Abdessadk Anagri
email: abdssadek.anagri@gmail.com

Elia Zgheib

Jerome Pulpytel

T T Mai Tran

Akram Alhussein

Farzaneh Arefi- Khonsari

Nanoindentation characterization of nanocomposites coating based on graphene and siloxane matrix deposited by dielectric barrier discharge plasma

Keywords: Thin film, Plasma nanocomposite, Graphene nanosheets, Nanoindentation, Viscoelastic behavior

Hexamethyldisiloxane (HMDSO)/Graphene nanosheets (GNs) nanocomposite coatings were deposited by a dielectric barrier discharge at atmospheric pressure. Commercially available GNs were incorporated into HMDSO precursor at different concentrations up to 2 wt%. The nanocomposite coatings were analyzed using scanning electron microscopy (SEM) and nanoindentation test. Based on SEM images, it was found that the GNs were well distributed and dispersed in the plasma polymerized hexamethyldisiloxane (ppHMDSO) matrix with no significant agglomeration. The results of nanoindentation tests performed with the continuous stiffness measurements mode showed an increase in the hardness and reduced Young's modulus with the increase of graphene nanosheets percentage. The maximum increase was two orders of magnitude for 2% GNs/ppHMDSO nanocomposite coatings compared to the homopolymer ppHMDSO. Furthermore, the creep compliance analysis showed that the nanocomposite coatings exhibited a viscoelastic behavior compared to the plasma polymer homopolymer.

Introduction

The development of nanocomposite thin films is increasing in the research sector and is attracting special worldwide attention [START_REF] Anandhan | Polymer nanocomposites: from synthesis to applications[END_REF][START_REF] Faupel | Metal-polymer nanocomposites for functional applications[END_REF][START_REF] Yang | Structural characteristics of diamond-like nanocomposite films grown by PECVD[END_REF][START_REF] Crosby | Polymer nanocomposites: the "nano" effect on mechanical properties[END_REF]. Through the combination of properties of at least two materials, nanocomposite layers offer an opportunity to fabricate new materials with unique and enhanced properties. Nowadays, nanocomposite layers are used in several fields thanks to their various properties for biomedical [START_REF] Kumar | Biocompatible responsive polypyrrole/GO nanocomposite coatings for biomedical applications[END_REF], mechanical [START_REF] Crosby | Polymer nanocomposites: the "nano" effect on mechanical properties[END_REF], and anticorrosion [START_REF] Yu | High-performance polystyrene/graphene-based nanocomposites with excellent anti-corrosion properties[END_REF] applications.

In recent years, graphene-based nanocomposites are one of the most studied families of nanocomposite materials, which is due to their potentials in combining the unique properties of graphene and metal, polymer, or ceramic matrix to improve the physical and chemical properties of the nanocomposite [START_REF] Algul | The effect of graphene content and sliding speed on the wear mechanism of nickel-graphene nanocomposites[END_REF][START_REF] Sahoo | Electrochemical characterization of in situ polypyrrole coated graphene nanocomposites[END_REF][START_REF] Murray | Alumina-graphene nanocomposite coatings fabricated by suspension high velocity oxy-fuel thermal spraying for ultra-low-wear[END_REF]. Among these different families of nanocomposites, graphene/polymer matrix has been extensively studied owing to the excellent synergy between these materials. Prior work has successfully demonstrated promising anticorrosion [START_REF] Anagri | Nanocomposite coatings based on graphene and siloxane polymers deposited by atmospheric pressure plasma. Application to corrosion protection of steel[END_REF], thermal [START_REF] Gresil | Thermal diffusivity mapping of graphene based polymer nanocomposites[END_REF], biosensing [START_REF] Sadasivuni | Graphene-based polymer nanocomposites in electronics[END_REF], and mechanical properties [START_REF] Papageorgiou | Mechanical properties of graphene and graphene-based nanocomposites[END_REF], with considerable performance improvement compared to graphene or pure polymer matrix.

Graphene/polymer nanocomposite materials can be prepared by different methods. The most commonly used one is the sol-gel preparation process [START_REF] Wang | Fabrication and characterization of graphene-reinforced waterborne polyurethane nanocomposite coatings by the sol-gel method[END_REF]. However, this wet process requires the use of solvent and it is carried out in several time-consuming steps. In addition, the residues of chemicals used and stabilization often affects the performance. Atmospheric plasma deposition is a promising alternative process for graphene/polymer nanocomposite fabrication due to its simple, fast, and solvent-free nature [START_REF] Anagri | Nanocomposite coatings based on graphene and siloxane polymers deposited by atmospheric pressure plasma. Application to corrosion protection of steel[END_REF]. However, the plasma process could be limited by the dispersion of the particles in the liquid precursor of the polymer matrix during the preparation of the colloidal suspension, which can cause non-uniformity of particles distribution in the matrix after plasma polymerization.

Recently, the mechanical properties of materials were studied using the nanoindentation technique. It is one of the competent techniques used in characterizing the mechanical behavior of metals [START_REF] Kim | Nanocrystallization during nanoindentation of a bulk amorphous metal alloy at room temperature[END_REF], ceramics [START_REF] Krell | Nanoindentation hardness of submicrometer alumina ceramics[END_REF], polymers [START_REF] Vanlandingham | Nanoindentation of polymers: an overview[END_REF], and more particularly thin films [START_REF] Fischer-Cripps | Nanoindentation of thin films and small volumes of materials[END_REF]. The main advantages of using the nanoindentation reveal in its high sensitivity to thin films rather than the macroscopic techniques. Despite the approximation used in the determination of the projected contact area (Ac), and the homogenization assumption owing to local measurement, the nanoindentation can be used to measure reliable mechanical properties compared to other techniques. The mechanical properties, e.g. hardness and reduced modulus, can be directly obtained from the simultaneous indentation load-displacement curves [START_REF] Nix | Elastic and plastic properties of thin films on substrates: nanoindentation techniques[END_REF]. Moreover, the viscoelastic behavior known as time-dependent properties can be assessed using this technique [START_REF] Fischer-Cripps | A simple phenomenological approach to nanoindentation creep[END_REF].

Nanoindentation has been widely used to characterize the mechanical behavior of graphene-based nanocomposites [START_REF] Alian | Molecular dynamics study of the reinforcement effect of graphene in multilayered polymer nanocomposites[END_REF][START_REF] Chang | Nanoindentation study of size effects in nickelgraphene nanocomposites[END_REF][START_REF] Patil | Nanoindentation of Graphene-Reinforced Silica Aerogel: A Molecular Dynamics Study[END_REF]. Shokrieh et al. [START_REF] Shokrieh | Nanoindentation and nanoscratch investigations on graphene-based nanocomposites[END_REF] determined the mechanical properties of Epoxy/graphene nano-platelets (GNP) at different percentages of GNP up to 0.5 wt%. They demonstrated that both the elastic modulus and hardness increased by adding 0.05 wt% of GNP. Moreover, nanoscratch tests showed less pile-ups and high wear resistance in the nanocomposite. However, up to now, nanoindentation has been used to characterize only graphene-polymer matrix composites, which were either bulk materials or thick layers of few micrometers.

Zhu et al. [START_REF] Zhu | Nanoindentation and thermal study of polyvinylalcohol/graphene oxide nanocomposite film through organic/inorganic assembly[END_REF] studied the mechanical and thermal properties of polyvinylalcohol (PVA) /graphene oxide (GO) nanocomposite films. They found that the addition of 0.5 wt% of GO was highly effective in improving the mechanical and scratch-resistance of PVA. Furthermore, the friction coefficients and scratch depths of PVA nanocomposites decreased with GO incorporation. Nowadays, in addition to the study of elastic properties, the research community is interested in the viscoelasticity or viscoplasticity of nanocomposites. Lu et al. [START_REF] Lu | Characterization of the linear viscoelastic behavior of single-wall carbon nanotube/polyelectrolyte multilayer nanocomposite film using nanoindentation[END_REF] investigated the linear viscoelastic behavior of single-wall carbon nanotube/polyelectrolyte multilayer nanocomposites film using nanoindentation. They fitted the creep compliance using a generalized Kelvin model and confirmed the validity of their method by comparing their results to those obtained from a small-scale tensile test conducted on the same nanocomposite.

Similarly, Chafidz et al. [START_REF] Chafidz | Polypropylene/multiwall carbon nanotubes nanocomposites: Nanoindentation, dynamic mechanical, and electrical properties[END_REF] used nanoindentation to determine the viscoelastic properties of polypropylene/multiwall carbon nanotubes nanocomposite. They fitted the nanoindentation creep results with an empirical equation and found that the creep rate and displacement of PP/MWCNTs nanocomposite were lower than those of neat polymer. Wang et al. [START_REF] Wang | Measurement of viscoelastic properties for polymers by nanoindentation[END_REF] fitted the experimental data using a three-element Maxwell model and the viscoelastic contact model to estimate the viscosity and the elastic modulus, which were related to the creep resistance.

Goodarzi et al. [START_REF] Goodarzi | Nanoindentation characterization of Glass/Epoxy composite for viscoelastic damage interlaminar modeling[END_REF] calculated the viscoelastic parameters by fitting the experimental nanoindentation data using the three-element Zener model. Arora and Pathak [START_REF] Arora | Nanoindentation characterization of polymer nanocomposites for elastic and viscoelastic properties: Experimental and mathematical approach[END_REF] tested the viscoelastic creep on three nanocomposites processed at different temperatures by fitting the nanoindentation results using a three elements Voigt model.

In our work, nanoindentation tests were performed for the assessment of the mechanical behavior of plasma nanocomposites coatings based on graphene nanosheets and siloxane matrix (GNs /ppHMDSO). The novelty of the work presented in this paper is the investigation of the effect of graphene incorporation into plasma polymerized HMDSO (ppHMDSO) on the mechanical behavior of the obtained nanocomposites. The elastic and viscoelastic properties were determined from the nanoindentation tests. Moreover, the plasticity index and depth recovery rate were calculated in order to quantify the plasticity of the plasma homopolymer coating and the nanocomposite one. Viscoelastic properties were also determined using a twoelement Maxwell model, a three-element Voigt model, and a combined Maxwell-Voigt fourelement model.

Materials and characterization methods

Samples preparation

Nanocomposites coatings with different GNs contents of 0, 0.1, 0.5, and 2.0 wt% were deposited on silicon wafers and mild steel E24 substrates by dielectric barrier discharge plasma at atmospheric pressure as reported in our previous paper [START_REF] Anagri | Nanocomposite coatings based on graphene and siloxane polymers deposited by atmospheric pressure plasma. Application to corrosion protection of steel[END_REF]. Typically, The graphene particles are dispersed in Hexamethyldisiloxane (HMDSO) liquid precursor using an ultrasonic homogenizer before injecting the suspension as an aerosol into the dielectric barrier discharge (DBD) plasma reactor.

In this way, 500 nm thick nanocoatings are obtained in one step in less than 6 min.

Scanning Electron Microscopy (SEM)

The morphology of the ppHMDSO/GNs nanocomposites coatings was observed by a SEM (SEM-FEG ULTRA 55 ZEISS) for coatings deposited on silicon wafers. SEM images were acquired in a high resolution in-lens secondary electron detector at 2 mm working distance, and electron accelerating voltage of 5 to 15 kV (extra high tension EHT). Since the composite layers are insulating, the samples were sputter-coated with a 5 nm thin layer of graphite.

Nanoindentation

The nanoindentation tests of ppHMDSO/GNs samples were performed using a TriboIndenter TI 980-Hysitron equipped with a Berkovich diamond tip (Ei = 1140 GPa, νi = 0.07). A polycarbonate glass specimen was used for calibration before performing tests. All the nanoindentation tests were conducted under the continuous stiffness measurement (CSM) option, which offers the direct measurement of the dynamic contact stiffness at each data point acquired during the indentation. The prescribed maximum displacement was kept equal to 350 nm. The hardness and reduced modulus of the ppHMDSO/GNs samples were determined from the average of ten indents with 30 µm spacing between each indent and a holding period of 15 seconds at maximum load. Nanoindentation was used first in 1992 by Oliver and Pharr [START_REF] Oliver | An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments[END_REF] to determine the hardness and modulus of different materials. The representative load-displacement curve in Fig. 1 is involved in the determination of the nano-mechanical properties of a material. The analysis of a nanoindentation load-unload curve allows the determination of the mechanical characteristics of the materials, namely the hardness H and the reduced Young's modulus Er. These quantities are obtained from the load charge (P) and the slope of the unloading curve (S) that can be directly determined from the nanoindentation curve (Fig. 1). The contact area Ac projected under the load cannot be directly read on the nanoindentation curve and thus constitutes the key parameter for the mechanical analysis of a material by nanoindentation. The nanohardness (H) is given by the ratio of maximum load (Pmax) and the contact area (Ac) at Pmax given in Eq. ( 1):

Mathematical background of nanoindentation measurements a) Hardness (H) and reduced modulus (Er)

𝐻 = 𝑃 𝑚𝑎𝑥 𝐴 𝑐 (1)
The reduced modulus (Er) can be calculated from the following Eq. ( 2):

1 𝐸 𝑟 = (1 -𝜈 𝑓 2 ) 𝐸 𝑓 + (1 -𝜈 𝑖 2 ) 𝐸 𝑖 (2)
where Ei and νi are respectively the Young's modulus and the Poisson's ratio of the indenter (indexed by "i") and Ef and νf are respectively the Young's modulus and the Poisson ratio of the film (indexed by "f"). The reduced modulus (Er) is also defined as follows:

𝐸 𝑟 = √𝜋 2β 𝑑𝑃 𝑑ℎ 1 √𝐴 𝑐 (3)
where β is a correction factor depending on the indenter geometry.

Following the observation of several experimental data, Oliver and Pharr [START_REF] Rar | On the measurement of creep by nanoindentation with continuous stiffness techniques[END_REF] noticed that, during a nanoindentation test, the unloading curve was rarely, linear (Fig. 1) even during the initial stage of the discharge. They showed that the behavior during the discharge is correctly described by a power-type law:

𝑃 = 𝐵(ℎ -ℎ 𝑝 ) 𝑚 ( 4
)
where P is the instantaneous load, h is the indentation depth, hp is the residual indentation depth after unloading, B and m are parameters depending on the tested material.

b) Plasticity Index

The plasticity index (ψ) is generally used to characterize the elastic-plastic response of the material under external stresses and strains. In the nanoindentation test, the plasticity index of a material can be calculated as follows (Eq. 5):

𝛹 = 𝐴 1 -𝐴 2 𝐴 1 (5)
where A1 and A2 are the areas under the loading and unloading curves, respectively (Fig. 1).

Knowing the residual depth, the depth coverage rate h can be calculated, which reflects the depth restoration rate of the material under the indenter. The restoration depth is given by the following equation:

 ℎ = ℎ 𝑚 -ℎ 𝑝 ℎ 𝑚 ( 6
)
where hm is the penetration depth at maximum load, hp is the residual depth after complete indenter removal, and (hm -hp) is the depth recovered after complete indenter removal.

Mathematical background for the viscoelastic behavior

Since many materials exhibit a mechanical behavior with time-dependent effects, one of the common and most efficient approaches to oversee the time-dependency and to estimate viscoelastic properties by nanoindentation is the application of a steady load and measurement of the depth (creep). Fischer-Gipps [START_REF] Fischer-Cripps | A simple phenomenological approach to nanoindentation creep[END_REF] used the same approach to predict the time-dependent effects and hence the linear-viscoelastic behavior of indented materials. The expression of an isotropic elastic material with homogeneous surface indented by a conical and rigid indenter (Berkovich type) for a steady applied load (P0) is given by Sneddon [START_REF] Sneddon | The relation between load and penetration in the axisymmetric Boussinesq problem for a punch of arbitrary profile[END_REF] according to the following equation:

𝑃 0 = 2 𝜋(1 -𝑣 2 ) tan 𝛼 𝐸 ℎ 2 (7)
where α is the angle between the cone generator and the substrate plan (Fig. 2). If we consider

E e = E /(1-ν 2 )
as the effective elastic modulus, which is the combination of the elastic modulus and Poisson's ratio of the material, Eq. ( 7) becomes:

ℎ 2 = 𝑃𝜋 2 𝐸 𝑒 tan 𝛼 or ℎ 2 = 𝑃𝜋 2 tan 𝛼 {𝑓 𝑒 } (8)
with f e = 1/E e is the effective elastic compliance.

By taking into account the change in the contact area between the indenter and the tested material over time, Lee and Radok [START_REF] Lee | The Contact Problem for Viscoelastic Bodies[END_REF] proposed an efficient approach, based on a time-dependent operator, time-dependent compliance i.e. 𝑓 𝑒 (𝑡), which is also known as creep compliance, thereby Eq. ( 8) can be written as:

ℎ 2 (𝑡) = 𝑃𝜋 2 tan 𝛼 {𝑓 𝑒 (𝑡)} (9)
In this work, we use three different models to assess the creep compliance 𝑓 𝑒 (𝑡), which appears in Eq. ( 9) and to predict h(t); a two-element Maxwell model (Fig. 3 shown in Table 1 [20]:

Table 1: The creep compliance 𝑓 𝑒 (𝑡) equations for the three models.

Model 𝑓 𝑒 (𝑡)

2-elements { 1 𝐸 1 𝑒 + 𝑡 µ } 3-elements { 1 𝐸 1 𝑒 + 1 𝐸 2 𝑒 (1 -𝑒 -𝐸 2 𝑒 µ 𝑡 )} 4-elements { 1 𝐸 1 𝑒 + 1 µ 1 + 1 𝐸 2 𝑒 (1 -𝑒 -𝐸 2 𝑒 µ 2 𝑡 )}
where 𝐸 1 𝑒 and 𝐸 2 𝑒 are effective elastic moduli, and µ 1 and µ 2 are creep viscosity constants.

Results and discussion

Characterization of GNs distribution incorporated in the ppHMDSO matrix

The distribution of GNs particles incorporated into the polymer matrix is observed using a SEM, as shown in Fig. 4. The dispersion of graphene nanosheets is fairly homogeneous across the surface.

The Feret diameter is calculated in one of the graphene sheets directions. It is defined as the distance between two parallel planes limiting the graphene sheet perpendicular to this direction. From the SEM micrographs of the three samples (Fig. 4), the Feret diameter of the particles was found between 1 µm and 12 µm, with 80% of the particles size ranging between 1 and 7 µm. The average diameter of graphene particles was 3.0, 3.2, and 3.7 μm for the different composite coatings deposited with 0.1, 0.5, and 2 wt% in the colloidal suspension nebulized in the reactor, respectively. However, the most prevalent diameter in the three coatings was around 1 µm, as shown in Fig. 5 (a). ImageJ software was used to estimate the area ratios of the graphene sheets in the matrix. For instance, coatings containing 0.5 wt% GNs present a surface fraction of the particles of about 0.7%.

Roughness analysis

In order to investigate the effect of surface roughness on the tribological properties of the nanocomposite coatings, surface roughness measurements were performed using an optical profilometer (AltiSurf 500, Altimet, Marin, France). Fig. 6 shows the scanned surface topography, depth histogram, and the bearing curve for the neat homopolymer ppHMDSO and nanocomposite coatings. The height parameters resulting from the Abbott-Firestone curve according to the ISO 25178 standard were estimated and presented in Table 2.

The variation in height of each point regarding the arithmetical mean of the surface is given by the arithmetic mean height (Sa), the highest value (0.4 µm) was obtained for 2%

GNs/ppHMDSO coating, and the lowest value (0.1 µm) was for the homopolymer. The observed difference can be explained by the random orientation of graphene nanosheets in the matrix as reported in previous work [START_REF] Anagri | Nanocomposite coatings based on graphene and siloxane polymers deposited by atmospheric pressure plasma. Application to corrosion protection of steel[END_REF]. A similar trend is observed for the root mean square height with a maximum value of 0.51 µm obtained for 2% GNs/ppHMDSO nanocomposite.

The skewness (Ssk) and the kurtosis (Sku) that represent, respectively, the asymmetry and the sharpness of the roughness profile were measured. For 0.1 wt% GNs and 2 wt% GNs, the values of skewness are close to zero, which indicate that the height distribution of peaks and valley in both coatings is symmetrical around the mean plane. However, the homopolymer and the 0.5 wt% GNs nanocomposite show a positive and negative values of skewness respectively, which means that the height distribution has more sharp peaks in ppHMDSO and deeper valley in 0.5 wt% GNs nanocomposite. For all the coatings, the kurtosis values are slightly higher than 3,which means that the height distribution exhibits a slightly sharp texture [START_REF] Kundrak | Roughness of ground and hard-turned surfaces on the basis of 3D parameters[END_REF].

Three other parameters: (Sp) defining the maximum peak height, (Sv) defining the maximum valley height, and their sum (Sz) were measured. The value of (Sz) was the highest for the 2 wt% GNs nanocomposite coating. This result can be explained by the increase in the percentage of graphene nanosheets as well as its random orientation in the polymer matrix.

Hardness (H) and reduced modulus (Er)

The effects of graphene content on the nanomechanical behavior of GNs/ppHMDSO nanocomposite coatings were evaluated from the nanoindentation measurements. Fig. 7 shows the typical loading/unloading curves of different coatings. The total penetration depth in the ppHMDSO homopolymer was approximately 450 nm, while the total penetration, with the same maximal load, in the 2 wt% GNs/ppHMDSO nanocomposite coating was approximately 300 nm (total thickness of thin films = 500 nm). With the CSM mode, the penetration depthdependent hardness (H) and reduced modulus (Er) of the coatings (Fig. 8) can be approximately divided into three zones. The first region characterizes an initial increase to a maximum value, followed by an asymptotic decrease towards a nearly constant value with a penetration depth between 100 and 225 nm. The last region is characterized by a quasi-linear increase in the values of H and Er with the penetration depth. In the first stage and at small penetration depth, the contact area measurements is usually influenced by the surface conditions of the tested sample, namely roughness and surface stress. In the third region, the increase of H and Er values with a penetration depth of more than 225 nm can be attributed to the substrate effect.

The constant values of H and Er obtained in the second stage indicate that the measured values correspond to the intrinsic properties of a single material. Hence, the hardness and reduced modulus of ppHMDSO and GNs/ppHMDSO coatings obtained in this region are shown in Fig. 9. In agreement with a previous work [START_REF] Shokrieh | Nanoindentation and nanoscratch investigations on graphene-based nanocomposites[END_REF], the incorporation of graphene particles in a polymer matrix led to a significant improvement of its mechanical properties. Further, the present work shows an increase of approximately two orders of magnitude of the hardness and reduced modulus of 2 % GNs/ppHMDSO compared to the homopolymer.

Plasticity index

The integrated areas under the load and unload curves were used to calculate the plasticity index as indicated in equation 5. Table 3 presents the plasticity index and depth coverage rate of coatings at two different maximum loads. The decrease in the recovery rate with increasing the maximum load from 450 to 950 µN was approximately 3%. This indicates that with a higher load, the ppHMDSO chains in contact with the graphene sheets had less time to elastically recover after the removal of the external load. Therefore, more residual plastic deformations remained after loading. These results are in agreement with the work of Shokrieh et al [START_REF] Shokrieh | Nanoindentation and nanoscratch investigations on graphene-based nanocomposites[END_REF] who studied the mechanical properties by nanoindentation of a nanocomposite composed of an epoxy-based polymer matrix and graphene nanosheets with different mass percentages ranging from 0.01 to 0.5% of graphene. The results showed that the hardness and elastic modulus of nanocomposites increased with the graphene percentage. However, the plasticity index decreased leading to an improvement in the elastic recovery of the nanocomposite.

Viscoelastic behavior of nanocomposites

Time-dependent creep compliance curves obtained from the load holding stage at 25 °C are shown in Fig. 10 (curves in scatters). For all different coatings, the form of the curves is remarkably similar. From this figure, we notice that the loading curves of coatings tested at 450 µN for 15 seconds holding period show two creep regions. The first transient creep stage is between 0 and 6 s noticed with an important increase in the penetration depth with time. After 6 s, the creep reached a steady stage and the penetration depth was quasi-constant with a small increase. The four curves do not show any stage of instantaneous acceleration of the penetration depth as a function of time. Hence, the creep curves can be expressed using viscoelastic creep models (without any plastic compound). The three viscoelastic models shown in Fig. 3 were used to fit the experimental data of creep compliance. The fitted curves are shown in Fig. 10 (curves in lines). The data were fitted using the nonlinear least square regression method with a correlation coefficient (R 2 ) greater than 0.99. The elastic (E e ) and viscous (µ) properties of ppHMDSO and GNs/ppHMDSO coatings calculated using the three models are tabulated in Table 4. Maxwell linear model cannot fit the transient creep stage, where all the coatings present a high penetration depth rate. Table 4 reports also the results obtained for the unknown parameters of the two non-linear models:

Voigt and combined Maxwell-Voigt models (Fig. 3). These two models had been accurately fitted for the ppHMDSO and GNs/ppHMDSO nanocomposites coatings.

According to the fitting results (Table 4), for the nonlinear models, firstly, for all nanocomposite GNs/ppHMDSO coatings, both moduli and viscosities values were higher than the ones obtained for the plasma homopolymer ppHMDSO except for the nanocomposite coating containing 0.5 wt% GNs, which had the highest penetration depth rate. Furthermore, the elastic moduli 𝐸 1 𝑒 and 𝐸 2 𝑒 , related to the instantaneous elastic deformation and the elasticity of the amorphous phase in the polymer matrix respectively, were enhanced by increasing the graphene wt%. A similar trend is noted for the viscosity with the increase of GNs wt% in the nanocomposite coating; both µ1 and µ2 were increased indicating higher resistance to the viscoplastic deformation.

Based on the moduli and viscosity values, one can note that all nanocomposite coatings containing GNs were more rigid compared to the homopolymer ppHMDSO. In addition, the viscosity of those nanocomposite coatings was significantly higher compared to the ppHMDSO. On the other hand, the plasticity index of the ppHMDSO sample was 0.22 and 0.25 obtained at a maximum loading of 450 µN and 900 µN, respectively. At these loads, the plasticity index of the 2 wt% GNs/ppHMDSO was found 0.19 and 0.21, respectively.

Considering the viscoelastic models used in this work and results obtained from the data fitting and based on the first low moduli values, we can say that the homopolymer ppHMDSO exhibited a viscoplastic behavior, while the 2 wt% GNs/ppHMDSO had a more viscoelastic behavior.

Conclusion

The goal of this work was to study the mechanical behavior of plasma nanocomposite coatings, based on graphene and siloxane plasma polymers, deposited on mild steel substrates mainly by nanoindentation tests. The latter has never been used to characterize the mechanical properties of sub-micrometric thick nanocomposite coatings. The viscoelastic behavior of the nanocomposite coatings was deeply investigated and the effect of graphene nanosheets was evaluated. From the obtained results, the main conclusions are the following:

-Nanocomposite coatings based on graphene and plasma polymers were synthesized on steel substrates using a DBD plasma, with a fairly homogeneous distribution of graphene nanosheets across the surface. The estimated area ratio of graphene sheets was found to be linearly proportional to the mass fraction incorporated in the colloidal solution.

-The investigation of the surface roughness showed a slightly higher roughness of the nanocomposite coatings surface compared to the homopolymer surface. The increase in the roughness can be explained by the random orientation of the graphene nanosheets in the polymer matrix.

-The incorporation of graphene nanosheets in the polymer matrix led to a significant improvement of its mechanical properties with a two orders of magnitude increase of the hardness and reduced modulus of 2 wt% GNs/ppHMDSO compared to the homopolymer. The increase of hardness and reduced modulus were associated with a decrease in the plasticity index value leading to an improvement in the elastic recovery of the nanocomposite.

-The time-dependent creep compliance curves were obtained from the load holding stage and was fitted using a three-element Voigt model, and a combined Maxwell-Voigt fourelement one. The parameters obtained from the fitting curve showed a significant increase in the elasticity as well as the resistance to the viscoplastic deformation of nanocomposite coatings compared to the homopolymer.

Figure 1 :

 1 Figure 1: Schematic representation of loaded and unloaded deformations of a specimen during indentation test.

Figure 2 :

 2 Figure 2: Schematic illustration of indentation.

Figure 3 :

 3 Figure 3: (a) Two-element Maxwell model, (b) three-element Voigt model and (C) combined Maxwell-Voigt four-element model for viscoelastic characterization of the material [20].

  (a)), a three-element Voigt model (Fig. 3 (b)) and a combined Maxwell-Voigt four-element model (Fig. 3 (c)) as

Fig. 5 (Fig. 4 :Figure 5 :

 545 Fig. 4: SEM images of a) ppHMDSO, b) 0.1 wt% GNs/ppHMDSO, c) 0.5 wt% GNs/ppHMDSO, and d) 2 wt% GNs/ppHMDSO.

Figure 6 :

 6 Figure 6: Surface topography (left) and depth histogram with bearing curve (right).

Figure 7 :Figure 8 :Figure 9 :

 789 Figure 7: Loading and unloading curves of coatings, deposited on steel substrates, obtained from nanoindentation tests.

Figure 10 :

 10 Figure 10: Creep response at 450 µN for 15 s holding period of A) ppHMDSO, B) 0.1 wt% GNs/ppHMDSO, C) 0.5 wt% GNs/ppHMDSO, and D) 2 wt% GNs/ppHMDSO coatings.

Table 2 :

 2 Surface topography parameters according to ISO 25178 standard. Sq, root mean square height; Sp, maximum peak height; Sv, maximum pit height; Sz, maximum peak height; Ssk, skewness; Sku, kurtosis.

	Plasma coatings			Height parameters
		Sa (µm)	Sq (µm)	Sp (µm)	Sv (µm)	Sz (µm)	Ssk	Sku
	ppHMDSO	0.10	0.12	2.60	1.78	4.38	0.78	4.15
	ppHMDSO	0.24	0.29	2.62	3.98	6.60	0.01	4.02
	0.1 wt% GNs
	ppHMDSO	0.28	0.36	1.53	5.45	6.98	-0.90	4.67
	0.5 wt% GNs
	ppHMDSO	0.40	0.51	2.90	6.78	9.68	0.10	3.95
	2 wt% GNs
	Sa, arithmetic mean height;

Table 3 :

 3 Plasticity index and depth coverage rate of coatings at two different maximum loads.

			Plasticity index (Si substrate)
	Load ppHMDSO	0.1% GNs/ppHMDSO	0.5% GNs/ppHMDSO 2% GNs/ppHMDSO
	450µN	0.22	0.21	0.20	0.19
	900µN	0.25	0.24	0.24	0.21
			Coverage rate (%)
	Load	ppHMDSO	0.1% GNs/ppHMDSO 0.5% GNs/ppHMDSO 2% GNs/ppHMDSO
	450µN	97.77	98.73	99.02	98.75
	900µN	94.97	95.94	96.12	96.01

Table 4 :

 4 Viscoelastic properties of ppHMDSO and GNs/ppHMDSO coatings using twoelement Maxwell model, three-element Voigt model, and combined Maxwell-Voigt fourelement model.

		2-element	3-element model		4-element model
		model
		𝑬 𝟏 𝒆	µ	𝑬 𝟏 𝒆	𝑬 𝟐 𝒆	µ	𝑬 𝟏 𝒆	𝑬 𝟐 𝒆	µ1	µ2
		(MPa)	(MPa.s)	(MPa)	(MPa)	(MPa.s)	(MPa)	(MPa)	(MPa.s)	(MPa.s)
	ppHMDSO	1.25	761.02	1.26 41.88 165.18 1.27 55.09 1980.07 130.81
	0.1 wt% GNs 1.66	853.93	1.68 47.20 207.16 1.68 68.70 1806.49 157.61
	0.5 wt% GNs 1.63	299.65	1.71 15.52 44.73	1.73 18.26 630.07	61.75
	2 wt% GNs	2.71 1041.64 2.76 57.01 221.26 2.77 73.12 2767.08 168.71

Acknowledgement

A. Anagri would like to acknowledge Sorbonne Université (France) for having granted his PhD fellowship.


Yang Si

Chloé Grazon

Gilles Clavier

Jean-Frédéric Audibert

Bianca Sclavi
email: bianca.sclavi@sorbonne-universite.fr

Rachel Méallet- Renault
email: rachel.meallet-renault@universite-paris

FRET-mediated Quenching of BODIPY Fluorescent Nanoparticles by Methylene Blue and its application to Bacterial imaging

Keywords: Fluorescent nanoparticles, methylene blue, fluorescence quenching, bioimaging, bacteria

High resolution and good signal to noise ratio is a requirement in cell imaging. However, after labelling with fluorescent entities, and besides several washing steps, there is often an unwanted fluorescent background that reduces the images resolution. For this purpose, we developed an approach to remove the signal from extra-cellular fluorescent nanoparticles (FNPs) during bacteria imaging, without any washing steps. Our idea is to use methylene blue to quench (>90%) the emission of BODIPY-based fluorescent polymer nanoparticle by a FRET process. This "Hide-and-Seek Game" approach offers a novel strategy to apply fluorescence quenching in bioimaging to improve image accuracy.

Introduction

Fluorescence bioimaging allows for the acquisition of images of whole cells or cell compartments in a quick and simple way [START_REF] Wolfbeis | An overview of nanoparticles commonly used in fluorescent bioimaging[END_REF]. For a long time it relied on luminescent molecules that can be tailor-made to target specific organelles, membranes [START_REF] Wang | BODIPY-Based Fluorescent Surfactant for Cell Membrane Imaging and Photodynamic Therapy[END_REF][START_REF] Perez | Supported Cell-Membrane Sheets for Functional Fluorescence Imaging of Membrane Proteins[END_REF], or bio macromolecules (e.g. DNA, proteins) [START_REF] Guseva | Mesosubstituted-BODIPY based fluorescent biomarker: Spectral characteristics, photostability and possibilities for practical application[END_REF][START_REF] Kowada | FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging[END_REF][START_REF] Guseva | Design, Spectral Characteristics, and Possibilities for Practical Application of BODIPY FL-Labeled Monoterpenoid[END_REF][START_REF] Guseva | Conjugate of meso-carboxysubstituted-BODIPY with thioterpenoid as an effective fluorescent probe: Synthesis, structure, spectral characteristics, and molecular docking[END_REF].

However, only a few of these chromophores are approved for use in the human body because of their potential toxicity [START_REF] Alford | Toxicity of Organic Fluorophores Used in Molecular Imaging: Literature Review[END_REF]. They also suffer from many drawbacks including poor water solubility [START_REF] Si | Fluorescent Copolymers for Bacterial Bioimaging and Viability Detection[END_REF], rapid photobleaching [START_REF] Wang | Luminescent nanomaterials for biological labelling[END_REF] or sensitivity of the fluorescence signal to the environment [START_REF] Xie | Epithelia transmembrane transport of orally administered ultrafine drug particles evidenced by environment sensitive fluorophores in cellular and animal studies[END_REF].

In this context, fluorescence nanoparticles (FNPs) have emerged as an appealing alternative owing to their excellent optical properties such as high photostability [START_REF] Ou-Yang | An infinite coordination polymer nanoparticlesbased near-infrared fluorescent probe with high photostability for endogenous alkaline phosphatase in vivo[END_REF][START_REF] Molkenova | Rapid synthesis of nontoxic and photostable carbon nanoparticles for bioimaging applications[END_REF], brightness [START_REF] Wr | Photoluminescent Nanoparticles for Chemical and Biological Analysis and Imaging[END_REF], and versatility of the emission wavelength [START_REF] Boschi | Overview of the optical properties of fluorescent nanoparticles for optical imaging[END_REF][START_REF] Cheruku | Fluorescent PCDTBT Nanoparticles with Tunable Size for Versatile Bioimaging[END_REF]. The design principle for any doped fluorescent nanoparticle is to gather as many fluorophores as possible together on the same matrix [START_REF] Pratiwi | FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging[END_REF]. Unlike single organic dyes, each nanoparticle can thus contain thousands of embedded fluorophores that are either self-aggregated [START_REF]Self-Assembled Organic Nanomaterials for Drug Delivery, Bioimaging, and Cancer Therapy[END_REF] or encapsulated in a matrix (polymer, silica) and protected from its environment [START_REF] Kohle | Dye Encapsulation in Fluorescent Core-Shell Silica Nanoparticles as Probed by Fluorescence Correlation Spectroscopy[END_REF][START_REF] Grazon | Core-shell polymeric nanoparticles comprising BODIPY and fluorescein as ultra-bright ratiometric fluorescent pH sensors[END_REF]. Due to the large number of fluorophores embedded in a single particle (up to thousands), FNPs are significantly brighter compared to individual organic dyes [START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF]. In the meantime, because they are encapsulated, the fluorophores are protected from the outside environment and are therefore more stable, as shown by reduced photobleaching and blinking [START_REF] Taylor | Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles[END_REF][START_REF] Si | Fluorescent Nanomaterials for Bioimaging and Biosensing : Application on E.coli Bacteria[END_REF].

Usually, for imaging cells are incubated with the fluorescent dyes or nanoparticles, followed by a washing step to remove excess fluorescent material [START_REF] Si | Fluorescent Copolymers for Bacterial Bioimaging and Viability Detection[END_REF][START_REF] Ea | A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging[END_REF]. However, because of residual non-specific binding it can be quite difficult to completely eradicate all the fluorescent nanoparticles, causing unwanted signal background and decreased detection accuracy. Alternate or additional strategies to washing can thus be necessary to discriminate the specific fluorescent signal.

We have previously described the development of ultra-bright organic BODIPY fluorescent nanoparticles (FNPs) [START_REF] Grazon | One-Pot Synthesis of Pegylated Fluorescent Nanoparticles by RAFT Miniemulsion Polymerization Using a Phase Inversion Process[END_REF]. In order to study their possible application for microbial imaging, we devised an approach based on cell electroporation to facilitate FNPs penetration in the cells. However, we faced a major issue: excess FNPs could not be completely removed by washing, and it was difficult to assert by conventional microscopy whether the FNPs were indeed internalized by the bacteria or just nonspecifically bound on the surface. We thus looked for a strategy to "hide" the FNPs located outside the trypan blue for 1 min. This technique was used to quench the signal from nanoparticles adsorbed to the cell surface for flow cytometry analysis [START_REF] Vranic | Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry[END_REF]. However, the quenching mechanism and the bioimaging application were not studied, and this technique doesn't allow for the analysis of fixed cells. Other examples of such an approach include the reversal of biotin-methylviologen quenching by avidin in aqueous solutions [START_REF] Chen | Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer[END_REF] or the intramolecular quenching of a fluorophore (2,3-diazobicylo [(2.2.2)]oct-2ene)-labeled oligomer by a terminal guanine residue in water [START_REF] Wang | Kinetics of end-to-end collision in short single-stranded nucleic acids[END_REF]. However, until now, few applications of energy transfer have been developed for biological applications especially for microorganism bioimaging.

Herein, we present a novel approach applying the fluorescence quenching process between BODIPY-based fluorescent nanoparticles (FNPs) and methylene blue (MB) to improve the imaging of the interaction between the FNPs and bacterial cells. Methylene Blue was chosen as a cationic blue dye since it is easily accessible, cheap and FDA approved [START_REF] Provayblue | Methylene Blue for Intravenous Administration): Uses, Dosage, Side Effects, Interactions, Warning[END_REF]. We conducted a photophysical study to reveal that the mechanisms behind the very high quenching (>90%) of FNPs by methylene blue is due to Förster Resonance Energy Transfer (FRET). Methylene blue was therefore applied in bioimaging approach of E. coli electroporated with FNPs to "hide" the excess nanoparticles that cannot be removed by the common washing protocol. Thanks to the improved resolution and accuracy of detection, this "Hide-and-Seek Game" approach offers a novel and promising strategy to apply fluorescence quenching to bioimaging, especially to precious samples where cells can be easily lost during the washing steps.

Results and discussions

The fluorescent nanoparticles (FNPs) were synthesized as previously described [START_REF] Grazon | One-Pot Synthesis of Pegylated Fluorescent Nanoparticles by RAFT Miniemulsion Polymerization Using a Phase Inversion Process[END_REF] (details in ESI). The FNPs are composed of a hydrophobic core composed of poly(styrene-co-BDπ) and a hydrophilic shell of poly(ethylene oxide)-block-poly(acrylic acid) that is negatively charged. The FNPs have a hydrodynamic diameter of 65nm, emit at 543nm with a quantum yield of 36% (Fig. S1). Thanks to the high number of BODIPY copolymerized in a single FNPs (n=2975), the overall brightness of the FNPs is 7.8x10 7 M -1 cm -1 . Methylene blue (MB) is a well-known cationic biocompatible dye [START_REF] Sabar | Preparation of sulfonated chitosan for enhanced adsorption of methylene blue from aqueous solution[END_REF], with a strong absorption band around 660 nm (ε=95000 L⋅mol -1 ⋅cm -1 ) [START_REF] Cenens | Visible Spectroscopy of Methylene Blue on Hectorite, Laponite B, and Barasym in Aqueous Suspension[END_REF]. We hypothesized that the overlap between the emission spectrum of FNPs and the absorption spectrum of MB can induce energy transfer (ET) where FNPs are quenched by MB (Fig. 1a). Moreover the cationic dye shall interact electrostatically with the anionic FNPs' shell thus respecting the ET distance criterion. Finally MB is known to have quite a low luminescence efficiency (22%) [START_REF] Shahinyan | Photophysical properties of methylene blue in water and in aqueous solutions of dimethylsulfoxide[END_REF], thus if sensitization by FRET occurs introduction of additional fluorescence signal will be limited. We made titration experiments to characterize the quenching process of FNPs by MB. We put a FNPs solution with a concentration of 0.5nM inside a cuvette and then added methylene blue solution gradually. Next, we recorded emission spectra after each addition of MB until the emission intensity of FNPs reach a plateau (Fig. 1b). After the addition of MB, we found out that the fluorescence intensity of FNPs decreases continually, at the same time, the fluorescence intensity of MB increases slightly. In the same conditions but without FNPs, MB does not exhibit fluorescence emission (Fig. S3). Thus the decrease in the fluorescence intensity of FNPs is correlated with the increase of the emission of MB indicative of an energy transfer from FNPs to MB. If photoinduced electron transfer (PET) was active, BODIPY would be quenched but MB emission would not be detected since MB would be reduced to its colorless reduced form, leukomethylene blue [START_REF] Kazemi | Photodegradation of methylene blue with a titanium dioxide/polyacrylamide photocatalyst under sunlight[END_REF].

In summary, the fluorescence intensity of FNPs decreases dramatically up to a loss of 90% upon addition of 2.6 nM to 1 µM of MB. The decrease in fluorescence reaches a plateau from 6 to 20 µM MB to reach 98% of quenching (Fig. S4a). To better understand the mechanism of the quenching process, the data are represented as Stern-Volmer plots (Fig. S4b). Generally speaking, a linear Stern-Volmer indicates a single class of dyes, all accessible to the quencher equally [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF][START_REF] Méallet-Renault | Polymer beads as nano-sensors[END_REF]. In our case, because of the Stern-Volmer plot gegatively deviates from linearity toward the x-axis, therefore, there are two or even more populations of Si, Y., Grazon, C., Clavier, G. et al. FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging. Photochem Photobiol Sci (2022). https://doi.org/10.1007/s43630-022-00215-1 fluorophores presented, one of which is buried that could not accessible to the quencher. We thus decided to analyze the data with a modified Stern-Volmer plot (Fig. 1c) according to equation S3 [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF][START_REF]A negative deviation from stern-volmer equation in fluorescence quenching[END_REF]. The plot is close to linearity, and the part of the initial fluorescence of FNPs accessible to MB quencher is 0.94, meaning that 94% of the initial FNPs fluorescence is quenched by MB.

To confirm this high quenching efficiency and obtain insight into the nature of the ET, we performed time-resolved fluorescence measurements of FNPs ([FNPs] = 0.5 nM in Milli-Q water) with increasing concentration of MB up to 2.63µM (Fig. 2a). Upon addition of MB to FNPs, we can see a clear shortening of the fluorescence lifetimes, indicative of a possible Förster Resonance Energy Transfer (FRET) involving FNPs as the donor and MB as the acceptor. Fluorescence decays were fitted by a sum of 3 exponentials to extract the average lifetime and thus calculate the FRET efficiency (E) (Fig. 2b, Equation S6). The FRET efficiency is very large and equals to 96% at its maximum, meaning that up to 96% of the FNPs fluorescence can be quenched due to MB in solution. This result corroborates our initial observation with static fluorescence quenching. There are two possible hypothesis for this efficient quenching: i) On one hand, methylene blue is a small, positively charged molecule and the shell of the FNPs contains acrylic acid units that are negatively charged at pH 7 (Fig. S1 cd). There might be an electrostatic interaction between the carboxylates moieties and the cationic dye, locating MB preferentially near the FNPs shell rather than in a homogenous distribution in solution. ii) On the other hand, the BODIPY fluorophores inside the particles are in distinct environments due to the core of the particle is composed of poly(styrene-co-BDπ). We previously demonstrated [START_REF] Grazon | Ultrabright BODIPY-Tagged Polystyrene Nanoparticles: Study of Concentration Effect on Photophysical Properties[END_REF] that these FNPs are constituted of two BDπ populations : a small percentage of isolated BDπ , mainly located deep in the core of the FNPs, and the majority of the BDπ forming aggregates and preferentially located on the outer part of the hydrophobic core (i.e. close to shell / water). Knowing this non homogeneous repartition of BDπ within FNPs, we assume that methylene blue can only quench the aggregated BDπ (majority) in the outer part of the core but not the ones deeply buried in the particle. Moreover, we know that the energy transfer between closely spaced BDπ is very efficient and fast. We thus believe that a single MB acceptor can quench multiple BDπ donors. Moreover, we observed a slight blue shift of the emission of the FNPs when the MB concentrations increased during the titration.(Fig. 1b). This is probably because of the selective quenching of the exposed BDπ versus the buried ones. These data reveal that BDπ aggregates radiating at longer wavelengths are more readily quenched than BDπ emitting at shorter wavelengths. These fluorescent spectra demonstrate that quenched BDπ (preferentially localized on the shell) present an emission maximum at 543 nm, while protected BDπ (embedded in the hydrophobic core) emit at 540 nm [START_REF] Si | Fluorescent Nanomaterials for Bioimaging and Biosensing : Application on E.coli Bacteria[END_REF].

The hydrodynamic diameter of the FNPs (~65nm, Fig. S1b) is too large to allow their transfer inside the bacteria by passive transport [START_REF] Si | Fluorescent Copolymers for Bacterial Bioimaging and Viability Detection[END_REF]. We therefore used electroporation for the internalization of FNPs into bacterial cells, by creating holes in their membranes. Fig. 3 shows how FNPs interacts with the bacteria after electroporation: Fig. 3a is the phase contrast image and Fig. 3b is the fluorescence image of the same bacterium. We observed green spots (FNPs) on the fluorescence image of this bacterium. To obtain a more clear visualization, we have overlapped image a and b(Fig. 3c), one can notice that all the particles are associated with the bacterial cell. Six Rounds of washing of the cells with PBS did not remove all the non-specifically bound FNPs. Therefore, in order to distinguish whether the FNPs had entered the bacterial cells or were sitting on its surface, we have used methylene blue when bioimaging the E.coli electroporated with FNPs to quench the fluorescence of the nanoparticles outside the cells. It has been reported that the outer membrane in gram negative bacteria such as E.coli avoids or slows down the uptake of MB in the cells [START_REF] Singh | Photo-inactivation of Escherichia coli and Enterococcus hirae using methylene blue and sodium anthraquinone-2-sulphonate: effect of process parameters[END_REF]. Therefore, E.coli cells that were electroporated with FNPs were placed inside an Eppendorf tube, MB was added and quickly mixed, and samples were immediately prepared for microscope analysis by placing them on a microscope slide.

Before the addition of methylene blue an excess of free nanoparticles could still be seen on the image even after six washing steps with PBS (Fig. 4ac). After the addition of MB, the free FNPs outside the bacteria were quenched, therefore could not be detected by the fluorescence microscope. Particles internalized by the bacteria were protected by the cell membrane and were not quenched by the MB, thus remaining fluorescent and visible under the microscope indicating a successful selective quenching process (Fig. 4bc). Using this quenching approach, we have successfully hidden the free FNPs on the fluorescence image, giving a clear background. The particles located inside the bacteria are thus easily distinguished (Fig. 4d), with an increase in the resolution and accuracy of detection as compared to the picture without MB quenching (Fig. 4c).

Conclusions

The fluorescent nanoparticles (FNPs) described here have a great potential for bioimaging applications thanks to a set of beneficial properties: large number of copolymerized BODIPY fluorophores (≈3000) in a hydrophobic environment, a hydrophilic and biocompatible shell and high brightness (approximately 1000 times larger than traditional organic dyes). However, after incubation with cells, extensive washing is not sufficient to remove excess FNPs in the surrounding media and may preclude accurate detection. We thus devised an original approach to "hide" the excess FNPs by selective fluorescence quenching of those found outside the cells and facilitate the observation of the ones that have entered the cells. Methylene blue was selected for its cationic nature, its reduced permeability to E.coli, its blue color (can act as an energy acceptor based on its good spectral overlap with BODIPY emission). MB thus acted as a quencher and its (limited) luminescence could be sensitized as demonstrated by titration studies.

They revealed that the FNPs emission is reduced by ~98%, demonstrating a very effective quenching process of FNPs by methylene blue due to FRET. There are two complementary explanations for this Si, Y., Grazon, C., Clavier, G. et al. FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging. Photochem Photobiol Sci (2022). https://doi.org/10.1007/s43630-022-00215-1 very efficient quenching: electrostatic interaction between the cationic MB and FNPs carboxylates that preferentially locates the MB in the FNPs shell and a dense population of BDπs close to the core-shell interface that is accessible to MB. FNPs were inserted in the bacteria by electroporation. Subsequently, quick addition of MB allowed to visualize only the internalized FNPs by fluorescence microscopy while "hiding" those left outside in the media or adsorbed non-specifically on the surface of the bacteria that were not removed by washing the samples with PBS. Particles within the bacteria were protected by the cell membrane and could not be quenched by MB, thus remaining fluorescent and visible by optical microscopy.

The FRET process is broadly used in bioimaging [START_REF] Algar | FRET as a biomolecular research toolunderstanding its potential while avoiding pitfalls[END_REF] or biosensing [START_REF]Booster, a Red-Shifted Genetically Encoded Förster Resonance Energy Transfer (FRET) Biosensor Compatible with Cyan Fluorescent Protein/Yellow Fluorescent Protein-Based FRET Biosensors and Blue Light-Responsive Optogenetic Tools[END_REF], especially to track the binding of proteins or DNA to specific ligands [START_REF] Sanders | Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins[END_REF]. However, there are fewer examples of the use of energy transfer to improve the contrast in bioimaging. Therefore, this "hiding" process is a novel and promising strategy to apply fluorescence quenching to bioimaging in bacteria, especially to precious samples where cells can be easily lost during the washing steps, which to our knowledge, has not been applied before. With the improved resolution and accuracy of detection, this "Hide-and-Seek" approach extends the applicability of our FNPs that could be further functionalized on their surface with labels that could target specific areas in cells such as mitochondria or specific proteins. The approach may also be easily extended to a broader range of luminescent nanoparticles e.g. in single-particle tracking application to study the dynamics in different biological systems such as cells, tissues, organisms, bacteria and other living systems in the future. The possibility to use this "hide-and-seek" approach with extension of BODIPY conjugation (red-shifted absorption and emission) is currently underway in our group. The pairing quencher shall have the following requirements: be cationic, be an energy acceptor (absorption spectrum overlapping red-BODIPY emission), be a limited cell permeant, have a limited luminescence efficiency (in order to avoid introducing additional fluorescent signal). Due to its wide absorption band, MB acceptor can be paired to a BODIPY donor with an emission up to 650 nm. On the other side, other membrane-impermeant dyes like cationic cyanine dimers (i.e.TOTO-1, JOJO-1…) could be envisioned as bright energy acceptors with such fluorescent nanoparticles donors.

  Si, Y., Grazon, C., Clavier, G. et al. FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging. Photochem Photobiol Sci (2022). https://doi.org/10.1007/s43630-022-00215-1 cells based on fluorescence quenching. Boland et al. found out that the fluorescence of the FITC-labeled nanoparticles outside eukaryotic cells (NCI-H292 cell line) is quenched when incubated with 0.11%

  Si, Y., Grazon, C., Clavier, G. et al. FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging. Photochem Photobiol Sci (2022). https://doi.org/10.1007/s43630-022-00215-1

Fig. 1 :

 1 Fig. 1: a. Absorption (dashed lines) and emission (continuous lines; exc=495 nm) spectra of FNPs (green) and MB (blue) in water. b. Fluorescence emission spectra (λexc= 495 nm) of a solution of FNPs at 0.5 nM upon titration of MB from 2.63 nM to 21 µM in Milli-Q water at room temperature. c. Relative fluorescence intensity of a solution of FNPs [FNPs]=0.5nM with increasing MB concentration: modified Stern-Volmer plot and linear fit (y-intercept = 1.07; slope = 0.031 µM).

Fig. 2 :

 2 Fig. 2: a. Fluorescence decays (λexc=495 nm, λF =542nm) of FNPs ([FNPs]=0.5 nM in Milli-Q water) upon sequential addition of MB (up to 2.63µM). b. FRET efficiency (black squares) calculated using lifetime measurements of FNPs upon sequential addition of MB. The continuous grey line is the fit of the experimental data by a Hill function.

  Si, Y., Grazon, C., Clavier, G. et al. FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging. Photochem Photobiol Sci (2022). https://doi.org/10.1007/s43630-022-00215-1

Fig. 3 :

 3 Fig. 3: Bioimaging of one E. coli cell interacting with FNPs after electroporation. a: phase contrast image, b: fluorescence image, c: overlap of images A and B (scale bar = 1 µm)[23].

  Si, Y., Grazon, C., Clavier, G. et al. FRET-mediated quenching of BODIPY fluorescent nanoparticles by methylene blue and its application to bacterial imaging. Photochem Photobiol Sci (2022). https://doi.org/10.1007/s43630-022-00215-1

Fig. 4 :

 4 Fig. 4: Electroporated E.coli interacting with FNPs before (a, c) and after (b, d) quenching with MB (a, b: phase contrast images; c, d: fluorescence images).

Acknowledgements

Authors are grateful to CNRS (Centre National de la Recherche Scientifique) of France for its support (Interface Physique Chimie Biologie Soutien à la prise de risque-and by Institut d'Alembert (FR 3242 CNRS-ENS de Cachan)). A PhD grant for Y.S. (01/10/2012 6466) and C.G. (01/10/2009 320) was provided by the MENESR (Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche) of France.

Competing Interests and Funding: There are no competing or conflicting interests to declare.

Supplementary Information

The online version contains supplementary material available.


Issaad Ghozlene

Berrebbah Houria

Djebar Mohammed-Reda

Rouabhi Rachid

Cellular investigation on the role Of Osmotic Pressure in Limiting the Toxic Effects of Pesticidesin Durum Wheat (Triticum durum Desf.)

Keywords: Durum Wheat, Glyphosate, Salinity, Oxidative Stress, Membrane Integrity, Catalase, Synergy, Antagonism

Soil salinity is considered a major limiting factor in plant development, given the importance of cereals as an essential nutritional material worldwide. The present study is interested in demonstrating the effects of the salt stress / herbicide interaction on the roots of a plant model: durum wheat (Triticum durum) of the WAHA variety. The results obtained show that wheat is a plant sensitive to the action of NaCl. However, we have observed morphophysiological and metabolic disturbances in seeds subjected to saline stress in increasing doses, after germination. The measurement of the enzymatic activity shows a stimulation of the Catalase activity (CAT) accompanied by a production of hydrogen peroxide at the origin of oxidative stress, this increase is proportional to the degree of stress induced. Also, this stress induced the release of electrolytes. In parallel, we were interested in the toxicity of glyphosate. The penetration of the herbicide inside the plant has led to remarkable morpho-physiological and biochemical disturbances. Regarding the NaCl / Glyphosate interaction, we note a clear improvement in the majority of the measured parameters, this can be considered as an antagonism effect between the two stresses allowing better root growth.

Introduction

Plants play a major role in the terrestrial ecosystem, an essential constituent of the biosphere; they are the source of the oxygen we breathe, transforming the CO2 in the atmosphere into O2. Soil salinity is a major problem in agriculture that limits plant growth and causes significant loss of crop productivity worldwide [START_REF] Allakhverdiev | Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp[END_REF][START_REF] Munns | Comparative physiology of salt and water stress[END_REF]. Salinity affects up to 20% and 50% of the total cultivated and irrigated land in the world, respectively [START_REF] Cheng | Combined effects of the plant growth-promoting bacterium Pseudomonas putida UW4 and salinity stress on the Brassica napus proteome[END_REF]. However, the use of saline water in agriculture is gradually increasing owing to shortage of fresh water. In the face of stress, plants develop adaptation strategies to respond to environmental changes by controlling and adjusting their metabolic systems. However, this is not without consequences leading to the appearance of oxidative stress characterized by the formation of reactive oxygen species (ROS). The uptake of pesticides by the roots varies between plants and chemicals and can be greatly influenced by adjuvants and environmental conditions. (Wanga and Liu,2007)

Materials and methods

Conduct of tests

The biological material used in this work is durum wheat from the Poaceae family more precisely (Triticum durum Desf). The organs chosen to carry out this study are the roots. The samples come from Sedrata (Souk Ahras), Algeria. We used the WAHA variety. The herbicide used is Glyphosate. Durum wheat seeds are rinsed with a solution of distilled water, then soak for 24 hours. The seeds are meticulously chosen before their use. (No breaks or apparent signs of abnormalities). To assess the effect of the interaction between salinity and herbicide on the durum wheat variety, we prepared watering solutions with the following concentrations: C1: 0 (control) C2: 20µM, C3: 50µM, C4: 100µM, C5: 200µM) of NaCl and a constant concentration of Glyphosate: C: 20µmol. For each dose of NaCl, Glyphosate and each interaction, five repetitions were performed. The treatment solutions are added through daily watering at the rate of 8 ml per Petri dish, during the germination phase (4 days), 40 ml of NaCl, 40 ml of glyphosate, 20 ml of NaCl and 20 ml of the herbicide (glyphosate) for those of interaction, after sowing seeds in pots containing potting soil.

Studied parameters The germination percentage

The percentage of germination is identified after 48, 72 and 96 hours of treatment. The number of seeds which have sprouted and whose length of at least one of their roots exceeds 2 mm is considered having sprouted [START_REF] Ben-Hamouda | Allelopathic effects of barley extracts on germination an ace ling growth of bread on durum wheats[END_REF].

Relative water content.

The relative water content is an indicator of the hydric state of the plant. The relative water turgor is determined by the percentage of water present in the excised roots after treatment. The calculation is made according to, Ladigues (1975), according to the following formula: RWC: Relative water content. FW: Freshweight. DW: Dry weight. TW: Turgid weight

Membrane integrity

The percentage of cellular integrity consists of measuring the release of electrolytes generated by the partial destruction of the plasma membrane. In this method we first took of 3 samples (stage 1 slip) from each level of stress, then rinsed them with distilled water, and then cut them into small pieces of 1cm and collected into test tubes containing 10ml of distilled water. The tubes are tightly closed with cotton and aluminum foil and maintained 24 hours at room temperature. After 24 hours, the first interpretation is performed using a conductivity meter while gently placing the probe in the tube after calibration of the device. The second reading is performed after 24 hours of the first time after tubes autoclaving at 80 ° C for 20 minutes. We take 0.5g of plant material (roots) which are placed in 20ml of distilled water for 48h at 25 ° C. They are left to cool. Then we add 0.5ml of H2SO4 and we let them in ice for 1h. The samples reading are carried out using a spectrometer at a wavelength of 365 nm. (Grieve and Grattan, 1983).

Enzymatic determination RWC = (FW-DW)/(TW-DW) x 100

Preparation of the enzymatic extract:

The enzymatic extract used in our work is prepared for the determination of catalase activity. After 14 days of treatment, the fresh roots of both varieties of T. durum (1g) are ground cold with a mortar in 5ml of phosphate buffer (50mM phosphate, pH 7.5). The homogenate is then filtered with an adequate cloth before cold centrifugation (12000 g for 20 min). The supernatant obtained is used as an extract determine the enzymatic activity. The method used is that of (Cakmak andHorst, 1991, Boscoloa andal., 2003). The decreasing in absorbance is recorded withing three minutes for a wavelength of 240 nm and a molar linear extinction coefficient ε = 39400 M-1.cm-1. L Reaching a final volume of 3 ml, the reaction mixture contains: 100 μl of the crude enzyme extract, 50 μl of 0.3% H2O2 hydrogen peroxide and 2850 μl of phosphate buffer (50 mM, pH7.2). The calibration of the device is done in the absence of the enzyme extract. The reaction is triggered by the addition of hydrogen peroxide. The catalase activity is expressed in nmol / min / mg of proteins. Determination of hydrogen peroxide. Grind 0.1g of roots and 5ml of 1% TCA Trichloroacetic Acid in an ice bath. The homogenate is centrifuged at 12000g for 15 minutes. 0.5ml of the supernatants are mixed with 0.5ml of regulator (potassium phosphate KOH) and 1 ml of potassium iodine (KI). The D.O is determined at 390nm using a spectrophotometer. [START_REF] Loreto | Isoprene Produced by Leaves Protects the Photosynthetic Apparatus against Ozone Damage, Quenches Ozone Products, and Reduces Lipid Peroxidation of Cellular Membranes[END_REF] Results.

Percentage of germination.

The figure shows the effect of salt stress on the percentage of germination of the treated seeds.

Figure1. Effect of salt stress on germination

Relative Water Content (RWC)

The figure represents the effect of salt stress on the relative water contents (RWC) of the roots.

Catalase activity (CAT).

The figure below shows the evolution of catalase activity in the roots of durum wheat treated with the different concentrations of NaCl.

NaCl µM

Figure 4 Effect of salinity on CAT activity

The figure above indicates the catalase activity is inhibited at the C3 and C4 concentration. This decrease is around 20% compared to the witness. On the other hand, we record a stimulation of the catalase activity at the highest concentration. This increase is around 10% Hydrogen peroxide content.

The figure shows the production of hydrogen peroxide in the roots under salt stress.

Figure 5 Effect of salinity on H2O2 concentration

The exposure of the durum wheat roots treated by the different concentrations reveals an increase in the production of H2O2 at the C3 concentration and which tends to decrease at the highest concentrations (C4 and C5)

Effect of glyphosate. Percentage of germination.

The figure shows the effect of glyphosate on the percentage germination of treated seeds.

Relative Water Content (RWC).

The figure shows the effect of Glyphosate on the relative water contents (RWC) of the roots.

Figure7. Effect of glyphosate on RWC

We see in the figure above that treatment with glyphosate tends to increase the relative water content compared to the control. Treatment with concentration (C2) records a significant increase, which is around 300% Membrane Integrity.

The figure illustrates the effect of glyphosate on the integrity of the root membrane.

Catalase activity (CAT).

The figure illustrates the evolution of catalase activity in roots subjected to increasing concentrations of glyphosate.

Glyphosate (µM

Control C1 C2 C3 C4 C5
Hydrogen peroxide content.

The figure illustrates the concentration of hydrogen peroxide in roots subjected to increasing doses of glyphosate

Figure10. Effect of glyphosate on H2O2 production

Exposure of durum wheat roots treated with different concentrations of glyphosate reveals an increase in the production of H2O2 at concentrations C3 and C4. This increase is around 90% and tends to decrease at the highest concentration (C5).

Effect of the interaction between salt stress and glyphosate. Percentage of germination.

The figure illustrates the NaCl / glyphosate cocktail effect on the germination percentage of the treated seeds. germination decreases with time and compared to the control. On the other hand, there is no effect recorded at 96 h in seeds treated with C3.

Figure11. NaCl /glyphosate cocktail effect on the germination percentage

Relative water content (TRE).

The figure illustrates the NaCl / glyphosate cocktail effect on the relative water contents (RWC) of the roots.

Figure12. NaCl / glyphosate cocktail effect on the RWC

We see in the figure above that the treatment with NaCl / glyphosate tends to increase the relative water content compared to the control. Treatment with concentration (C3) records a significant increase, which is around 600% Membrane Integrity.

The figure illustrates the NaCl / glyphosate cocktail effect on the integrity of the root membrane.

Catalase activity.

The figure illustrates the activity of catalase in the roots subjected to the NaCl / glyphosate cocktail effect.

Figure14. Effect of NaCl / glyphosate cocktail on CAT activity

The figure above indicates that the catalase activity is stimulated in the seeds treated with the different concentrations of NaCl / glyphosate. The stimulation is important at the C5 concentration, of the order of 500% compared to the control.

Hydrogen peroxide content.

The figure shows the evolution of the average hydrogen peroxide content in the roots subjected to the salt stress / glyphosate interaction.

Figure15. NaCl / glyphosate cocktail effect on H2O2 concentration

The exposure of durum wheat roots treated by the different concentrations of NaCl / glyphosate reveals an increase in the production of H2O2. This increase is proportional to the stress induced. Discussion:

We will proceed to a general discussion of the results which have just been presented. This discussion will consist of three parts: The first will focus on the effects of salt stress, the second, those of glyphosate, while the third will focus on the interaction of the two molecules. Salt stress.

The study of physiological parameters shows that salinity affects the relative water content and the percentage of germination. According to our analyzes, the relative water content of the treated seeds has decreased significantly to avoid water loss. According to Hassani, and al., 2008 the absorption of water is maintained at a sufficient level to avoid dehydration of the plant tissues, to establish the phenomenon of succulence and to be able to dilute as many osmolytes as possible. The more the stress increases, the more the relative water content (RWC) drops. This seemingly obvious result is confirmed by numerous studies by Ali Dib, 1992.

The results reported in our study show that wheat is a plant sensitive to the action of NaCl, at the germination stage; the germination of the seeds diminishes when the salt concentration increases, which causes its inhibition. Our results clearly show that the seeds germinate better in the absence of salinity. The same results are reported by [START_REF] Bliss | Osmotic sensibility in relation to salt sensitivity in germinating barley seeds[END_REF], which demonstrate that the delay in seed germination is due to the time necessary for the seed to set up mechanisms allowing it to adjust its internal osmotic pressure. Also the results obtained on the biochemical parameters prove that the salinity has great repercussions on the membrane integrity, the enzymatic activity of catalase and the concentration of hydrogen peroxide. The integrity of the membrane increases with increasing doses of NaCl. The cell membrane of our plant model is indeed a main site of salt damage causing its dysfunction which leads to an increase in the leakage of ions causing lipid peroxidation, results confirmed by [START_REF] Mansour | Cell permeability under salt stress In[END_REF], who concluded that l he harmful effect of salinity on the plasma membrane is mainly due to the action of salt ions.

Continuing with the data collected on the CAT assay and the H2O2 concentration, where we observed a great complementarity of the results which reveal a dose-dependent production of hydrogen peroxide. The plants have developed an antioxidant system confirmed by catalase activity which plays an important role in the elimination of H2O2 and this in order to restore cellular homeostasis impaired by salinity [START_REF] Saraf | Enhancement of Catalase Activity under Salt Stress in Germinating Seeds of Vigna radiata[END_REF][START_REF] Rouabhi | Toxic Effect of a Pesticide, Diflubenzuron on Freshwater Microinvertebrate (Tetrahymena pyriformis)[END_REF][START_REF] Henine | Oxidative stress status, caspase-3, stromal enzymes and mitochondrial respiration and swelling of Paramecium caudatum in responding to the toxicity of Fe 3 O 4 nanoparticles[END_REF] Glyphosate.

Herbicides are frequently and abruptly used in intensive cropping systems to optimize agricultural production following accumulation in the soil and subsequent uptake by plants. Herbicides are toxic to many crops, including grains. Among cereals, wheat is considered important because of its high protein nutritional value. [START_REF] Mohammed | Glyphosate induced toxicity to chickpea plants and stress alleviation by herbicide tolerant phosphate solubilizing Burkholderia Cepacia PSBB1 carrying multifarious plant growth promoting activities[END_REF] In our experience, the penetration of glyphosate inside the plant has induced morpho-physiological and biochemical disturbances.

According to the study by [START_REF] Zablotowicz | Nitrogenase activity, nitrogen content, and yield responses to glyphosate in glyphosate-resistant soybean[END_REF], herbicides considerably reduced the nitrogenase activity of rhizobium (an enzyme complex allowing the symbiosis of rhizobium bacteria with plants through the formation of nodules). Under the effect of increasing concentration of glyphosate the symbiotic events are reached (the formation of nodules; true organs of metabolic exchange). As a result, the symbiotic events decrease, leading to a decrease in the formation of nodules responsible for the metabolic exchange between bacteria and plants.

According to Satchivi, and al., 2000, glyphosate enters tissue and reaches active metabolic sites, such as the plant's meristems, after being translocated through vascular tissue. Similarly, the herbicide can also be transferred to various plant tissues (Ready, et al., 2004). As a result, plant organs exhibit high rates of metabolism and growth which represent important sinks for glyphosate [START_REF] Hetherington | The absorption, translocation and distribution of the herbicide glyphosate in maize expressing the CP-4 transgene[END_REF].

The results relating to the different percentages of the germination of treated seeds show a significant decrease observed following the treatments with increasing concentrations of glyphosate after 96 hours. Thus, some authors link the number of seeds germinated to the rate of proteins synthesized, which suggests that oxidative damage to the roots may be responsible for the observed germination delay. [START_REF] Berova | Effect of paclobutrazol on wheat seedlings under low temperature stress[END_REF][START_REF] Willekens | Catalase is a sink for H2O2 and is indispensable for stress defense in plants[END_REF]. However, at low doses the herbicide weakly affects this parameter Regarding the relative water content, our results demonstrated that at low dose (C2) glyphosate has a stimulating effect on the development of the root system leading to a very high relative water content compared to controls, or the doses superiors used have a depressive effect on root growth which explains the low water levels observed. According to Boreva and Stoeva., 2000, in the unfavorable conditions of the surrounding environment, the plant is weakened and therefore incapable of making the most of its environment.

The biochemical parameters are also subject to the repercussions of glyphosate. Our results demonstrate that the increase in hydrogen peroxide is accompanied by the increase in membrane integrity, which is the cause of lipid peroxidation, which led to the stimulation of the enzyme trapping ROS: catalase. These parameters are frequently used as indicators of oxidative stress in plants. [START_REF] Gunes | Silicon mediates changes to some physiological and enzymatic parameters symptomatic for oxidative stress in spinach (Spinacia oleracea L) rown under B toxicity[END_REF] In a gene expression analysis, Ahsan, et al., Found that the application of glyphosate generates hydrogen peroxide (H2O2), resulting in peroxidation and destruction of lipids in Oryzasativa rice leaves Glyphosate / salt cocktail effect. The environmental environment of plants is frequently subjected to all types of aggression affecting their growth and development, among these aggressions, biotic (Herbicide) and abiotic (Salt) stress, the flagship subject of our study.

In accordance with the results that we have collected on the plant; the interaction between salinization and glyphosate had a beneficial effect on our treated roots compared to the results obtained for each of the stresses separately (salt and glyphosate). We suspect that this result is due to an antagonism effect, which means that one of the two stresses applied has block the effect of the other. The salt stress may have been prevented the penetration of the different solutes which explains the reduction in the harmful effects of glyphosate.

Regarding the physiological parameters, we note a clear improvement in the percentage of germination under the effect of the interaction, this can be considered as an antagonism effect between the two stresses allowing better root growth. The relative water content is slightly affected by this interaction because by reaching the highest doses of combined stress, we notice a regression in the rate of hydration of the roots. This can be explained by the combined action of NaCl and glyphosate which produce an overall toxic effect by synergy.

Concerning the biochemical parameters, our observations always remain in agreement with the results that we obtained later from each of the stresses exerted individually, in fact the increase in hydrogen peroxide is accompanied by the increase in membrane integrity thus disturbing catalase activity. The toxic combination of salt stress / glyphosate works in synergy.

Figure ( 01

 01 Figure (01) represents the germination percentage of durum wheat seeds of the WAHA variety measured during 4 days (48h, 72h and 96h). Treatment with NaCl shows that the percentage of germination increases with time and decreases compared to the control. The statistical study reveals a significant decrease in the percentage of germination of seeds treated with NaCl, at 24 h and 72 h. This drop reached 70% at the highest concentration.

Figure2.

  Figure2. Effect of salinity on the RWC

Figure3.

  Figure3. Effect of salinity on MI

Figure 6 .

 6 Figure 6. Effect of glyphosate on germination

Figure8.

  Figure8. Effect of glyphosate on IM

Figure 9 .

 9 Figure 9.Effect of glyphosate on CAT activity

Figure ( 11 )

 11 Figure[START_REF] Hassani | Effet de la Salinite sur L'eau et Certains Osmolytes Chez L'orge (Hordeum Vulgare)[END_REF] represents the germination percentage of durum wheat seeds of the WAHA variety measured during 4 days (48h, 72h and 96h). The cocktail effect shows that the percentage of

Figure13.

  Figure13. NaCl / glyphosate cocktail effect on IM

Annals of the Romanian Society for Cell Biology ISSN: 1583-6258 Review Letter

Dear Author,

Manuscript Submission ID: Annals_2022_1006

Manuscript Title: Cellular investigation on the role Of Osmotic Pressure in Limiting the Toxic Effects of Pesticides in Durum Wheat (Triticum durum Desf.) On behalf of Editorial Team of the "Annals of the Romanian Society for Cell Biology", we are pleased to inform you that your manuscript has been accepted publication in Current Issue of 2022 as per the recommendations given by the peer review group of experts.

The blind peer review process results are given below 1. Originality: 85% 2. Related of scope: 72% 3. Clearly and concisely written: Yes 4. Results: Satisfactory 5. References are Cited Properly: Yes

Final Decision: Accepted

For any further query feel free to contact us.

Regards

Editorial Team, Annals of the Romanian Society for Cell Biology