blank assay was associated with a biosensing response of [∆I/I]Blank = 0.08 ± 0.04. Otherwise, the immunosensor response with Legionella at 10 CFU/mL ([∆I/I]10 CFU/mL), obtained as the mean of three simultaneously measurements, was 0.08 ± 0.08. Thus, both of the [∆I/I]Blank and [∆I/I]10 CFU/mL responses were nearly the same.

This result suggests that 10 CFU/mL corresponds to the limit of detection of Legionella under static conditions, and this is in agreement with the obtained LOD (3 x SD). Nevertheless, due to the weakness of the response value and the relatively high SD, it would not be easy to detect 10 CFU/mL every time. Thus, increasing the response value of 10 CFU/mL by changing the detection conditions could be of great interest. Such an improvement would result in a more efficient quantification of 10 CFU/mL.

Log [CFU/mL]

-0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0

-0,3 -0,2 -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0

3.3.Performance of the dynamic biosensor a-Electrochemical characterization

The biosensor described above was used to perform electrochemical measurements to detect and quantify Legionella present in circulating solutions (dynamic conditions). The aim was to achieve a faster response time and amplified signal sensing compared to measurements performed under static conditions as discussed previously.

The biosensor was coupled to the microfluidic system as described in Section 2.6. The biosensor measurements were performed under dynamic conditions with an antibody concentration of 0.1 mg/mL. A plot of ΔI/I values vs. the logarithm of a Legionella concentration ranging from 10 to 10 6 CFU/mL is shown in Fig. 4 (red curve). It can be seen that, compared to the sensor performance under static conditions (blue curve), the performance under dynamic conditions (red curve) indicates an amplified biosensing response. Furthermore, a linear increase in the ΔI/I values was only observed up to 10 3 CFU/mL. Between 10 3 CFU/mL and 10 6 CFU/mL the representative curve trended downward and tended to plateau, whereas the ΔI/I values measured under static conditions revealed a linear increase over the entire 10 to 10 8 CFU/mL bacterial concentration range. This finding illustrates the precocious appearance of a saturation phenomenon when the biosensor was used under dynamic conditions. Moreover, as can be seen in Fig. 4, the linear part of the curve established under dynamic conditions (R 2 = 0.991) was almost parallel to the linear representation obtained under static conditions. Considering that biosensor sensitivity is defined as the slope of the linear part of the calibration curve, our results suggest that the sensitivity of the biosensor under dynamic conditions remains almost the same as the sensitivity observed under static conditions. Circulation of each of the three bacterial concentrations (10, 10 2 , and 10 3 CFU/mL) led to a nearly 4.5-fold increase in the ΔI/I values relative to the ΔI/I values obtained under static conditions., namely [∆I/I]10 CFU/mL = 0.361 ± 0.04. The enhancement of the immunosensing response by the use of this microfluidic system suggests that it should be possible to use this biosensor under dynamic conditions to quantify Legionella at very low concentrations ranging from 10 to 10 3 CFU/mL. Due to the 4.5-fold increase in the immunosensing response achieved with circulation, a bacterial concentration of 10 CFU/mL can more readily be detected under dynamic conditions. In light of this, our method based on the use of circulating bacteria appears to be more suitable for quantification

of Legionella pneumophila at a concentration of 10 CFU/mL. In addition, it seems likely that the detection limit should be significantly lower compared to the LOD obtained under static conditions. Furthermore, the incubation time with the bacteria was reduced from 180 min under static conditions to 100 min under dynamic conditions. The enhancement in the immunosensing response under dynamic conditions may be attributed to the increase in the number of cells captured by recirculation of the Legionella samples. This is, on the one hand, directly linked to a higher probability of contact between the bacteria cells and the functionalized surface and, on the other hand, it explains the occurrence of a precocious saturation phenomenon under dynamic conditions.

Moreover, linearity was not sought for bacterial samples at high concentrations since the aim of this study was to detect and quantify Legionella at very low concentrations. In France, the law of the decree of February 1 st , 2010 (Arrêté du 1 er février 2010, n.d.) specifies that target values of 10 3 CFU/L in public entities and 10 CFU/L in healthcare institutions should be used for Legionella risk assessment.

b-Physiological state of Legionella under dynamic conditions

In order to evaluate the impact of dynamic conditions on the physiological state of Legionella, culture and flow cytometry measurements were performed.

FCA (as described in Section 2.7), was applied to a bacterial concentration of 10 5 CFU/mL.

The percentage of dead cells before and after sample circulation was almost the same: 27%

for the bacterial cell input (the precirculated aqueous sample) and 29% after dynamic detection. Thus, these results indicate that the microfluidic system did not affect the viability of the Legionella cells under dynamic conditions. Moreover, similar to the results obtained by the culture method, the FCA results confirm, as in our previous study [START_REF] Sboui | Characterisation of electrochemical immunosensor for detection of viable not-culturable forms of Legionellla pneumophila in water samples[END_REF], that viable and culturable cells as well as viable but nonculturable cells were captured by the biosensor with the same degree of efficacy.

Table 2 presents the number of colonies for the three Legionella concentrations ranging from 10 to 10 3 CFU/mL. When performed with the precirculated aqueous samples, 15, 96, and 945 colonies were enumerated for the corresponding theoretical concentration of 10, 10 2 , and 10 3 CFU/mL, respectively. For these three bacterial concentrations, the plates incubated with recirculated solutions had 5, 45, and 601 enumerated colonies, respectively. Thus, these results indicate a decrease in the number of enumerated colonies when the cultures were performed with recirculated solutions compared to cultures performed prior to circulation.

Percentage-wise, these decreases were 67%, 53%, and 36%, respectively. The decrease in Legionella cells in the circulated solutions could result in more bacterial cells being captured, thereby leading to enhancement of the biosensing signals with regard to ΔI/I values.

As described above, a similar linear relationship was found between the ΔI/I values and the logarithm of the bacterial concentration ranging from 10 to 10 3 CFU/mL. Moreover, we found a linear relationship by plotting the percentage of captured cells and the logarithm of the corresponding concentrations (R 2 = 0.996). These two linear relationships suggest that the ΔI/I values were correlated to the percentage decrease in the number of colonies (R 2 = 0.977).

Therefore, it can be assumed that the increase in ΔI/I values was due to the greater extent of Legionella capture under dynamic conditions. An electrochemical method was developed by coupling a functionalized biosensor with a microfluidic cell for the detection of Legionella pneumophila sg1 in water samples. The main functionalization steps of the biosensor were performed by covalent linkage of a selfassembled monolayer of 16-amino-1-hexadecanethiol (16-AHT) onto a gold surface to immobilize an anti-L. pneumophila monoclonal antibody (mAb). Characterization by electrochemical methods and microscopic imaging techniques indicated immobilization of both the mAb and the targeted bacteria. Under static conditions, the biosensor exhibited a wide linear response range from 10 to 10 8 CFU/mL and a detection limit of 10 CFU/mL. Using a microfluidic system, a linear biosensing signal was obtained between 10 and 10 3 CFU/mL, with a response amplification by a factor of 4.5 compared to the results under static conditions. Thus, the observed enhancement of the biosensor response should result in a better detection limit: less than 10 CFU/mL. Therefore, the use of this combination for electrochemical detection of Legionella cells has several advantages such as sensitivity, good response amplification, an easy fabrication process, a faster method, real-time detection, and portability. There is hence ample potential to expand the application of this biosensor configuration to assess the risk of Legionella bacterial contamination in environmental water systems, and a real-time biosensing signal could be of practical value in this field, especially during epidemics.

Figure 1 .

 1 Figure 1. SEM images of bare Gold (a); 16-AHT/Gold (b); mAb/16-AHT/Gold (c), and mAb-BSA/16-AHT/Gold (d)

Figure 2 .

 2 Figure 2. Square-wave voltammetry (a) and cyclic voltammetry (b) of the modified electrode of bare Gold (red), 16-AHT/Gold (blue), and mAb-BSA/16-AHT/Gold (green)

  recorded signals permitted us to calculate the ∆I/I as [I (mAb-BSA) -I (detection)] / I (mAb-BSA). For each of the three antibody concentrations, a plot of the ∆I/I values versus the logarithm of the Legionella concentration is represented in Figure 3. As expected, there was a decrease in I detection due to bacterial capture, and thus an increase in the ∆I/I values as the bacterial concentration increased. The observed increase in the ∆I/I

Figure 3 .

 3 Figure 3. SWV net currents and sensor performance for antibody concentrations of 0.01 mg/mL (a), 0.05 mg/mL (b), and 0.1 mg/mL (c) under static conditions. A CLSM image of captured GFP-L. pneumophila (left) and a CLSM image of captured GFP-L. pneumophila with reflectance mode (right) using an antibody concentration of 0.1 mg/mL (d)

Figure 4 .

 4 Figure 4. Sensor performance: performance comparison between dynamic conditions (red curve) and static conditions (blue curve)

Table 1 .

 1 RMS value evaluation based on the AFM results (2 x 2 µm 2 )

	Surface	RMS value ± SD (nm)
	Bare gold	164 ± 75
	16-AHT/Gold	149 ± 55
	mAb/16-AHT /Gold	148 ± 73
	mAb-BSA/16-AHT/Gold	131 ± 54

Table 2 .

 2 Post-quantitative assessment of Legionella in recirculated artificially contaminated samples

	by culture

Acknowledgments

The authors gratefully acknowledge financial support from the Region Rhône Alpes -CMIRA program. A special thanks to the late Prof. Serge RIFFARD who played a key role in spearheading this work.

The authors also thank the laboratory team of the GIMAP and Hubert Curien (Saint-Étienne, France) for their technical assistance.