Jazia Awassa

Samantha Soulé

Damien Cornu

Christian Ruby

Sofiane El-Kirat-Chatel

Sofiane El-Kirat
email: elkirat1@univ-lorraine.fr

Understanding the Role of Surface Interactions in the Antibacterial Activity of Layered Double Hydroxide Nanoparticles by Atomic Force Microscopy

Understanding the mechanisms of interactions between zinc-based layered double hydroxides (LDHs) and bacterial surfaces becomes of great importance to improve the efficiency of these antibiotic-free antibacterial agents. In fact, the role of surface interactions in the antibacterial activity of zinc-based LDH nanoparticles compared to that of dissolution and generation of reactive oxygen species (ROS) is still not well documented. In this study we show that synthesized ZnAl LDH nanoparticles exhibit a strong antibacterial effect against Staphylococcus aureus and cause serious cell wall damages as revealed by growth test and atomic force microscopy (AFM)

Introduction

Despite the success achieved by the use of antibiotics to fight pathogenic bacteria, bacterial contamination and subsequent infections still present a significant threat on the global healthcare. 1 Moreover, the excessive use of traditional antibacterial drugs, e.g. antibiotics, led to the emergence of antibiotic-resistant bacteria. 2,3 Recent development in the field of material science and nanotechnology opened up new channels for the development of new inorganic antibiotic-free antibacterial agents as metallic nanoparticles (NPs), 4,5 silica NPs, 6,7 metal oxides NPs 8,9 and layered double hydroxides. 10,11 Layered double hydroxides (LDHs) are clay lamellar solids consisting of stacks of positively charged brucite-like sheets comprising divalent and trivalent metal hydroxides. This net positive charge is actually compensated by the presence of negatively charged intercalating anions present in the interlamellar region alongside with water molecules. [12][13][START_REF] Rives | Layered double hydroxides: present and future[END_REF][START_REF] Theiss | [END_REF] Due to their interesting properties including their tunable composition, chemical stability, high anion exchangeability and biocompatibility, LDHs were extensively studied as efficient antibacterial agents in different potential antibacterial applications such as water purification, 16 food packaging, 17,18 wound dressings, 19 and textile industries. 20 4 Recent studies proved that pristine unmodified LDHs, in particular zinc-based LDHs, could induce an efficient antimicrobial effect without any further chemical modification. 16,[21][22][23][24][25] In particular, zinc-based pristine LDHs were found to provide an efficient antibacterial activity against various bacterial strains. We have recently reviewed the different antibacterial mechanisms of pristine LDHs. 26 The three main mechanisms proposed in the literature are: (1) membrane and cell wall damages by direct interactions with bacterial surfaces; 16,22,27,28 (2) release of constituent divalent metal ions; 16,21,22,24,[27][28][29] and (3) generation of reactive oxygen species (ROS) which are well known to be toxic to the bacteria. 22,24,26,27,29 Despite the fact these three hypotheses were proposed to explain the antimicrobial effect of pristine LDHs, the role of each antibacterial mechanism in the total antimicrobial action is not determined, because the proof of evidence by real experimental set up is scarce. In our recent study, 21 we confirmed the contribution of the amount of released zinc metal ions out of Zn-based LDHs to their antibacterial activity (Mechanism 2). Zn-based LDHs were able to release Zn 2+ in broth media, and LDH samples having higher release profile of Zn 2+ possessed the highest antibacterial activity. In fact, an increase in Zn 2+ ions concentration may disturb the permeability of bacterial membranes by changing their active normal transport system thus leading to their damage. 30 Although our 5 previous finding supported the first suggested antibacterial mode of action of pristine LDHs by release of divalent ions, this does not eliminate the possibility of the contribution of the other two suggested mechanisms, particularly, direct surface interactions with bacterial surfaces (Mechanism 1).

Bacterial adhesion to LDHs was previously investigated using standard adsorption isotherm, density-gradient separation techniques as well as scanning and tunneling electron microscopy imaging. 19,[31][32][33][34] Although these investigations admitted to the presence of direct surface interactions between LDHs and bacteria, almost none of them correlated such interactions to the antibacterial activity of LDHs, as no antibacterial tests were carried out. In other words, adsorption or adhesion cannot itself explain antibacterial activity. Moreover, only few adhesion forces between bacteria and antibacterial clay materials or inorganic NPs were successfully measured using atomic for microscopy (AFM) technique, 35,36 and as far as our knowledge none of them was performed to particularly decipher the antibacterial mechanisms of LDHs.

AFM is considered advantageous compared to other high-resolution techniques, because it allows analysis of samples in liquid, thus mimicking physiologically relevant conditions. 37 Thanks to the development of AFM-based force spectroscopy (FS) approaches, AFM was not only limited to be 6 a powerful tool for high resolution imaging of bacterial cells but also used to probe bacterial adhesion phenomena. 38,39 In this context, AFM provides a quantitative evaluation of adhesion interactions between bacteria and Zn-based antimicrobial LDHs and a potential correlation to their antibacterial properties.

In order to measure LDH NPs interactions with bacterial walls using AFM-based force spectroscopy (FS), two contact surfaces need to be created: a NPs-functionalized AFM probe surface, and a bacterial substrate surface to be probed. In fact, some studies reported the attachment of NPs on AFM probes using commercial glue adhesives. 40 However, if LDH NPs were to be attached to AFM probe using any type of glue, contamination may take place due to the possibility of glue adsorption on the surface thus leading to false adhesion force measurements. Another way to fix NPs on AFM tips is by chemical covalent functionalization using covalent linkers. [41][42][43] However, these chemical modifications may alter the LDHs chemical nature and thus may in turn influence their interactions with bacteria. In-situ deposition of NPs on the tips under electron beam irradiation, evanescent wave illumination and short current-limited bias voltage were also reported. [44][45][46] Nevertheless, the advanced devices required in such approaches may not be readily available, which hamper their generalization.

7

Therefore, a new suitable method to functionalize AFM tips with LDHs is necessary to probe their interactions with bacteria and to understand their antibacterial mechanism. For this purpose, we designed an original approach to fabricate LDH-functionalized AFM tips by in-situ growth of LDH films directly on the surface of AFM tips. These LDH-functionalized tips were used to probe adhesion interactions with Staphylococcus aureus bacteria, a Gram-positive bacterium with multiple resistance strains able to infects humans leading to severe morbidity and mortality. 47,48 The antimicrobial activity of ZnAl LDH NPs against S. aureus bacteria was determined using agar disk diffusion and broth microdilution methods and compared to the effect of MgAl LDH NPs and micron-sized ZnAl LDHs. The comparison between the antibacterial properties of nano-sized and micron-sized ZnAl LDHs showed that the antibacterial properties of ZnAl LDH NPs go beyond the simple dissolution of Zn 2+ ions, where it was also related to surface contact interactions. Furthermore, AFM imaging revealed that growth inhibition by ZnAl LDH NPs was associated to severe morphological cell wall changes whereas cells treated with MgAl LDH NP remained intact.

Then, we took advantage of our original approach to grow LDH films in situ on AFM tips and to probe the interaction between LDHs and living S. aureus cells using AFM-based FS approach. The physico-chemical characterizations based on X-ray diffraction (XRD) and Fourier-Transfer Infra-red (FT-IR) spectroscopy revealed that LDH films presented properties comparable to those of LDH NPs. As compared to the controls, the FS adhesion maps recorded with ZnAl LDHfunctionalized tips presented strong adhesion (hundreds of pN) at high frequency which suggest that the antimicrobial effect of Zn-based LDH NPs is at least partly due to specific and direct interactions with the bacterial cell wall. This study provided valuable insights on the correlation between the specific ability of Zn-based LDHs to adhere on bacterial surfaces and their capability to inhibit the growth of bacteria.

Results and discussion

Structural and textural properties of ZnAl and MgAl LDH NPs. Nitrate-intercalated ZnAl and

MgAl LDH NPs were prepared by coprecipitation-hydrothermal method, described elsewhere. 49 The M II :Al III molar ratio of ZnAl and MgAl LDH NPs evaluated using inductively coupled plasma mass spectroscopy (ICP-MS) analysis were found to be 1.94 and 1.97, respectively. Such obtained values were very close to the expected value of 2, thus corresponding potentially to ideal arrangements of the M II and Al III cations in the brucite-like sheets. Figure 1a shows the XRD patterns of the prepared MgAl and ZnAl LDH NPs, both having the characteristic reflections of LDH materials exhibiting a hexagonal lattice with rhombohedral 3R 10 symmetry and no further crystalline impurities. 50 The interlayer spacing of both LDH NPs extracted from diffraction peaks at (003) and (006) basal reflections was 0.89 ± 0.01 nm, which is in good agreement with the interlayer spacing values for nitrate-intercalated LDHs. 51,52 Similarly, the FT-IR spectra of both LDH NPs showed characteristic peaks of nitrate-intercalated LDHs corresponding to O-H stretching vibrations at 3446 cm -1 and NO 3 -vibration 1350 cm -1 (Figure 1b). 53 No additional vibration bands which could be assigned to amorphous impurity phases had been detected, suggesting the high purity of our prepared NPs. X-ray photoelectron spectroscopy (XPS) was further used to confirm the chemical composition and bonding states of MgAl and ZnAl LDH NPs (Figures S1 andS2). N1s, Al2p, and O1s peaks located respectively at about 406.5, 74.5 and 532 eV, were found in both MgAl and ZnAl LDH NPs (Figures S1a andS2a). The presence of N1s peak is ascribed to NO 3 -intercalating anions. 54 The Al 2p region (Figures S1b andS2b) illustrates the presence of Al(III) species in both samples. 55 The O1s signal peak can be separated into three peaks located at about 530 eV, 532 eV and 533 eV (Figures S1c andS2c), which are attributed to the lattice oxygen, hydroxyl groups and water molecules, respectively. [55][56][57] Moreover, the XPS spectra of MgAl NPs shows the presence of Mg2p peak at ~50.5 eV (Figure S1d) accounting for the constituent Mg(II) divalent metal. 58 On 11 the other hand, the presence of Zn(II) constituent divalent metals in ZnAl NPs was confirmed by the presence of Zn2p 1/2 and Zn2p 3/2 located at ~1044 and ~1021 eV respectively. 54 Therefore, such configurations accounts for typical MgAl and ZnAl LDH structures as suggested by XRD and FT-IR analysis.

Both MgAl and ZnAl NPs presented a hexagonal lamellar structure as revealed by their SEM and AFM images (Figure 1). Both LDH NPs possessed similar average particle sizes (obtained as an average particle size of 100 LDH NPs) extracted from their SEM images (172± 31 nm and 176 ± 44 nm, for MgAl and ZnAl NPs, respectively). Similar average particle size values (obtained as *Data for micron-sized ZnAl LDH are adapted from our previous study. 21 ZnAl LDH NPs showed an efficient antibacterial effect against S. aureus with an average inhibition zone diameter of 20.6 mm and MIC value of 93.8 μg/mL. On the other hand, MgAl LDH NPs did not provide any antibacterial activity revealed by any of the two antibacterial tests, and thus played the role of a negative control in this case. The superior antibacterial activity of ZnAl LDH NPs 13 could be attributed to the nature of their constituent divalent metal, e.g. Zn 2+ . 21 In fact, it was previously reported that zinc-based NPs have a strong antibacterial effect towards a broad range of bacteria. 25 Such an effect may be achieved by adhering to bacteria, changing its charge distribution and blocking the transport of nutrients in and out of the membrane, or else by binding to bacterial intracellular DNA leading to their denaturation. 25,60 The antibacterial activity of the prepared ZnAl LDH NPs against S. aureus was slightly higher than those reported by Dutta et al. for ~250 nm-sized ZnAl LDHs having a MIC value of 200 μg/mL against S. aureus, 22 μg/mL against the same bacterial strain, which is superior to that of our ZnAl LDH NPs. 16 Such a difference in the antibacterial activity should be interpreted very cautiously, since many parameters play a role in the antibacterial properties of LDHs including their different physicochemical properties strains and to the different followed experimental procedures used to demonstrate the antibacterial test assays. In our previous study performed with identical antibacterial tests experimental procedures, 21 we showed that the antibacterial activity of micron-sized ZnAl LDHs (particle size ~ 1 μm) was related to released Zn 2+ ions (Mechanism 2) which was found to be 3.3% relative to the total amount of metal zinc ions initially present in the nitrate-intercalated ZnAl LDHs. Consequently, the tested LDH sample showed an inhibition zone diameter of 11 mm. On the other hand, the partial dissolution profile Zn 2+ released out of ZnAl LDH NPs (particle size~ 167 nm ) used in this study was found to be 6%. Interestingly, the increase in inhibition zone diameters for ZnAl LDH NPs used in this study was almost in the same order (2 times) of the increased amount of released zinc ions. The fact that these augmentations fall in the same range indicates that the enhanced antibacterial performance for ZnAl NPs in agar disk diffusion test can be related to Zn 2+ release in broth media. By design, inhibition zones are related to the diffusion of released cations, 26 which is in agreement with such finding.

On the contrary, the MIC value can be impacted by all the antimicrobial mechanisms, i.e. surface interactions and ROS generation. 26 In this case, the MIC of ZnAl LDH NPs (93.8 μg/mL) used in this study was almost 16 times greater than that obtained previously for micron-sized ZnAl LDH (MIC = 1500 μg/mL). 21 Taking into consideration that similar antibacterial tests assay protocols 15 against the same strain were adopted in both studies, the increase in Zn 2+ release by a factor of 2 cannot by itself explain the enhanced antibacterial activity of ZnAl LDH NPs. In fact, this seems to be more related to the lateral crystal size of LDHs which was reduced by almost 6 times in the case of ZnAl NPs compared to micron-sized LDHs used previously. Additionally, the great difference in MIC values between micron-sized particles and our newly synthesized NPs could also be related to the ability of NPs to provide a stable mobile suspension in solution, whereas micron-sized LDH particles were sedimented. This provides an evidence concerning the role of surface interactions in the antibacterial effect of LDHs (Mechanism 1). In this context, smaller size LDHs were able to induce an enhanced antibacterial effect due to their larger surface area (in this case, the surface is 36 times greater for the NPs) and a higher mobility of the NPs in a stable suspension promoting more accessible surface and contact with the tested S. aureus bacteria. 26,29,61 Finally, it is important to mention that the contribution of ROS generation to the antibacterial On the other hand, cells treated with antibacterial ZnAl LDH NPs were completely distorted with highly corrugated surfaces presenting hollows and crests (Figure 3c). Similar cells damage observations were previously reported. [63][64][65] Moreover, cells treated with ZnAl LDH NPs were found to have 3 times rougher surfaces (Rq = 49.5 ± 0.7 nm) than untreated and MgAl LDH NPs treated cells. The increase in surface roughness of bacteria suggests the existence of cell wall 18 damages induced by ZnAl LDH NPs, 62,63 which could be eventually followed by cell lysis and subsequent leakage of intracellular components. 65 To examine the morphological impacts of antibacterial ZnAl LDH on a greater fraction of cells, S. aureus were immobilized on mica substrates and imaged in air (Figure S3). Although imaging in air is not physiologically relevant, it can enhance morphological features and help to differentiate topographical differences between cells. 66 Untreated and MgAl NPs treated S. aureus cells appeared on the mica surface as grape-like clusters of round cocci with homogeneous cellular shapes and regular surfaces The diameter of cells observed in clusters varied from 600 to 1080 nm and the average value was 752 ± 115 nm, typical for this type of microorganism. 67,68 Upon treating S. aureus with antibacterial ZnAl LDH NPs, nearly all cells presented abnormal shapes and irregular surfaces. Some cells were enlarged in diameter (~1400 nm) suggesting cellular swelling due to ZnAl NPs treatment. Other cells appeared completely distorted with deep grooves, crests, hollows and even some split surfaces. Finally, some cells were completely disrupted or lysed leaving behind some cell debris covering the mica substrate.

The corresponding height profiles of bacterial cells immobilized on mica substrate were also determined (Figure S4). Cells treated with MgAl NPs showed a height profile of 480 ± 45 nm quite similar to that of healthy untreated cells (445 ± 19 nm). In contrary, S. aureus cells treated with ZnAl LDH NPs presented a lower height profile (180 ± 77 nm), suggesting their collapse.

Consequently, cells treated with antibacterial ZnAl LDH NPs did not only exhibit a different morphology than those of healthy untreated ones but also different height profiles as well. Similar effect was previously noted for damaged Escherichia coli cells. 65 All these observations suggest that ZnAl LDH NPs induce an antibacterial effect on S. aureus cells by targeting their cell wall leading to their damage, swelling or collapse, leakage of intracellular components and subsequently to the death of the microorganism. [63][64][65] AFM tips can be functionalized with LDH Films. Surface interactions occurring at the interface between bacteria and ZnAl LDH NPs are probably a main contributor to the antibacterial effect.

In order to decipher the role of surface interactions in the antibacterial activity of ZnAl LDH NPs by AFM-based FS, AFM tips were functionalized with LDHs. This was performed by growing LDH films directly on the surface of silicon nitride AFM tips. However, since it is quite difficult to perform LDH characterizations directly on AFM tips, the characterization was done on LDH films initially grown on silicon wafers. ZnAl and MgAl LDH films were synthesized on aluminum coated silicon wafers by in-situ growth method described elsewhere. 69 The obtained M II :Al III molar ratios evaluated by ICP-MS analysis for ZnAl and MgAl LDH films were 2.1 and 2.05, respectively. These values correspond again very well to the expected M II :Al III molar ratio of 2, similar to those for nano-sized and micron-sized 21 ZnAl and MgAl LDHs.

The XRD patterns of the synthesized ZnAl and MgAl LDH films exhibited the characteristic (003) and (006) basal reflections of the LDH structure, which confirmed the formation of the LDH film on the silicon wafer (Figure S5a). The interlayer spacing of both LDH films was 0.89 ± 0.01 nm, which is in perfect agreement with the interlayer spacing values for nitrate-intercalated LDHs and their previously synthesized NPs counterparts. 51,52 The quasi-absence of non-basal reflections at high angles implied the c-oriented growth of LDH crystallites in the film. 70,71 Similarly, the FT-IR spectra of ZnAl and MgAl films also confirmed the successful growth of nitrate-intercalated LDH films without any amorphous impurity phases (Figure S3b).

The morphology of the LDH films and LDH-functionalized AFM tips were examined by SEM imaging. As shown in Figure 4a and b, MgAl and ZnAl LDHs were successfully grown on the surface of silicon wafer with their c-axis mostly perpendicular to the substrate. The morphology of the observed structures showed a "cauliflower-like" shape, very similar to that reported by other groups, 72,73 using comparable in-situ grown wet-chemistry synthesis method. Electron dispersive 21 X-ray spectroscopy (EDS) was further performed on the LDH films (Figure S6) which ensured the growth of LDH films on silicon wafers with a M II :Al III molar ratio similar to that determined by ICP analysis (Table S1).

22

The thickness of the formed films was determined by cross-sectional SEM imaging and was found to be 312 ± 24 nm and 315 ± 25 nm for MgAl film and ZnAl LDH films, respectively (Figure S3c,e). Scratch method was also used to determine the thickness of the obtained films by AFM imaging and the obtained values (314 ± 10 nm and 316 ± 12 nm for MgAl film and ZnAl LDH films, respectively) were in good agreement with those found by SEM imaging (Figure S3d,f).

Similar synthesis conditions were adapted to grow LDH films on silicon nitride AFM tips, where bare silicon nitride tip was first coated with aluminum (Figure 4c andd). ZnAl and MgAl LDH films were then grown directly on the surface of aluminum-coated tips (Figure 4e andf). The morphology of LDH films grown on the tips was similar to those grown on silicon wafers, however, the hexagonal particles in this case seemed to be less defined probably due to the pyramidal geometry of tip. The successful functionalization of AFM tips with MgAl and ZnAl LDH films with a M II :Al III molar ratio ~2 similar to that obtained on LDH films grown on silicon wafers was also confirmed by EDS analysis (Figure S7 and Table S1). an adhesion signature with a frequency ranging from 63 to 93% (Figure 5b and Figure 6b). The variation in adhesion frequencies among the probed cells may be attributed to the slight heterogeneity in cell wall microarchitecture and composition or else to the position of ZnAlfunctionalized AFM tips during measurements. Measured adhesion forces of all cells mainly ranged from 180 to 800 pN (Figure 5b and Figure 6b) with rupture lengths ranging between 10 and 300 nm (Figure 5c). Such finding indicates that ZnAl tips were able to recognize S. aureus cell wall with high adhesion frequency, thus suggesting the presence of surface interactions occurring at the interface between ZnAl LDHs and bacterial cell wall.

Force spectroscopy reveals specific surface interactions between antibacterial zinc-based

To determine whether the presented surface interactions between ZnAl LDH and S. aureus are specifically related to their antibacterial properties, the adhesion behavior of ZnAl-functionalized tips was compared to that of control Al-coated tips as well as to that of non-antibacterial MgAlfunctionalized ones. For this purpose, individual living S. aureus cells trapped in porous membranes (Figure 6a) were probed using the three different AFM tips, e.g. ZnAl-functionalized, MgAl-functionalized and Al-functionalized tips.

Upon probing the same cells (Figure 6a) using Al-functionalized tips, no considerable adhesion between aluminum and S. aureus was noticed, where the adhesion frequencies and force magnitudes did not exceed 11% and 280 pN (Figure 6b), respectively. This indicates that constituent aluminum trivalent metal ions do not contribute to the observed adhesion surface interactions between ZnAl LDH and rthe cell wall of S. aureus.

MgAl-functionalized tips adopting quite similar morphologies to those of ZnAl-functionalized ones (Figure 4e,f) were also used to probe the cell surfaces of S. aureus. This was actually done for two main reasons. First, to eliminate the possibility of the contribution of the LDH-functionalized tips morphology to the reliability of our force measurements. Second, to determine whether the observed surface interactions between the cell wall of S. aureus and ZnAl LDHs are specific to Zn-based LDHs and to further link them to their demonstrated antibacterial properties.

Although cells probed with MgAl-functionalized tips showed slightly higher adhesion frequencies (13-21%) and force magnitudes (150-300 pN) in comparison to Al-functionalized tips, their adhesion behavior in terms of both adhesion frequencies and force magnitudes were remarkably At the end of each experiment, both LDH-functionalized tips were washed with ethanol, dried with nitrogen and imaged again using SEM. The functionalized tips seemed to be intact after being in contact with the bacteria during the experiments (Figure S8) thus confirming the robust functionalization protocol used.

We had previously established in this work that ZnAl LDH NPs possessed specific and superior antibacterial properties in comparison to non-antibacterial MgAl LDH NPs, which was validated by S. aureus growth inhibition (Figure 2) and imposing serious morphological impacts to their cell walls (Figure 3 and Figure S1). Therefore, the specific adhesion interactions observed for antibacterial ZnAl LDHs provided a first insight about the contribution of direct contact interactions to the antibacterial activity of Zn-based LDHs (Mechanism 1). In fact, the sensitivity 27 of microorganisms to different divalent metals-based materials is actually dependent on several factors, including the role each metal plays in normal metabolism, and the presence of specific binding sites for each metal. 74,75 S. aureus cells were highly sensitive to ZnAl LDHs probably due to the antibacterial role played by their constituent zinc components where they showed strong surface adhesion interactions with the tested bacterial membranes. In this context, ZnAl LDH NPs were specifically able to recognize and adhere to S. aureus cells thus imposing serious damages to their cell walls by direct surface interactions. nm 2 ) obtained on the top of cells using ZnAl-functionalized AFM tip (b) MgAl-functionalized AFM tip (c) Al-functionalized AFM tip (d). White/grey pixel correspond to adhesive events and black pixels correspond to non-adhesive events (scale 0-1000 pN). Data were obtained from four independent experiments using different tips and independent cell cultures.

Conclusion

In the present study, the role of surface interactions in the antibacterial activity of ZnAl LDH NPs dishes using sterile cotton tipped swabs (COPAN) to prepare bacterial lawns monolayers.

Antibiotic filter disks of (diameter = 6mm, Dominique Dutscher) were placed on the dishes and 20 μL of LDH dispersions (10 mg/mL) were loaded on the respective disks. Petri dishes were then incubated at 37°C for 24 h. Afterwards, bacterial inhibition zones were observed around the disks.

The diameter of the circular zones was measured using a centimeter ruler and mean values of three repeated trials were recorded and represented in millimeters.

Broth microdilution turbidimetric method was used to determine the minimum inhibitory For imaging in air, cells were centrifuged, washed twice and resuspended in ultra-pure distilled water then deposited on mica substrates. After 2 hours, substrates were gently rinsed in ultrapure distilled water and dried overnight at 30 °C. For both air and liquid conditions, images were acquired in peak-force tapping mode using SNL-C tips.

38

For force measurements on cells using AFM-based force spectroscopy, stationary phase cultures of S. aureus were trapped in porous membranes in a similar procedure used for cells imaging in MOPS. First, bare MLCT-C tips were used to localize and image individual cells. Then, the tips were replaced by aluminum-coated tips, MgAl and ZnAl LDH-functionalized tips. Adhesion force maps and histograms were obtained by recording 32 × 32 force-distance curves on areas of 400 x 400 nm 2 on cells surfaces, calculating the adhesion force of the last adhesion peak for each force curve, and displaying the value as a grey pixel. All curves were recorded using a maximum applied force of 250 pN, a contact time of 250 ms, and constant approach and retraction speeds of 1 µm/s. Data were processed using the commercial Nanoscope analysis (Bruker) and MATLAB (The MathWorks, Natick, MA) softwares.

At the end of each experiment, both LDH-functionalized tips were washed with ethanol, dried with nitrogen and imaged again using SEM.

21

 21

Figure 1 .

 1 Figure 1. NPs characterization. (a) XRD patterns of MgAl and ZnAl NPs. (b) FT-IR spectra of MgAl and

  an average particle size of 100 LDH NPs) of 169 ± 35 nm for MgAl NPs and 171 ± 38 nm for ZnAl NPs were obtained from their AFM height images. The height profiles revealed that our synthesized NPs also had a similar average whole thickness (obtained as an average thickness of 100 LDH NPs) of 10.6 ± 0.4 and 10.4 ± 0.7 nm, for MgAl and ZnAl LDH NPs, respectively . Such a thickness unambiguously implied the multilamellar nature (~12 layers) of the synthesized NPs. 59 ZnAl LDH NPs impose an efficient antibacterial effect against S. aureus. The antibacterial activity of ZnAl and MgAl LDH NPs against S. aureus was first evaluated using agar disk diffusion method. The generated inhibition zone diameters and their mean values obtained from three 12 repetitive trials are presented in Figure 2a and b. Broth microdilution turbidimetric tests were also carried out to determine the minimum inhibitory concentration (MIC) required to inhibit the growth of S. aureus by ZnAl and MgAl LDHs (Figure 2c).

Figure 2 .

 2 Figure 2. NPs antibacterial activity against S. aureus. (a) Agar disk diffusion test using 10mg/mL ZnAl and

  yet remarkably superior to those of micron-sized Zn-based LDHs prepared by Lobo-Sanchez et al.(MIC value of 10,000 μg/mL). 24 On the other hand, Zn-based LDH NPs having a particle size ~50nm reported by Moaty et al. presented a MIC value of 15.6

  Open Access Article. Published on 27 June 2022. Downloaded on 6/27/2022 12:02:51 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online DOI: 10.1039/D2NR02395D 14

  activity of our ZnAl LDH NPs (Mechanism 3) could be eliminated since all experiments were performed inside an incubator in the absence of light and the production of ROS by LDHs is only pronounced in the presence of a suitable light source.26 ZnAl LDH NPs affect the morphology of S. aureus cells. AFM imaging was used to visualize the surface topography of S. aureus cells before and after treatment with ZnAl and MgAl LDH NPs at concentration of 93.8 μg/mL, corresponding to MIC value of ZnAl LDH NPs. Additionally, the roughness parameter Rq (root mean square of the heights) was obtained from AFM images performed on the top of bacteria at a high magnification of 400 × 400 nm 2 scanning areas. The measured roughness parameter for all treated/untreated cells was performed on a similar area to that of the high magnification AFM images, e.g. 400 × 400 nm 2 (512 samples/line; 262,144 data points).As shown in Figure3aand b, untreated cells as well as those treated with non-antibacterial MgAl LDH NPs presented regular spheroid morphologies and smooth surfaces (Rq = 15.9 ± 0.6 nm, 16.8± 0.8 nm, for untreated and MgAl NPs-treated cells, respectively). The obtained Rq values seemed to be greater than those previously obtained in the literature,62,63 probably due to the different bacterial immobilization method, selected area of measurement and/or used AFM imaging conditions. Page 16 of 42 Nanoscale Nanoscale Accepted Manuscript Open Access Article. Published on 27 June 2022. Downloaded on 6/27/2022 12:02:51 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.

Figure 3 .

 3 Figure 3. AFM deflection images of S. aureus in liquid (scale bar = 250 nm). (a) Untreated S. aureus cells.

Figure 4 .

 4 Figure 4. SEM images of LDH films on silicon wafers and AFM tips (scale bar = 1 μm). (a) MgAl LDH

  LDHs and S. aureus. Single living S. aureus cells were probed by ZnAl-functionalized tips using AFMbased FS. Figure 5a-c and Figure 6b shows adhesion maps, adhesion force and rupture length 23 histograms as well as representative force-distance (FD) curves obtained between different S. aureus cells and ZnAl tips. Adhesive pixels were randomly distributed on the maps suggesting the homogeneous adhesion of ZnAl LDH to the host cell surface. Most recorded FD curves showed

Figure 5 .

 5 Figure 5. Force spectroscopy on live S. aureus cells with ZnAl-functionalized AFM tips. (a) Adhesion maps

Figure 6 .

 6 Figure 6. Force spectroscopy with functionalized tips reveals specific interaction for ZnAl tips. (a)

  was studied. ZnAl LDH NPs exhibited specific antibacterial activity against S. aureus in comparison to non-antibacterial MgAl LDH. An evidence of the contribution of surface interactions to the antibacterial activity of LDHs was pronounced by the superior antibacterial activity of nano-sized ZnAl LDH NPs compared to micron-sized ZnAl LDHs. For similar quantities, agar disk diffusion showed an inhibition zone two time larger and broth microdilution antibacterial assays showed that the MIC was 16 times lower for ZnAl NPs, showing an effect beyond the simple dissolution into Zn 2+ antibacterial ions. Therefore, direct contact interactions have to be considered, which is evidenced by serious morphological impacts including membrane distortion, swelling and dissociation into cell debris which were observed on the surface of S. aureus cells upon being treated with ZnAl LDH NPs at MIC. In addition, LDH-functionalized AFM tips were prepared and used to probe surface adhesion interactions between the cell surface and LDHs using AFM-based FS. ZnAl-functionalized AFM tips were able to recognize the cell wall of S. aureus cells with high adhesion frequencies. Additionally, comparison of the adhesion behavior of three different AFM tips, e.g. Al-coated, MgAl functionalized and ZnAl-functionalized tips, indicated the presence of specific surface interactions between ZnAl LDHs and S. aureus cells. Such finding suggested the presence of a strong correlation between the specific ability of zinc-based LDHs to adhere and damage bacterial cell walls, thus supporting the previously suggested antibacterial mechanism of pristine LDHs by direct surface interactions (Mechanism 1). Experimental part LDH NPs synthesis: Nitrate-intercalated ZnAl and MgAl LDHs were synthesized by coprecipitation-hydrothermal method, adapted from Xu et al.. 49 Briefly, a mixture of 2 mmol M(NO 3 ) 2 .6H 2 O (M = Zn, Mg; SIGMA-ALDRICH) and 1 mmol Al(NO 3 ) 3 •9H 2 O (SIGMA-ALDRICH )with a total volume of 10 mL was very quickly added to 40 mL of 0.15 M NaOH solution (VWR Chemicals), under nitrogen atmosphere and vigorous stirring for 10-30 minutes. The LDH slurry was obtained by centrifugation at 10,000 rpm, washed twice and dispersed again 50 mL decarbonated water. The resultant suspension was then transferred to a stainless-steel autoclave with a Teflon lining and heated at 80 °C for 24 h. LDH NPs powders were then obtained by centrifugation and air drying at 80 °C.Synthesis of LDH films and LDH-functionalized AFM tips: Nitrate-intercalated ZnAl and MgAlLDH films were synthesized on aluminum coated silicon wafers by in-situ growth method adapted from Zhang et al.,69 with some slight modifications. Silicon wafers were first cleaned with piranha solution, washed thoroughly with water and ethanol, and then coated by plasma sputtering (Q15OT S, Quorum) with a thin layer of aluminum (~10 nm). M(NO 3 ) 2 •6H 2 O (M = Zn, Mg; 2.5 mmol) and NH 4 NO 3 (30 mmol, SIGMA-ALDRICH) were dissolved in deionized water (50 mL), and 1% ammonia solution was then slowly added until the pH reached 6.5 and 9.5 for ZnAl and MgAl LDH films respectively. This solution was transferred to a stainless-steel autoclave with a Teflon lining where coated aluminum samples were immersed. The autoclave was heated for 24 h at 80 °C. The obtained LDH films grown on silicon wafers were then washed with distilled water and ethanol then dried in air at room temperature. Similar procedure was followed to synthesize LDHfunctionalized AFM silicon nitride probes, by which LDHs were in-situ grown on MLCT-C (Bruker corporation) AFM tips.M II :Al III molar ratio determination. M II :Al III molar ratios of LDH NPs and films were measured by Induced Couple Plasma Mass Spectroscopy (ICP-MS Agilent-7800). LDH NPs and films were dissolved into 1% HNO 3 . The detected isotopes were:27 Al,66 Zn, and26 Mg.X-ray diffraction measurements. X-ray diffraction (XRD) characterizations were conducted using a Panalytical X'Pert Pro MPD diffractometer in reflection geometry using Cu radiation (Kα 1 = 1.5406 Å), a Ge(111) incident-beam monochromator, Soller slits of 0.02 rad, programmable divergence and anti-scatter slits. The irradiated area was fixed to 10 x 10 mm 2 , and an X'Celerator detector was used. All diffraction data were collected using the same procedure, i.e. at the same scattering angle range (between 5 and 60°) with a 0.0167° step and a speed of 1°/min. The interlamellar distance of both LDH NPs and films was extracted from (003) and (006) basal reflections which is equal to d(003) and d(006)/2. Infrared spectroscopy Infrared spectra of LDH NPs were recorded in the midinfrared range on a Bruker Vertex 70v spectrometer equipped with a KBr beam-splitter and a deuterated triglycine sulfate (DTGS) detector. An attenuated total reflection (ATR) accessory (Platinum ATR with a single reflection diamond crystal, Bruker) was used and the spectral resolution was 4 cm -1 and the accumulation time was 1 min (100 scans). Compartments containing the detector and the ATR accessory were under vacuum (<1 hPa). Infrared spectra of LDH films were recorded under nitrogen flow in a transmission mode on a FT-IR Nicolet 8700 spectrometer equipped with a KBr beam splitter and a DTGS thermal detector. The reference was the aluminum coated Si wafer for LDH films. LDH films spectra were recorded and processed using OMNIC 8.1.0.10 software (Thermo Fisher Scientific Inc.).

76 LDH 37 For

 7637 concentration (MIC) of the prepared ZnAl and MgAl LDH powders. S. aureus cells were precultured in 5 mL TSB overnight at 37°C. An inoculum of S. aureus was then prepared and diluted to obtain an optical density of 0.1 at λ = 600 nm (OD 600 = 0.1). ZnAl and MgAl LDH powders were dispersed in TSB and two-fold dilution series were then prepared in a 96-well microtiter plate. Each well included 25 μL of the diluted LDH powder and 175 μL of bacterial inoculum. Un-inoculated broth wells containing the different used LDH dispersion concentrations were used as a negative standard growth control. As a positive control, 12 wells were prepared with 175 μL of bacterial inoculum and 25 μL of TSB. The micro-titer plate was then sealed with parafilm and 36 incubated at 37°C under agitation for 48 hours. The MIC value for both LDH samples was recognized as the lowest concentration that had no visible turbidity. Partial dissolution of ZnAl NPs. Partial dissolution profile of the ZnAl LDH NPs into their Zn 2+ was evaluated by dispersing 10 mg/mL of the NPs in Tryptic Soy Broth (TSB). After a given incubation time of 24 hours at 37°C, the supernatants were collected by centrifugation. Finally, the metal concentration in the supernatant was measured by ICP-MS (Agilent-7800).Atomic force microscopy (AFM) imaging and force spectroscopy. All AFM experiments were performed at room temperature using Bioscope Resolve AFM (Bruker corporation, Santa Barbara, CA) and the spring constants of the used cantilevers were determined using the thermal noise method. NPs immobilized on silicon substrates and LDH films were imaged in peak-force tapping mode using SNL-C tips (Bruker corporation) in air. LDH NPs were dispersed in an ethanol solution, sonicated for 30 min and deposited on clean silicon substrates and dried at 30°C overnight. The average particle size and thickness of MgAl and ZnAl LDH nanoparticles was obtained from their respective AFM height images, calculated as an average particle size of 100 LDH NPs. topography images on S. aureus, cells were cultivated alone (untreated) or with (treated) MIC of ZnAl and MgAl LDH NPs during 24 h in TSB at 37°C. For imaging in MOPS ((3-(Nmorpholino)propane sulfonic acid, Sigma) buffer solution, stationary phase cultures of S. aureus were centrifuged, washed twice in MOPS and filtered into porous polycarbonate membranes (pore size = 0.8 μm, Millipore). After filtration and rough rinsing, a 1 x 1 cm 2 piece of membrane was cut and attached with double sided adhesive tape at the bottom of a Petri dish and immersed in MOPS. The roughness parameter Rq (root mean square of the heights) of the cells was calculated using the commercial Nanoscope analysis (Bruker) from AFM images performed on the top of bacteria at a high magnification of 400 × 400 nm 2 scanning areas. The measured roughness parameter for all treated/untreated cells was performed on a similar area to that of the high magnification AFM images, e.g. 400 × 400 nm 2 (512 samples/line; 262,144 data points).

View Article Online DOI: 10.1039/D2NR02395D

Acknowledgments

Page 38 of 42 Nanoscale We would like to acknowledge the spectroscopy and microscopy Service Facility of SMI LCPME (Université de Lorraine-CNRS-http://www.lcpme.cnrs-nancy.fr). Claire Genois from LCPME (Université de Lorraine-CNRS-http://www.lcpme.cnrs-nancy.fr) is particularly acknowledged for the ICP-MS analysis. We would also like to thank the plateforme PMD²X de l'Institut Jean Barriol de l'Université de Lorraine , in particular Pierrick Durand for the XRD analysis. Jazia Awassa acknowledges the French Ministry of Higher Education for her PhD grant.

Funding

Financial support was received from the French Ministry of Higher Education (MESR), the French National Scientific Centre (CNRS).

X-ray photoelectron spectroscopy. A photoelectron spectrometer (Kratos Axis Ultra DLD, Manchester, Royaume-Uni) equipped with a degassing chamber under ultra-vacuum, a storage room and an analysis chamber UHV under ultra-vacuum was used to obtain the XPS spectra of LDH NPs. Samples were deposited on a conductive copper belt, inserted on the transfer bar, introduced into a degassing chamber and then to the analysis chamber which is equipped with a monochromatic X-ray source Al Kα emission at 1486.6 eV. Electron counting and detection were performed by a delay line detector DLD (Delay-Line Detector, 115 channels). Spectra were recorded at a normal angle (90 o ) between the surface of the sample and the direction of ejected electrons. The acquisition of low-resolution spectra has been carried out with pass energy of 160 eV at steps of 1 eV and the high-resolution spectra with pass energy of 20 eV at steps of 0.5 eV.

The analyzed area for all samples was fixed at 0.3 × 0.7 mm 2 . Samples were corrected for charging effects using the C 1s peak. The spectra were adjusted by the Shirley baseline by two models of peak compounds: 70 % of Gaussian function and 30 % of Lorentzian function.

Scanning electron microscopy imaging. The morphology of LDH NPs, films and LDHfunctionalized AFM tips was examined by Scanning electron microscopy (SEM) performed on a JEOL JSM-IT500HR combined to an electron-dispersive X-ray spectrometer (EDS), with a Field Emission Gun (FEG) and a voltage of analysis situated in between 2kV and 10 kV. The analysis was performed under high vacuum, and 60 μm diaphragm aperture was settled. Secondary Electron Detector (SED) was used for imaging. In order to determine the thickness of the formed LDH films, similar SEM imaging conditions were applied to image lateral cross-sections of LDH films.

Conflicts of interest

There are no conflicts of interest to declare.


Gwenolé Corre

Karim Boudergui

Guillaume Sannié

Vladimir Kondrasovs

Neutron detection with large plastic scintillators for RPM applications

Neutron Detection with Large Plastic Scintillators for RPM Applications G. Corre, K. Boudergui, G. Sannie, V. Kondrasovs  Abstract-Homeland security requests the use Radiation Portal Monitor (RPM) to detect and differentiate gamma and neutron radiation. Gamma detection is required for illicit transportation of radioactive matter detection. Neutron detection is important to control nonproliferation of enriched material. Manufacturers worldwide propose sensors based on 3 He which give the actual state of art in term of neutron detection. The price fluctuations due to the announcement of the shortage of 3 He forces manufacturers to find viable alternative. From 10 years sensors providers have the challenge to replace previous 3 He detectors that are known to be the most commonly deployed neutron sensor. As 3 He detectors can only detect neutron, they must be completed with gamma detector. The proposed approach is based on pulse time correlation between adjacent sensors from signal collected by EJ200 plastic scintillators to detect gamma and neutron. Results obtained during FP7 Scintilla project test campaigns show the system relevance for replacement of today's 3 He detectors.

Index Terms-Nuclear measurements, neutrons, gamma rays, Solid scintillation detectors, Security

I. INTRODUCTION

HE Radiation Portal Monitors (RPMs) detect and differentiate gamma and neutron radiation. They are required to control illicit transportation of radioactive matter detection and nonproliferation of enriched material. The system is based on EJ200 plastic scintillator set and a dedicated digital signal processing. This signal processing is used for discrimination of neutron and gamma pulses, since these signals are not inherently well separated by the scintillator itself. These methods include pulse timing, pulse shape, coincidence and dual-detector subtraction algorithms. The scintillators set-up and embedded signal processing are designed to answer to the ANSI [START_REF]American National Standard for Evaluation and Performance of Radiation Detection Portal Monitors for Use in Homeland Security[END_REF] and IEC [START_REF] Iec | Radiation protection instrumentation -installed radiation monitors for the detection of radioactive and special nuclear materials at national borders[END_REF] specifications for RPM applications. The section II describes related works based on plastic scintillators to solve the shortage of 3 He. The system and its digital signal processing are detailed in section III. Results obtained in laboratory and during the benchmark campaign on the Scintilla project are discussed in section IV.

This work was supported in part by the European Commission under FP7 Scintilla Grant.

G. Corre, G. Sannie, V. Kondrasovs, K. Boudergui are with the CEA, LIST, Laboratoire Capteurs et Architectures Electroniques, F-91191 Gif Sur Yvette, France (telephone: 33(0)169082785, e-mail: gwenole.corre@cea.fr).

II. RELATED WORKS

A wide range of materials, known to be very sensitive to neutron, were developed and tested, BF3 tubes, B lined tubes, Li Glass Fiber, Non-Scintillating Plastic Fiber [START_REF] Hansen | Neutron-gamma discrimination in plastic scintillators[END_REF], [START_REF] Dallimore | The development of a scalable He-3 free neutron detection technology and its potential use in nuclear security and physical protection applications[END_REF]. Since 15 years, works on gamma/neutron discrimination with nonloaded plastics scintillators appear in the literature. They allow performing simple, low cost and robust detector sets without any export restriction. A large number of technics are studied to improved detectors sensibility for example by adding Gd sandwich to enhance neutron interaction to gamma interaction [START_REF] Peerani | Testing on novel neutron detectors as alternative to 3He for security applications[END_REF], [START_REF] Alemberti | SCINTILLA. A European project for the development of scintillation detectors and new technologies for nuclear security[END_REF]. In [START_REF] Vanier | Calibration and testing of a large-area fast-neutron directional detector[END_REF] and [START_REF] Söderström | Digital pulse-shape discrimination of fast neutrons and γ rays[END_REF] two possible approaches to neutron/gamma discrimination are described; one based on digital pulse processing to differentiate pulse types and the other based on low density scintillators to lengthen the time interval between multiple interactions. Results show that these techniques cannot be applied in the context of RPM applications without any improvement. Some works try to change the properties of plastic by introducing other components. In [START_REF] Dallimore | The development of a scalable He-3 free neutron detection technology and its potential use in nuclear security and physical protection applications[END_REF], the sensor contains scintillation plate with the mixture of the crystals of znS: Ag (Cu): (6) LiF of those dispersed into the optically transparent medium. The discrimination is done on the shape of pulse. In [START_REF] Peerani | Testing on novel neutron detectors as alternative to 3He for security applications[END_REF], a good overview of existing technologies and discrimination techniques is done. These methods include pulse timing, pulse shape, coincidence and dual-detector subtraction algorithms. Pulse shape discrimination (PSD) is the most common method to distinguish between pulses produced by gamma rays and neutrons in scintillator detectors. This technic is impractical in non-loaded plastic scintillator since these signals are not inherently well separated by the sensor material itself. The digital signal processing proposed in [START_REF] Söderström | Digital pulse-shape discrimination of fast neutrons and γ rays[END_REF] shows that pulse shape discrimination can be performed on non-loaded scintillator if the signal is well digitized. The main limitation is the sensitivity of distortion introduced by the use of large plastic scintillators or a photomultiplier [START_REF] Normand | MA-NRBC: First successful attempt for neutron gamma discrimination in plastic scintillator[END_REF] which not allows its applications on RPM detection system. These limitations are bypassed with the use of multi-scintillator time amplitude analysis as shown in the following section.

T III. SYSTEM OVERVIEW

A. System synopsis

The DAQ electronics is designed for the detection of PMT signals (pulse time duration of the order of tens of nanoseconds and total charge of tens of Pico-Coulomb). This is achieved by using multi-ways fast digitizer boards that samples the input signals at 200 MS/s. The digital samples are treated in real time to find the arrival time and amplitude of each pulse. The electronic acquisition system is controlled by an embedded CPU. The pulses induced by gamma and neutron particles in each of the four scintillators are analyzed by adjacent couple of scintillators to evaluate the gamma and neutron counting rates. The system is composed of the following elements:

-four 100x100x1000 mm² EJ 200 plastic scintillators to offer an important detection surface, -Photomultipliers -a cadmium slide between each scintillator -Dedicated acquisition board -Embedded CPU -Remote end users PC

The scintillator geometry 100x100x1000 mm 3 offers a large detection surface. The interface between two scintillators allows placing 1x100x1000 mm 3 Cadmium. Fig. 1 describes the setting of one pillar for a vehicle configuration. The use of four pillars is needed to reach the neutron detection performance fixed in the RPM norm. In operating conditions, infrared sensors are added. The computer used for GUI can provide N42.42 file to a distant server at the end of each vehicle move.

B. General principle

Our digital signal processing can distinguish the different kind of interaction when they occur in two adjacent scintillators. The process can discriminate gamma, neutron and muon. The detection is based on interaction described in Fig. 2. The neutron interaction can be detected with the recoil proton which ionized atomic layers of plastic atoms and create light. The mean energy of recoil proton is around from 100 Kev to 400 KeV. Neutrons can also generate gamma flash due to high cross section of material such as Cadmium or Gadolinium. The time between a neutron interaction with plastic and a gamma generated by Cadmium is around 20 nanoseconds. The energy level of these two interactions is very different. Detect gamma flash improves the detection of neutrons. A gamma with energy less than 200KeV creates an interaction in only one plastic scintillator. A gamma with higher energy and a muon can interact with two or more scintillators. The time detection between two interaction caused by gamma (or muon) are very close, generally, less than 2 nanoseconds.

C. Neutron/gamma discrimination implementation

The neutron/gamma discrimination is performed into the FPGA. The discrimination is done pulse by pulse in real time and reduces the dead time of the system according to the duration of a pulse. The dead time falls down to 30 nanoseconds. The process discrimination provides real time neutron count rate. Laboratory and ISPRA's test bench show that neutron answer is still full operating, even if the total count rate reaches 2 million counts per second. The algorithm is shown in Fig. 3. The algorithm is described for a pair of scintillator and is duplicated for each pair used in the detection setting. When two pulses arrive in the defined time window, the discrimination process is performed.

if (T(j) -T(i)) < Time_window then if A(i) or A(j) > Amp_threshold then DeltaA=abs(A(j)-A(i))

if A(i) < A(j) then if (DeltaA>=low_threshold) Consider that a pulse P(i) of amplitude A(i) is created at T(i) in a detector and a pulse P(j) of amplitude A(j) is created at T(j) in its adjacent detector. P(i) is arrived before P(j). If all conditions are met, the number of neutrons is incremented. The neutron counting is read each 100 ms and a moving average is performed to provide the neutron count rate in cps/s. This treatment is realized on each pair of detectors. In the vehicle configuration, there are 3 adjacent pairs of detectors. The overall neutron count rate is the sum of the results of the three treatments.

IV. RESULTS

A. Preliminary results

The detector system neutron counting rate background is compare to theoretical counting. The study of influence of gamma and muon is performed to verify the neutron counting rate background. The gamma impact on neutron counting rate is realized with a cobalt 60 which generates 1.2 Mcps on four scintillators. There are around 1200 gamma per scintillator that appear in the timing window used to performed neutron gamma discrimination. This high rate does not disturb neutron counting. The muons particles impact is also tested by vary the position of the detection set. The time/amplitude analysis rejects muons and limits their effect on neutron counting rate. In a second step, a mobile source carrier test performed in the laboratory uses a Californium 252 of 10 000 c/s/4π. Two pillars are positioned on each side of the transit source, face to face. The laboratory tests objectives were to find the fine tuning for background threshold to satisfied 1/10 000 rates of alarm or 1 alarm in 2 hours (IEC standards) or 1 in 8 hours as required by ANSI standard. Results of laboratory tests are summarized in Table I.

1) Neutron detection test

When a testing neutron source passes through a portal at the constant reference speed (as stated above), it should trigger an alarm in 59 cases out of 60 trials. The reference neutron testing source is a Cf-252 source with emission of 20'000 n/s (±20%) shielded with 1 cm steel and 0.5 cm lead. The source effectively used (NC5549) had an emission of 21000 n/s at the date of the tests. The test has been executed also in nonstandard conditions, in particular, for lower neutron emission 13'000 n/s and 7'000 n/s, with a 8cm HDPE moderator, and for different source heights, 2.1 m and 2.7 m. The benchmark results show that the RPM detection system reaches the requirement of ANSI for vehicle. The low counting rate of the system limits their performance because of statistical distribution. The system is 3 times less efficient with the HDPE moderator. The height increase shows that the system has to adapt the pillars detector position to match to another kind of application such as container scanning.

2) False alarm

The false alarm rate in a background measurement condition is relatively close to the rate defined in [START_REF]American National Standard for Evaluation and Performance of Radiation Detection Portal Monitors for Use in Homeland Security[END_REF] and [START_REF] Iec | Radiation protection instrumentation -installed radiation monitors for the detection of radioactive and special nuclear materials at national borders[END_REF]. Tests are carried out to verify if strong gamma source do not create false alarm in absence of neutron and do not disturb neutron detection in presence of neutron. A 137Cs source is used and do not create neutron detection disturbing until 2 million cps.

V. CONCLUSION

This work shows that it is possible to use standard plastic scintillators for RPM applications. The proposed approach based on a multi-scintillators EJ-200 and its dedicated timeamplitude analysis provides good results compared to RPM requirement. Some additional works on identification are currently done. This system would then become a real and complete alternative to the 3 He. It is also less expensive than boron lined systems, and safer than BF 3 system. This kind of systems can be used in a wide range of security applications.

Fig. 1 .

 1 Fig. 1. Detection system synopsis (the set scintillators + embedded acquisition board in duplicated for each additional pillar used)

Fig. 4 .

 4 Fig. 4. Detection success rate in function of 252Cf activity

Fig. 5 .

 5 Fig. 5. Detection success rate in function the 252Cf height

TABLE I

 I All tests are based on the ANSI and IEC standards for RPM. The General testing conditions are the following. Dynamic test are performed at a source speed of 8 km/h for vehicle. The portal configuration (distance from source) is full-size double-sided vehicular portals with 5 m distance between pillars.The responses to neutron radiation for RPMtype of equipment is generally performed in dynamic conditions.

			. BLIND TEST RESULTS
	Speed	Number of	Number of Neutron	% of neutron
	(m/s)	passages	detections	detections
	1,2	294	260	88
	1,7	168	122	73
	1,95	90	65	72
	2,16	60	44	73
	B. Blind test at ITRAP+10 Facility, JRC

ACKNOWLEDGMENT

The authors would like to thank all the members of ITRAP+10 Facility from JRC, Ispra for their fruitful help during the different benchmark campaigns of the FP7 Scintilla project.