C Madzak

M C Mimmi

E Caminade

A Brault

S Baumberger

P Briozzo

C Mougin

C Jolivalt

Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis

Keywords: fungal laccase, pH-dependence, polyphenol oxidase, site directed mutagenesis, Yarrowia lipolytica expression system

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for kcat between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate.

Nevertheless, the Asn mutation led to a significant shift of the pH (pH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

Introduction

Laccases (EC 1.10.3.2) are polyphenoloxidases performing the reduction of oxygen to water while oxidizing organic substrates by a one-electron redox process. Laccase activity was first described in plants (Rhus vernicifera) by Yoshida in 1884 and more recently enzymes were evidenced in bacteria (Diamantidis et al., 2000). However, laccases are mainly produced by filamentous fungi (ascomycetes and mainly wood-decaying basidiomycetes) [START_REF] Xu | Encyclopedia of Bioprocessing Technology: Fermentation, Biocatalysis and Bioseparations[END_REF]. In these organisms, they are involved in several biological processes such as lignin degradation (Eggert et al., 1997), morphogenesis and pathogenesis. In addition to lignocellulosic substrates, an extensive list of xenobiotics [pesticides, polycyclic aromatic hydro-carbons (PAHs), dyes, etc.] often containing phenolic or aromatic amino groups are transformed by laccases (Gianfreda et al., 1999;Jolivalt et al., 1999) either by bond cleavage or by oxidative coupling.

Owing to the great variety of reactions catalysed by laccase, this enzyme has attracted considerable interest in various fields of research, e.g. development of oxygen cathodes in biofuel cells, green biodegradation of xenobiotics, biosensors, organic synthesis, labelling immunoassays (Mayer and Staples, 2002). This ability to oxidize such a large range of substrates seems to be related to the non-specific free radical mechanisms which have evolved to degrade lignin and is greatly favoured by the high redox potential of the laccases, ranging from 430 mV for tree laccase from Rhus vernicifera to 780 mV for fungal laccase from Polyporus versicolor (Xu et al., 1999).

We demonstrated also that fungal laccases were efficient tools for bioprocesses leading to polluted water cleanup (Jolivalt et al., 2000) and soil bioremediation (Rama et al., 2001). Nevertheless, laccase-mediated biotransformation of xenobiotics in natural media suffers from two main limitations of the enzyme: an acidic optimal pH for activity and the requirement in several cases (e.g. PAHs) for a redox mediator. The modification of these laccase properties should be achieved through a site-directed mutagenesis strategy.

Based on sequence alignments without precise information concerning both the substrate cavity geometry and the interactions between amino acids and substrate, the pioneering work of [START_REF] Xu | [END_REF]Xu et al. ( , 1999) ) suggested that a non-ligating tripeptide in the vicinity of the active site was involved in the redox potential level. Although the redox potentials were not significantly altered, the triple mutants had a phenoloxidase activity whose pH optimum shifted 1 unit lower or higher while the kinetic parameters were greatly changed. These results were interpreted as possible mutation-induced structural perturbations of the molecular recognition between the reducing substrate and the enzyme.

In recent years, crystal structures of free laccases from Coprinus cinereus (Ducros et al., 1998), Trametes versicolor (Piontek et al., 2002), Melanocarpus albomyces (Hakulinen et al., 2002) and Rigidoporus lignosus (Garavaglia. et al., 2004) have been published. They revealed a distorted trigonal planar Cu geometry of the T1 centre at the active site of the enzyme, with three strong and highly conserved ligands, two histidines and a cysteine and a relatively distant axial ligand, generally leucine or phenylalanine in fungal laccases. In addition, three-dimensional structures of laccases have been obtained in the presence of a reducing substrate [START_REF] Bertrand | [END_REF]Enguita et al., 2004). Our group showed that the cavity of the laccase from T. versicolor was fairly wide and that it can accommodate a large variety of substrates which were not tightly buried in it [START_REF] Bertrand | [END_REF]. The presence of an arylamine (2,5dimethylbenzeneamine or 2,5-xylidine) at the T1 active site of the enzyme revealed two important residues for the interaction between the amino group of the reducing substrate and the enzyme. The first is a histidine (458) that also coordinates the T1 copper and acts as the primary electron acceptor from the substrate. This histidine is highly con-served among laccases. The second, aspartate 206, is hydrogen bonded via the terminal oxygen of its side chain to the amino group of 2,5-xylidine. Asp206 appears relatively buried in the cavity, surrounded by a crown of hydrophobic residues which lie in a more external part (Figure 1). All these residues con-tribute to the positioning of the substrate. Moreover, it was found for phenolic substrates such as phenylurea derivatives that a pH increase from 3 to 5 generally induced a decrease in the Km values, which can be interpreted as a better affinity between the substrate and the enzyme. As the protonation state of phenolic derivatives remains unchanged in this pH range, such an alteration in the interaction between the substrate and the enzyme could be a consequence of the deprotonation of one of the amino acid side chains interacting with the substrate, suggesting the involvement of an acidic amino acid, possibly Asp206.

That new hypothesis led us to develop in the present work a mutagenesis approach performed on a T.versicolor laccase (AF414109) considering the Asp206 residue. Because different biological functions are generally attributed to laccases depending on their origin (white-rot fungal laccases are involved in the degradation of lignin whereas plant laccases participate in the initial stages of its synthesis), we decided to mutate Asp206 into three amino acids: (i) a non polar amino acid, alanine, (ii) glutamate, which can be found among ascomycetes whereas aspartate seems fully conserved in basidiomycetes, and (iii) asparagine, found in all plant laccase sequences (Figure 2).

Here we had three objectives: (i) to determine how the site-directed mutagenesis would impact the pH dependence of enzymatic activity, (ii) to investigate the possibility of increasing the efficiency of the catalytic activity of the enzyme towards selected substrates and (iii) to enlarge the permissivity of laccase at the T1 site towards xenobiotics recalcitrant to laccase-catalysed oxidation. 3,4,4, was purchased from Aldrich, 2,2'-azinobis(3ethylbenzthiazoline-6-sulfonic) acid (ABTS) and 2,5-xylidine from Sigma and 2,6-dimethoxyphenol (2,6-DMP) from Acros. Coniferyl alcohol was synthesized according to the method of Ludley and Ralph (1996). Chemicals used as buffers were obtained from VWR (Prolabo, Rectapur grade) and solvents for HPLC and HPSEC from Carlo Erba (RS grade). The structures of the substrates of laccases used in this study are shown in Figure 3.

Materials and methods

Chemicals

Bacteria and yeast strains used

Escherichia coli DH5F' strain (Life Technology was used for the construction and routine propagation of vectors. Yarrowia lipolytica Po1g strain (Madzak et al., 2000) was used as the host for heterologous laccase expression. The isogenic Y. lipolytica Po1t prototrophic strain (Madzak, 2003) was used as a nonproducing control.

Plasmid design and site-directed mutagenesis

The plasmids used in this study are listed in Table I. The previously described laccase-expressing vector p69TVLB is designed to integrate at the pBR322 docking platform of the Y. lipolytica Po1g recipient strain (Jolivalt et al., 2005). This vector uses the native signal peptide to direct the secretion of active recombinant laccase IIIb into the culture medium (Jolivalt et al., 2005). Our purpose was to introduce into T. versicolor laccase ORF a point mutation leading to the desired amino acid change at position 206. The ORF codon corresponding to this position is located within a 65 bp region delimited by XmaI and ClaI restriction sites. Our strategy for directed mutagenesis of laccase IIIb was to replace this DNA fragment from p69TVLB vector by a synthetic DNA fragment carrying the desired point mutation. The presence of a second ClaI restriction site in p69TVLB vector (located at 1400 bp from the former, between XPR2 terminator and LEU2 selection marker gene) obliged us to perform first a partial digest of p69TVLB vector DNA using ClaI restriction enzyme. One of the products of this first step was a linearized p69TVLB vector DNA, which was purified on agarose gel. We performed on this DNA a second restriction, using XmaI enzyme. Among the products of this second step was an 8.8 kbp XmaI-ClaI DNA fragment corresponding to p69TVLB vector deleted from its 65 bp XmaI-ClaI region. This deleted vector was purified on agarose gel and ligated to one of three different synthetic sticky-ended (XmaI-ClaI) DNA fragments carrying the desired point mutation. Each point mutation was chosen so that the new codon fitted the peculiar Y. lipolytica codon bias. Each of the three synthetic fragments was obtained by hybridization of a couple of oligonucleotides (sequences of sticky ends in bold, codon 206 in bold Prior to yeast transformation, p69TVLB-D206A, -D206N and -D206E plasmids were linearized with NotI restriction endonuclease, in order to direct their integration at the pBR322 docking platform of the recipient strain (see review by Madzak et al., 2004). Transformation of Y. lipolytica Po1g strain with mutated laccase-expressing vectors was per-formed according to Xuan et al. (1988). The transformants were selected on YNB minimal medium plates, as described previously (Madzak et al., 2005). For each construction, several transformants were checked for correct integration of a single copy of the vector at the pBR322 docking plat-form, using polymerase chain reaction (PCR) analysis (data not shown) and one of each was selected for further analysis.

Cultivation conditions and laccase isolation

The transformants obtained from Po1g strain using p69TVLB-D206A, -D206N or -D206E plasmids were checked for active laccase production on agar plates supplemented with CuSO4 (0.1 mM) and ABTS (0.2 mM), as described previously (Jolivalt et al., 2005). Plates were incubated in the dark at 28°C for 3 days and checked for the development of a green colour.

For assaying laccase activity, transformants were cultivated on modified PPB liquid medium (glucose 20 g/l, yeast extract 1.32 g/l, NH4Cl 1.32 g/l, MgSO4•7H2O 0.24 g/l, CuSO4 0.025 g/l, thiamine 0.33 mg/l) under vigorous shaking (180 r.p.m.), in baffled 2 l Erlenmeyer flasks containing 200 ml of medium, at 28°C. Modified PPB medium was buffered using 50 mM phosphate buffer (pH 6.3). Inoculation was performed using one-day cultures to give an initial OD600 of 0.1. Yeasts were cultivated for 4-5 days.

After centrifugation (6000 r.p.m. for 10 min), culture supernatants were filtered through glass-fibre filters of decreasing porosity (1.6, 0.7 and 0.22 mM) and then concentrated in an ultrafiltration cell (Amicon) equipped with a YM30 membrane (Millipore). The concentrate was dialysed in the same cell against 50 mM citrate-phosphate buffer (pH 6.8). The 4 ml of enzyme solution (11 U/ml) was stored at -20°C with 30% (w/v) glycerol.

Laccase activity measurements using spectrophotometric methods

Standard assays for laccase activity were performed by measurement of enzymatic oxidation of ABTS at 420 nm (e = 3.6 x 10 4 cm -1 M -1 for the oxidation product). The reaction mixture contained 100 mL concentrated extracellular fluid (prepared as described above) and 900 mL ABTS (1 mM) in Na2HPO 4 /citric acid buffer (0.1 M, pH 3.0) at 30°C. The buffer solution was saturated with air by bubbling prior to the experiment. One unit of enzyme activity is defined as the amount of enzyme that oxidizes one mmole ABTS in one min. Kinetics analysis of the mutants were performed with ABTS 1 mM at pH 3 in citrate/phosphate buffer 0.1 M. pH activity dependence of the mutants was tested with both ABTS and 2,6-DMP. Experiments were performed in the conditions described above for ABTS or with 2 mM 2,6-DMP in Na2HPO4/citric acid buffer (0.1 M, pH 3.4). 2,6-DMP oxidation product was detected at 468 nm (e = 4.96 x 10 4 cm -1 M -1 ). Enzymatic transformation of xenobiotics followed by HPLC and HPSEC analysis 2,5-Xylidine. Triplicate samples of 2,5-xylidine (0.08 mM) in 40 mM citrate-phosphate buffer (pH 5) were incubated at 30oC using 1 unit of laccase activity. After a 24 h incubation, 20% (v/v) acetonitrile was added to stop the enzymatic reaction and the reaction mixture was centrifuged (6000 r.p.m.) for 10 min at 14°C. Analysis of the supernatant was performed on a Varian HPLC system equipped with a Model 9010 solvent delivery system, a Rheodyne Model 7125 injection valve with a 20 ml sample loop and a Model 9050 absorbance detector operating at 254 nm. A TSK gel (250• 4.6 mm i.d.) C18 column was used. The mobile phase, at a flow-rate of 1 ml min -1 , was composed of water (A) and acetonitrile (B), initially set at 15% A-85% B for 5 min, then ramped to 100% B in 13 min and maintained at 100% B for 7 min.

Coniferyl alcohol. Coniferyl alcohol transformation was assayed in the following reaction mixture: 0.6 ml of 25 mM citrate-phosphate buffer (pH 4.5) containing 0.67 mM coniferyl alcohol and 0.33 mM 3,4,5-TMB, used as an internal standard for the subsequent analytical determination of the reaction extent. The solution was aerated for 15 min. The reaction was initiated by adding 0.0015 units of laccase activity, as measured following the protocol for laccase activity measurements described above, and incubated at 30oC for 4 h. The reaction mixture was acidified with 0.05 ml of 0.1 M HCl to enhance phenol solubilization in organic solvents and extracted with 1.5 ml of dichloromethane on an ice-bath. The organic phase was dried with Na2SO4 before solvent evaporation and dissolution of the solid residue in 400 ml of stabilized tetrahydrofuran prior to high-performance size-exclusion chromatography (HPSEC) [START_REF] Baumberger | Proceedings of the 12 th International Symposium on Wood and Pulping Chemistry[END_REF]. Samples were ultrafiltered on a 0.45 mm PTFE filter (Millipore) before injection of an aliquot (5 ml) on a styrene-divinylbenzene copolymer gel column (PL-gel, Polymer Laboratories, mm, 100 A˚, 600 x 7.5 mm i.d.) with THF as eluent (1 ml min -1 ) and UV detection (280 nm). Calibration was performed using commercial polyethylene oxide standards (Igepal, Aldrich) and purified coniferyl alcohol dimers and tetramer. LC-MS analysis of the samples was performed after evaporation to dryness and dissolution in methanol with an ion trap mass spectrometer (Thermofinnigan LCQ Deca, Finnigan MAT, San Jose, CA) equipped with an electrospray interface set in the negative mode (sprayer needle voltage 4 kV, temperature of the heated capillary 350°C).

Results

Expression of the Asp206 mutants

Ligninolytic enzymes are generally difficult to express heterologously in active form (Jönsson et al., 1997). No active recombinant laccase expression has been reported yet in prokaryotic systems such as Escherichia coli, probably owing to the lack of post-traductional modifications such as glycosylation which are involved in the enzyme stabilization against proteolysis (Yoshitake et al., 1993). In the present work, the yeast Y. lipolytica was chosen as a host for laccase expression.

The plasmids used in this study (Table I) were obtained by inserting the wild-type or site-directed mutant sequence of T. versicolor laccase IIIb into the monocopy integrative pBR-based vector pINA1269, from the Y. lipolytica expression system, using the recombinant hp4d promoter (Madzak et al., 2000). As a host, Y. lipolytica is particularly suitable for the genetic engineering of heterologous genes. Indeed, pBRbased Y. lipolytica expression vectors offer (i) a very high transformation efficiency (105 transformants per mg) and (ii) precise targeting of monocopy integration events at the pBR322 docking platform of recipient cells, leading to transformants similar in terms of copy number and integration locus [for reviews, see Madzak (2003) and Madzak et al. (2004)].

The constructs were successfully expressed in Y. lipolytica. The mutant proteins were secreted in an active form, as checked by growing transformants on plates supplemented with ABTS which was oxidized into a green radical cation by excreted laccase activity (data not shown).

Owing to the targeted monocopy integration of the constructs, the effect of different site-directed mutations can be compared directly in culture supernatants, provided that yeasts are grown under similar conditions and that the laccase secretion level is the same for all of the mutants. In our experiments, transformants were grown simultaneously following the same protocol and we checked, using optical density (OD600) measurements of cultures, that growth variations between transformants did not exceed 10% (data not shown). In parallel, activity measurements were performed using ABTS as a substrate during the time course of the culture. No activity was detected during the first 48 h, which was consistent with the growth-phase dependence of hp4d promoter, which began to exert its full activity at the end of the growing phase (Nicaud et al., 2002). On the second day, significant levels of activity were detected for each of the transformants, but different levels were observed depending on the mutation, in the decreasing order Asp206Ala > Asp206Asn > Asp206Glu (Figure 4). The activity levels increased during the subsequent days while their respective order remained the same during the whole culture time course. The culture was stopped after 5 days. The laccase production level remained rather low (around 1 mg/l) and the quantity of enzyme available was not sufficient to undergo an extended purification process. The crude supernatant was only filtered, concentrated and dialysed against pH 6.8 phosphate buffer. This neutral pH was chosen to preserve catalytic activity.

Kinetic parameters determination

Kinetic parameters were determined using a spectrophotometric method for two substrates, ABTS at pH 3 and 2,6-DMP at pH 3.4, both in 0.1 M citrate-phosphate buffer. As the culture supernatants were only concentrated by ultrafiltration and dialysed without any further purification step, kcat values were calculated using the total protein concentration in the enzyme solution studied. This method leads to an under-estimation of the kcat values. However, the calculated kcat values obtained for ABTS as a substrate (Table II) led to the same ranking of the mutants as obtained for the time course profile of activity measurements in the different mutant culture supernatants (Figure 4). It can therefore be considered that, even if they are underestimated, the kcat values provide an accurate comparison of the relative mutant catalytic rates.

For ABTS as a reducing substrate, the data given in Table II clearly show that the Asp to Ala mutation induced the most significant changes in the enzymatic properties of laccase. Indeed, the substitution of Asp206 with Ala led to a 5-fold decrease in the Km value (10 mM for the mutant compared with 50 mM for wild-type enzyme). This mutation also resulted in a significant increase in the kcat value: 4.3 s -1 for the Ala mutant versus 1.3 s -1 for the wild-type recombinant laccase. The lowest change in the enzymatic properties was observed for the substitution with Glu: Km decreased 2-fold compared with the wild-type enzyme, whereas there was no significant change in the reaction rate. The Asn mutant exhibited intermediate values between wild-type laccase and Ala mutant for both kinetic parameters.

The kinetic parameters were determined for 2,6-DMP at pH 3.4, i.e. the pH with maximum activity for the recombinant wild-type laccase. Under such conditions, the efficiency of all the laccases tested was lower (by a factor of at least 10) for 2,6-DMP than for ABTS, mostly due to higher values of the Michaelis constant Km (Table II), whereas only marginal changes were observed for kcat values between the mutants and the recombinant wild-type laccase. Compared with its value for the wild-type laccase, a significant enhancement of Km was found for both Asp206Asn and Asp206Ala mutants. with 17-and 8fold increases, respectively.

Transformation of 2,5-xylidine, a non-phenolic substrate

After a rough purification of the culture supernatant consisting of centrifugation and dialysis against citrate-phosphate buffer (pH 6.8), followed by sample concentration, the extent of transformation of 2,5xylidine by wild-type and mutated lac-cases was measured. For each laccase, the amount of enzyme used in the experiment was normalized with respect to its activity towards ABTS. Experiments were performed at pH 5. Table III provides details of the 2,5-xylidine transformation efficiency for wild-type and mutated laccases. Both the native laccase expressed by T. versicolor and the recombinant wild-type laccase expressed by Y. lipolytica exhibited a similar trans-formation extent of around 55%. Experimental conditions were chosen to maximize the sensitivity of the experiment while allowing the comparison between all the mutants tested: the most efficient mutant, Asp206Glu, almost fully oxidized 2,5-xylidine (94%). 2,5-Xylidine is therefore almost 2-fold more efficiently transformed by the Asp206Glu mutant than by the wild-type enzyme. In contrast, replacement of an aspartic amino acid by a non-charged amino acid leads to a lowering of the oxidation rate of the substrate: the loss remains limited for the Asn mutant (45%, instead of 58% for the wild-type) whereas the Ala mutant is 2-fold less active than wild-type laccase. To summarize the results, the order of transformation rates is Asp206Glu > wild-type > Asp206Asn > Asp206Ala.

Transformation of coniferyl alcohol, a phenolic substrate Wild-type and mutated laccases were assayed for their efficiency in oxidizing coniferyl alcohol, which is a precursor in lignin formation (Brunow et al., 1998). Incubation conditions similar to these used for 2,5xylidine, but with a pH of 4.5, were used. The T. versicolor laccase expressed in Y. lipolytica exhibited the highest transformation rate compared with the mutants, whose reaction rates decreased in the order wildtype > Asp206Glu > Asp206Ala > Asp206Asn (Table III).

HPSEC analysis of the reaction mixture showed that incubation of the enzymes with coniferyl alcohol led in all cases to the formation of dimeric species (Figure 5) and LC-MS revealed that the oxidation products exhibited similar compositions. The main dimers were identified by LC-MS according to their retention time and mass spectra compared with purified standards. They consist in three dehydrodimers resulting from the radical coupling of oxidized coniferyl alcohol through pinoresinol -, phenylcoumaran -5 and arylglycerol -0-4 bonds (Brunow et al., 1998). Together with the dimers, traces of trimers showing molar masses of 538 were detected in allcases. They were tentatively assigned to a guaiacylglycerol-,bisconiferyl ether structure (De Angelis et al., 1999).

Effect of pH on laccase activity

Experiments conducted with ABTS showed that the pH-activity profile of the mutants was very similar to that observed for the recombinant wild-type laccase (Figure 6A). Assayed solutions were prepared from concentrated stock solutions in order to exhibit a similar activity at pH 3 (12-13 U/ml). The activity of all wild-type and mutated laccases decreased in a monotonic manner on varying the pH from 3 to 6 in citratephosphate buffer, with very similar profiles. Only slight differences could be noticed. In particular, Glu mutant activity remained systematically lower than all others until pH 5, whereas wild-type laccase exhibited the highest activity, especially at pH higher than 4.5 where the difference became significant.

Ala and Asn mutants showed very similar activity over the whole pH range studied.

The effect of the mutations appeared in a much more marked way when we examined the influence of pH on the oxidation of a phenolic substrate, 2,6-DMP. In that case, we observed a bell-shaped profile of the activity as a function of pH for all the laccases tested, whether mutated or not (Figure 6B). Both wild-type recombinant and Asp206Glu mutant laccases showed very similar profiles, with maximum activity at pH 3.4. The only observed slight difference was that, at pH above 3.4, the activity loss of the Glu mutant was more rapid than for the wild-type laccase. When laccase was mutated with an uncharged amino acid at position 206, a shift of the maximum activity towards higher pH values was observed. Compared with the wild-type laccase behaviour, the most affected mutant was that of Asn, which exhibited a shift of the maximum from 3.4 to 4.8, whereas the Ala mutant showed maximum activity at an intermediate pH value of ~4.

Discussion

The catalytic properties of laccases mutated in position 206 were assayed for their transformation efficiency towards different substrates and for the nature of the transformed products of a ligninolytic compound. The laccase sequence alignment emphasized that in fungal laccases an Asp or Glu residue is found in position 206 whereas Asn is highly conserved in this position in plant laccases. One of the objectives of this work was to test whether this difference could determine the bio-logical function of the enzyme. Indeed, fungal laccases are involved in lignin depolymerization whereas plant laccases would rather participate in the early stages of lignin polymerization. However, preliminary tests with synthetic dimers or tetramers did not show any significant degradation with T. versicolor laccase. It is worth mentioning that in vitro assays with fungal laccases give contrasting results (Solomon et al., 1996). As far as the opposite reaction, e.g. the polymerization stage, is concerned, coniferyl alcohol was chosen as a model precursor of lignin in order to test the efficiency of both wild-type and mutated laccases towards the lignin formation process. Accordingly, all the laccases investigated here led to the same dimers and trimers in similar proportions. The only observed difference between the variants studied concerns the conversion rate of the phenolic substrate, which is lowered by the mutations. These results were obtained under conditions very different from natural ones (in the presence of lignin and in synergy with other fungal enzymes and mediators), which do not allow any extrapolation on the in vivo biological function of fungus or plant laccases.

From a more quantitative point of view, the study of the catalytic properties of Asn206-mutated laccases with ABTS as a substrate clearly showed that the replacement of an acidic residue by a non-polar Ala significantly enhanced the efficiency (kcat/Km) of the enzyme by a factor of 15. Enguita et al. (2004) reported the crystallographic structure of an endospore coat laccase from Bacillus subtilis in the presence of ABTS. Because of the low occupation factor of the ABTS atoms, the substrate binding site could not be precisely described. However, it was suggested that ABTS binds in a large pocket mainly formed by apolar residues. In such an environment, where interactions between aromatic groups dominate, it is therefore consistent to note that the substitution of Asp206 leads to a catalytic efficiency increase inversely proportional to the substituted residue polarity. This increase in efficiency is due to both an increase in kcat and to a decrease in Km, indicating a concomitant increase in the affinity for the substrate and in the kinetics of electron transfer, all the more marked as the amino acid in position 206 is less polar.

Further, the contribution of the 206 residue to the pH-activity profiles seems to be very weak since the curves corresponding to the different mutants are very similar. We would nevertheless expect that the replacement of an amino acid carrying a negatively charged lateral chain at a pH above its pKa (~5 for carboxylic acids) could contribute to reducing the unfavourable electrostatic interactions with a negatively charged substrate (due to the presence of two sulfonate groups in ABTS). How-ever, pH variations could not evidence such an interaction.

The study of the pH dependence of the activity profile of the mutants with 2,6-DMP as a substrate indicated that the nature of the amino acid at position 206 significantly impacts the value of the observed maximum (pHopt). For the Asn mutant, a pHopt shift towards higher pH (of 1.4 units) for the oxidation of 2,6-DMP was observed. For all mutants studied, the activity curve exhibits a bell-shaped profile, as observed previously for phenolic substrates. Xu (1997) postulated that this bell-shaped profile is a consequence of two opposite effects: the ascending part of the curve at acidic pH is generated by the redox potential difference between the reducing substrate and the type 1 copper of laccase and is favoured, for a phenolic substrate, by higher pH. The descending part is generated by the binding of a hydroxide anion to the type 2/type 3 coppers of laccase, which inhibits the activity at higher pH, combined with a decrease in the oxygen reduction potential with increase in pH. The resulting bell-shaped profile of the pH dependence of laccase activity is almost identical for both non-mutated laccase and Asp206Glu mutant, showing that the replacement of the aspartic residue by another carboxylic acid has a negligible influence on the enzyme catalytic properties. However, a significant shift of the maximum activity towards higher pH is observed for the neutral mutants, the shift being greater for Asp206Asn than for Asp206Ala. Interestingly, a pH shift similar to that observed for Asp206Asn was also obtained with a recombinant laccase carrying a double mutation (Asp206Asn and Ser212Cys; data not shown), which rein-forces the causal link between this shift and the Asp206Asn mutation. Assuming that the descending part of the bell-shaped pH profile of the pH dependence of activity results from the inhibition of laccase activity b hydroxide binding at the type 2/type 3 site and that this interaction is not affected by the mutations at the 206 position because of its distance from the mutation site, the observed pHopt shift must be attributed to a corresponding shift of the ascending part of the curve. According to Xu's conclusions, a shift towards more acidic pH can be induced by a favoured deprotonation of the reducing substrate. As the pKa of 2,6-DMP is 9.9, deprotonation of 2,6-DMP does not occur spontaneously at acidic pH. However, the vicinity of a carboxylic residue interacting with the reducing substrate in the active site, such as an Asp or a Glu residue at the 206 position, is likely to favour its deprotonation. Such a protic event cannot take place when mutating the Asp 206 residue into Ala or Asn, as confirmed by the weak docking of 2,6-DMP with these neutral mutants, compared with Asp 206 and Asp206Glu, as deduced from their respective Km values (Table II). For coniferyl alcohol, also a phenolic substrate, the transformation rates at pH 4.5 follow the order As Glu > Ala > Asn, which is similar to that observed in the ascending part of the pH activity profile of 2,6-DMP, suggesting that the same behaviour could b generalized to all phenolic substrates.

For 2,5-xylidine, a slightly different transformation efficiency order was observed: Glu > Asp > Asn > Ala. In this case, we suggest that the stabilization of the radical formed after electron transfer to the type 1 copper could account for this result. Indeed, the carboxylate lateral chain at position 206 is likely to provide an electron-rich environment. The amide group of Asn could also provide a stabilization of the radical due to the presence of a negative charge on oxygen atom in its mesomeric form, but is less favourable owing to the concomitant presence of the positive charge on the nitrogen, whereas the Ala mutant lacks electrons able to enhance radical stabilization.

Conclusion

The present work emphasizes the influence of the residue at position 206 on the catalytic properties of fungal laccases. Substrates with different structures and functional groups were tested. For all the substrates, mutation-induced modification of their transformation rate with ABTS remains within the same range, around 3-fold. Ranking the wild-type and the mutated laccases according to their transformation efficiencies depends on the substrate and the results could be explained on the basis of interactions between the substrate and the mutated residue at the catalytic site of the enzyme. The influence of the mutations on the pH-activity profile of laccase with 2,6-DMP showed that the Asn mutation led to a significant shift (pH = 1.4) of the optimum towards higher pH. The same tendency is expected for other phenolic substrates such as pesticide derivatives and nonylphenol, which are known to be environmental pollutants and so could contribute to an optimized degradation of these molecules in situ in soils at neutral pH.

Table I. Laccase-expressing integrative vectors used for studying the effects of different mutations in T.

versicolor laccase codon 206 Table II. Kinetic parameters of the T. versicolor laccase expressed in Y. lipolytica (YL4) and of the mutants at the 206 position a a Assays were performed with ABTS and with 2,6-DMP as substrates in 0.1 M phosphate buffer, pH 3 and 3.4, respectively. kcat was calculated versus the total protein concentration in solution.

  underlined): (i) for p69TVLB-D206A plasmid, 50P-Ph-CCG GGT AAA CGC TAC CGT TTC CGC CTG GTG TCC CTG TCG TGC GCC CCC AAC TAC ACG TTC AGC AT-30 and 50-Ph-C GAT GCT GAA CGT GTA GTT GGG GGC GCA CGA CAG GGA CAC CAG GCG GAA ACG GTA GCG TTT AC-30; (ii) for p69TVLB-D206N plasmid, 50-Ph-CCG GGT AAA CGC TAC CGT TTC CGC CTG GTG TCC CTG TCG TGC AAC CCC AAC TAC ACG TTC AGC AT-30 and 50-Ph-C GAT GCT GAA CGT GTA GTT GGG GTT GCA CGA CAG GGA CAC CAG GCG GAA ACG GTA GCG TTT AC; (iii) for p69TVLB-D206E plasmid, 50-Ph-CCG GGT AAA CGC TAC CGT TTC CGC CTG GTG TCC CTG TCG TGC GAG CCC AAC TAC ACG TTC AGC AT-30 and 50-Ph-C GAT GCT GAA CGT GTA GTT GGG CTC GCA CGA CAG GGA CAC CAG GCG GAA ACG GTA GCG TTT AC-30. These constructions were checked by sequencing (Genaxis).

Fig. 1 .

 1 Fig. 1. Orthogonal views of the binding site for xylidine. Left: view from the entrance of the cavity. Right: orthogonal view obtained by a 90o rotation around a vertical axis. The enzyme residues and the substrate are shown in ball-and-stick form and the copper from the type 1 site (Cu1) as a sphere. Carbon atoms are grey, nitrogen atoms light grey and oxygen atoms dark grey. The 2,5-xylidine substrate (Xyl) backbone is black. The surface of those residues that contribute to hydrophobic interactions with the substrate is shown. Hydrogen bonds with the nitrogen atom from xylidine are shown by dotted lines and the coordination of Cu1 with His458 by a black line. The figure was drawn with PyMOL (DeLano, 2002).

Fig. 3 .

 3 Fig. 3. Structures of laccase substrates used in this work.

Fig. 5 .

 5 Fig. 5. Chromatographic analysis of coniferyl alcohol transformation by wild-type and mutated laccases expressed in Y. lipolytica. YL4, wild-type recombinant laccase expressed in Y. lipolytica.

Fig. 6 .

 6 Fig. 6. Activity of wild-type and mutated laccases produced in Y. lipolytica as a function of pH, with ABTS (A) or 2,6-DMP (B) as substrates. Open diamond, wild-type recombinant laccase expressed in Y. lipolytica (YL4); filled diamond, Asp206Ala; filled square, Asp206Asn; filled triangle, Asp206Glu.

Acknowledgements

We wish to thank B. Pollet for her technical assistance with LC-MS analysis of the coniferyl alcohol oxidation products. We are grateful to Dr J.-M. Beckerich for his continuing interest in this work.


Davide Fabiani

Gian Carlo Montanari

Leonard A Dissado

Christian Laurent

Gilbert Teyssedre

Peter H F Morshuis

Riccardo Bodega

Alfred Campus

Ulf H Nilsson

Effect of semicon-insulation interface on space charge formation in HVDC polymeric cables

Keywords: HVDC, polyethylene, semiconducting material, insulating material, charge injection, space charge accumulation threshold

come   L'archive ouverte pluridisciplinaire

INTRODUCTION

Much effort has been spent to expand the use of polymeric cables (usually limited to HVAC) to HVDC power links [START_REF] Campus | Material solutions for extruded HVDC Cables[END_REF]. This would provide tangible environmental and economic benefits with respect to mass-impregnated cables traditionally used in HVDC applications. Polymeric cables, however, seem to be greatly affected by space charge build-up, particularly when DC voltage is applied [START_REF] Zhang | Evidence of strong correlation between space charge buildup and breakdown in cable insulation[END_REF][START_REF] Dissado | The role of trapped space charges in the electrical ageing of insulation materials[END_REF][START_REF] Montanari | The electrical degradation threshold of polyethylene investigated by space charge and conduction current measurements[END_REF]. Space charge accumulation is considered, indeed, to be one of the main factors responsible for ageing acceleration in cable insulation, due to local electric field enhancement produced by accumulated charge, particularly in the presence of voltage polarity inversions.

Space charge build up is a complex mechanism that is yet to be fully explained. Generally, if the electric field is larger than a given threshold, i.e. the threshold for space charge accumulation, charge injection from the electrodes, promoted by field and temperature, may prevail over charge extraction/recombination [START_REF] Montanari | The electrical degradation threshold of polyethylene investigated by space charge and conduction current measurements[END_REF]. This causes accumulation of space charge in traps located in the insulation bulk that hold the excess charge injected by the electrodes for times depending on trap depth which, in turn, may be associated with chemical/morphological structure of the insulating material. It has been observed that the amount of charge injection and accumulation may depend significantly on the contact between electrode and insulation. In particular, since a semiconductive layer is interposed between metal electrodes and insulation of a polymeric cable, the interface between semicon and insulation is thought to have a significant effect on space charge build-up in insulation. Experimental tests were performed on mini-cable specimens having different combinations of insulation (INS1, INS2) and semiconductive materials (SC1, SC2), with the aim of investigating the way the semicon-insulation interface affects space charge accumulation. In particular, space charge measurements were carried out on these specimens at different electric field and temperature values, to obtain the characteristics of charge accumulation as a function of field and temperature.

TEST PROCEDURES

In order to measure space charge accumulation characteristics for each insulation system, space charge measurements were performed by means of the Pulsed Electro Acoustic (PEA) technique on mini-cable and plaque specimens [START_REF] Montanari | Investigating ac space charge accumulation in polymers by PEA measurements[END_REF]. Before testing, a thermal treatment was applied to cable models and plaques for 5 days at 80°C and for 3 days at 70°C, respectively, with the aim of expelling in principle all crosslinking by-products [START_REF] Nilsson | The use of model cables for evaluation of dc properties of polymeric cable materials[END_REF].

Two kinds of specimens, made by insulating/semiconducting materials were considered for testing:

1) Press-moulded plaques, consisting of a two-layer sandwiches, one insulating layer, 0.5 mm thick, and one semiconducting layer, 0.5 mm thick. 2) Cable models, reproducing HV cables on a reduced-scale (mini-cables), consisting of three layers: inner semicon (0.7 mm thick), insulation layer (1.5mm thick) and outer semicon (0.15 mm thick). The conductor diameter is 2.8 mm (Fig. 1). Space charge tests were performed by poling both kinds of specimens at electric fields ranging from 2 to 40 kV/mm (average field) for 10000s and at different temperatures from 25°C to 70°C, under isothermal conditions, i.e., through heating uniformly the specimens inside an oven. Isothermal conditions were needed in order to avoid the formation of a temperature gradient across cable insulation, which may have an effect on space charge accumulation [START_REF] Fabiani | The Effect of Temperature Gradient on Space Charge and Electric Field Distribution of HVDC Cable Models[END_REF]. Depolarisation for at least 3600 s followed after polarisation. A quantity associated with the space charge accumulation in the insulation bulk is the medium absolute charge density, Qmed, which can be calculated directly from the space charge profiles by means of following equation [START_REF] Montanari | The electrical degradation threshold of polyethylene investigated by space charge and conduction current measurements[END_REF]:

   2 1 ) ( 1 1 2 x x med dx x x x Q  (1)
where ρ(x) is the space charge density detected at the beginning of the depolarisation phase (conventionally, after 2s from voltage removal), x1 and x2 are the electrode radial positions.

EXPERIMENTAL RESULTS

Space charge patterns reported in Figs. 2-5 represent a 3dimensional plot where the extent of charge amount, as a function of time, is characterized by a colour scale (positive charge = warm colours and negative charge = cold colours). The white colour stands for saturation of colour scale (> 10 C/m 3 ), thus indicating an out-of-scale amount of space charge (as can occur, for example, for electrode charge). Anode and cathode are the inner and the outer semicon electrodes, respectively, while the horizontal and vertical axis represent time and position in the insulation bulk.

Figure 2 shows the space charge patterns relevant to tests at average poling field of 13 kV/mm on INS1-SC1 minicables at 25 °C (Fig. 2A) and 70°C (Fig. 2B). As can be seen, a small amount of positive charge can be observed in the insulation bulk at room temperature (Fig. 2A), because the average field is not too far from the threshold for space charge accumulation (7 kV/mm). As temperature increases, the accumulated charge density rises and, moreover, an homocharge distribution close to the cathode builds up (Fig. 2B). Increasing the average field to 40 kV/mm, space charge patterns on INS1-SC1 mini-cables show more and more accumulated charge (Fig. 3). At room temperature (Fig. 3A) negative fast charge packets, i.e., injected charge travelling from the cathode to the anode, are observable in the insulation bulk, eventually accumulating as heterocharge close to the anode. At the same time, positive charge packets bring charge accumulation near the cathode. At 70°C (Fig. 3B), a more evident distribution of heterocharge close to both electrodes can be seen already from the start of the polarisation phase. As for mini-cables, Figs. 4 and5 show that the extent of space charge accumulation in the insulation bulk increases on rising electric field and/or temperature. At room temperature homocharge can be seen close to both electrodes in steady state. At 70°C, heterocharge accumulates close to the cathode and homocharge near the anode. The next section will try to provide an explanation of the behaviour regarding space charge accumulation showed by mini-cables and plaques.

Similar observations can be made from space charge patterns of different combinations of semicon/insulating materials. For the sake of brevity, these patterns, reported elsewhere [START_REF] Fabiani | Polymeric HVDC cable design and space charge accumulation. Part 1: Effect of semicon/insulation interface[END_REF], are omitted in this paper. However, a summary of all the tests performed on all the combinations of insulation systems is provided by the values of medium charge density, Qmed, calculated by eq.

(1) and plotted in Fig. 6 as a function of average geometric electric field, obtaining the so-called threshold characteristics.

It can be noted that at 25°C INS2-SC1 shows the smallest amount of charge, i.e., the largest threshold, followed by INS1-SC1, INS2-SC2 and INS1-SC2, which seem to have similar characteristics at room temperature (Fig. 6A).

An increased space charge accumulation can be observed at 70°C (Fig. 6B) for all the insulation systems. This time, INS2-SC2 is the insulation system accumulating the lower amount of charge, followed by INS1-SC2, INS1-SC1 and finally INS2-SC1.

It is noteworthy that, as expected, the threshold for space charge accumulation (indicated by arrows in Fig. 6) diminishes as temperature increases owing to injection of excess charge from electrodes favoured by temperature increase. In particular, the values of threshold are halved for all the tested combinations passing from 25°C to 70 °C. It should be pointed out, moreover, that the largest thresholds at 25°C and 70°C is shown by INS2-SC1 (8 kV/mm, average field) and INS2-SC2 (4 kV/mm, average field), respectively. From these results, specimens constituted by SC2 seem to accumulate less charge than SC1 at high temperature, while the opposite occurs at room temperature. The role of the semiconducting layer regarding charge accumulation as a function of temperature, thus, needs to be better analyzed. This will be done in the next Section.

DISCUSSION

First of all, we discuss the heterocharge build-up seen in mini-cable specimens at both electrodes and in plaques at the cathode. Evidence of relatively fast charge packets traveling in the insulation bulk can be observed, particularly at high fields (see, e.g., Fig. 3). Since mobility of this charge increases significantly with temperature, charge packets can be detected precisely only by employing an ultra-fast acquisition system (see [START_REF] Delpino | Fast charge packet dynamics in XLPE insulated cable models[END_REF]). The presence of fast charge packets gives rise to heterocharge formation close to the electrodes, just a few instants after voltage application. The charge injected from one semicon electrode, in fact, travels through the insulation bulk and remains trapped at the other semicon interface, acting as a partially-blocking electrode. This could explain why heterocharge accumulates symmetrically at both electrodes in minicables, but not in plaques, where heterocharge is observed only at the cathode. In mini-cables, in fact, both electrodes have a semicon interface, while plaques have only one semicon layer close to the cathode. It should be emphasized that the heterocharge extent increases with temperature, as expected, since temperature gives rise to injection and, thus, to charge packets accumulating close to the opposite electrode (see Fig. 3).

Referring to space charge amount and comparing space charge characteristics of the different combinations of semicon/insulation reported in Fig. 6, it could be concluded that the best insulation system at room temperature (Fig. 6A) might be that involving INS2 and SC1, since the combination INS2-SC1 shows very good space charge features (i.e., high threshold field, 8 kV/mm, and small trapped charge as the applied field exceeds threshold). The worst combination, on the contrary, is INS1-SC2, showing a significantly larger amount of charge and a smaller threshold than INS2-SC1.

As temperature increases (see Fig. 6B), semicon materials seem to behave differently. The best combination in terms of accumulated space charge contains always INS2 material, but SC2 in place of SC1. The second best combination involves again SC2 (INS1-SC2), while the largest amount of accumulated space charge can be associated with SC1 (INS2-SC1).

Summarizing, experimental results showed that even if the insulating material INS2 accumulates less charge than INS1, at high temperature the nature of the semiconducting material seems to govern space charge accumulation. All the combinations including SC1 and SC2 show, in fact, the larger and the smaller charge amount, respectively, independently of the insulating material. This could be explained considering that semiconducting materials may show a different temperature dependence regarding charge injection. Material SC1, in fact, is more easily injecting than SC2 at high temperatures, while the opposite occurs at room temperature. Since the behaviour of SC2 seems to depend less on temperature (in terms of space charge accumulation) HVDC cables designed with the combination INS2-SC2 would be subjected to an electric field profile less dependent on temperature, thereby improving cable reliability.

CONCLUSIONS

The experimental results here reported show that the nature of semiconducting material have an important role on space charge accumulation in the insulation bulk, affecting injection and extraction of charges at the electrode/semicon/insulation interface. These results can be successfully exploited for HVDC polymeric cable manufacturing. Comparing different semicon-insulation candidates, in fact, space charge measurements can provide fundamental information for improving cable design by choosing the best combination of semicon-insulation, i.e., the one showing the smallest amount of charge combined with the mildest dependence of accumulated charge on temperature, as well as for optimizing cable manufacturing by developing improved technological processes.

Figures 4

 4 Figures 4 and 5 report results obtained on flat specimens (plaques) of INS1-SC1, at 20 and 40 kV/mm, respectively, for temperature levels, 25 °C (A) and 70°C (B). The semiconductive layer acts as cathode while an Al foil is used as anode.

Figure 2 :

 2 Figure 2: Space charge patterns on INS1-SC1 minicables at 13kV/mm (average field). Test temperature: 25°C (A) and 70°C (B).

Figure 3 :

 3 Figure 3: Space charge patterns on INS1-SC1 minicables at 40kV/mm (average field). Test temperature: 25°C (A) and 70°C (B).

  Cite as: D Fabiani, G.C.

Figure 4 :

 4 Figure 4: Space charge patterns on INS1-SC1 plaques at uniform field of 20kV/mm. Test temperature: 25°C (A) and 70°C (B).

Figure 5 :

 5 Figure 5: Space charge patterns on INS1-SC1 plaques at uniform field of 40kV/mm. Test temperature: 25°C (A) and 70°C (B).

Figure 6 :

 6 Figure 6: Space charge characteristics for mini-cables at 25°C (A) and 70 °C (B). The thresholds for space charge accumulation are indicated by arrows.

Figure 1: Mini-cable specimen. Conductor diameter = 2.8 mm; Inner semicon = 0.7 mm; Insulation thickness = 1.5 mm; Outer semicon = 0.15 mm.

	Two different XLPE-based insulating materials (INS1 and
	INS2) and semiconducting materials (SC1 and SC2) were
	evaluated, giving rise to insulation systems with different
	permutations of semicon/insulating materials. In particular,
	the combinations INS1-SC1, INS2-SC1, INS2-SC2 for the
	plaques and INS1-SC1, INS2-SC1, INS1-SC2, INS2-SC2
	for the mini-cables were used for experimental testing.
			Stranded copper
			conductor
			Inner semicon
			Insulation
			Outer semicon
	50 cm	10 cm	50 cm

Cite as: D Fabiani, G.C.

Montanari

, L.A. Dissado, C. Laurent, G. Teyssedre, P.H.F. Morshuis, R. Bodega, A. Campus, U.H. Nilsson, "Effect of semicon-insulation interface on space charge formation in HVDC polymeric cables", 7 th Int. Conf. on Insulated Power Cables (JiCable), Versailles, 24-28 June 2007. Proc. Jicable'07, pp. 470-474, 2007.

Acknowledgments

This research was performed within the European Project (of the 5 th Framework-program) HVDC, "Benefits of HVDC Links in the European Power Electrical System and Improved HVDC Technology".


Francesco Gullo

T Christen

Christina Villeneuve-Faure

H Hillborg

Christian Laurent

Séverine Le Roy

Gilbert Teyssedre

Dependence of the field and charge distribution at semicon/polyethylene interface on the press-molding process derived from Kelvin Probe Force Microscopy

I. INTRODUCTION

Electronic properties of interfaces between conducting/semiconducting electrodes and a dielectric layer can strongly influence charge transfer [START_REF] Jones | The Contribution of Field-Induced Morphological Change to the Electrical Aging and Breakdown of Polyethylene[END_REF][START_REF] Mizutani | Barrier heights and surface states of metal-polymer (PET) contacts[END_REF][START_REF] Christen | HVDC Insulation Boundary Conditions for Modeling and Simulation[END_REF]. The theoretical contact potential barrier (metal work function minus dielectric electron affinity) cannot explain the measurements [START_REF] Taleb | Modeling of Charge Injection and Extraction in a Metal/Polymer Interface through an Exponential Distribution of Surface States[END_REF], and it has been proposed that the interfacial electronic properties control the charge transfer. Recent approaches focus on ab initio modeling in order to unravel the nature of these states in model situations [START_REF] Huzayyin | Quantum mechanical study of charge injection at the interface of polyethylene and platinum[END_REF][START_REF] Chen | First-principles study of aluminum-polyethylene interfaces[END_REF]. The discrepancy between theoretical potential barriers and those obtained through charge transport modeling with fluid models [START_REF] Roy | Description of charge transport in polyethylene using a fluid model with a constant mobility: fitting model and experiments[END_REF] is another evidence that the description of interfacial properties is not satisfactory. In spite of this, very few studies aimed at investigating experimentally the interfacial electronic properties in situations of interest for electrical engineering design. Owing to the increasing development of HVDC technologies, especially polyethylene insulated power cables for energy transport, we focus on carbon black (CB) filled host polymer (SC) / PE interfaces.

We recently reported on chemical, structural and dielectric nanoscale properties of such interfaces [START_REF] Gullo | Nano-scale characterization of dielectric-semicon contacts[END_REF][START_REF] Gullo | Impact of press-molding process on chemical, structural and dielectric properties of insulating polymers[END_REF]. The bare PE was studied by Photoluminescence (PL), Infrared Spectroscopy (IR) and Atomic Force Microscopy (AFM) to characterize the morphology and the chemical properties of the films. Macroscopic electrical properties were also investigated by charging/discharging current and space charge measurements.

It appears that when using polyethylene terephthalate (PET) as protective cover layer during press-molding process, extra signatures appear in PL and IR spectra related to diffusion of oxidized groupspresumably decomposition products from PET into the PE surface. Also changes in surface roughness appear, depending on the nature of the cover layer, PET or Al. However, the different press-molding conditions did not reveal substantial changes in the formation of space charge, and the nature of electrode remains the most influencing factor for space charge and conduction features. In this work we show that press-molding conditions have an impact on the charges near the SC-PE contact.

II. EXPERIMENTAL

A. Peak Force -Quantitative Nano Mechanics and Kelvin Probe Force Microscopy

The interfacial properties at SC/PE interfaces were investigated by Peak Force Amplitude Modulation Kelvin Probe Force Microscopy (PF-AM-KPFM) using the AFM Multimode 8 from Bruker which combines two different AFM modes: Peak-Force Quantitative NanoMechanical (PF-QNM) which probes the topography and mechanical properties (adhesion, deformation, Young modulus) [START_REF] Young | The use of the PeakForce quantitative nanomechanical mapping AFM-based method for high-resolution Young's modulus measurement of polymers[END_REF] and Kelvin Probe Force Microscopy (KPFM) which probes the surface potential [START_REF] Zerweck | Accuracy and resolution limits of Kelvin probe force microscopy[END_REF]. A PtIr coated Si tip and a lift of 40 nm were used.

B. Sample preparation

The sample preparation procedure is described in [START_REF] Gullo | Nano-scale characterization of dielectric-semicon contacts[END_REF]. We used a nonstabilized low-density polyethylene from ExxonMobil™ (LDPE LD 101BA). The SC was produced by mixing acetylene black nanoparticles to the PE compressed into granules. These materials are used to realize a three layered sandwich-like structure SC-PE-SC with a thickness of 150 m. Each layer is 50 m thick and produced in a hot-press by heating at 140°C. To avoid the films sticking to the metallic surface of the mold, we introduced a cover layer of PET or Al. The three layers are subsequently heated together to 110°C under a small pressure to ensure an intimate contact. Then, cross sections of 200 nm in thickness are taken from the sandwich structure using ultra-cryo-microtomy. The semicon is grounded during KFM measurements.

III. RESULTS AND DISCUSSION

A. PFQNM and KPFM profiles

Fig. 1 gives surface potential profiles for the sandwich whose PE was processed using an Al protecting-layer. The magnitude of the surface potential increases slowly from SC/PE interface to bulk PE to reach a maximum value. Strong variation of surface potential maximum from one cross-section to another one acquired on the same sandwich is observed. However, the variation range is more important for the cross-section processed with PET protecting layer when compared to Al. This can be due to local variation in surface chemistry (cf. FTIR and PL results reported in [START_REF] Gullo | Impact of press-molding process on chemical, structural and dielectric properties of insulating polymers[END_REF]), inducing differences between crosssections coming from different sandwich areas.

Owing to the difference between PE and SC, we would expect a sharp transition of the surface potential at interfaces instead of this "bell-like" shape. This smooth variation of surface potential may have different origins. First, a smooth transition could be the consequence of KPFM spatial resolution resulting from parasitic capacitance between AFM probe cantilever and sample [START_REF] Jacobs | Resolution and contrast in Kelvin probe force microscopy[END_REF].

To identify the impact of this factor in our experimental conditions, the surface potential profile was measured over a reference sample with Au and Al electrode deposited over silicon nitride [START_REF] Villeneuve-Faure | Interface Properties in Dielectrics: a Crosssection Analysis by Atomic Force Microscopy[END_REF]. In this structure, the surface potential profile presents a sharp transition (2 µm width) at Au/Si and Si/Al interface and the difference of Al and Au surface potential (1.1eV) corresponds to the difference between their work functions. This shows that even if the surface potential absolute value is unattainable under ambient conditions, relative measurements are possible. The origin of the smooth transition observed in our sample is therefore not due to the limitation in spatial resolution of the KPFM. Such smooth interface was already observed in P-N junction [START_REF] Chen | Insight into interfaces and junction of polycrystalline silicon solar cells by kelvin probe force microscopy[END_REF]. In this case non-abrupt interface was attributed either to doping (i.e. electric charges) [START_REF] Robin | Investigation of the cleaved surface of a PIN laser using Kelvin probe force microscopy and two-dimensional physical simulations[END_REF] or material diffusion from one layer to another [START_REF] Kryvchenkova | Nondestructive method for mapping metal contact diffusion in InO3 thin film transistors[END_REF]. In our case, we can exclude diffusion of carbon black particles in the SC/PE interface as shown by the adhesion contrast in Fig. 1(a). This suggests that other sources of charges are involved in this phenomenon. A methodology to extract charge profiles from surface potential measurements has been developed to confirm this analysis.

B. Interface charge quantification

To determine the charge density ρ from the surface potential, we need to assume that it reproduces the bulk potential. The validity of this hypothesis was demonstrated qualitatively by Emmerich et al. [START_REF] Emmerich | Real-space measurement of potential distribution in PECVD ONO electrets by KPFM[END_REF] when charges are uniformly distributed in the volume. The charge density distribution can be obtained from the Maxwell-Gauss law:

) ( 0 P E D     
where D is the electric displacement, 0 the vacuum dielectric constant, E the electric field, P the polarization.

This equation can be rewritten, inserting the potential, as:

∇ 2 V = -(free + pol) /0
where free and pol stands for free and polarization charge respectively.

We intend to apply this second derivative to the KPFM surface potential map which consists of a matrix of numbers which represent the value of the surface potential in different points. To calculate the first and second differential order of the potential map shown in Fig. 1b, Savitzky-Golay smoothing method in two dimensions was implemented [START_REF] Savitzky | Smoothing and differentiation of data by simplified least squares procedures[END_REF] using a third order polynomial to fit experimental data. Due to the symmetry of the sample and in order to simplify the calculation, the derivative has been calculated only across the perpendicular direction of the interfaces. Results in terms of charge density profile are presented in Fig. 2(c). Close to each interface a negative charge density cloud (free and polarization charges) is present in PE with corresponding positive image charges in the SC.

Cite as: F. Gullo, T. Christen

C. Process influence on charge distribution

Fig. 2 depicts the electric field and charge density profiles for PET and Al as cover layers. In both cases, negative charge accumulation is seen at the PE/SC interface with its positive image charge in SC. Whatever the process, negative or positive charge clouds spread over around 2-3 µm in PE. However, the maximum density is strongly variable depending on the process.

D. Polarization influence on the charge distribution

An external electric field was applied on PE by keeping the SC electrodes on different potentials. Two kinds of configuration are tested. In one case, the left electrode was on 2V, while the right electrode was on -2V (fig. 3(a)). In the other case the polarity was reversed (fig. 3(d)).

Fig. 3 shows the surface potential profile for the two cases. From the calculated charge density profile one concludes that negative charges are located close to the cathode and positive charges close to the anode. In both cases of PET/Al cover layers, charges appear within a few microns from the interface. For the Al layer a wellreproducible negative charge is observed, while for PET both polarities are detected with variation from sample to sample. This might be due to the presence of chemical species transferred to the PE surface during press molding, as revealed by infra-red and photoluminescence spectroscopy [START_REF] Gullo | Impact of press-molding process on chemical, structural and dielectric properties of insulating polymers[END_REF]. The contact charges are not affected by applying a field of a few hundred V/mm when measured after polarization.

E. Discussion

All the samples we have tested exhibit the same sharp contrast of the adhesion force between the PE matrix and the SC. It has also been found [START_REF] Gullo | Nano-scale characterization of dielectric-semicon contacts[END_REF] that the PE films processed with Al protecting layers exhibit higher interface roughness than the one processed with PET, consistently with our initial surface roughness conditions established before sandwich processing. No diffusion of CB from the SC into the LDPE has been observed. Comparing adhesion and surface potential maps shows that, even if the physical interface is abrupt, the surface potential decreases comparatively slowly going from SC to PE. The potential variation is over a range of around 15 µm and has a bell-shaped form for all types of protecting layer. The surface potential maximum varies from one cross-section to another one in the same sample, particularly for the sample prepared using PET as protecting layer. This can be due to surface chemistry changes which could be non-homogenous over the surface.

Whatever the process, negative or positive charge clouds (depending on cover layers during the molding process) spread over around 2-3 µm in LDPE. The maximum density is strongly impacted by the fabrication process changing from -7 C/m 3 to 0.7 C/m 3 for the PET layer, or, in terms of surface charge density, from -8 to 0.9 µC/m². This value is much lower than the value obtained theoretically for the contact charge density as estimated by Density Functional Theory in model situation [START_REF] Roy | Coupling molecular modeling, fluid modeling, and KPFM measurements to characterize an interface at the nanoscale[END_REF]. This is probably due to KPFM lateral resolution which is for the best equal to 10 nm and usually around to 100 nm in atmospherically condition.

The charge density located close to interfaces is higher for PE processed with PET than with Al. This could be linked to the presence of oxidized species diffusing from the PET cover layer. However, there is a strong variation in the amount and sign of charge in the case of the PET cover layer depending on process conditions. It is not clear whether this behavior is a consequence of the interaction of the PE matter flow with PET surface during processing.

Finally, it has been found that the surface potential profile is influenced by the lateral bias applied to the SC electrode. Obviously, in this way, the initial surface potential is superposed to the linear one induced by the bias applied on the SC. This internal electric field induced by applying the dc voltage appears proportional to the applied voltage and there is no apparent evolution of the initial charge distribution under this moderate applied field (under 80 V/mm): this charge appears therefore relatively stable. As future perspective of this work, the application of higher electric fields to inject charges in LDPE will be crucial to investigate at a local scale the charge injection and to compare injected charge density determined by KPFM and PEA (Pulse Electro Acoustic) methods.

IV. CONCLUSION

The synergetic use of AFM-based techniques, such as PF-QNM and KPFM, has been demonstrated to represent not only a novel approach for the most complete and thorough characterization of insulating material interfaces, but also to constitute a valid method for the study of electrical phenomena at the contact, such as space charge accumulation and injection, allowing for the simultaneous correlation of the electrical properties to the physical interface characteristics, like roughness. The scatter in the results of surface potential profiles obtained by KPFM in case of PE processed with PET protective layer are suspected to be linked to the dispersion in amount of surface contamination, and, to a major extent, to the influence of the protecting layer using in sample preparation (e.g. contamination from the protecting layer). A future continuation of this work should include the challenging task of measuring the microscopic potential after charge injection at high fields and to compare injected charge density determined by KPFM and PEA method.

  Cite as: F. Gullo, T. Christen, C. Villeneuve-Faure, H. Hillborg, C. Laurent, S. Le Roy, G. Teyssedre, "Dependence of the field and charge distribution at semicon/polyethylene interface on the press-molding process derived from Kelvin Probe Force Microscopy", IEEE Int. Conf. on Electrical Insulation and Dielectric Phenomena (CEIDP), Cancun, Mexico, 21-24 Oct. 2018. Proc. CEIDP-18, pp. 469-472, 2018. DOI: 10.1109/CEIDP.2018.8544910

Fig. 1 .

 1 Fig. 1. Comparison of (a) adhesion, (b) surface potential map and (c) surface potential profile for sandwich whose PE was processed using Al. Grey areas in (c) represent SC and "other cross-section" corresponds to other sample (same sandwich which was cut at different position).

Fig. 2 .

 2 Fig.2. Comparison of surface potential, electric field, and charge density maps (top to bottom) for sandwich whose PE was processed a), b) and c) using Al,and d), e) and f) PET cover layers. The charge density is computed using Savitzky-Golay 2D method.

Fig. 3 (

 3 Fig.3 (a-d). Surface potential (a-b), electric field (c-d) and charge density profiles (e-f) in SC/PE/SC sandwich with PE processed with Al cover layer measured applying polarization on SC layer.

  Cite as: F. Gullo, T.Christen, C. Villeneuve-Faure, H. Hillborg, C. Laurent, S. Le Roy, G. Teyssedre, "Dependence of the field and charge distribution at semicon/polyethylene interface on the press-molding process derived from Kelvin Probe Force Microscopy", IEEE Int. Conf. on Electrical Insulation and Dielectric Phenomena (CEIDP), Cancun, Mexico, 21-24 Oct. 2018. Proc. CEIDP-18, pp. 469-472, 2018. DOI: 10.1109/CEIDP.2018.8544910


Simon Le Corgne

Emmanuelle Duwez

Malick Nam

Mathieu Olivier

From face-to-face to mobile Internet: replicate the French ESS questionnaire on the ELIPSS panel

 The Socio-Political Data Center (CDSP) is responsible for the data collection of the ELIPSS panel and the ESS.

 A part of the ESS 7th round was administered to the ELIPSS panel.

 Each survey process has his specificities (mobile Internet vs. face-to-face).

Context and objectives

What is the influence of the administration mode?

Elipss in a few words



  Online survey instrument for the scientific community  Aim: to address the lack of questionnaire survey tools in humanities and social sciences  Sampling: probability-based  Periodicity: each month  Duration: 30 minutes maximum  Administration: self-administered 7-inch touchscreen tablet & 3G subscription ESS in a few words  Academically cross-national survey conducted every two years across Europe since 2001  Aim: to chart stability and change in the social structure, conditions and attitudes in Europe  Sampling: probability-based  Periodicity: biennial  Duration: 1 hour  Administration: face-to-face


F Gullo
email: gullo@laplace.univ-tlse.fr

C Villeneuve-Faure

S Le Roy

C Laurent

G Teyssedre

T Christen

C Törnkvist

H Hillborg

Nano-scale characterization of insulator-semicon contacts

Keywords: Polymer; HVDC cable insulation, electrical contact, atomic and Kelvin probe micrsocopy I

A main challenge in the development of polymerinsulated HVDC power cables is to achieve a robust insulation, because the accumulation of space charge under electrical and/or thermal stresses can significantly reduce the cable reliability. The space charge formation depends on the electrode-insulation contact which refers to the transition of carbon-black filled host polymer ('semicon', SC) and unfilled polymer. The fundamental processes which govern the contact physics and their dependence on the microstructure properties of the polymer and the interface are still poorly understood. In this paper we present a methodology to probe SC-insulator contact properties on a nanometer scale and to investigate the impact of material processing.

INTRODUCTION

The development of HVDC cable technology faces the problem of material design and optimization in a context where phenomena that play a role in dielectrics under DC stress are far from being under control [START_REF] Hanley | A general review of polymeric insulation for use in HVDC cables[END_REF] [START_REF] Christen | Characterization and Robustness of HVDC insulation[END_REF]. Contrary to AC stress, where the electric field distribution is governed by the permittivity, under DC, it is essentially controlled by resistivity or conductivity [START_REF] Vu | Electric field profile measurement and modeling in multi-dielectrics for HVDC application[END_REF]. The resistivity in dielectrics is temperature and field dependent (in contrast to the much less dependent dielectric permittivity). Therefore, the electric field distribution under DC stress is largely determined by the operating conditions. In particular, space charge effects may change the contact behavior dependent on the interface type (metal/insulator, semicon/ insulator, dielectric/ insulator) and make difficult to estimate the electric field distribution both in transient and steady state conditions [START_REF] Christen | HVDC insulation boundary conditions for modeling and simulation[END_REF]. The prediction of the field distribution is, however, the most important pre-requisite for designing insulation devices like cables and cable accessories (i.e. joints and terminations).

Progress in this frame can be carried out by systematic materials characterization through space charge or conductivity measurements [START_REF] Vu | Electric field profile measurement and modeling in multi-dielectrics for HVDC application[END_REF] for understanding and modelling, which provides help towards greater control of the HVDC insulation behavior. However, in order to take into account the processes at the interfaces, boundary conditions need to be settled [START_REF] Christen | HVDC insulation boundary conditions for modeling and simulation[END_REF]. Such boundary conditions encompass for example charges exchange at metal/electrode interface [START_REF] Taleb | Modeling of charge injection and extraction in a metal/polymer interface through an exponential distribution of surface states[END_REF], or possibly electrochemical processes due to ion drift from the bulk to the surface [START_REF] Teyssedre | Charge transport modeling in insulating polymers: From molecular to macroscopic scale[END_REF]. Since, in extra clean insulation, the majority carriers are not intrinsic but usually injected, it is obvious that the conductance of insulation is strongly governed by the contact boundary conditions. To determine them, the electrodeinsulator interface properties must be known. Unfortunately, space charge measurement methods like Pulsed Electro Acoustic (PEA) method [START_REF] Takada | Space Charge Measurement in Dielectrics and Insulating Materials[END_REF], which may provide spatial information, exhibit an upper limit of the resolution of a few microns, which is insufficient for reasonable interface characterization.

This article discusses appropriate methodologies for understanding the semicon (SC) -polymer insulation contact properties with the help of Fourier Transform Infrared (FTIR) Spectroscopy, Atomic Force Microscopy (AFM) and Kelvin Probe Force Microscopy (KPFM).

II. MATERIALS AND EQUIPMENT

A. Sample preparation

The materials were chosen to minimize the complexity of the matter. As a base insulation material we used a nonstabilized low-density polyethylene from ExxonMobil™ (LDPE LD 101BA), which is designed for Medium/High Voltage insulation, and which can be peroxide crosslinked.

To avoid that contact properties be influenced by diffusion of additives and impurities, the SC was produced by adding to the LDPE carbon black (CB) nanoparticles compressed into granules. The CB is an acetylene black compound for electrical applications (Denka Black 50%). The mixing was done with a Brabender internal mixer for 15 minutes at 15 rpm and 130°C with a ratio of 50/50 wt.% LDPE/CB. These materials are used to realize sandwich-like samples in 3 layers SC-LDPE-SC (3x50µm) through steps depicted in Fig. 1. Each single layer is produced in a hot-press by heating at 140°C and compressing the proper amount of pellets in the mold. To avoid that these films stick to the mold surface, a covering layer is used. Previous studies suggest that the material used for this cover layer induces different properties on the film [START_REF] Ghorbani | Heat-treatment and physical properties of high voltage DC XLPE cable insulation material[END_REF] [START_REF] Ghorbani | Characterization of conduction and polarization properties of HVDC cable XLPE Insulation Materials[END_REF]. For this reason, we will compare samples produced with aluminum and produced with PET (Mylar) as cover layer. The single layer films can be analyzed in this form or represent the first step to make the sandwich SC-LDPE-SC (Fig. 1a). By using a special mask, the three layers can be melted together with heating to 110° C and applying a small pressure (Fig. 1b). In this manner, samples with two flat LDPE-SC contacts are produced. To analyze this sample in cross-section, it is cut a small triangle, as shown in Fig. 1c. Using an ultracryo-microtome a flat surface about 150µm width and few hundred microns length is obtained from the tip of the triangle by removing thin layers of about 200 nm in thickness (Fig. 1d). After this treatment the surface is flat enough to measure the contact properties in cross-section.

B. Equipement

Different techniques were used to characterize the insulator-SC contact. First, Fourier Transform Infrared Spectroscopy (FTIR) in ATR (Attenuated Total Reflection) mode was performed using a diamond crystal [START_REF] Rabek | Polymer photodegradation: mechanisms and experimental methods[END_REF]. In this configuration, the penetration depth (inverse of absorption coefficient [START_REF] Fowles | Introduction to Modern Optics[END_REF]) is between 0.5 and 2 µm according to the wavelength. With this technique the chemical bonds present in the single layer LDPE, known to play an important role in the contact properties [START_REF] Khabbaz | Chemical and morphological changes of environmentally degradable polyethylene films exposed to thermo-oxidation[END_REF], present in the single layer LDPE were determined.

To characterize interface properties, the Multimode 8 from Bruker is used. Peak Force Amplitude Modulation Kelvin Probe Force Microscopy (PF-AM-KPFM) is the AFM technique performed. It combines two different modes: Peak-Force Quantitative NanoMechanical (PF-QNM) to probe mechanical properties [START_REF] Young | The use of the PeakForce quantitative nanomechanical mapping AFM-based method for high-resolution Young's modulus measurement of polymers[END_REF] and Kelvin Probe Force Microscopy (KPFM) to probe surface potential [START_REF] Zerweck | Accuracy and resolution limits of Kelvin probe force microscopy[END_REF]. PF-QNM consists in acquiring topography and mechanical properties (adhesion, deformation, Young modulus…) and KPFM (with fixed cantilever lift from the surface) consists in acquiring the surface potential. The AFM tip has a PtIr coating (SCM-PIT) and nominal spring constant (3±2)N/m. For surface potential measurement a lift of 40 nm is used.

To determine mechanical properties from Peak Force measurement, an AFM-tip calibration is needed. For this, in a first step we determined the photodetector sensitivity using Force Distance Curve Measurement. Then we computed the cantilever spring constant using the Thermal Tune mode [START_REF] Hutter | Calibration of atomic-force microscope tips[END_REF]. A spring constant of 1.66 N/m was found in this way.

III. RESULTS AND DISCUSSION

In this section samples produced with aluminum or PET cover layers will be compared. First, the LDPE was characterized with FTIR, afterwards the SC-LDPE-SC sandwich was investigated with atomic probe microscopies.

A. FTIR results

Fig. 2 compares the ATR-FTIR spectra obtained on LDPE single layer processed using either PET or Aluminum cover layers during samples press-molding. Classical LDPE vibration modes are observed for both samples. The essential differences between the two samples are found in the region 1000 to 1800 cm -1 . Higher absorption is found at 1722 cm -1 and 1263 cm -1 for the sample processed with PET. These represent the vibration modes, respectively for double and single carbon-oxygen bonds. This could be interpreted as a more advanced oxidative stage, or more likely to some diffusion of moieties from the PET layer when in contact with the material.

B. Cross-sections of SC-LDPE-SC sandwiches using different AFM imaging modes

Two sandwiches, processed with aluminum and PET cover layers, were prepared to characterize the cross-section surface with AFM. The images shown in Fig. 3 were obtained on a sample processed with Al as cover layer. They correspond to the 3 modes: topography, mechanical response in adhesion, and surface potential. Note that the three images were realized exactly on the identical sample area during the same measurement. The surface topography map (Fig. 3a) shows the height difference between different areas. The topography is not flat, and it is noticeable that the central part, which is slightly hollowed, has a lower height compared to the rest of the sample. The surface topography can also measure the surface roughness. The average roughness takes two different values, about 10 nm for the LDPE and about 20 nm for the SC, due to the protruding dots. Fig. 3b corresponds to the surface adhesion map. This quantity gives the greatest contrast between LDPE and SC, which reveals the separation between the two materials. The image clearly shows that there are no defects like voided areas at the interface and that the two materials are smoothly bonded together. Fig. 3c presents the surface potential map and Fig. 3d the potential profile on a line perpendicular to the interfaces (cf. Fig. 3c). The vertical lines on Fig. 3d define the physical interfaces between LDPE and SC as identified from the adhesion map image. Note that according to the Poisson's equation, the space charge density is proportional to the negative second space-derivative (a measure for the curvature) of the potential. The figures show that from the electrical point of view the interface is not as sharp as for the adhesion map, but the potential gradually decreases within LDPE. The potential is almost symmetrical, with the exception of a peak present near the right contact, probably due to a defect close to the surface. In Fig. 3d the potential profile on a line, perpendicular to the interfaces, was added as a reference. The vertical lines define the physical interfaces between LDPE and SC as identified from the adhesion map image.

A similar analysis for the sample prepared using PET cover layer is represented in Fig. 4. The surface topography (Fig. 4a) seems smoother compared to the one of Fig. 3a. The adhesion map (Fig. 4b) are however comparable. The main difference in the surface potential map is the existence of an almost zero surface potential in the whole insulator and also in the SC, when the samples are processed PET. For metals the surface potential is usually considered as the work function difference between the AFM tip and the material being probed. For semiconductors, it is the difference with respect to the Fermi levels [START_REF] Ishii | Static states and dynamic behaviour of charges: observation and control by scanning probe microscopy[END_REF]. The contrast obtained by scanning the surface of heterogeneous materials would then be related to variations in work function or Fermi levels. For organic insulators, however, the interpretation is not so straightforward. Indeed, they are usually considered like semiconductors, but their HOMO-LUMO gap is typically large (for LDPE it is about 8 eV), therefore the concentration of thermally excited carriers is extremely small. The issue of KPFM measurements on insulating surfaces was addressed, e.g., by Ishii et al. [START_REF] Ishii | Kelvin Probe Study of Band Bending at Organic Semiconductor / Metal Interfaces : Examination of Fermi Level Alignment[END_REF]. The observed potential reflects the charge distribution in the sample layer and the substrate surface. However, the present results are obtained with the ground on the lateral electrode, which does greatly change the limit conditions. As the potential profiles are through the interface, it may reflect energy level equalization, with charge exchange between the two materials. Sharp profiles have been reported for example in case of PZT/Pt/SiO2 interfaces [START_REF] Eng | Nanoinspection of Dielectric and Polarization Properties at Inner and Outer Interfaces in Functional Ferroelectric PZT Thin Films[END_REF]. The relative spreading of the potential can therefore be related to the spreading of the space charge associated to band bending, or to the specific geometry of the system.

Concerning the difference in potential profiles for the two materials, two possible hypotheses are  greater surface oxidation in case of PET cover layer, and polarization processes  impact of the sample thickness: realizing that the sample made with PET cover-layer is substantially thinner (28µm vs. 41µm), two interfaces regions would be in interaction providing a homogenization of the potential, cf. Fig. 3c vs. Fig. 4c.

Previous images were obtained by scanning the surface over a length of 70 µm to give an overview of the sandwich structure. This represents a very large scale for the AFM and a narrower view is presented in Fig. 5. At this scale, the SC is no longer uniform, and it is possible to distinguish the CB nanodots in topography (Fig. 5.a and 5.c) and adhesion (Fig. 5.b and 5.d) maps. What is considered as nanodots or clusters of nanodots appear with less adhesion force compared to the polymeric matrix.

Imaging the surface with greater magnification allows estimating the size of these nanodots or clusters at around 80 nm. The SC-LDPE interface, which appeared flat at bigger scale, now shows an appreciable nano-roughness in adhesion. This zoom demonstrates that there is no carbon black migration in the LDPE volume. The interface roughness is characterized by peak-to-valley distance around 200 nm. However, considering the current images, the interface between the SC and LDPE appears sharp, without evident accumulation or spreading of the CB at the interface.

IV. CONCLUSIONS

A technique was developed for preparing small SC-LDPE-SC sandwich samples. Their interface roughness profiles were characterized with a peak-to-valley distance of around 200 nm. With an ultra-cryo-microtome, a cross-section surface with an average roughness of less than 20 nm was achieved. For these samples it is possible to characterize the electrode-insulator contact on a microscopic scale, which is of large relevance for HVDC cable insulation with SC-PE interfaces.

The FTIR analysis, suggests that the sample processed using PET as cover-layer presents more oxidized moieties on its surface. Cover-layers are not present during cable and cable accessory production, but they are present during production of small insulation samples, which are usually used for determining the electrical properties of a (new) cable or accessory material. A difference between the samples made with Al and PET cover-layers is also revealed in the surface potential profile. The potential varies smoothly at both interfaces of the sandwich and the shape of the variation points to negative charge build-up over several micrometers within the LDPE layer. From the surface mechanical properties, it is possible to extract information on the sharpness of the interfaces at the larger scales, and on the distribution of CB at the smaller scales. Since the polymer host matrix is the same for SC and insulation, the only way to distinguish the interface is to observe the presence of the nanodots.

In summary, we demonstrated a suitable methodology to perform a comprehensive characterization of an insulator-SC interface down to the nanoscale. Although some significant further effort is needed, this will eventually help us to produce optimized SC-LDPE contacts.

Fig. 2 FTIR

 2 Fig. 2 FTIR spectra of LDPE single layer, using different cover layers, in ATR with a diamond crystal and a internal reflection angle of 45°: (a) full range and (b) details on impurities.

Fig. 1 .

 1 Fig. 1. The four steps for the realization of a sandwich SC-LDPE-SC for AFM measurement in cross-section: (a) press of the single layers; (b) agregation of the three layers; (c) cut of a small triangle; (d) cut a tip with an ultra-cryomicrotome to obtain a flat surface. The cover layer coloured in light blue in (ab), depending on the sample, is made of aluminum or PET.

Fig. 3

 3 Fig. 3 Images of SC-LDPE-SC cross-sections processed using aluminum cover layer, analyzed with PF-KPFM-AM mode. The maps represent different surface properties: (a) surface topography, (b) surface adhesion, (c) surface potential (d) surface potential on a single line. Vertical lines in (d) are physical interfaces identified by adhesion measurements. The colour bar on maps represents the scale of measured quantities.

Fig. 4

 4 Fig.4Images of SC-LDPE-SC cross-sections processed using PET cover layer, analyzed using PF-KPFM-AM mode. The maps represent the same surface properties as described in Fig.3.

Fig. 5

 5 Fig. 5 Zoom on the SC-LDPE interface processed with aluminum (a,b) and PET (c,d) cover layers, and analyzed with PF-KPFM-AM mode. The images represent: (a,c) surface topography, (b,d) surface adhesion.

ACKNOWLEDGMENTS

Authors thanks S. Balor of IBCG-biotoul for ultra-cryomicrotomography.


Elina Casas

Mélanie Nicolas Landrein

Nicolas Elinacasas

Mélanie Bonhivers Landrein

Mfp

Ultra Expansion microscopy protocol with improved setup for upright and inverted microscopes

Keywords: Immuno-labelling, fluorescence microscopy, expansion microscopy, ultra expansion microscopy, U-ExM, upright microscope, inverted microscope Ultra Expansion microscopy Ultra Expansion microscopy

Ultra Expansion microscopy protocol with improved

setup for upright and inverted microscopes. v1

CITATION

This setup allows the imaging on upright and inverted microscopes. Also, the gels do not dehydrate during long microscopy sessions and can be further stored t 4°C.

  Isch C, Majneri P, Landrein N, Pivovarova Y, Lesigang J, Lauruol F, Robinson DR, Dong G, Bonhivers M (2021). Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar.. PLoS pathogens. LINK https://doi.

  Preparation coverslips ( 24mm ) coated with poly-L-lysine for mounting -Place the coverslips on Parafilm and cover each of them with 400 µL of poly-L-lysine 00:30:00 -recover and store the poly-L-lysine solution -wash the slides 2 times in a water bath -Dry the coverslips up on adsorbing paper. -Pre-cool overnight at -20°C a metallic tube holder -If required, prepare your cells on 24 mm coverslips to grow. Day 1-Sample preparatio, first expansion and primary antibody incubation Day 1-Sample preparatio, first expansion and primary antibody incubation 1-Prepare cells on 12 mm coverslips 1-Prepare cells on 12 mm coverslips In 24-well plates, prepare coverslips with your favourite cells. The cells density should not be too high to avoid nonisotropic expansion (non-confluent cells). Wash the cells with PBS. 2-Activation 2-Activation -Place the coverslips in 24-well plate, cells up, and load 1 mL of FA/AA solution -Fill up the empty wells with water U-ExM Monomer solution (MS) U-ExM Monomer solution (MS) Denaturation solution (DS) Denaturation solution (DS) protocols.io | https://dx.doi.org/10.17504/protocols.io.the MS + TEMED + APS solution and gel polymerization -Add 5 µL of TEMED 10% into the 90 ul MS aliquote, then add 5 µL of APS 10% -Vortex 2 sec and load on the parafilm 2 drops of 35 µL not more . (Do 2 drops at once only as the polymerization is very rapid). -Immediately, adsorb the excess FA/AA solution on a tissue and transfer the coverslips on the drop, cells side facing down. -Incubate On ice 00:05:00 , then at 37 °C without shaking without shaking 01:00:00 . During this step, prepare step 4. c-Gel detachment -After the polymerization and in a 6-well plate, add 1 mL of denaturation solution. -Transfer the coverslip+polymerized gel (gel facing up ) to the 6-well plate -Incubate Room temperature 00:15:00 100 rpm or until the gel detaches from the coverslip. DS per sample in an eppendorf tube. -Collect the detached gel and load it into the DS. -Fill up the tube with heated DS and incubate at 95°C 01:30:00 . OR Change the water bath at least once and incubate Overnight 4 °C ) -Measure the diameter of the gel (expect of a >4x expansion compared to the 12mm diameter of the coverslip) rpm a-In a 24-well plate, per sample, add 1 mL of PBS, 2% BSA, 0.2% Tween-20. protocols.io | https://dx.doi.org/10.17504/protocols.io.bvwqn7dw -Fill up the empty wells with water -Gel cutting -Use a 1 mL Tips to cut a circular piece of the gel ( usually 4 pieces can easily be cut from one gel). -Place each piece of gel into the 24-well plate -Incubate in PBS, BSA, Tween-20 00:10:00 or more 37 °C 100 rpm -Primary antibody(ies) incubation -Remove the PBS, BSA, Tween-20 solution and replace it with 350 µL of primary antibody(ies) diluted in PBS, BSA, Tween-20 -Incubate 37 °C Overnight 100 rpm , the lid sealed closed to avoid evaporation. Day-2 Secondary incubation, second expansion and gel mount Day-2 Secondary incubation, second expansion and gel mount 1-Secondary antibody incubation 1-Secondary antibody incubation -Wash 3 times in 350 µL PBS, BSA, Tween-20 37 °C -Remove the PBS, BSA, Tween-20 solution and replace it with 350 µL of secondary antibody(ies) and Hoechst Hoechst ( 5μg/mL ) diluted in PBS, BSA, Tween-20 -Incubate 37 °C 03:00:00 100 rpm , the lid sealed closed to avoid evaporation, in the dark. diameter of the gel, it should be consistent with the first expansion ( You can store the gels water in water at 4°C) 3-Mounting and imaging 3-Mounting and imaging

  org/10.1371/journal.ppat.1009329 Make 90 ul aliquotes and store at -20°C for up to 2-3 weeks Make 90 µL aliquots and store at -20°C for up to 2-3 weeks maximum. Note: the solution does not freeze.

	Reagents Equipment Stock solution		Company Company Stock solution	Reference Reference Volume to prepare per sample Final concentration Comment Comments
	milliQ Water Thermoblock		Eppendorf		0.2 um filtered
	N,N'-Methylenbisacrylamide Vortex 2% PBS Parafilm	Euromedex	EU0560	store at 4°C 0.2 um filtered
	SDS 20% Tris-Base Equipment Equipment Reagent	Stock concentration Volume / Weight Final concentration
	NaCl TRIS-Base	-			0.6 g	stock solution 5M 50 mM
	Sodium Acrylate 38% HCl (fuming?) -	AK Scientific 97-99%, R624 store the powder at -20°C pH to 9.0
	TEMED compound ddH2O Tween-20 Formaldehyde 37% -SDS 20% (694 mM) Euromedex volume 10 mL 50406 Sigma P-7949 19 ul 28.82 mL Acrylamide 40% 25 ul NaCl 5 M 4 mL	Final concentration 0.7% 200 mM store RT 1% 200 mM
	Reagents Reagents PBS 1x milliQ water			956 ul	qsp 100 mL
	Prepare just before use
	Activation solution (FA/AA) Activation solution (FA/AA)
	Equipment		Company		Reference	Comments
	12 mm round coverslips		VWR			6530021	Comments
	Metal block Shaker Reagent Incubator 37°C homemade spatula Sodium acrylate	B		pre-cooled -20°C Final concentration Comment RT shaker or that can accomodate a shaker with a rigid plastic cover 38% w/v Dissolve little by little in milliQH2O and on ice under the fume hood. 0.22 um filtered.This solution is critical. Use only solution that appears clear or very slightly yellow.
	1.5 ml tubes
	Reagents 24 mm round coverslips Sodium acrylate stock solution (SA) Company Sodium acrylate stock solution (SA)	Reference	Comment
	Absolute ethanol 24-well plates	NUNC
	Acrylamide 40% 6-well plates	Euromedex NUNC	EU0060-A	store at 4°C
	Ammonium persulfate Glass slides Themoscientific J1800 AMN7 Euromedex EU0009 Stock solution Stock solution Volume to prepare per sample Final concentration store as 10% in H2O at -20°C (APS) Formaldehyde 37% Sigma 252549 Laboratory wipes Sodium Acrylate (SA) 38% (W/W) 500ul 23% (W/V)
	HCl Petri dishes Acrylamide (AA)	40%		250ul	1N solution	10% (W/V)
	HOECHST (bisBenzimide H 33342 trihydrochloride) Bis-acrylamide (BIS) 2% Sigma Plumbing joints PBS 10x		B2261 50 ul 20x27 N14 100 ul	0.1% (W/V) make 5 mg/mL in H2O. Store at -20°C as aliquotes 1x
	protocols.io | https://dx.doi.org/10.17504/protocols.io.bvwqn7dw	3

protocols.io | https://dx.doi.org/10.17504/protocols.io.bvwqn7dw protocols.io | https://dx.doi.org/10.17504/protocols.io.bvwqn7dw

protocols.io | https://dx.doi.org/10.17504/protocols.io.bvwqn7dw