Ines Muguet

Ali Maziz

Fabrice Mathieu

Laurent Mazenq

Guilhem Larrieu
email: guilhem.larrieu@laas.fr

Combining PEDOT:PSS Polymer Coating with Metallic 3D Nanowires Electrodes to Achieve High Electrochemical Performances for Neuronal Interfacing Applications

Keywords: charge injection limit, charge storage capacity, electrochemistry, electrophysiology, impedance, interfaces, nanoelectrodes, nanowires, PE-DOT:PSS

This study presents a novel approach to improve the performance of microelectrode arrays (MEAs) used for electrophysiological studies of neuronal networks. The integration of 3D nanowires (NWs) with MEAs increases the surface-to-volume ratio, which enables subcellular interactions and high-resolution neuronal signal recording. However, these devices suffer from high initial interface impedance and limited charge transfer capacity due to their small effective area. To overcome these limitations, the integration of conductive polymer coatings, poly(3,4-ethylenedioxythiophene)poly(styrenesulfonate) (PEDOT:PSS) is investigated as a mean of improving the charge transfer capacity and biocompatibility of MEAs. The study combines platinum silicide-based metallic 3D nanowires electrodes with electrodeposited PEDOT:PSS coatings to deposit ultra-thin (<50 nm) layers of conductive polymer onto metallic electrodes with very high selectivity. The polymer-coated electrodes were fully characterized electrochemically and morphologically to establish a direct relationship between synthesis conditions, morphology, and conductive features. Results show that PEDOT-coated electrodes exhibit thickness-dependent improved stimulation and recording performances, offering new perspectives for neuronal interfacing with optimal cell engulfment to enable the study of neuronal activity with acute spatial and signal resolution at the sub-cellular level.

Introduction

Recording and stimulating the electrical activity of neurons with acute spatiotemporal resolution is a fundamental step toward an in-depth understanding of the mechanisms involved in their small effective area, high initial impedance, and low chargetransfer capacity. Moreover, the mechanical mismatch between the stiff metallic electrode and the surrounding soft neural tissue may trigger immune responses, which lead to encapsulation of the electrodes, neuronal degeneration, and subsequent device failure. [34,35] Consequently, 3D-electrode performances must be improved through surface modification at the nanoscale to enable precise recordings and maximum charge transfer, as well as keeping the optimal designs of the electrodes, while being minimally troublesome to the tissue. Metallic and organic coatings on the electrode active site, such as IrOx, [36][37][START_REF] Zeng | IEEE Intl. Conf. on Cyborg and Bionic Systems (CBS)[END_REF][START_REF] Peixoto | Ann. Intl. Conf. of the IEEE Engineering in Medicine and Biology Society[END_REF] Pt black, [START_REF] Fujishiro | [END_REF] graphene oxide, [41,[START_REF] Batrisya Ismail | IEEE-EMBS Conf. on Biomedical Engineering and Sciences (IECBES)[END_REF] carbon nanotubes, [START_REF] Saunier | [END_REF] or conductive polymers such as Poly (3,4-ethylenedioxythiophene) (PEDOT) [7,10,[44][45][46] have been gaining considerable interest for interfacing with neurons as they can improve charge transfer and reduce the electrode impedance. PEDOT:PSS possesses well-known properties such as high conductivity, biocompatibility, and excellent stability. Last, due to its soft nature, PEDOT:PSS has the potential to reduce the mechanical mismatch at the electrode-cell interface. [34] Therefore, it is envisioned as minimally disturbing to the tissue and is an ideal candidate for improving 3D-structured electrodes performances.

Here, we combine metallic nanowire-based electrodes , made of platinum silicide (PtSi), with the remarkable properties of polystyrene sulfonate-doped PEDOT (PEDOT:PSS). We demonstrate for the first time the ability to deposit, with high selectivity, a nanoscale layer of conductive polymer coating onto the NWs using electrodeposition. The deposition was first optimized on PtSi planar microelectrodes with diameters ranging from 80 to 5 μm, for surface scaling, comparison with the literature, and investigation, as diameters under 10 μm are not commonly studied. Electrochemical depositions were then performed on PtSi NWs, and parameters were tuned to obtain nanolayers of PEDOT:PSS. All coated probes were electrochemically assessed, and PtSi electrodes were tested as controls. Electrochemical impedance measurements were used to evaluate the improvement of the recording performances, and cyclic voltammetry measurements were performed to evaluate the charge storage capacity. Voltage transient experiments were completed to investigate the electrodes behavior under electrical stimulation conditions. All coated electrodes were also morphologically characterized using scanning electron microscopy, in order to establish a direct relationship between the synthesis conditions, the layer morphology, and the electrical features.

Results and Discussion

PEDOT:PSS Coating Optimization on Planar Microelectrodes

For the first part of the study, we used a microfabricated MEA device composed of planar platinum-silicide microelectrodes, structured on a silicon-on-insulator substrate, with diameters ranging from 5 to 80 μm. All access lines are insulated from the liquid media; however, it is worth mentioning that part of them is exposed, which thus adds 4 μm 2 of conductive surface to each electrode, as shown in Figure 1a. This additional active area has been considered in all experiments and calculations. Details about the microfabrication can be found in the experimental section. All PEDOT:PSS electrochemical depositions were optimized on this planar design first prior to coating nanowire-based electrodes to facilitate the implementation on 3D-nanostructures by targeting the same range of surfaces, study the effect of surface scaling, and make the positioning with literature easier.

The electrodeposition route is an important parameter, as it affects the growth of the polymer, its morphology, and its electrochemical properties. In this study, we investigated two electrochemical depositions, each performed through an oxidation process by either the application of a constant current (galvanostatic mode) or a potential scanning through cyclic voltammetry (potentiodynamic mode). Both depositions were performed in aqueous media using a conventional three-electrode cell configuration, as illustrated in Figure 1b, in which the microelectrodes were coated individually. The PtSi microelectrode was used as the working electrode (WE), a thick Pt wire was used as the counter electrode (CE), and a commercial Ag/AgCl electrode was used as the reference electrode (RE).

Galvanostatic (GS) deposition was carried out at a current density of 10pA μm -2 and a temperature of 21 ± 1°C until a total charge of 1.9 nC μm -2 was consumed. A relatively low synthesis potential at the initial stage of the GS synthesis (750 mV) suggests the polymerization of the adsorbed oxidized monomer layer. The GS electropolymerization process has not yet been fully elucidated; nonetheless, several mechanisms are commonly accepted. [45,[47][48][49] In such mechanisms, the initial potential rise is caused by the evolution of the monomer concentration gradient, and is followed by a primary nucleation process divided between the induction period (monomer oxidation and oligomerization) and progressive steady nucleation, followed by 2D and 3D growths of the conductive polymer on the PtSi surface. This forms a positively charged PEDOT polymer structure, with a negatively charged PSS doping agent acting as a counter ion. In potentiodynamic deposition (PD), electrodes were cycled from -0.7 to 1 V, at a scan rate of 10 mV s -1 for two cycles. The cyclic voltammogram (CV) shown in Figure 1d is typical of what has been reported elsewhere in PD electropolymerization conditions. [48] The oxidation current is observed at positive potentials higher than 750 mV. This feature is attributed to the oxidation of the monomer prior to polymerization through mass transport and charge transfer processes. A nucleation loop is then observed between the forward scan and reverse scan in the CV, which is associated with the initial stage of the nucleation process of the PE-DOT:PSS film.

The morphologies of the PEDOT:PSS coatings obtained by both GS and PD modes have been inspected using optical and scanning electron microscopy (SEM). Both electrochemical deposition routes provide an individual, precisely localized coating onto the conductive part of the electrode, as seen in Figure 1e. SEM images in Figure 1f demonstrate that all electrode diameters, from 5 to 80 μm, were uniformly covered in PEDOT:PSS. However, the surface morphology was found to be dependent on the deposition route used for the PEDOT synthesis. The structure of PEDOT in GS conditions appears thin and smooth, with a very localized and uniform surface. This is due to the low polymerization rate and low deposition charge, which induce a controlled deposition. On the other hand, PEDOT:PSS deposited in PD conditions produced a highly porous globular 3D structure with an uncontrolled and inhomogeneous distribution. In addition, the polymer quantity deposited onto small electrodes (∅ < 20 μm) exceeds the electrode diameter, making it difficult to evaluate the real active surface. The micron-scale surface structure of PtSi, GS-coated electrodes has been characterized and compared with Atomic Force Microscopy (AFM). Results are represented in Figure 1g, and the rugosity has been calculated (RMS Sq, 2.811 nm, 4.683 nm for PtSi and GS-coated electrodes, respectively). A slight increase in the rugosity of the surface, hence an augmentation in the active area when coated, can be observed in the case of GS-coated electrodes. The quantity of PEDOT:PSS deposited via PD ∅5 μm electrodes has been estimated with a profilometer scan, as exposed in Figure 1h,i. The PEDOT:PSS coating is ≈50 μm long and 3.5 μm high, which greatly exceeds the initial planar dimensions of the electrode and opens new perspectives for 3D-microstructurations of planar microelectrodes.

Both deposition routes present very different features, almost opposite. PD-deposited PEDOT:PSS layers tend to form 3D micro-structures, which exceptionally increase the active area of the electrode. On the other hand, the GS is more controlled, homogeneous, and precise, which likely makes it more compatible with and integrated with nanowires. V a and V p represent the access potential and the polarization potential respectively. Here, V p , the polarization potential, equals the cathodic water window limit value, meaning it reaches the CIL. h) Current injection limit as a function of diameter for uncoated and coated electrodes. i) Charge Injection Limit as a function of diameter for uncoated and coated electrodes.

Recording and Stimulation Performances of Planar Microelectrodes

The effect of PEDOT:PSS coating on PtSi planar electrodes was investigated through Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV), and Voltage transient experiments (see details in the Experimental Section). Uncoated PtSi electrodes of identical diameters were also tested as controls. EIS was performed on coated electrodes to assess their recording performance. Data were acquired in a two-electrode configuration using a Pt electrode as the counter electrode in the frequency range 10 0 -10 5 Hz with an amplitude of 20 mV in a 0.9%w NaCl solution. to a lack of sensitivity of the equipment in low frequency range.

The phase angle within the frequency range 10 2 -10 5 indicates a mixed capacitive-ohmic-like behavior, which is similar to what has been observed on Pt electrodes. [46] As expected, the presence of a PEDOT:PSS layer induces a lower mean impedance, as well as a change in the phase shape, implying an increase in the conductivity and electroactive area of the electrode. Thus, the transition from the capacitive regime to the series-resistance dominant regime happens at lower frequencies (from above 10 5 Hz for PtSi, to 10 3 and 10 Hz for GS-coated and PD-coated electrodes, respectively). The GS-deposited PEDOT:PSS shows a lower impedance in the capacitive regime of approximately an order of magnitude compared to bare PtSi. Due to its cauliflower-like shape, the PD deposited PEDOT: PSS layer not only augments the effective area but broadens the real electrode area as well. Hence, it exhibits an even lower mean impedance in the capacitive regime (almost two orders of magnitude lower than bare PtSi), and the earliest shift to the series-resistance dominant area. Figure 2c represents the impedance values at 1 kHz, the frequency of an action potential commonly used as a reference. The data have been fitted using allometric power functions. Fitted data demonstrate that PDcoating the electrodes lowers the impedance of about ten times across the entire diameter range. GS-coating, however, lowers it by ten times for small electrodes (from 1 to 0.1 MOhm for Ø5 μm electrodes), by 20 times for medium-sized electrodes (Ø20 μm)

and by 60 times for bigger electrodes, from 0.3 to 0.005 MΩ for Ø80 μm. Similar studies have been conducted on PEDOT:PSS/Pt electrodes with diameters of 2.5-20μm [33] resulting in impedance reductions of ≈2.2 times for small diameters (5 μm). Electrodes with 20 μm diameters and above, composed of PEDOT:PSS on Au, Pt, and nanostructured Pt, have been reported in the literature. Waafi et al. have reported an impedance reduction of 15 times for Ø30 μm PEDOT:PSS covered Pt electrodes. [50] Ganji et al. have observed a reduction of the impedance of 25 times for medium-sized electrodes (Ø20 μm); however, they reported a reduction of only 20 times for bigger electrodes (Ø80 μm). [46] The impedance reduction we report for all diameters, along with the calculated specific impedance (see Table 1), indicate that our PEDOT:PSS coated electrodes can be considered at the state-ofthe-art when compared to the literature.

To assess the coating influence on the electrical stimulation performances of the electrodes, cyclic voltammetry and voltage transient experiments were performed, in order to determine the cathodal Charge Storage Capacity (CSC) and Charge Injection Limit (CIL). The CSC, which represents the maximum charge available that could be reversibly injected during a cathodic pulse, is defined as the time integral under the cathodic current within the cycled region. [START_REF] Merrill | Somatosensory Feedback for Neuroprosthetics[END_REF] The potential range used for cycling is preferably within the water electrolysis window, whose limits are defined by the water reduction potential (cathodal limit) and the water oxidation potential (anodal limit). Outside those limits, irreversible faradaic reactions take place, which may be deleterious for the neuronal tissue or the electrode itself. [START_REF] Ganji | [END_REF] The cathodic CIL is a subset of the CSC, which represents the real available charge that can be reversibly injected during cathodic pulsing. The CIL is determined using voltage transient experiments, during which the maximum injected current is determined before polarizing the electrode potential beyond the cathodal water window limit. A large CSC and CIL, and therefore a large amount of injectable charge, are desirable for maximum stimulation performance.

CV measurements were conducted using the conventional three-electrode setup mentioned above. The uncoated PtSi CV curve in Figure 2d has been obtained on a Ø30 μm electrode, within the -0.9 and 1.2 V potential range, at a scan rate of 200 mV s -1 in a 0.9%w NaCl solution. The water electrolysis window of the PtSi is considered to be -0.9/1.1 V, which is similar to the typical Pt water window when cycled in PBS. [53] The typical hydrogen adsorption and desorption peaks appear between -0.5 and -0.8 V. Nonetheless, it is interesting to notice the absence of a surface oxidation area and oxide reduction peak when compared to the conventional Pt CV shape. [53] Figure 2e illustrates the CV curve of PtSi, PEDOT:PSS/PtSi (GS coated), and PEDOT:PSS/PtSi (PD-coated) Ø30 μm electrodes characterized between -0.5 and 0.6 V at 200 mV s -1 to avoid any undesirable faradaic reactions. Indeed, organic electrodes usually exhibit a smaller water electrolysis window, from -0.9 to 0.6 V in the case of PEDOT:PSS/Pt or PEDOT:PSS/Au. [START_REF] Ganji | [END_REF] As expected, a great increase in the electroactive area can be observed. In accordance with the coating morphology, PD-coated electrodes feature a larger effective area than GS-coated probes. Additionally, the symmetry of the PEDOT:PSS/PtSi cyclic voltammograms implies that the doping process is highly reversible, which indicates promising properties regarding the ionic to electronic charge transfer at the electrode interface. [START_REF] Saunier | [END_REF] The cathodic CSC values of the probes has been extracted from these data, considering the initial planar area of the electrode, and results, which have been fitted using allometric power functions, and are displayed in Figure 2f. As anticipated, the CSC of the PEDOT:PSS coated electrodes is significantly higher than bare electrodes. PD-coating the electrodes increases the CSC up to 10 times for small electrodes, with 176 mC cm -2 for Ø5 μm PD-coated electrodes compared to 16.3 mC cm -2 for Ø5 μm PtSi electrodes. The CSC is increased up to five times for larger electrodes, with 4.16 mC cm -2 for Ø80 μm compared to 0.25 mC cm -2 for Ø80 μm PtSi electrodes. GS-coated electrodes demonstrate a distinct behavior when compared to PD-coated electrodes. They exhibit a slight increase in CSC for small electrodes (Ø5 μm), reaching 19.7 mC cm -2 , while for larger electrodes (Ø80 μm), the increase is magnified up to 52 times, resulting in a CSC of 13.03 mC cm -2 .We demonstrated a very prominent improvement for small PD-coated electrodes; nonetheless, the morphology of the coating is extremely rough, and the coating exceeds the initial planar electrode active area. The improvement appears less significant in the case of small GS-coated electrodes <Ø20 μm due to the thin morphology of the coating. Nonetheless, it is important to note that the CSC can only be regarded as a relative value, as it relies on the electrode material, shape, electrolyte composition, and experiment conditions (potential range and scan rate). In addition, when cycled, the electrode is subjected to a slow voltage ramp, which contrasts with the usual neuronal stimulation conditions.

Consequently, voltage transient experiments are more likely to represent the true electrode behavior during cell stimulation, as the probe is exposed to multiple cathodal-first, biphasic current pulses. Here, we applied symmetric cathodal-first biphasic pulses of a millisecond, with an interphase gap of 10 μs, and an interpulse delay of 95 ms. [START_REF] Ganji | [END_REF][54][55][56] An example of the voltage transient response to injected current of a Ø60 μm PtSi electrode is displayed in Figure 2g. Two slopes can be observed. The first voltage drop corresponds to the access potential (V a ), associated with the ohmic resistance of the liquid media. The second slope, which indicates a gradual polarization at the electrode interface, is designated as the polarization potential (V p ). Interestingly, the PtSi voltage transient is similar to the usual Pt response. [26,[START_REF] Ganji | [END_REF] The maximum injectable current determined to calculate the CIL is considered to be when ΔV p reached the cathodic water window limit value [START_REF] Ganji | [END_REF] and is plotted in Figure 2h. Coated probes reach their limit at higher current (examples of voltage transient response for PEDOT:PSS-coated probes are displayed in Section S2, Supporting Information), meaning more charge can be injected before creating undesirable faradic reactions that are deleterious for the cell membrane or electrode itself. The corresponding CIL values have been calculated, and the results are exposed in Figure 2i. The CIL of PtSi electrodes ranges between 0.5 and 0.03 mC cm -2 , and analogous results have been reported on Pt microelectrodes [START_REF] Ganji | [END_REF] (CIL 0.83 mC cm -2 ). The CIL of GS-coated probes ranges between 8 and 5 mC cm -2 and PD-coated electrodes exhibit a CIL ranging from 50 to 5 mC cm -2 for Ø5-Ø80 μm, respectively. Optimized PEDOT-based coatings have been reported in a similar range between 1.2 and 8.4 mC cm -2 . [START_REF] Ganji | [END_REF]57] Yet, in the case of small electrodes (Ø5 μm), a GS-coating increases the CIL by 16 times, and this increase is greater than what has been observed on PEDOT:PSS-coated Pt electrodes of similar diameters. [START_REF] Ganji | [END_REF] The improvement is even greater with PD-coated results. Indeed, PD-coating increases the CIL of small electrodes by 100 times, which is particularly boosted by the cauliflower-like morphology.

The improvement of the performances induced by PE-DOT:PSS coating is highly beneficial for sub 10 μm diameter electrodes, which were until now difficult to implement in MEA devices, due to the strong degradation of the global performance (impedance, SNR, current injection…) linked to the miniaturization of the surface. This opens new perspectives for electrophysiology studies with high resolution.

PEDOT:PSS Coating Optimization on 3D-Nanostructured Microelectrodes

For the second part of the study, we used a microfabricated device called a Nanostructured Electrode Array (NEA), [17] as shown in Figure 3a, where the sensing part is composed of seven interconnected vertical core-shell type PtSi nanowires of 3.5 μm height and 350 nm diameter each. However, the base of each nanowire remains insulated and only the upper part is exposed (further details can be found in Section S1 and Figure S2, Supporting Information), hence the recording site is 3 μm high, as seen on Figure 3b. The total conductive surface per electrode is estimated to be 23.8 μm 2 . Details about the fabrication process can be found in the Experimental Section. The key aspect ratio of the probes contributes to achieving a high interaction with cells [18,19,23,58,59] and thus reaching high signal resolution, therefore it has to be maintained. For that matter, nanoprobes were coated using only galvanostatic deposition to obtain a thin and conformal coating. Indeed, as expected, the PD-route leads to a rough morphology of the layer, which is incompatible with an integration on nanostructures, as illustrated in Section S1 (Figure S1, Supporting Information). Electrochemical deposition was performed in the same three-electrodes configuration as detailed above (Figure 1b), in the same electrolyte solution of 10 mm EDOT and 10 mm Na:PSS. The conductive part of the probes was successfully coated, as presented in Figure 3b. The scanning transmission electron microscopy with high-angle annular dark-field image (STEM-HAADF) showing the cross-section confirms that the PEDOT:PSS layer is dense, homogeneous, and wraps around the nanostructure tightly. Energy Dispersive X-Ray Analysis (EDX) has been performed on the coated NWs and results can be found in Section S1, Figure S2 (Supporting Infor-mation). The conductive polymer thickness has been tuned by applying currents between 10 and 5 pA μm -2 and total consumed charges between 60 and 2 nC per electrode, which corresponds to charges varying between 2.52 and 0.084 nC μm -2 (Figure 3c). As a result, PEDOT:PSS coatings with a thickness range between 700 and 40 nm have been obtained, as represented in Figure 3d. Thin coatings were successfully achieved, meaning the preservation of the key aspect ratio of the NWs, which would imply optimal cell engulfment. Thin PEDOT:PSS depositions (the reported layer thickness is 100 nm) on 1 μm high mushroom-shaped microelectrodes have been reported [10] as well; however, to the best of our knowledge, layers thinner than 100 nm have not yet been demonstrated. PSS coated electrodes using a simple 5-elements circuit, composed of an electrolyte resistance (R s ), a charge-transfer resistance (R ct ), a double layer capacitance (C dl ), as well as a system capacitance (C p ) and system resistance (R p ). e) Graph representing the experimental and modelized impedance modulus and phase of a PtSi NWs electrode. f) Graph representing the experimental and modelized impedance modulus and phase of a coated NWs electrode (410 nm thick). g) Graph exposing the variation of Cdl and Cp with respect to the coating thickness. h) Graph exposing the variation of R ct and R s with respect to the coating thickness. In all the panel, the diameter of PtSi NWs is 350 nm.

Electrochemical Impedance Spectroscopy

Similarly

as controls. As previously explained, EIS data were acquired in a two-electrode configuration using a Pt electrode as the counter electrode in the frequency range 1-10 6 Hz with an amplitude of 20 mV in a 0.9%w NaCl solution. The measured impedance modulus and phase spectra for bare PtSi and coated NWs, over the frequency range 10 1 -10 5 are represented in Figure 4a,b. In accordance with what has been observed on planar electrodes, the mean impedance of PtSi NWs decreases proportionally with the coating thickness. The phase angle of bare NWs exhibits a high variability between 10 1 and 10 2 , which, corresponds to noise induced by the potentiat. The phase angle is close to -90°across the frequency range 10 2 -10 5 Hz, which indicates a highly capacitive behavior. When coated, the phase angle changes correspondingly with the coating thickness, which demonstrates an increase in the effective area and a shift to an ohmic-like behavior. Analogous trends have already been obtained on PEDOT-coated microthreads, [60] PEDOT-coated 3D-structured microelectrodes, [10] and PEDOT nanotubescovered electrodes. [61] The thermal noise can be extracted from these impedance measurements, for PtSi-and PEDOT:PSScoated electrodes (layer thickness 400 nm) following the method described in Section S3 (Supporting Information). The thermal noise is found to be slightly lower with PEDOT: PSS coated electrodes (4.10 μV ± 0.10 μV) that PtSi NWs-based electrodes (4.37 μV ± 0.29 μV). Impedance values at 1 kHz have been extracted, and the data have been fitted using a linear function, as represented in Figure 4c. The impedance of bare PtSi NWs is 0.9 MOhm, and, as expected, the impedance value decreases when coated. Fitted trends indicate that thick coatings (700 nm) can extensively reduce the impedance by 18 times, from 0.9 MOhm for PtSi NWs to under 0.05 MOhm. However, thick coatings are less desirable for neuronal interfacing, as they affect the aspect ratio of the probes and may result in lower cell engulfment. Therefore, thinner coatings (<300 nm) are more relevant, already reducing the impedance at 1 kHz by three times to 0.3 MOhm. Similar trends have been observed on coated gold 3D-microelectrodes, [10] yet Jones et al. reported a maximal impedance decrease of only six times. The thinnest layer we obtained (40 nm) can also reduce the impedance at 1 kHz by almost half its value, from 0.9 to 0.5 MOhm. This is an excellent and promising result for applications in need of remarkably thin layers of PEDOT:PSS.

Equivalent Circuit Model

To further interpret these results, we conducted a theoretical analysis of the equivalent circuit model corresponding to our system, as in Figure 4d. Here, we demonstrate that our data can be fairly well described and modeled using a simple fiveelements circuit, composed of an electrolyte resistance (R s ), a charge-transfer resistance (R ct ), a double layer capacitance (C dl ), as well as a system capacitance (C p ) and system resistance (R p ) [54] (see Section S4, Supporting Information, for the resulting equation). The electrolyte resistance, R s , is measured between the working electrode and the platinum reference electrode; hence, it also includes all resistances originating from the access lines and contact pads. [33,62] R ct and C dl are both associated with phenomenon at the electrode/electrolyte interface. R ct reflects the ability of the electrode-electrolyte interface circuit to resist the flow of charges from the ionic solution to the electronic conductor while C dl accounts for the stored charge in the double-layer region, at the electrode-electrolyte junction. R p , as well as C p , are associated with the entire setup, comprising the initial contribution of the NEA regarding resistive and capacitive effects, as well as the measurement equipment, that is, connecting wires and a potentiostat. data are accurately fitted (r 2 > 0.99), over the frequency range 10 2 -3.10 5 Hz, to avoid the impact of noise and artefacts caused by the potentiostat to the fitting algorithm, and examples of fitted data for bare PtSi and PEDOT:PSS coated NWs (410 nm layer) are displayed in (Figure 4e,f). Results indicated that R p is very high (>50MOhm), and its effect is mainly prominent over low frequencies (<10 2 Hz), where background noise also affects the signal; hence, we chose to disregard this parameter in our calculations.

Parameter values with respect to the coating thickness are plotted in Figure 4g,h. The trends in Figure 4g show that C p remains constant (average value 100 ± 9.5 pF), and is lower than C dl , which confirms that it represents the contribution of the set-up. Results demonstrate that C dl is significantly greater than C p , and C p only affects the current flow starting around frequencies of 30 kHz. Therefore, system capacitances should not disturb neuronal recordings as events happen at lower frequencies. A sharp, thickness-dependent increase of C dl value can be observed when the electrodes are coated, in comparison with bare PtSi (variation up to 11 764% measured between the PtSi and 620 nm-thick PEDOT:PSS layer). This demonstrates an important increase in the effective area when coated, as the capacitance value is directly proportional to the surface, as well as reflects the charge storage properties of PEDOT:PSS. Indeed, a higher capacitance value implies that more charge can be stored at the interface. On the other hand, Figure 4h indicates a decrease in R ct , proportionally to the coating thickness, (96.5% decrease measured between the PtSi and 620 nm-thick PEDOT:PSS layer). This illustrates that the addition of PEDOT:PSS onto the probes promotes ionic to electronic charge transfers between the electrode and electrolyte, as well as confirms the increase in effective area as R ct is inversely proportional to the surface. Hence, this study highlighted that the improvement in performance is induced by a combined effect of the PEDOT:PSS intrinsic properties and the increase in effective area, in particular the potential inherent porosity of the PEDOT:PSS layer. [57] Finally, R s remains constant (average value 536 ± 48 Ω) and is lower than R ct , in accordance with the fact that R s represents the electrolyte resistance. The slight variations could be linked to the working electrode/counter electrode distance, as the placement of both is manual.

As a conclusion, we demonstrated that the circuit components match the physical characteristics of the system and can therefore be used as a baseline for further design optimization of the device and result interpretation.

Stimulation Performances of Nanowires-Based Probes

CV measurements were conducted to assess the charge storage capacity of the electrodes, using the conventional three-electrodes setup mentioned above. Figure 5a illustrates the cyclic voltammogram of uncoated PtSi NWs, within the -0.9 and 1.2 V potential range, at a scan rate of 200 mV s -1 in a 0.9%w NaCl solution. The water electrolysis window (-0.9/1.1 V) remains unchanged compared to planar configurations and is similar to the nanostructured Pt water window when cycled in PBS. [63] Coated NWs were characterized between -0.5 and 0.6 V at 200 mV s -1 to avoid any undesirable faradaic reactions, as seen in Figure 5b. As expected, the presence of a coating increases the electroactive area and improves the charge storage properties, as confirmed in Figure 5c. Additionally, the coated NWs CV in Figure 5b indicate a high reversibility in the doping process, which implies beneficial properties with regard to ionic to electronic charge transfer at the electrode-electrolyte interface. [START_REF] Saunier | [END_REF] The cathodic CSC of the NWsbased electrodes has been calculated for different thicknesses, and the data have been fitted using linear functions, as represented in Figure 5c. As predicted, the CSC of the PEDOT:PSS coated electrodes is thickness-dependent. Fitted trends reveal that thick coatings exhibit a CSC up to six times higher than bare electrodes, from 6 to 30 mC cm -2 for bare and 700 nm coatings, respectively. 300 nm coatings double the CSC to 15 mC cm -2 while (<100 nm) do not significantly improve the CSC.

The alteration of the PEDOT coating on the underlying PtSi nanowires was assessed via prolonged redox CV scanning within the -0.5 to 0.6 V potential range, at a scan rate of 200 mV s -1 in a 0.9%w NaCl solution. In Section S5, Figure S4 (Supporting Information) shows the evolution of the CV scans and the corresponding CSCs values during the time of redox charge sweeping over 3000 redox cycles. For comparison, the CSCs of bare PtSi controls are provided. For PEDOT-coated nanowires, a slight increase in CSCc during the redox cycling can be seen, which is associated with the initial reorganization of the polymer upon CV sweeping. [64,65] The corresponding CSCc value stays constant along the redox cycles window. In addition, SEM images of PEDOT-coated nanowires and bare PtSi controls after the redox CV stimulations, suggested that all nanoelectrodes exhibited no physical delamination or degradation even after thousands of redox cycles, confirming excellent structural control and longevity. Robustness over prolonged CV redox scanning demonstrates excellent polymer stability under CV stimulation conditions and suggests longer survival of the nanoelectrodes for recording applications.

To assess the NWs behavior during neuronal electrical stimulation, we performed voltage transient experiments. Bare and coated NWs were exposed to multiple cathodal-first, biphasic current pulses of a millisecond, with an interphase gap of 10μs, and an interpulse delay of 95 ms. An example of voltage transient response is represented in Figure 5d. Interestingly, NW-based electrodes exhibit a very small access potential, which is a common behavior in metallic microelectrodes. [32,[START_REF] Ganji | [END_REF] Results show that, for a same injected current of 0.08 μA, PtSi NWs present a higher V p than PEDOT:PSS/PtSi NWs. This implies that coated probes reach their limit at higher currents; therefore, the CIL should be higher. This result is confirmed in Figure 5e,f. Indeed, the current injection limit and correspondent charge injection limit are greater for PEDOT:PSS coated NWs. Bare PtSi NWs present a CIL of 0.34 mC cm -2 . In accordance with what has been observed on the CSC graph, 300 nm coatings already increase the CIL by 4× (from 0.34 mC cm -2 for PtSi NWs to 1.4 mC cm -2 ), and the CIL is increased up to 10×, to 3.53 mC cm -2 at 700 nm. PEDOT:PSS coatings therefore improve both the recording and stimulation performances of PtSi 3D-nanoelectrodes. As anticipated, thicker coatings enhance the properties more in comparison with ultrathin coatings; however, the morphology of the layer can possibly interfere with cell engulfment for layers >300 nm.

To further position these results among other research groups developments, we compared the electrochemical performances of both our PEDOT:PSS coated planar MEA and nanowires configurations, with comparable neuronal interfacing configurations reported in the literature (see Table 1). Among the three categories, it is only the 3D nanostructures (third category) that significantly enhance cell engulfment on the electrodes, which is crucial for achieving high-resolution signal recording. We consider configurations to be comparable to ours when homogeneous layers of conductive coatings are deposited on metallic electrodes, indicating possible compatibility with an integration on nanostructures. We compared the results for ∅20 μm GScoated electrodes and 300nm-coated NWs, the latter allowing us to keep the high aspect ratio of the electrodes. In both cases, we obtained specific impedance values at the state-of-the-art, with 6.36 MΩ μm -2 for planar electrodes and 7.14 MΩ μm -2 for NWs. Regarding the CIL values, planar electrodes also exhibit state-ofthe-art performances with 6 mC cm -2 while NWs possess a CIL of 1.4 mC cm -2 . The exact contribution of the 3D-configuration in the CIL value decrease when compared to planar configuration remains under investigation. Nonetheless, the CIL value for NWs is within the range of reported values (from 0.9 to 6.2 mC cm -2 ) for a noticeably small surface area.

Conclusion

PtSi planar microelectrodes and NWs were successfully coated in PEDOT:PSS using electrodeposition. Various simple and reproducible polymerization routes have been investigated, from galvanostatic to potentiodynamic, showing a large range of layer morphology and performances. Electrochemical properties of bare and coated microelectrodes were assessed with EIS measurements, CV analysis, and voltage transient experiment, showing improvement for all diameters, notably for small diameters (down to 5 μm), where the state-of the art performance could envision the use of miniaturized MEA for studying the neuronal activity with much higher spatial resolution. Optimal deposition conditions were applied to 3D nanostructured electrodes based on NWs, resulting in conformal and homogeneous layers of PE-DOT:PSS with thicknesses ranging from 700 to 40 nm, demonstrating the feasibility of selectively integrating ultra-thin layers of conducting polymer onto 3D-nano objects. Hence, the optimal cell engulfment should be maintained, as the aspect ratio of the NWs is kept for thicknesses of 300 nm and under. Electrochemical properties analysis showed that the presence of a conductive polymer coating leads to remarkable thickness-dependent characteristics combining low impedance, high charge storage capacity, and high charge injection limit. Ultra-thin layers of PE-DOT:PSS, of thicknesses 100 nm and under, effectively maintain the nanometric dimensions of the structure, minimizing intrusiveness and perturbation of the sensing structure toward the cell. Moreover, it enhances the surface performance by reducing impedance by 50% at 1 kHz and provides a reduced mechanical mismatch at the electrode/cell interface due to the soft nature of the PEDOT:PSS material. Layers that are several hundreds of nanometers thick, with an optimal thickness of 300 nm for the PEDOT:PSS layer, exhibit an impedance at 1 kHz that is three times lower than that of bare electrodes. Additionally, they demonstrate a CSC that is twice as high and CIL that is four times higher. Furthermore, these layers ensure the impeccable alignment of cells on the nanostructures, which promotes optimal electrode/cell interactions.

By combining, for the first time, nanometric high aspect ratio geometry with a high performance coated surface, PEDOTcoated 3D-nanostructured devices open new perspectives for neuronal interfacing, as they should enable the stimulation and recording of neuronal activity with acute spatial and signal resolution, while not impeding optimal cell engulfment.

Experimental Section

NEAs Fabrication: The 3D-nanostructured electrodes presented in this paper were fabricated using a CMOS-compatible process, similar to previously published. [17] A summary is included here for completeness (see Figure S5 in Section S6, Supporting Information). First, the nanowires were patterned on a 4″ silicon-on-insulator wafer using a large-scale and cost-effective top-down approach through photolithography (i-line stepper) and anisotropic plasma reactive ionic etching steps. The aspect ratio of the probes could be tuned by adjusting the etch duration and performing sacrificial oxidation. Then, following the same protocol, the electrode base and conducting lines were structured down to the oxide layer. It is important to note that this structuration step was the first step of the process when fabricating planar microelectrodes. Next, a conformal platinum (Pt) layer was deposited on the wafer by Physical Vapor Deposition, by annealing to create a stable platinum silicide (PtSi) layer of ≈90 nm, forming a core-shell type nanostructure. The unreacted Pt layer was then removed using a selective chemical etching process. Next, the wafer was covered in conformal oxide layers to ensure the insulation of the electrode structures from any liquid environment. Last, the upper part of the probes was opened, creating vertical and conductive nanostructures. Finally, the chip was integrated onto a printed circuit board using the flip-chip method, resulting in a conventional MEA-like device with external contacts. Additionally, a glass cylinder was introduced for conducting electrochemical experiments or cell cultures.

Electrochemical Characterization: All electrochemical characterization and depositions were performed with a VMP3 Multichannel Potentiostat (Biologic Sciences Instruments). Cyclic voltammetry measurements were conducted with a Platinum counter electrode and a commercial Ag/AgCl reference electrode (LF-1.6-48, Innovative Instruments, Inc.) in 0.9%w NaCl solution. Impedance spectra were acquired from 1 Hz to 100 kHz with an amplitude of 20 mV, using a platinum counter electrode in 0.9%w NaCl solution (Sodium Chloride 99%, Sigma-Aldrich). PEDOT:PSS depositions were achieved using the same three-electrode setup, in a 10 mm 3,4-Ethylenedioxythiophene (EDOT) (Sigma-Aldrich), 10 mm Sodium poly(4styrenesulfonate) (Na:PSS) (Mw 70 000, Sigma-Aldrich) in deionized water prepared from a Millipore Milli-Q A10 system (18.2 MW cm -1 resistivity). Voltage transient experiments were done using the conventional three electrode configuration, in a SP-240 potentiostat (Biologic Sciences Instruments) in Dulbecco's phosphate buffer saline solution.

Morphological Characterization: Focused-ion beam (FIB) milling and scanning electron microscopy (SEM) were performed with a Helios NanoLab 600i Dual Beam (FEI). AFM scans were performed using Dimension ICON (BRUCKER), and profilometer analysis was conducted on a Tencor P16+ (KLA TENCOR).

Figure 1 .

 1 Figure 1. Coating optimization on planar electrodes. a) Picture of the MEA device with SEM images showing the electrodes design. b) Schematic of the conventional three electrodes system, showing the working electrode (WE), the counter electrode (CE) and the reference electrode (RE). c) Galvanostatic deposition curve represented as potential versus time. d) Graph showing the cyclic voltammetry deposition. e) SEM picture showing an electrode array in which bare, GS coated and PD-coated electrodes can be observed. This demonstrates the selectivity of the deposition. f) SEM images of GS-coated electrodes (blue) and PD-coated electrodes (red) with diameters ranging from 80 to 5 μm. g) AFM measurements of PtSi and PEDOT:PSS (GS coated) surfaces. h) SEM image showing the scan path (red) of the profilometer on a ∅5 μm PD-coated electrode. i) Profilometer trace of a ∅5 μm PD-coated electrode showing the height and length of the deposition.

Figure 2 .

 2 Figure 2. Complete electrochemical characterization of planar electrodes. a) Impedance modulus versus frequency of a bare, PD-coated, and GS-coated ∅30 μm electrode. b) Phase versus frequency of a bare, PD-coated, and GS-coated ∅30 μm electrode. c) Impedance modulus value at 1 kHz as a function of diameter for bare, PD-coated, and GS-coated electrodes. d) Cyclic voltammogram of an uncoated PtSi ∅30 μm electrode. e) Cyclic voltammograms of a bare, PD-coated, and GS-coated ∅30 μm electrode. f) Charge Storage Capacity values of bare, PD-coated, and GS-coated electrodes as a function of diameter. g) Voltage transient of a ∅60 μm PtSi electrode at 4 μA injection.V a and V p represent the access potential and the polarization potential respectively. Here, V p , the polarization potential, equals the cathodic water window limit value, meaning it reaches the CIL. h) Current injection limit as a function of diameter for uncoated and coated electrodes. i) Charge Injection Limit as a function of diameter for uncoated and coated electrodes.

  Figure 2a,b shows the impedance modulus (|Z|) and phase spectra of bare PtSi, GS-coated, and PD-coated Ø30 μm electrodes. The PtSi electrode possesses a high impedance modulus, and fluctuations within the frequency range 10 0 -10 2 are due Adv. Mater. 2023, 35, 2302472 2302472 (4 of 13) © 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH 15214095, 2023, 39, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adma.202302472 by Guilhem Larrieu -Cochrane France , Wiley Online Library on [10/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

  Adv. Mater. 2023, 35, 2302472 2302472 (5 of 13) © 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH

  Authors. Advanced Materials published by Wiley-VCH GmbH

Figure 3 .

 3 Figure 3. Coating thickness optimization of NWs-based electrodes. a) Picture of the NEA device with SEM images showing the electrodes design. b) SEM images of a 200 nm PEDOT:PSS coating with a high-angle annular dark-field imaging STEM-HAADF image showing the cross section of a coated nanowire. c) Graph showing the obtained PEDOT:PSS layer thickness as a function of the applied deposition charge per electrode. d) SEM images showing the coating optimization, with thicknesses varying from 700 to 40 nm.

  , to what has been done with planar electrodes, the influence of PEDOT:PSS coatings on PtSi nanowires has been measured through EIS, CV and Voltage transient experiments (see Experimental Section), and uncoated PtSi electrodes were tested Adv. Mater. 2023, 35, 2302472 2302472 (7 of 13)

Figure 4 .

 4 Figure 4. Complete impedance characterization of NWs-based electrodes a) Impedance modulus versus frequency of bare and PE-DOT:PSS coated NWs. b) Phase versus frequency bare and PEDOT:PSS coated NWs. c) Impedance modulus value at 1 kHz as a function of PEDOT:PSS coating thickness. d) Equivalent circuit model for bare PtSi electrodes and PEDOT:PSS coated electrodes using a simple 5-elements circuit, composed of an electrolyte resistance (R s ), a charge-transfer resistance (R ct ), a double layer capacitance (C dl ), as well as a system capacitance (C p ) and system resistance (R p ). e) Graph representing the experimental and modelized impedance modulus and phase of a PtSi NWs electrode. f) Graph representing the experimental and modelized impedance modulus and phase of a coated NWs electrode (410 nm thick). g) Graph exposing the variation of Cdl and Cp with respect to the coating thickness. h) Graph exposing the variation of R ct and R s with respect to the coating thickness. In all the panel, the diameter of PtSi NWs is 350 nm.

  Authors. Advanced Materials published by Wiley-VCH GmbH 15214095, 2023, 39, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adma.202302472 by Guilhem Larrieu -Cochrane France , Wiley Online Library on [10/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Figure 5 .

 5 Figure 5. Complete electrochemical characterization of NWs-based electrodes regarding the stimulation performances. a) Cyclic voltammogram of PtSi NWs. b) Cyclic voltammograms of NWs before and after coating. The coating thickness here is 500 nm. c) Charge Storage Capacity values of bare and coated NWs as a function of coating thickness. d) Voltage transient comparison between PtSi and coated NWs (300 nm) at 0.08 μA injection. V a and V p represent the access potential and the polarization potential, respectively. e) Current injection limit as a function of coating thickness. f) Charge Injection Limit as a function of coating thickness.

  Authors. Advanced Materials published by Wiley-VCH GmbH

Table 1 .

 1 Comparison of the electrochemical performances of both our PEDOT:PSS coated planar MEA and nanowires configuration, with comparable neuronal interfacing configurations (i.e., homogeneous conductive coating layers) reported in the literature.

	Material	Shape	Electrode surface area			Reference
			[μm 2 ]
	PLANAR MEA
	Pt	Disk	7850	424	0.2	[66]
	Pt	Disk	7850	1570	0.83	[46,52]
	Au	Disk	7850	1570	0.2	[46,52]
	PtIr	Wire	4500	405	0.13	[67]
	IrOx/NPG	Disk	706	6.8	2.3	[68]
	PEDOT:PSS/Au	Disk	61 575	121	2.5	[69]
	PEDOT:PSS/Au	Disk	7850	170	0.9	[70]
	PEDOT:PSS/Au	Disk	314	15.7	1.9	[46,52]
	PEDOT:PSS/Au	Disk	314	9.42	6.2	[57]
	PEDOT:ClO 4 /Au	Disk	177	70.8	/	[7]
	PEDOT:CNT/Au	Disk	706	10.6	/	[71]
	PEDOT:PSS/Pt	Disk	314	15.7	/	[72]
	PEDOT:PSS/Pt	Disk	314	18.84	2.71	[46,52]
	PEDOT:PSS/PtSi	Disk	318	6.36	6	This Work
	3D MICRO-ELECTRODES
	Au	Mushroom	20	60	/	[10]
	PEDOT:PSS/Au	Mushroom	20	8.84	/	[10]
	(thickness 100 nm)
	3D NANO-ELECTRODES
	TiO 2	Nanostraw-nanocavity	96	31	/	[16]
	Au	Nanopillar	4.86	422	/	[15]
	IrOx	Nanotube	5.13	29.7	/	[15]
	Pt/Ti	Ultra-sharp Nanowire	0.628 * estimated	9.54	0.6	[73]
	PEDOT:PSS/PtSi (thickness 300 nm)	Nanowires	23.8	7.14	1.4	This Work

Specific |Z| @1 kHz [MΩ μm -2 ]

CIL [mC cm

-2 ]

Adv. Mater. 2023, 35, 2302472 2302472 (3 of 13) © 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH 15214095, 2023, 39, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adma.202302472 by Guilhem Larrieu -Cochrane France , Wiley Online Library on [10/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH 15214095, 2023, 39, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adma.202302472 by Guilhem Larrieu -Cochrane France , Wiley Online Library on [10/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Adv. Mater. 2023, 35, 2302472 2302472 (12 of 13) © 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH 15214095, 2023, 39, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adma.202302472 by Guilhem Larrieu -Cochrane France , Wiley Online Library on [10/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Adv. Mater. 2023, 35, 2302472 2302472 (13 of 13) © 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH 15214095, 2023, 39, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adma.202302472 by Guilhem Larrieu -Cochrane France , Wiley Online Library on [10/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Acknowledgements

This work was supported by the by the European Commission through the NEUREKA project (grant agreement number 863245) and by the LAAS-CNRS micro and nanotechnologies platform, a member of the Renatech French national network. The authors would like to thank Dr. Aurélie Lecestre and Adrian Laborde for their help during the fabrication process, Samuel Charlot for his assistance with the packaging of the NEAs, Dr. Venkata Suresh Reddy Vajrala for his guidance during electrochemical characterizations and Matthieu Sagot for his experience in data analysis.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Conflict of Interest

The authors declare no conflict of interest.


Mounia Lahfa

André Padilla

Karine De Guillen

Joana Pissarra

Mouna Raji

Stella Cesari

Thomas Kroj

Pierre Gladieux

Christian Roumestand
email: christian.roumestand@cbs.cnrs.fr

Philippe Barthe

Magnaporthe Oryzae Mounia Lahfa

H, 13C, 15 N backbone and side-chain NMR assignments for three MAX effectors from Magnaporthe oryzae

published or not. The documents may come

Biological Context

Phytopathogenic fungi cause ravaging diseases that constitute a threat to agriculture and food security. Among the large variety of pathogens, the ascomycete Magnaporthe oryzae causes the rice blast, the most destructive disease of rice worldwide [START_REF] Ou | Pathogen Variability and Host Resistance in Rice Blast Disease[END_REF]. This pathogen is a hemi biotrophic organism, starting its infection by growth and colonization of the plant and then entering a necrotic phase [START_REF] Dean | The Top 10 fungal pathogens in molecular plant pathology: Top 10 fungal pathogens[END_REF][START_REF] Fernandez | Rise of a Cereal Killer: The Biology of Magnaporthe oryzae Biotrophic Growth[END_REF] responsible for the crop's damages and losses [START_REF] Hogenhout | Emerging Concepts in Effector Biology of Plant-Associated Organisms[END_REF][START_REF] Jones | The plant immune system[END_REF][START_REF] Doehlemann | Reprogramming of plant cells by filamentous plantcolonizing microbes[END_REF]. Beside rice, several other cereal cultures are impacted, including wheat, barley and millet [START_REF] Couch | Origins of Host-Specific Populations of the Blast Pathogen Magnaporthe oryzae in Crop Domestication With Subsequent Expansion of Pandemic Clones on Rice and Weeds of Rice[END_REF].

Even if resistance appears in some cultures, the fungus generally escapes this blast resistance [START_REF] Araújo | Development of blast resistant somaclones of the upland rice cultivar araguaia[END_REF] by secreting during infection an extended repertoire of small proteins (size under 200 amino acids) named effectors, whose specific targets remain only partially known. Effectors show very divergent sequences, sharing a signal peptide on the N-terminal end. This signal peptide is essential for their secretion and then their translocation inside the plant cell [START_REF] Petre | How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells?[END_REF]. These translocation mechanisms remain mostly unknown but it has been shown that effector secretion can be dependent on the conventional secretory pathway of the plant, or dependent on another secretion system from the pathogen itself (Ribot et al. 2012;[START_REF] Giraldo | Two distinct secretion systems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzae[END_REF][START_REF] Eseola | Investigating the cell and developmental biology of plant infection by the rice blast fungus Magnaporthe oryzae[END_REF]. They constitute a way to escape basal resistance based on PAMP/MAMP recognition by extra-cellular cell surface receptors [START_REF] Wang | The role of effectors and host immunity in plant-necrotrophic fungal interactions[END_REF]. Some effectors can also prevent recognition of effectors by specific resistance proteins, hindering the protecting immune response [START_REF] Houterman | Suppression of Plant Resistance Gene-Based Immunity by a Fungal Effector[END_REF].

In 2015, a new family of effectors, called MAX-effectors (for Magnaporthe Avrs and ToxB like) was identified from bioinformatic analysis [START_REF] De Guillen | Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi[END_REF] and the 3D structure has been solved for several of them (Zhang et al. 2013;[START_REF] Nyarko | Solution NMR Structures of Pyrenophora tritici-repentis ToxB and Its Inactive Homolog Reveal Potential Determinants of Toxin Activity[END_REF][START_REF] Maqbool | Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor[END_REF][START_REF] De Guillen | Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi[END_REF][START_REF] De La Concepcion | Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen[END_REF], 2021;[START_REF] Zhang | A positive-charged patch and stabilized hydrophobic core are essential for avirulence function of AvrPib in the rice blast fungus[END_REF][START_REF] Maidment | Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense[END_REF][START_REF] Bentham | A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum[END_REF]. Despite having low sequence identity (generally under 25 %), they share a conserved structure and topology consisting of a sandwich made of five to six antiparallel β strands (Zhang et al. 2013;[START_REF] Nyarko | Solution NMR Structures of Pyrenophora tritici-repentis ToxB and Its Inactive Homolog Reveal Potential Determinants of Toxin Activity[END_REF]). These MAX-effectors also show two highly conserved cysteines forming a disulphide bond between the two sheets (de [START_REF] De Guillen | Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi[END_REF]. Restricting the analysis to the genomes of Magnaporthe genus, where most of MAX effectors were identified, a new prediction pipeline (to be published elsewhere) allows us to extract new MAX candidates, among them MAX47, MAX60 and MAX67. We report here the backbone and side-chain resonance assignments of these three effectors. These assignments will provide the foundation for their 3D structure determination, and will allow interaction studies with their putative targets.

Methods and Experiments

Recombinant protein expression and purification.

Uniformly labeled ( 15 N and 15 N/ 13 C) proteins (MAX47, MAX60 and MAX67) were expressed in E. coli BL21 (DE3) cells (Invitrogen, Thermo Fisher Scientific, Waltham, USA) from a homemade plasmid pDB-his-CCDB-3C (courtesy of Frederic Allemand, CBS Montpellier, France) allowing expression of the recombinant fusion proteins with a His 6 -tag cleavable by the protease HRV 3C. In the three constructs, the N-terminal signal peptide has been removed from the original sequence, yielding final proteins of 95, 89 and 60 residues long for MAX47, MAX60 and MAX67, respectively. Protein expression was carried out in 15 NH 4 Cl (1 g/l) or 15 NH 4 Cl (1 g/l) / 13 C-Glucose (3 g/l) enriched M9 medium. Cells were grown at 37°C until reaching an OD 600 = 0.8 and then, expression proceeded overnight at 30°C after induction by addition of 0.3 mM IPTG. Cells were harvested by centrifugation, resuspended in denaturing buffer (50 mM Tris, 300 mM NaCl, 1mM DTT (dithiothréitol), 8M urea, pH 8) and lysed by ultrasonication. The supernatant containing the unfolded protein was applied to a HisTrap HP 5 ml affinity column (Cytiva, Freiburg im Breisgau, Germany). The His 6 -tagged protein was eluted in 50 mM Tris, 300 mM NaCl, 1mM DTT, 8M urea, pH 8 with an imidazole gradient up to 500 mM. At this step, MAX67 was directly dialyzed against 25 mM Na Acetate, 2 mM DTT, pH 4.6 buffer in order to remove imidazole and urea, allowing the refolding of the protein. The His 6 -tag was then cleaved off using the HRV 3C protease. For MAX60 and MAX47 refolding, the His-tagged proteins were dialyzed at 4°C in 0.18% ammonia solution, to avoid protein precipitation during the re-folding process at pH 4.6. The His 6 tag was then cleaved out directly at pH 8. Ammonia was removed by lyophilization and the lyophilized samples were dissolved into 25 mM Na Acetate, 2 mM DTT, pH 4.6 buffer. All samples were then concentrated using Amicon Ultra Centrifugal Filter Devices (MW cutoff 3,000 Da), (Merck Millipore, Burlington, USA) prior to size exclusion chromatography (SEC) using HiLoad 16/600 Superdex 75 pg column (Cytiva). Fractions containing proteins were pooled, concentrated to 0.5 mM and stored at -20 °C.

NMR spectroscopy.

Samples for NMR measurements (200 µL) were prepared at about 0.5 mM protein concentration in 25 mM Sodium Acetate buffer (pH 4.6), and 5% of D 2 O was added for the deuterium lock. 1 H, 15 N double-resonance and 1 H, 15 N, 13 C triple-resonance NMR experiments were performed at 32°C on Bruker AVANCE III 700 MHz and 800 MHz spectrometers equipped with a cryogenic Z-gradient 1 H-13 C-15 N 5 mm probe head. Backbone and Cß resonance assignments were made using HNCA, HNCACB, CBCA(CO)NH, HNCO and HN(CA)CO standard experiments [START_REF] Sattler | Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients[END_REF]) performed on the uniformly 15 N/ 13 C labeled samples. Side chains and Hα proton resonance assignments were obtained through 3D [ 1 H, 15 N] NOESY-HSQC (mixing time 150 ms) and TOCSY-HSQC (isotropic mixing: 60 ms) experiments. Water suppression was achieved with the WATERGATE sequence [START_REF] Piotto | Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions[END_REF]). 1 H chemical shifts were directly referenced to the methyl resonance of DSS (2,2-Dimethyl-2-silapentane-5-sulfonate, Sodium salt), while 13 C and 15 N chemical shifts were referenced indirectly to the absolute frequency ratios 15 N/ 1 H = 0.101329118 and 13 C/ 1 H = 0.251449530.

Extent of Assignments and data deposition.

The data sets allowed the assignment of 90.9%, 97.3% and 99.7% of the proton resonances, 95.5%, 98.8% and 98.3% of the nitrogen resonances (amide groups), and 94.0%, 98.1% and 99.4% of the carbon resonances (Cα, Cβ and C') for MAX47, MAX60 and MAX67, respectively. The assignments are deposited in the BMRB under accession numbers 34731 (MAX47), 34730 (MAX60) and 34729 (MAX67). The good signal dispersion observed in the [ 1 H, 15 N] HSQC for these three proteins (Fig. 1) is consistent with well-folded 3D structures. Interestingly, we observed two cross-peaks for each residue in the C-terminal segment (E98-W106) of MAX60, with relative intensities of 0.45:0.55. Probably, the cis-trans isomerization of one or two of the proline residues that flank W106 in the C-terminus (P104-F105-W106-P107) induces two different orientations for the tryptophan aromatic side-chain, yielding two different chemical environments for the neighboring residues. TALOS-N [START_REF] Shen | Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks[END_REF] analysis suggests a high content in extended ß-strands in the 3D structure of the three proteins, consistent with the secondary structure of the MAX-effectors (Fig. 1). In addition, Φ/Ψ values determined for residues Y91-E102 in MAX60 indicate the presence of an α-helix in the C-terminal end of the protein. These conclusions are further supported by CSI 3.0 analysis [START_REF] Hafsa | CSI 3.0: a web server for identifying secondary and super-secondary structure in proteins using NMR chemical shifts[END_REF]. The consensus chemical shift index derived from 1 Hα, 13 Cα, 13 Cβ, 13 C' and 15 N 1 H chemical shifts also supports the presence of five ßstrands in the 3D structure of the three proteins, compatible with the ß-sandwich structure shared by MAX effectors. Also, CSI analysis supports the presence of an α-helix in the C-terminal end of MAX60 (See Supplementary Material, Fig. S1). At this step, we have no precise idea about the role of this C-terminal helical extension in the function of the effector, even if we suspect that it might confer some target specificity to MAX60 with regard to others MAX effectors. This assumption has to be further supported with NMR in-vitro interaction studies with potential effector targets, once the solution structure of this effector will be solved.

Figure 1 :

 1 Figure 1: (Left) 2D [ 1 H-15 N] HSQC spectra of MAX47, MAX60 and MAX67 (from top to bottom, as indicated) recorded at 700 MHz on 15 N-uniformly labeled samples dissolved in 25 mM Sodium Acetate buffer pH 4.6 at 32°C. Cross peak assignments are indicated using the one-letter amino acid code and number. The sequence

This project was funded by the ANR project MagMAX (ANR-18-CE20-0016-02) and supported by French Infrastructure for Integrated Structural Biology (FRISBI) grant No. ANR-10-INSB-05. *Authors' contributions: M.L., K.d.G., J.

Assignments have been deposited to and are available from the BMRB data bank.


Paweł Piotr Michałowski
email: pawel.michalowski@imif.lukasiewicz.gov.pl

Jonas Müller

Chiara Rossi

Alexander Burenkov

Eberhard Bär

Guilhem Larrieu

Peter Pichler

Secondary ion mass spectrometry quantification of boron distribution in an array of silicon nanowires

Keywords:

The development of non-planar structures such as arrays of nanowires (NWs), poses a significant challenge for dopant concentration determination. Techniques that can be readily used for 3D structures usually lack the desired sensitivity whereas secondary ion mass spectrometry (SIMS), known for its excellent detection limits, is designed to analyze flat samples. In this work, we overcome the limitation of standard SIMS approaches. NWs are covered with photoresist forming a flat surface. For high incident angle bombardment, the sputtering process becomes self-flattening, i.e. the ions collide with the sidewalls of the exposed tips of NWs at much lower angles and sputter them significantly faster. Thus, reliable information about the dopant distribution along the height of NWs can be obtained. The SIMS analysis can be performed on an array of 1000 x 1000 nanowires with a detection limit of about 5 x 10 16 atoms/cm 3 and a reasonable signal-to-noise ratio of about 10 dB.

Introduction

Nanowires (NWs) are receiving global recognition due to their quasi-one dimensional geometry and resulting unique properties, such as high area-to-volume ratio, high optical output, low defect density, and waveguiding properties [START_REF] Joyce | A review of the electrical properties of semiconductor nanowires: insights gained from terahertz conductivity spectroscopy[END_REF][START_REF] Barrigón | Synthesis and applications of III-V nanowires[END_REF][START_REF] Wang | A comprehensive review of template-synthesized multi-component nanowires: From interfacial design to sensing and actuation applications[END_REF]. Their potential applications are ranging from photonics [START_REF] Quan | Nanowires for photonics[END_REF], electronics [START_REF] Jia | Nanowire electronics: From nanoscale to macroscale[END_REF], optoelectronics [START_REF] Li | Nanowire electronic and optoelectronic devices[END_REF], sensing [START_REF] Patolsky | Nanowire nanosensors[END_REF], photovoltaics [START_REF] Mokkapati | Review on photonic properties of nanowires for photovoltaics[END_REF], and energy storage [START_REF] Zhou | Nanowires for electrochemical energy storage[END_REF], to name just a few.

To further develop NWs based devices it is essential to assess their chemical composition, particularly dopant distribution. For uniformly doped NWs charge concentration can be determined using Kelvin probe force microscopy [START_REF] Orrù | A roadmap for controlled and efficient n-type doping of self-assisted GaAs nanowires grown by molecular beam epitaxy[END_REF], photoluminescence [START_REF] Orrù | A roadmap for controlled and efficient n-type doping of self-assisted GaAs nanowires grown by molecular beam epitaxy[END_REF], Raman spectroscopy [START_REF] Suomalainen | Te-doping of self-catalyzed GaAs nanowires[END_REF] or off-axis electron holography [START_REF] Wolf | Electron holographic tomography for mapping the three-dimensional distribution of electrostatic potential in III-V semiconductor nanowires[END_REF]. However, most of these techniques have poor detection limits.

Additional complications arise when the dopant distribution in not uniform. One such example is an array of boron-doped silicon NWs which are fabricated in a top-down manner by patterning and anisotropic etching of a doped silicon substrate [START_REF] Wu | High aspect ratio silicon etch: A review[END_REF][START_REF] Huang | Metal-assisted chemical etching of silicon: A review[END_REF][START_REF] Dasgupta | 25Th anniversary article: Semiconductor nanowires -synthesis, characterization, and applications[END_REF]. To fabricate the gate-all-around nanowire field-effect transistors (GAA-NW-FETs) several oxidation processes are usually performed [START_REF] Kwong | Vertical silicon nanowire platform for low power electronics and clean energy applications[END_REF][START_REF] Larrieu | Vertical nanowire array-based field effect transistors for ultimate scaling[END_REF]. Wet-thermal oxidation, or a prolonged dry-thermal oxidation, and subsequent oxide removal are used to obtain the desired NW diameter also creating a new defect-free interface [START_REF] Han | Modelling and engineering of stress based controlled oxidation effects for silicon nanostructure patterning[END_REF]. Finally, a short dry oxidation creates a thin gate oxide. It is well known that during these oxidation processes boron atoms preferentially segregate into the growing oxide and, thus, the concentration in the remaining silicon core may significantly decrease [START_REF] Kato | Redistribution of diffused boron in silicon by thermal oxidation, Japan[END_REF][START_REF] Fukata | Segregation behaviors and radial distribution of dopant atoms in silicon nanowires[END_REF][START_REF] Koffel | On an improved boron segregation calibration from a particularly sensitive power MOS process[END_REF].

Atom probe tomography (APT) may be considered a perfect candidate to analyze dopant distributions in a nanowire, particularly because the shape of a sample is already similar to the tip-shaped specimen used in the APT analysis. It should be, however, noted that most APT analyses of p-type silicon have been performed on samples with boron concentrations exceeding 10 20 atoms/cm 3 and the background/noise level is usually in the order of 1-2 x 10 19 atoms/cm 3 [START_REF] Nomoto | Atom probe tomography of phosphorus-and boron-doped silicon nanocrystals with various compositions of silicon rich oxide[END_REF][START_REF] Nomoto | Atom probe tomography analysis of boron and/or phosphorus distribution in doped silicon nanocrystals[END_REF][START_REF] Tu | Influence of laser power on atom probe tomographic analysis of boron distribution in silicon[END_REF]. For the GAA-NW-FET device much lower concentrations are required and, thus, APT analysis becomes not feasible. Furthermore, the sample preparation and analysis are both time-consuming and performed on a single NW, not a full array.

On the other hand, secondary ion mass spectrometry (SIMS) provides a much bigger analysis area and is well-known for its excellent detection limits of trace elements [START_REF] Wittmaack | High-sensitivity depth profiling of arsenic and phosphorus in silicon by means of SIMS[END_REF][START_REF] Ber | Secondary ion mass spectroscopy investigations of magnesium and carbon doped gallium nitride films grown by molecular beam epitaxy[END_REF][START_REF] Chiou | Secondary ion mass spectrometry analysis of in-doped p-type GaN films[END_REF][START_REF] Emziane | In situ oxygen incorporation and related issues in CdTe/CdS photovoltaic devices[END_REF][START_REF] Matsunaga | Secondary ion yields of C, Si, Ge and Cs surface density and concentration in SIMS[END_REF][START_REF] Gnaser | SIMS detection in the 10 12 atoms 𝑐𝑚 -3 range[END_REF], even in the 10 12 atoms/cm 3 range [START_REF] Gnaser | SIMS detection in the 10 12 atoms 𝑐𝑚 -3 range[END_REF]. Particularly, SIMS profiling of boron dopants is widely discussed in metrology-related journals and proceedings of SIMS conferences for more than forty years [START_REF] Wittmaack | Profile distortions and atomic mixing in SIMS analysis using oxygen primary ions[END_REF][START_REF] Clegg | A comparative study of SIMS depth profiling of boron in silicon[END_REF][START_REF] Vandervorst | High resolution SIMS and neutron depth profiling of boron through oxide-silicon interfaces[END_REF][START_REF] Gautier | SIMS depth profile correction for the study of the first step of the diffusion of boron in silicon[END_REF][START_REF] Shao | Response function during oxygen sputter profiling and its application to deconvolution of ultrashallow b depth profiles in Si[END_REF][START_REF] Merkulov | Advanced SIMS quantification in the first few nm of B, P and As ultrashallow implants[END_REF][START_REF] Merkulov | Improvement of extra low impact energy SIMS data reduction algorithm for process control[END_REF][START_REF] Kumar | Evaluation of secondary electron intensities for dopant profiling in ion implanted semiconductors: a correlative study combining SE, SIMS and ECV methods[END_REF]. However, standard SIMS analysis is performed on a flat sample and cannot be directly used https://doi.org/ In this work, we present a procedure to overcome this limitation. Prior to the measurement silicon NWs are embedded in organic material. In standard SIMS, the difference in the etching rate of these materials would result in a non-uniform sputtering process. However, high incident angle bombardment ensures that the sputtering process is self-flattening, i.e. when the tips of NWs are exposed, the incoming primary ions collide with the sidewalls of the NWs directly and thus these tips are sputtered much faster than the organic material. The result of a measurement recreates the distribution of boron along the height of NWs.

Experimental setup

Sample preparation

Large Arrays of vertical nanowires (100 × 100) were fabricated by a top down approach on highly doped p-type silicon (boron-doped at 3.88 × 10 19 cm 3 ) [START_REF] Han | Realization of ultra dense arrays of vertical silicon nanowires with defect free surface and perfect anisotropy using a top-down approach[END_REF][START_REF] Guerfi | High resolution HSQ nanopillar arrays with low energy electron beam lithography[END_REF][START_REF] Larrieu | Sub-15 nm gate-all-around field effect transistors on vertical silicon nanowires[END_REF]. An initial mask of a negative-tone resist (hydrogen sylesquioxane) was patterned on the substrate through low voltage (30 kV) electron beam lithography using a Raith-150 system. The highly vertical nanopattern was successively transferred into the substrate by fluorine-based anisotropic reactive ion etching in an Alcatel AMS4200 system, creating perfectly vertical nanostructures of 270 nm height. The remaining resist was removed in diluted hydrofluoric acid, followed by a low-power oxygen plasma cleaning step. The as-fabricated NWs were then oxidized by either wet-or dry-thermal oxidation in a Centronic E1550HT tube furnace. Wet-thermal oxidation was carried out at 850 • C for 5 min to create 20.6 nm of oxide while an equally thick oxide layer of 19.2 nm could be achieved through dry oxidation at 860 • C for 66 min. This sacrificial oxide layer was removed by wet chemical etching in a buffered oxide etchant, reducing the size of the NWs. Finally, a thin dry-oxide layer of few nm was created by dry-thermal oxidation at 725 • C for 20 min and removed in buffered oxide etchant. All samples, having undergone the exactly same structuring process (lithography & etching) but different oxidation procedures, were embedded in an organic photoresist Microposit S1805. A thick layer of several hundred nm was spin-coated on the samples and etched down in an oxygen plasma until the surface was approximately 20 nm above the NWs' heads.

Secondary ion mass spectrometry

A CAMECA IMS SC Ultra instrument was used in all experiments. The incident angle of the cesium primary beam was varied between 20 and 75°. The instrument did not allow direct control over the incident angle but different values could be achieved by changing voltages applied to the accelerator, sample holder, and the floating voltage applied to the primary column. The impact energy was 500 eV while the primary current was 10 nA, and the beam was rastered over 150 × 150 μm 2 whereas the analysis area was limited to 70 × 70 μm 2 . Positive detector polarity was used and thus all signals were registered as CsX + cluster ions. Quantification was based on ion-implanted reference samples.

Complementary characterization techniques

Scanning electron microscopy (SEM) micrographs were obtained with a FEI Helios 600i double beam system to inspect samples before and after the photoresist deposition to determine the size of the NWs.

Atomic force microscopy (AFM) measurements were performed by a Bruker Dimension FastScan with ScanAsyst using silicon probes (Bruker OTESPA-R3 model) with a typical nominal tip radius of 7 nm. The depth of SIMS craters was determined and the root-mean-square (RMS) roughness of the samples was measured over a scan area of 30 × 30 μm 2 .

Results and discussion

Fig. 1 presents a scanning electron microscopy (SEM) overview of the array of as-fabricated NWs confirming their high quality and uniformity. As mentioned, arrays of NWs usually undergo several oxidation processes and, thus, a series of samples is needed to evaluate boron segregation during each step. Four different arrays of NWs will be considered, as presented in Table 1.

It is important to emphasize that the lateral resolution of the SIMS technique is not enough to distinguish the oxidized and not oxidized parts of the NWs and, thus, the oxide has always been removed before the analysis (even the gate oxide). In this way, it is possible to assess the distribution of boron that remains in the silicon core of a nanowire.

Even though many SIMS instruments are equipped with positionsensitive detectors, it is impossible to perform a direct measurement on NWs. The ion bombardment of the three-dimensional structures leads to a non-uniform and impossible to predict sputtering process and thus the secondary ions would be emitted simultaneously from the substrate and the NWs (along their full height). A hypothetical solution would be to fill the space between the nanowires of NWs with undoped silicon forming a flat sample -a depth profile would reflect the distribution of dopant along the height of NWs. However, it is not feasible to uniformly deposit silicon, avoiding forming voids and rough surfaces. It is, however, possible for organic materials such as photoresists, as shown as set-in in Fig. 1. Unfortunately, a significantly different etching rate of these materials would once again result in a non-uniform sputtering process.

It should be, however, noted that the etching rate depends not only on the material but also on the incident angle of the primary ions, and, thus, additional experiments have been devised -for each incident angle silicon and S1805 photoresist material has been sputtered for 20 min and the depth of the crater has been measured with AFM, hence the sputtering rate has been calculated, as presented in Fig. 2A. It can be immediately noted that:

• For all incident angles the etching rate of the photoresist is almost an order of magnitude higher than for silicon; • The etching rate does not change significantly in the range of 20-55°for both materials and starts to decrease for higher angles; • For the 75°incident angle the etching rate of both materials decreased more than two orders of magnitude.

It can be therefore concluded that for the 75°incident angle the etching rate of the photoresist is more than an order of magnitude lower than for silicon in standard incident angles (20-55°). Thus, the sputtering process for 75°incident angle may be considered self-flattening, as schematically shown in Fig. 2B. Due to a much higher etching rate of the photoresist, at some point, the tips of the NWs will be exposed. However, the primary ions will collide with the sidewalls of the exposed NWs at much smaller angles and thus they will be etched much faster than the remaining photoresist and the sample will become flat again. A depth profile obtained in such non-trivial conditions will reflect the boron distribution along the height of the NWs.

To verify the self-flattening property of the measurement procedure AFM topography scans have been performed at the surface of a sample, and after sputtering of 10, 175, and 350 nm, i.e. still in the photoresist, in the middle of NWs, and in the substrate, respectively. The resulting RMS roughness is 1.20, 0.92, 1.11, and 0.82 nm, respectively. It can be noted that the roughness decreases after the sputtering of 10 nm of photoresist and increases after reaching the NWs but it is still lower than at the surface of the sample. The lowest value has been obtained for the silicon substrate. AFM topography images are presented in Figures S2-5 in the Supplementary Materials.

As shown in Fig. 3A, a matrix effect and/or agglomeration of cesium at the interface between the organic material and the silicon results in artificial enhancements of the CsSi + and CsB + signals. To obtain the realistic distributions it is essential to perform point-to-point normalizations to the Cs + signal, as shown in Fig. 3B.

The analysis of the point-to-point normalized silicon signals yields important information about the validity of the method. As it is shown in Fig. 4A, the intensity of the silicon and boron signals coming from the substrate are the same for all samples but differ for the NWs region. The intensities of signals coming from the substrate correspond to the total field of view which has been set to 70×70 μm 2 . The intensity of the silicon signal coming from the NWs scales linearly with the total area of the NWs in the array and, thus, it is possible to determine the diameter of the NWs directly from the SIMS measurements. It equals 70.1 ± 0.6, 59.9 ± 0.7, 57.8 ± 0.7, and 52.9 ± 0.8 nm for samples after etching, as well as after dry, wet, and gate oxidations, respectively. These values are in perfect agreement with the actual size of the NWs, as presented in Table 1.

The change of the boron concentration from the tips of the NWs towards and into the substrate is presented in Fig. 4B. As expected, for the sample after etching there is no difference between NWs and the substrate regions but a clear depletion is observed for other samples, reaching up to about 200 nm into the substrate for the dry oxidized sample. Not surprisingly, the effect is significantly more pronounced for NWs as boron atoms can segregate into the growing oxide from all directions whereas the resupply of dopant from the bulk is less effective. Table 2 summarizes how much lower the boron concentration is on average and at the tip, middle (approximately 120 nm of height), and foot of the NWs when compared to the as fabricated NWs. The average ratio is ranging from 4.6 to 21.0 and the effect is significantly more pronounced at the tip of the NWs (ranging from factor 6.6 to 56.7).

It can be noted that the boron concentration is constant at the depths of 150-250 nm (NWs region) and 550-650 nm (substrate region) for all samples. Thus, it is possible to calculate the signal-to-noise ratio (SNR) for these regions as ratio of the mean intensity and maximum absolute deviation. The SNR for the substrate region is the same for all samples and equals 17.6 dB while for the NWs it is 15.6, 8.3, 15.4, and 9.4 dB for the as fabricated, dry, wet, and gate samples, respectively. It may seem surprising that the SNR for the substrate

Table 2

Factor by which the boron concentration in NWs is lower when compared to the sample after etching. The columns show the average value, and the values registered at the tip, middle, and foot of the NWs. region is only moderately higher than for the NWs region of the as fabricated sample. It should be, however, noted that the intensity of both, silicon and boron signals in the substrate region is very high (around one million counts per second, as shown in Fig. 3) which is very close to the upper limit of the electron multiplier detector and the error introduced by its dead time is relatively high. The SNR for the NWs region remains reasonably high for all samples but, as expected, it decreases for samples with lower boron concentration. Further decreases of the boron concentration (either by longer oxidation processes or lower initial dopant level) or NW diameters may result in an unacceptable signal-to-noise ratio. In such a case a larger array of NWs should be fabricated for the SIMS analysis.

Conclusions

By embedding the array of vertical nanowires in a photoresist matrix and using a high incident primary ion bombardment makes the measurement procedure to be self-flattening and ensures a uniform sputtering process, despite the difference in the etching rate of both materials. Thus, the proposed method allows the precise quantification of boron distributions in arrays of silicon nanowires with reasonable SNR even for concentration below 10 18 atoms/cm 3 . The sensitivity of the method is directly proportional to the total area of silicon NWs and, thus, it can be expected that for larger arrays (1000 × 1000 NWs) a detection limit of about 5 × 10 16 atoms/cm 3 can be achieved with the SNR around 10 dB. This is significantly better than APT analysis can offer. In theory, oxygen primary ions should be more suitable for the analysis of boron dopants. However, preliminary experiments have shown that the differences in the sputtering rate for silicon and photoresist are not significant and, thus, the analysis will no longer be self-flattening.

The measurement has revealed that during the oxidation of silicon NWs the majority of the boron atoms segregate into the growing oxide and their concentration may decrease even more than one order of magnitude, especially at the tip of the NWs. This change significantly alters the operation of p-type doped GAA-NW-FET devices (Figure S6 in Supplementary Materials) and the knowledge about the non-homogeneous distribution of boron dopants along the height of nanowires may prove invaluable for the further optimization of GAA-NW-FET devices [START_REF] Rossi | Performance of vertical gate-all-around nanowire p-MOS transistors determined by boron depletion during oxidation[END_REF].

Declaration of competing interest

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

Fig. 1 .

 1 Fig. 1. SEM micrographs of large Si-nanowire arrays (100 × 100) after their top-down fabrication, using electron beam lithography and highly anisotropic reactive ion etching (Part A and D) and after embedding in an organic resist matrix (Part B and C).

Fig. 2 .

 2 Fig. 2.Part A: the sputtering rate of silicon and organic material determined for different primary ions incident angles. Each data point is based on six separate measurements. Part B: the schematic concept of the self-flattening analysis. If the tips of NWs are exposed, the primary ions will collide with them at a much lower angle and sputter them at a much faster rate than the rest of the sample.

Fig. 3 .

 3 Fig. 3. Depth profiles obtained for the wet sample. Part A: raw data indicate significant agglomeration of cesium at the interface between organic material and silicon. Part B: silicon and boron signals point-to-point normalized to the cesium signal.

Fig. 4 .

 4 Fig. 4. Part A: Comparison of the silicon signals for all samples. The intensity scales linearly with the total area of the NWs in the array and thus it is possible to determine the diameter of each NW directly from the SIMS measurement. Part B: Comparison of the boron concentrations for all samples. A clear boron depletion is observed after the oxidation processes and subsequent oxide removal.

  : Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing -original draft, Writing -review & editing, Visualization, Funding acquisition. Jonas Müller: Investigation, Formal analysis, Visualization, Writing -review & editing. Chiara Rossi: Software, Data curation, Formal analysis, Visualization, Writing -review & editing. Alexander Burenkov: Formal analysis, Writing -review & editing. Eberhard Bär: Formal analysis, Writing -review & editing. Guilhem Larrieu: Resources, Formal analysis, Writing -review & editing, Supervision. Peter Pichler: Conceptualization, Formal analysis, Writing -review & editing, Supervision, Funding acquisition.

  10.1016/j.measurement.2023.112630 Received 20 December 2022; Received in revised form 4 February 2023; Accepted 17 February 2023 to characterize 3D nanostructures (cf. Figure S1 in Supplementary Materials).

Table 1

 1 A summary of samples used in the experiment.

	Name	Oxidation	Temperature	Time	NW diameter	NW height
			( • C)	(min)	(nm)	(nm)
	After etching	-	-	-	70	270
	Dry	Dry	860	66	60	240
	Wet	Wet	850	5	58	247
	Gate	Wet	850	5
		+ dry	725	20	53	241

Acknowledgments

The research leading to these results has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 871813 MUNDFAB and the National Centre for Research and Development, Poland under grant agreement No. LIDER/8/0055/L-12/20/NCBR/2021. This work was supported by the LAAS-CNRS micro and nanotechnologies platform, a member of the Renatech french national network.

Data availability

Data will be made available on request.

Appendix A. Supplementary data

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.measurement.2023.112630.