Fernando Lopitz-Otsoa

Sofia Lachiondo-Ortega

Teresa Cardoso Delgado

Mikel Azkargorta
email: mazkargorta@cicbiogune.es

Felix Elortza
email: felortza@cicbiogune.es

Manuel S Rodríguez
email: manuel.rodriguez@itav.fr

María Luz

María Luz Martínez-Chantar

Ubicare

SUMO-binding entities (SUBEs

Keywords: SUMO, SUBEs, Liver cancer, Hepatocellular carcinoma, hepatoma, Mass-Spectrometry

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INTRODUCTION

Liver cancer is the sixth most prevailing cancer worldwide [START_REF] Forner | Hepatocellular carcinoma[END_REF]. Hepatocellular Carcinoma (HCC) is the most common form of primary liver cancer (70-85% of total liver cancer). Common risk factors for the development of HCC include chronic hepatitis B or C infection, metabolic syndrome, Type 2 diabetes and alcohol consumption [START_REF] Gerbes | Gut roundtable meeting paper: selected recent advances in hepatocellular carcinoma[END_REF]. HCC is considered phenotypically and genetically a very heterogeneous cancer being HCC pathogenesis characterized by disruption of a complex network of signaling pathways.

Although our knowledge of the major molecular pathways implicated in the pathogenesis of HCC has increased dramatically in the last years, one of the main difficulties when treating HCC is that many pathways are activated and inhibiting one generally drives compensation by other pathways. Thus, a more global mechanism, such as post-translational modifications of proteins that can simultaneously regulate multiple disrupted signaling pathways may provide a valuable therapeutic approach for liver cancer clinical management.

Post-translational modifications are considered key mechanisms regulating protein homeostasis and function in eukaryotic cells. These modifications extend the diversity of the proteome by inducing structural and functional changes in proteins through different mechanisms like covalent binding of functional groups, cleavage of regulatory subunits and degradation of other proteins. The most common post-translational modifications include phosphorylation, methylation, acetylation, glycosylation, ubiquitination and ubiquitinlike proteins (UbLs). Among all UbLs, protein modification by SUMO (Small Ubiquitin MOdifier) has attracted particular attention due to its critical roles in a variety of cellular processes, including transcription, cellular localization, DNA repair, and cell cycle progression (REF). In liver cancer, SUMOylation appears to be altered [START_REF] Seeler | SUMO and the robustness of cancer[END_REF][START_REF] Tomasi | S-adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers[END_REF][START_REF] Li | Cbx4 governs HIF-1alpha to potentiate angiogenesis of hepatocellular carcinoma by its SUMO E3 ligase activity[END_REF][START_REF] Zubiete-Franco | SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer[END_REF], and changes in the SUMOylation of specific proteins has been identified to play a role in the progression of the cancer related diseases [START_REF] Zubiete-Franco | SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer[END_REF].

In mammals, there are five SUMO paralogues, being SUMO-1, -2 and -3 more studied, compared to SUMO-4 and -5 [START_REF] Sarge | SUMO and its role in human diseases[END_REF][START_REF] Silva-Ferrada | Strategies to Identify Recognition Signals and Targets of SUMOylation[END_REF][START_REF] Liang | SUMO5, a Novel Poly-SUMO Isoform, Regulates PML Nuclear Bodies[END_REF]. SUMOylation in mammals is carried out by an enzymatic thiol-ester cascade involving three enzymes, the heterodimeric SUMO activating enzyme SEA1/SEA2 or E1, the SUMO conjugating enzyme Ubc9 or E2 and a SUMO-E3-ligase specific for each target protein. Several families of SUMO E3s have been reported whose action appears to be in a dynamic equilibrium with hyperactive SUMO-specific proteases known as SUSPs or SENPs (REF). Indeed, isolating endogenous SUMOylated proteins in vivo is rather challengingly mainly due to the presence of these active SENPs which remove SUMO from protein substrates making this reaction with high reversibility (REF).

Initial studies to study SUMOylation in vivo were based on the detection by Western-blot of specific SUMOylated proteins using antibodies against the protein of interest. First, the protein was immunoprecipitated using specific antibodies, and then analyzed by PAGE-Western blot detection with anti-SUMO antibodies. However, antibodies generally made with non-modified recombinant protein, in many cases, do not immunoprecipitate the SUMOylated form of a protein. Another approach to study SUMOylation has been the nickel chromatography using the different Histidinylated (His6) versions of SUMO molecules. Preliminary experiments can be set up by transiently expressing His6-SUMO molecules together with target proteins of interest. However, it will be more convenient to detect SUMO-modified forms from cells stably expressing His6-SUMO (REF). Even though this approach is attractive, it is limited to cultured cells and cannot be applied for example to the study of the SUMOylated proteome in tissue biopsies. To overcome these limitations, SUMO-binding entities (SUBEs), also known as SUMO traps, were developed (REF). Briefly, SUBEs are recombinant proteins that comprise tandem repeats of SUMOinteracting motifs (SIMs) thereby recognizing SUMO molecules on modified proteins with an increase in the overall affinity for SUMO substrates. SUMO-traps were engineered by introducing RNF4-derived SIM2 and SIM3 motifs in tandem, into a glutathione S-transferase (GST) vector tag, an heterologous carrier protein (REF). Overall, this method provides a tool to facilitate the purification and identification of SUMO target proteins in liver cancer samples that include both hepatoma cells and mouse liver biopsies.

PROTOCOL

An overall scheme of the protocol described in this manuscript is sown in Figure 1.

Cell Preparation and Lysis

1.1) Maintain cells in P100 plates in standard grown media at 37 °C in a humidified atmosphere of 5% CO2-95% humidity. Huh-7 (human hepatoma cell lines) and THLE2 (human hepatic cell line), were used.

Huh-7 was cultured in DMEM supplemented with 10% FBS, 1% PSA and 1% glutamine. THLE2 cells should be grown in BEBM supplemented with 10% FBS, 5 ng/ml EGF and 70 ng/ml phosphoetanolamine.

1.2) Grow cells in P100 plates to a density of approximately 1.2-1.5 x 10 6 cells per time/condition point.

NOTE: Do not use too many cells otherwise you can saturate the SUBEs and recover only highly abundant

SUMOylated proteins (and loose the low abundant ones).

1.3) Using a sterile tissue-culture flow hood, aspirate the media from the flask and wash cells with 5 mL of sterile 1x phosphate-buffered saline (PBS).

1.4) Lyse cells directly on the plate (which is placed on ice). For one P100 plates, use 500 μL lysis buffer.

Using cell scraper, gently scrape the cells off the bottom of the plate into the Lysis media. Check all the cells have come off by inspecting the base of the plate before moving on. Alternatively, cells may be harvested by trypsination and pelleted in order to reduce the volume of lysis buffer and amount of SUBEs and GST. In the case of trypsination, aspirate the PBS and add 1 mL of 1x (0.05%) trypsin-EDTA to the plate, making sure that cells are covered by trypsin. Place plate in the incubator maintained at 37°C, 5%

CO2, and 95% humidity until all cells have detached from the plate (approximately 5 min). Add 2 mL of pre-warmed growth media to the plate to deactivate the trypsin-EDTA.

1.5) Clarify the lysate by centrifugation in a microfuge at 15500g and 4°C for 10 min.

NOTE:

The protocol can be paused here.

Tissue Preparation and Lysis

All experiments were approved by the respective institutional committees for animal care and handling. All efforts were made to minimize animal suffering and to reduce the number of animals used.

2.1)

For the extraction in tissues, wash with cold PBS, and freeze immediately in liquid nitrogen. All samples are stored at -80°C.

2.2)

Approximately, 75 mg fragments from snap-frozen or fresh livers were homogenized in 1 mL of icecold lysis buffer with the Precellys 24 homogenizer. Run the Precellys 24 at 6500 rpm, 2x60 sec each, 30 sec pause. Transfer the supernatant to another tube and discard the pellet. Alternatively, triturate frozen tissues in liquid nitrogen and recover in 1 mL of lysis buffer.

2.3) Clarify the lysate by centrifugation in a microfuge at 15500g and 4°C for 10 min.

NOTE:

The protocol can be paused here.

Binding of GST-SUBEs or GST control to glutathione-agarose beads

3.1) Preparation of glutathione beads: Reconstitute 10 mL lyophilized glutathione-agarose beads in deionized water overnight at 4°C (or minimally for 30 min at room temperature). After swelling, the agarose has to be washed thoroughly with ten volumes of de-ionized water or PBS three times by centrifugation at 1000 RPM during 5 min to remove lactose or ethanol (present in the lyophilized powder to preserve agarose beads). At the end, beads are suspended in PBS to obtain 50% (v/v) slurry.

3.2) Add 100 μg of GST-SUBEs or GST control to 100μL glutathione beads slurry. This reaction take place in a tube with PBS (500µl).

NOTE:

The amount of GST-SUBEs used for pull-downs depends on the relative abundance of the SUMOylated proteins of interest. It is recommended to set up conditions analyzing by Western blot the input, bound, and unbound material using anti-SUMO2/3 antibodies or against your proteins of interest.

3.3) Incubate GST-SUBEs or GST control with beads, slowly rotating (EQUIPMENT NAME AND BRANDT) at 4°C for at least 2 h (slow binding reaction).

NOTE: Adding 1 mM dithiothreitol (DTT) improves GST binding to glutathione column.

3.4) After incubation, recover the agarose beads by centrifugation at 1000 RPM during 5 min. At the end, the beads are suspended in PBS to obtain 50% (v/v) slurry.

NOTE:

The protocol can be paused here. 4.5) Elute the sample with 30 μL 1:1 3X boiling buffer and the respective lysis buffer. This is called the BOUND Fraction.

GST Pull

Identification and characterization of SUMO targets and/or SUMOylated proteome

5.1) Perform Western blot analysis using an anti-SUMO2/3 antibody or the specific antibody of your choice (Fig. 2B).

5.2) In the case of

REPRESENTATIVE RESULTS

Identification of a specific SUMOylation substrate in liver tumor biopsies

Liver Kinase B1 (LKB1) SUMOylation has been recently shown to be an important oncogenic driver in liver cancer [START_REF] Zubiete-Franco | SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer[END_REF]. SUBEs were used to enrich and isolate the SUMOylated proteins both in glycine Nmethyltransferase (Gnmt) deficient (Gnmt -/-) liver cancer mice and wild type (Gnmt +/+ ) mice. Figure 2 shows an example of images and results obtained from the SUBEs capture in non-tumor and tumor liver of wild type and Gnmt -/-hepatocellular carcinoma mice. In Figure 2a are included (Ponceau S staining), the three different fractions (input, FT and BOUND) obtained in the SUBEs pull-down assay. A Ponceau S stain is useful to control a possible deleterious effect on the charge of blotted proteins to be evaluated by Western blots. In Figure 2b a Western blot analysis of LKB1 by using SUBEs to capture endogenous SUMOylated LKB1 is shown. The levels of the SUMOylation of LKB1 are augmented in liver tumors. In the case of Western blot analysis, equal charge and transferred proteins was observed by Ponceau staining in the input fraction and was not significantly altered after washes (flow through fraction). The amount of protein captured with SUBEs was significantly higher, in particular in the tumors. (Figure 2a).

Alternatively, Coomassie staining of a duplicated gel can provide similar information. Sticky proteins such as p53 or SUMOylated forms of some proteins might bind to the GST control. To remove background, lowdensity agarose beads, BSA coating, or additional washes can be considered. However, this could affect applications such as mass spectrometry and might result in loss of information.

Characterization of the SUMOylated interactome in human hepatoma cells

To investigate the capacity of the SUMO-trap to interact with naturally SUMOylated proteins Huh-7 (human hepatoma) and THLE2 (human hepatic) cell lines, were used. Normally, in the case of specific protein SUMOylation, the modification process is induced by challenging/treating cells with a specific agent and/or condition. Depending on the cellular context, the use of SUBEs resolve this problem, since it captures SUMOylated proteins (SUMOylome) at basal level Figure3. The first step is the visualization of the total material captured with SUBEs and using GST as negative control. For this purpose we can use conventional protein staining protocols Figure3a. Protein identification and abundance calculation were carried out using PEAKS software (Bioinformatics Solutions), and data was further loaded onto Perseus platform (REF) for statistical analysis and heatmap generation. Proteins identified with at least two different peptides were considered in the final analysis. A permutation-based FDR-corrected T-test was applied for the comparison of the abundances, and proteins with a q < 0.05 and a SUBE/GST ratio greater than 2 were considered as enriched and kept for further discussion Figure3b.

FIGURES AND TABLES FIGURE AND TABLE LEGENDS:

DISCUSSION

JoVE is a methods-based journal. Thus, the Discussion section of the article should be focused on the protocol and not the representative results.

This section should discuss the following with citations:

• Critical steps in the protocol • Modifications and troubleshooting of the method • Limitations of the method • The significance of the method with respect to existing/alternative methods • Future applications or directions of the method Herein, we have provided a complete and detailed description of the methodology reporting the use of SUBEs for the enrichment, isolation and identification and characterization of the SUMOylated proteins in in vivo models of liver cancer. Both in mouse liver tumors and human hepatoma cells, we were able to correctly isolate and identify SUMOylated proteins of interest or to perform a high-throughput characterization of the SUMOylated proteome and interactome. Even though the synthesis of SUBEs is outside the scope of this manuscript, for further information the following references should be looked at (REFs). The protocol described is fast and very sensitive and the critical step of the protocol include the SUBEs are recombinant proteins that comprise tandem repeats of SIMs thereby recognizing SUMO molecules on modified proteins with an increase in the overall affinity for SUMO substrates. Due to its high specificity and sensitivity, the use of SUBEs for the isolation of the SUMOylated proteome is advantageous relative to other approaches in literature such as the detection by Western-blot of specific SUMOylated proteins using antibodies against the protein of interest or the nickel chromatography using the different histidinylated versions of SUMO molecules. However, it must be taken into consideration that as the SUBEs protocol is performed under non denaturing conditions, maintaining the interaction between SUMOylated proteins and interactors. Therefore we obtain information about the SUMO interactome rather than only a list of SUMOylated target proteins. Thus, further experiments are necessary to confirm if a protein identified is a target of SUMO or an interacting factor. Other limitation of the SUBEs is the fact that control GST-tramps used are able to capture many background proteins related to oxidative stress.

This issue is especially relevant during the Mass-Spectrometry analysis due to high sensitivity of the technique. In order to overcome these limitations, biotinylated SUMO-traps (bioSUBEs) have been Also methods for detection and quantification of SUMOylated proteins can be considered with both GST and bioSUBEs versions such as it was done with the TUBEs (REF).

Overall, the use of SUBEs for the isolation and characterization of the SUMOylated proteome relevant in liver cancer is a fast and sensitive method providing vast information on the still rather unknown role of SUMOylation pathway in liver cancer.

ACKNOWLEDGMENTS:

This

Figure 1 .

 1 Figure 1. Schematic diagram of the protocol flow chart used for the enrichment, isolation and identification and characterization of the SUMOylated proteome in vivo for the study of liver cancer.

Figure 2 .

 2 Figure 2. Liver Kinase B1 (LKB1) is modified by SUMO-2 in mouse models of Hepatocellular Carcinoma. (A) Ponceau S staining of the three different fractions (input, FT and BOUND) obtained in the

Figure 3 .

 3 Figure 3. Heatmap depicting the differentially enriched proteins in Huh7 and Thle2 SUBE samples. (A) Protein stain of captured material, with GST (Negative control) and SUBEs. (B) Heatmap depicting the differentially enriched proteins in Huh7 and Thle2 SUBE samples. Proteins with a q<0.05 and a SUBE/GST ratio>2 were considered as SUBE-enriched proteins in each cell line. The heatmap displays the abundances of those proteins enriched only in one of the cell lines under analysis.

  use of SENPs inhibitors (PR619? Any other?) or chemical isopeptidase inhibitors such as NEM (N-Ethylmaleimide) and IAA (2-Iodoacetamide) in the lysis buffer. OTHER CRITICAL STEPS Do you see SUMO signatures? (SUMO chains?) You have to check this in the MS data.

  developed (REF).Other applications of this technology include the combination of SUBEs technology with Real-time Surface Plasmon Resonance (SPR) allowing the real-time interactions with SUMOylated proteins from cell extracts(REF) MORE APLLICATIONSThe bioSUBE version can also be used to detect by florescence SUMOylated proteins in living cells since streptavidin-labelled with distinct florescent dies can be combined with distinct antibodies for specific target proteins.

  work was supported by grants from Institut National du Cancer, FRANCE, INCa grant PLBIO16-251 (PLBIO16-251), CONACyT-SRE (Mexico) grant 0280365 and the REPERE program of Occitanie, France (M.S.R.). Also, NIH (US Department of Health and Human services)-R01AR001576-11A1, Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco, MINECO: SAF2017-87301-R integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013-2016 cofinanciado con Fondos FEDER, BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C), Asociación Española contra el Cáncer (T.C.D, M.L.M-C), Daniel Alagille award from EASL (to T.C.D), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M), La Caixa Foundation Program (to M.L.M). We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNEStimuli

  Spin down beads in a microfuge (1000 RPM for 5 min) and collect supernatant for analysis. take 1/10 of total volume (e.g., 50 μL) and dilute in equal volume of 3X boiling buffer. This fraction is considered as the flow-through (FT) fraction.

	Down Assay
	4.1) Following step 1.5 or 2.3, take 1/10 of total volume (e.g., 50 μL) and dilute in equal volume of 3X
	boiling buffer. This fraction is considered as INPUT.
	4.2) Add 450 μL of clarified lysate to 100 μ L glutathione beads slurry. Incubate lysate with beads, slowly
	rotating at 4°C for at least 2 h (slow binding reaction).
	4.3)

4.4) Wash three times with 1 mL ice-cold PBS, 0.05% Tween 20 (MARCA), spin down in a microfuge at 4°C and 1000 RPM for 1 min. Aspirate carefully until no liquid remains. The beads correspond to SUBEs BOUND (SB) fraction.

DISCLOSURES

Dr. Martínez-Chantar advises for Mitotherapeutix LLC.


Pierre Bauer
email: pierre.bauer05@orange.fr

Karine Mougin

Delphine Faye

Arnaud Buch

Pierre Ponthiaux

Vincent Vignal

K Pierre

Synthesis of 3D Dendritic Gold Nanostructures Assisted by a Templated Growth Process: Application to the Detection of Traces of Molecules

Complex architectures like 3D gold dendritic nanostructures were synthesized by an in situ templated growth method using a thin film of a block copolymer [polystyrene-b-poly(4-vinylpyridine)] deposited onto silicon substrates. The overall study has demonstrated the strong link between the morphology, size, and distribution of the structures and the synthetic physicochemical parameters, such as pH, reaction temperature, concentration, and nature of reactants. A nonequilibirum state of the medium has been required to create a fractal growth of the gold structures onto a prepatterned gold-seeded surface and has led to a better control of the structures' surface coverage rate. Those as-prepared nanodendrites have also exhibited high electrocatalytic activity toward a significant enhancement factor, as well as important sensitivity, thanks to tip effects. The electrochemical experiment results have demonstrated efficient adsorption and quantification of very low traces of specific molecules like glutathione or hexadecanethiol.

■ INTRODUCTION

Noble metal nanoparticles (NPs) have been extensively studied due to their fascinating optical, electronic, or catalytic properties and their promising applications in photoluminescence, [START_REF] Hu | Fabrication of Dendrite-like Au Nanostructures and their Enhanced Photoluminescence Emission[END_REF] superhydrophobic surfaces, 2 sensors/biosensors, [START_REF] Pingarron | Gold Nanoparticle-based Electrochemical Biosensors[END_REF][START_REF] Shu | Direct Electrodeposition of Gold Nanostructures onto Glassy Carbon Electrodes for Non-Enzymatic Detection of Glucose[END_REF][START_REF] Zen | Amino Acid Analysis using Disposable Copper Nanoparticle Plated Electrodes[END_REF] catalysis, [START_REF] Ye | Fluoride-Assisted Galvanic Replacement Synthesis of Ag and Au Dendrites on Aluminium Foil with Enhanced SERS and Catalytic Activities[END_REF] or surface enhanced raman scattering spectroscopy (SERS). [START_REF] Lu | Synthesis and Characterization of 3D Dendritic Gold Nanostructures and their use as Substrates for SERS[END_REF][START_REF] Huang | Controlled Synthesis of Dendritic Gold Nanostructures Assisted by Supramolecular Complexes of Surfactant with Cyclodextrin[END_REF][START_REF] Ye | Electrochemical Growth of Flower-like Gold Nanoparticles on Polydopamine Modified ITO Glass for SERS Application[END_REF] Indeed, the long-range and large-scale arrangement of metal NPs represents an important issue in the development of nanostructured materials. Especially gold nanoparticles (AuNPs) have attracted a great deal of interest. Due to their high stability (which is an advantage compared to other metals), AuNPs possess unique properties, such as excellent electrical and heat conductivity and strong adsorption in the visible and near-infrared wavelength region (380-750 nm).

Great efforts have been devoted to synthesize gold nanomaterials with novel morphologies. The control of the shape, size, and distribution of those particular structures has become a major challenge, since it considerably influences their intrinsic properties. Among various shapes, such as rods, [START_REF] Xu | Shape Change and Color Gamut in Gold Nanorods, Dumbbels, and Dog Bones[END_REF] prisms, [START_REF] Gou | Solution-Phase Synthesis of Cu2O Nanocubes[END_REF] cubes, [START_REF] Zhang | Synthesis and SERS Properties of Nanocrystalline Gold Octahedra Generated from Thermal Decomposition of HAuCl4 in Block Copolymer[END_REF] stars, [START_REF] Kumar | M High-yield Synthesis of Optical Response of Gold Nanostars[END_REF][START_REF] Wang | PH-dependent Evolution of Five-star Gold Nanostructures: An Experimental and Computational Study[END_REF] or dendrites, [START_REF] Lee | Seeding Method for Three-dimensional Dendritic Growth of Gold Nanoparticles Stabilized by Hexatrimethylammonium Bromide[END_REF][START_REF] Ren | Controlled Synthesis of Golf Nanoflowers Assisted by Poly(vinyl pyrrolidone)-Sodium Dodecyl Sulfate Aggregations[END_REF] dendritic nanostructures have attracted much attention because of their high effective surface-to-volume ratio, high sensitivity, and special texture with sharp tips and nanoscale junctions. Because of the highly symmetric face-centered cubic (fcc) crystal structure of Au, difficulties appeared in forming well-defined dendritic nanostructures with a homogeneous distribution. A nonequilibrium state is usually required to adjust reaction parameters (such as solvent, reactant concentration, temperature, pH) and to select the growth method. [START_REF] Huang | Facile Synthesis of Dendritic Gold Nanostructures with Hyperbranched Architectures and their Electrocatalytic Activity toward Ethanol Oxidation[END_REF] Various methods have been developed to fabricate those specific structures, such as electrodeposition, [START_REF] Hau | Aminosilane-Assisted Electrodeposition of Gold Nanodendrites and their Catalytic Properties[END_REF][START_REF] Zhang | Polyelectrolyte Multilayer as Matrix for Electrochemical Deposition of Gold Clusters: toward Super hydrophobic Surface[END_REF] hydrothermal reduction, [START_REF] Tang | Poly(N-vinyl-2 pyrrolidone) (PVP)-Capped Dendritic Gold Nanoparticles by One-step Hydrothermal Route and their High SERS Effect[END_REF] seed-mediated growth, [START_REF] Jana | Seed-Mediated Growth Approach for Shape controlled Synthesis of Spheroidal and Rod-like Gold Nanoparticles using a Surfactant Template[END_REF][START_REF] Pan | 3D Dendritic Gold Nanostructures: Seeded Growth of a Multi-Generation Fractal Architecture[END_REF] or the wet chemical way. [START_REF] Huang | Ultrafast Preparation of Threedimensional Dendritic Gold Nanostructures in Aqueous Solution and their Applications in Catalysis and SERS[END_REF][START_REF] Han | Ultrafast Growth of Dendritic Gold Nanostructures and their Applications in Methanol Electro-oxidation and SERS[END_REF] Lv and co-workers have reported several facile and controlled electrochemical routes to prepare three-dimensional hierarchical dendritic gold nanostructures with different amino-capping agents. [START_REF] Lv | Shaped-controlled Electrosynthesis of Gold Nanodendrites for Highly Selective and Sensitive SERS Detection of Formaldehyde[END_REF][START_REF] Lv | Facile and Controlled Electrochemical Route to Three-dimensional Hierarchical Dendritic Gold Nanostructures[END_REF] However, the seed-mediated growth can offer an alternative process to improve the surface coverage rate of nanostructures relative to the Frens method. [START_REF] Frens | Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions[END_REF] In order to control the nucleation density in 2D arrays, Park et al. have worked on the fabrication of a nanoporous template made of a diblock copolymer such as polystyrene-b-poly(2-vinylpyridine) (PS-b-P4VP). [START_REF] Park | A Simple Route to Highly Oriented and Ordered Nanoporous Block Copolymer Templates[END_REF][START_REF] Park | From Nanorings to Nanodots by Patterning with Block Copolymer[END_REF] This method has allowed an optimization of the distribution of gold dendritic nanocrystals onto the surface. However, despite all these efforts to improve the quality and variability in the synthesis of flower-like structures, the detailed mechanism through which they evolve from spherical seeds has not been yet completely understood.

A significant amount of research has been devoted to the development of effective analytical protocols for measuring traces of molecules. A wide range of detection strategies is now available, such as fluorescence, [START_REF] Burns | Fluorescent Core-shell Silica Nanoparticles: towards "Lab on a Particle" Architectures for Nanobiotechnology[END_REF] mass spectroscopy, or electrochemistry. [START_REF] Naik | Towards Single Molecule Nanomechanical Mass Spectrometry[END_REF] Among all, electrochemical methods propose certain advantages, such as simplicity, rapidity, lower cost, and higher limit of detection. The groups of Wang and Coussot have reported the sensitive quantification of aminoacids and glucose using glassy carbon electrodes modified with multiwalled carbon nanotubes and copper microparticles. [START_REF] Deo | Electrochemical Detection of Amino Acids at Carbon Nanotube and Nickel-carbon Nanotube Modified Electrodes[END_REF][START_REF] Coussot | A Rapid and Reversible Colorimetric Assay for the Characterization of Aminated Solid Surfaces[END_REF] Dehdari Vais et al. have recorded cyclic voltammograms of Au pyramidal electrodes in phosphate-buffered saline (PBS) solution in the absence and presence of azathioprine. 34 More recently, Liu and co-workers have worked on the development of an electrochemical DNA biosensor via an electrode modified with dendritic gold nanostructures to gain ultrasensitivity (fM) and a dynamic detection range (from 1 fM to 1 nM). [START_REF] Li | Development of an Electrochemical DNA Biosensor with a High Sensitivity of fM by Dendritic Gold Nanostructure Modified Electrode[END_REF] In this work, we describe the development of an electrochemical and optical sensor for the detection and quantification of traces of organic molecules. The active platform has been structured with gold flower-like crystals (AuNFs) grown onto a nanopatterned stainless steel substrate. Relevant and effective reaction conditions have been discussed to obtain well-defined gold dendritic structures with controlled symmetries. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) have allowed studying the shape, size, coverage rate, and crystallography of the metallic nanostructures. Then, quantification of adsorbed probe molecules (glutathione and hexadecanethiol) on gold structures has been carried out by local and global electrochemistry methods. Results were encouraging, showing the possibility to both discriminate and quantify adsorbed molecules at very low concentrations. Correlated localized surface plasmon resonance properties of the sensor device have also been investigated, showing its high sensitivity. Overall, the proposed process itself represents a highly interesting and promising route for producing a sensitive sensor using a very simple method, and more importantly, it will also contribute to a better understanding of the growth mechanism of anisotropic metal nanoparticles.

■ MATERIALS AND METHODS

Materials. Tetrachloroauric acid trihydrate (HAuCl 4 •3H 2 O, ≥99.9%), cethyl trimethylammonium bromide (CTAB, ≥99.9%), hydrazine hydrate (N 2 H 4 , 50-60%), and glutathione (GSH, ≥99%) were all purchased from Sigma-Aldrich. Hydroxylamine hydrochloric (NH 2 OH, ≥99% trace metal basis) and methylene blue (MB, ≥ 99%) were purchased from Fluka. Polystyrene-b-poly(4-vinylpyridine) (PS 4 0 0 0 0b -P4VP 5 6 0 0 with M w /M n = 1.10) and 2-(4hydroxyphenylazo)benzoic acid (HABA, ≥98%) were purchased from Interchim. Coomassie brilliant blue (CBB, ≥99%) was supplied by Cap Alpha. Buffer T1 (5% ice-cold acetic acid, 10% methanol, 85% Milli-Q water), buffer T2 (0.25 M aqueous sodium carbonate 50% diluted in methanol, pH 11.25), and buffer T2HCl (92% of buffer T2 with 8% of 3 N HCl ) were prepared in the lab. Water was purified using a Milli-Q system.

Template Fabrication. The multistep fabrication of nanostructured films started by a mechanical polish (Mecapol P320 Presi Device) of 304L stainless steel plates (10 × 15 × 1 mm 3 ) to control their roughness (S a = 0.34). Samples were finally washed twice in dichloromethane. Then, thin films of the diblock copolymer PS-b-P4VP mixed with HABA into chloroform were deposited by dip-coating over a large area onto cleaned stainless steel surfaces. The thickness of the dip-coated film on the surface was evaluated as 45 ± 2 nm, measured by ellipsometry (Multiskop OPTREL). The (PS-b-P4VP + HABA) phase separation was then enhanced by 4 days of solvent annealing in a saturated vapor of 1,4-dioxane. HABA was then removed by dipping the substrate in a methanol bath for 5 min, and sample was dried under a nitrogen flow. The nanoporous film sample was placed under UV light (λ = 254 nm) for 30 min in order to strengthen the mask against hydrolysis, but also to stabilize it over the time. Argon plasma treatment was used (power, 90 W; pressure, 1 mbar) during 60 s to burn residues of HABA or P4VP coating the bottom of the nanopores.

Synthesis of Au Dendritic Nanostructures. Samples were immersed in 5 mL of colloidal gold aqueous solution (0.001% wt) for 1 h and then reduced by a solution of hydrazine hydrate (10 μL). In a typical synthesis of Au dendritic structures, samples (recovered with PS-b-P4VP and gold seeds) were immersed in 5 mL of NH 2 OH aqueous solution (20 mM) for 45 min under controlled temperature and pH. Then 50 μL of HAuCl 4 aqueous solution (1% wt) was added and the solution was kept under controlled conditions without stirring for 1 h. Samples were then washed with distilled water and dried under nitrogen flow. The obtained gold nanostructures were characterized by SEM and XRD.

Multifractal Analysis. Fractals have revolutionized the geometric description of real objects and they have been applied in various fields, like geophysics, materials sciences, ecology, and medical science. The concept of fractals enables description of complex physical objects that have irregular shapes or fragmentated structures and that can be assigned to noninteger dimensions. Although multifractals provide a powerful mathematical model for complex structures, applied sciences were more often using a single fractal dimension to describe non-Euclidean objects. [START_REF] Adler | Multifractal Characterization of Pharmaceutical Hot Melt Extrudates[END_REF] Multifractal analysis can be used as an extension of the monofractal analysis and decomposes self-similar measures into intertwined fractal sets that are characterized by their singularity strength and fractal dimension. Multifractals can therefore be described as a superposition of homogeneous fractal objects. In multifractal analysis, the probability (P i ) of finding the object pixel in the ith box is determined by

P( ) i i ε ε ∼ α (1)
where α i is the Lipschitz-Holder exponent corresponding to the density in the ith box. The number of boxes [N(α)] where P i has singularity strengths between α and α + dα is found to scale as

N( ) f ( ) α ε ∼ α - (2)
where f(α) is the Hausdorff fractal dimension of the set of boxes with singularities α. The box counting method determines the partition function X(q,ε), which can be considered as the probability to find the object in the ith box for different moments q varying in the [-∞, ∞] interval. The partition function (X) is expressed as

X q p ( , ) ( ) i N i q q D 1 ( ) ( 1) q ∑ ε ε ε = ∼ ε = - (3)
with D q being the generalized dimensions corresponding to the scaling exponents for the qth moment of the measure. D q is finally defined as

D q p 1 1 lim log ( ) log q i N i q 0 1 ( ) ∑ ε ε = -ε ε → = (4)

Langmuir

Three generalized dimensions were of particular interest: (1) D 0 , the capacity dimension, which describes how a multifractal system covers the observed domain; (2) D 1 , the information dimension (or Shanon entropy), which characterizes the degree of disorder in a distribution; and (3) D 2 , the correlation dimension, which indicates the degree of clustering. Higher D 0 values indicate higher degrees of space coverage, while higher D 1 values correspond to higher disorder and lower D 2 values suggest a higher clustering level. Figure 1 depicts the general shape of a multifractal system for which D 2 < D 1 < D 0.

Optical Measurements. Surface-enhanced raman scattering (SERS) spectra were acquired using a Raman spectrometer (Labram BX40 Horiba) equipped with a CCD detector with a spectral resolution of 5 cm -1 . Prepatterned samples were illuminated by a linearly polarized He-Ne source at 633 nm with an output power P = 50 mW through a 100× objective lens, which is also used to detect the backscattered light (Rayleigh and Raman signals). In such a configuration, the laser spot size has been estimated as 80 μm 2 . A notch filter of optical density 6 was placed at the entrance of the spectrometer to reflect the Rayleigh signal. Each spectrum (300-3000 cm -1 ) was the average of five scans with a total acquisition time of 10 s. The wavelength of the camera was calibrated using the Raman band of silicon (100) wafers.

The reference sample was prepared by evaporation of gold onto a stainless steel plate (50 nm), immersion into a MB aqueous solution, and then washing with distilled water and drying under atmospheric conditions. Samples were prepared by immersing the prepatterned surface with gold dendritic structures in variable concentrations (from 10 -9 up to 10 -3 M) of MB aqueous solutions. They were then washed with distilled water and dried in a desiccator. Measurements at different positions were carried out for each sample to analyze both the homogeneity of the distribution of the structures on the substrates and the reproducibility of the nanostructures' plasmon bands response.

ADECA Colorimetric Method. The amino density estimation by colorimetric assay (ADECA) colorimetric method is a process to quantify the amount of adsorbed molecules on the surface. [START_REF] Ji | Size Control of Gold Nanocrystals in Citrate Reduction: The Third Role of Citrate[END_REF] This technique has to be calibrated in order to link the number of active sites on the substrates with the absorbance of adsorbed free dyes by UV spectroscopy (PerkinElmer Lambda 750). Different concentrations of CBB solutions have been prepared (2.5, 5, 10, 20, 30, 40, 50, and 60 mg/L) by dilution of a mother one. A 3 mL portion of CBB solution and 240 μL of T2HCl buffer were mixed together in disposable cuvettes (12.5 × 12.5 × 45 mm 3 ). The curve A = f([CBB]) can be then plotted according to Beer-Lambert's law as depicted in Figure 2.

In a 25/2 proportion of CBB and HCl, two forms of the dye molecule are visible in Figure 2a (pH 8.4). The acidic form seems to absorb at 450 nm and the basic form at 650 nm. As expected, a linear increase of absorbance is observable with the increase of CBB concentration, validating Beer-Lambert's law (Figure 2b). This fitting of the curve gives us the equation A = 0.059[CBB] + 0.066 ≈ 0.059[CBB] in first approximation. Hence, this system allows use of relatively low concentrations of free dye for free site detection. Then, it is possible to link the absorbance and the number of active sites by the equation

N AN V M 0.059 a CBB = ( 5
)
where N is the number of active sites on the sample, A is the maximum absorbance of the free dye, N a is the Avogadro number, V is the volume of dye released (3 mL + 240 μL), M CBB is the molecular weight of the dye (M = 855.02 g/mol), and 0.059 is the slope of the Beer-Lambert's curve.

A first step of conditioning was performed by immersing samples into 4 mL of colorized buffer T1 during 10 min. Then, staining in 2 mL of CBB coloration solution was realized during an appropriate time. Samples were washed three times in T1 buffer and distilled water during 10 min. Finally, after this step of washing, samples were immersed in 3 mL of decolorized buffer T2 for 30 min. Spectroscopic measurements have been recorded with 3 mL of solution and an appropriate amount (240 μL) of reading buffer T2HCl.

Local and Global Electrochemical Experiments. The local and global electrochemical measurements were performed with a potentiostat Biologic SP200. For the local voltamperometry, the microcell was composed of a working electrode, which corresponded to the stainless steel plate coated with gold nanoflowers (AuNFs) connected to the potentiostat and a 200 μm diameter capillary made of silicon, a reference electrode of Ag/AgCl, and a platinium counter electrode. [START_REF] Krawiec | Use of the Electrochemical Microcell Technique and the SVET for Monitoring Pitting Corrosion at MnS Inclusions[END_REF] The experiments have been carried out in a 0.1 M Na 2 SO 4 solution.

For global voltamperometry, a classical three-electrode device with a prepatterned sample as working electrode, a platinum counter electrode, and a Ag/AgCl reference electrode was settled upon. The electrolyte was 0.1 M Na 2 SO 4 solution.

■ RESULTS AND DISCUSSION

Growth Mechanism and Characterization of the Au Flower-like Structures. Au dendritic nanostructures have been synthesized using a solution-based reduction approach. These nanocrystals have been grown onto stainless steel plates templated with a nanostructured block copolymer (PS-b-P4VP) film recovered with gold seeds. The overall synthesis protocol is illustrated in Figure 3.

The substrates were first immersed into a colloidal gold solution followed by the addition of N 2 H 4 reducing agent. In a typical reaction, gold salts are subsequently reduced from Au 3+ to Au 0 . After this seeding step, gold nanoparticles (AuNPs) are covered by negative charges coming from hydroxyl ions (HO -). [START_REF] Nishimura | Role of Base in the Formation of Silver Nanoparticles Synthesized Using Sodium Acrylate as a Dual Reducing and Encapsulating Agent[END_REF] Then, a structuring agent was used to reduce HAuCl 4 and to structure both the morphology and size of these specific Au structures. According to Mahksin et al., in this seeding growth method, hydroxylamine molecules play the role of reductant, but also accelerate the reduction of Au 3+ on the surface of the seeds. After immersing gold-seeded plates into an aqueous solution of NH 2 OH, molecules are rapidly and selectively adsorbed on the specific Au crystal planes, covering them with NH 3 O -groups. Larger sized AuNPs are formed covered with NH 2 -groups via addition and diffusion of Au 3+ ions. [START_REF] Makhsin | The Effect of Size and Synthesis Methods of Gold Nanoparticleconjugated MαHIgG4 for use in an Immunochromatographic Strip Test to Detect Brugian Filariasis[END_REF] These groups act as shape-directing agents for initial branching, providing specific binding at some specific locations. According to Lv and co-workers, [START_REF] Lv | Facile and Controlled Electrochemical Route to Three-dimensional Hierarchical Dendritic Gold Nanostructures[END_REF] the -NH 2 groups of cytosine molecules are likely to produce branched nanostructrures and could increase the branching power of structuring agents. Hence, the following reactions might be considered as a possible mechanism occurring during the gold nanoflowers (AuNFs) growth: Figure 4 shows typical scanning electron microscopy (SEM) images of Au dendritic structures at different magnifications. As is shown in Figure 4a, aggregated flower-like structures were formed with an average size of 500 nm. Higher magnification (Figure 4b) reveals that hierarchical gold structures close to fivepointed stars have been obtained. They were composed of tips, which were evenly extended out, with the angle between each adjacent tip of ∼68°(Figure 4b).

4Au 3N H 12HO 4Au 3N 12H O 3 2 4 0 2 2 + + ↔ + + + - (6)
Au AuCl 4HO NH OH 2Au NO 4Cl 4H O 4 3 2 + + + ↔ + + + + - - + - (7)
HRTEM was carried out to determine the crystal orientation of the obtained gold dendrites. As a result, they have been deposited on a gold grid. Figure 5a shows HRTEM images of a trunk with leaves with its corresponding selected area electron diffraction (SAED) patterns. The dendrites consist of a trunk with rod-like perpendicular leaves. The SAED pattern shows a perfect straight line of diffraction spots, which verifies that one dendrite contains a single crystalline face. HRTEM measurements also demonstrated this single crystalline nature at the tip areas of the branch, as shown in Figure 5b. The atomic lattice exhibits a d spacing of 0.24 nm in the axial direction of the branch, with the d spacing of (111) planes for the fcc Au crystal. The average angle between branches is on the order of 68°, as depicted in Figure 5c.

The XRD spectrum was also plotted to determine the crystal orientation of the obtained gold dendrite. Figure 5e shows a typical XRD spectrum of the hierarchical gold five-pointed star structures and exhibits sharp diffraction peaks located at 38.4°, 44.5°, and 62.7°corresponding to {111}, {200}, and {220}, respectively. They can be attributed to Au crystals with the fcc crystal system, indicating that dendrites are well-crystallized. Afterward, Figure 5d shows the EBSD orientation map of an overall gold nanoflower. On the mapping image, different crystallographic colors appear for each tip. This important result shows perfectly that each branch has its own crystallographic orientation of (101) or (111), which proves the growth of threedimensional Au nanoflowers.

Effect of the Reaction Conditions. Effect of pH on Gold Structures' Morphology. Investigations have been realized to evaluate the pH-dependence of HAuCl 4 reduction by hydroxylamine on the control of gold nanostructures' morphology growth at room temperature. Prepatterned substrates were immersed into 5 mL of NH 2 OH aqueous solution with pH varying from 4 to 8 for 45 min, and then 50 μL of 1% wt gold salts was added to initiate the reduction reaction. Figure 6 presents F heterogenic with coexistence of four-tipped quadraedric stars and eight-tipped octaedric stars. With a further increase of the pH up to 5, more aggregated flower-like gold structures were observed with an average diameter of 2 μm. It is clear that the developed flower-like structures exhibit several trunks with numerous rod-like leaves. At higher pH (6 or 7), more hyperbranched gold flower-like structures are formed. Figure 6c shows that gold structures start to connect to each other with formation of a tapered shape closer to a bush-like structure, as seen at an advanced stage in Figure 6d. Finally, Figure 6e shows that, at a basic pH, several cracks in the copolymer thin film have appeared and anisotropic gold nanostructures have replaced the flower-shaped structures. The Ffractal dimension of each flowerlike structure has also been investigated. Results show that by increasing the pH up to 7 or more, the structures tend to grow in a 2D diffusion-limited aggregation cluster (Brownian tree with D = 1.7), whereas other structures grow in a 3D polymer motion without self-intersection (D = 1.66). In both cases, particles, during the growth process, follow a Brownian motion with haphazard and disorderly diffusion. [START_REF] Grassberger | Generalizations of the Hausdorff Dimension of Fractal Measures[END_REF] These above results suggest that the acidity and basicity of reaction solutions can kinetically favor or inhibit the reduction rate of gold ions, which can affect the initial gold nucleation process. [START_REF] Thambi | Synthesis of Complex Nanoparticle Geometries via PH-Controlled Overgrowth of Gold Nanorods[END_REF] It is known that the reductive activity of hydroxylamine increases with a higher pH value, which could explain the appearance of longer branches at this pH. Moreover, Zhao and co-workers suggest in their experiments that chlorine ions have also a key effect on the intraparticle ripening process of the nanoflowers. [START_REF] Zhao | State of the Art in Gold Nanoparticle Synthesis[END_REF] Hence, under neutral or basic conditions, the possible binding of the chlorine ions with gold atoms is mainly forbidden due to the increase of the hydroxyl groups concentration. This results in facilitating the movement of gold atoms from one facet to another facet and allowing the growth of specific branched gold structures. On the contrary, at high acidity, the reduction of gold ions could be inhibited and may induce delays in the branch expansion, leading to the growth of 2D planar structures.

Finally, pH represents one of the key factors to grow typical branched gold nanocrystals. In addition, a sufficient reductive activity of hydroxylamine molecules represents a precondition to ensure the proper arrangement of ions at the surface of particles, which allows the growth of dendritic-like structures.

This study has perfectly shown that the reduction is much faster at higher pH (close to neutral) contrary to lower pH, proving the pH-dependence of the reduction reactions and the structuring effect of the molecules at the surface during the growth of flowerlike structures.

Effect of the Reaction Temperature. Besides the pH effect, an appropriate reaction temperature remains also essential for the formation of Au dendritic nanocrystals, as this parameter greatly influences the reductive activity of the structuring agents and the system diffusion constant. [START_REF] Lin | Facile Synthesis of Catalytically Active Platinum Nanosponges, Nanonetworks, and Nanodendrites[END_REF] Experiments at different temperatures, varying from 20 to 90 °C with a 10 °C step, have been investigated. Temperature has been controlled with a closed double-jacketed glass cell and a heating bath. Figure 7 displays SEM images of the structured surfaces obtained at four different reaction temperatures.

It is observable that the reaction temperature does not really impact the hierarchical architecture of the individual flowers. Nevertheless, a low reaction temperature seems to form both alternative flowers and spheres, while only gold flower-like structures are visible at high reaction temperatures above 60 °C (Figure 7c). In addition, the observation of the SEM images shows that the nanoparticles' coverage rate seems to be mainly similar between 20 and 60 °C (Figure 7a-c), while an increase of the reaction temperature up to 90 °C causes a deep enhancement of the distribution of Au nanostructures (Figure 7e). It could be explained by the convergence of the reaction temperature close to the boiling point of the solvent (water). In this case, this phenomenon could cause a strong rise of the mixture entropy, which will enable a rapid reduction of the AuCl 4

-ions to Au 0 on the prepatterned samples. Previous work realized by Zhang et al. has shown that the reaction temperature impacts both the reducing power of the structuring agent and diffusion rate of additives in the medium. A low temperature seems to cause too slow of a reduction of small gold particles to ensure the three-dimensional growth of nuclei, while higher temperature increases the possibility to ensure three-dimensional growth of branched gold nanostructures. [START_REF] Zhang | Controllable Synthesis of Three-dimensional Branched Gold Nanocrystals assisted by Cationic Surfactant Poly(diallyldimethylammonium) Chloride in Acidic Aqueous Solution[END_REF] Consequently, the temperature represents an essential parameter to avoid aggregation of the structures and ensure the hierarchical arborescence of flowers. In particular, the high diffusion rate allows one to improve the seed density and separation of the structures, one from another. Control of the reaction temperature during the entire reaction allows formation of more homogeneous and well-dispersed nanostructures at the sensor surface.

In order to quantify these data, multifractal analysis has been realized. This analysis has allowed comparing of D 0 , D 1 , and D 2 values calculated for each SEM image of AuNFs surfaces realized at different reaction temperatures varying from 20 to 80 °C, as depicted in Figure 8. By comparing sample surfaces at each temperature, it has been observed that a higher temperature corresponds to a higher D 0 , which means that the surface coverage rate increases with temperature. Indeed, D 0 increases from 20 to 80 °C, from 1.27 up to 1.39; this corresponds to a higher apparent space coverage of flower-like structures, as observed in the SEM images. Then, D 1 is correlated to the Shanon entropy. A higher D 1 value meant that the degree of disorder increased as well. The disorder is increasing with the increase of surface coverage until 80 °C. Finally, the correlation dimension D 2 increases, too, with temperature; this behavior corresponds to a decrease of the Au clustering level (nanoflower aggregation).

The multifractal analysis has provided a unique tool to gain a better understanding of the microstructure growth and distribution onto the surface of samples. By studying the influence of temperature on the Au flower-like structures growth, information on and indirect quantification of the coverage rate, homogeneity, and cluster level on the surface have been obtained. Finally, a 60/70 °C reaction temperature has been resolved to grow flower-like structures onto prepatterned substrates. The distribution has been then optimized, leading to the formation of hyperbranched nanoflowers through a selforganization process under nonequilibrium conditions.

Effect of Gold Salt Concentration. The pH-dependent method offers highly interesting perspectives for the formation of Au flower-like structures. However, the very low coverage rate of the sensor surface by AuNFs after addition of a few tens of microliters of HAuCl 4 shows us the limit of the crystal growth process. However, by varying the amount of gold salts in the solution, the final distribution of the flowers could have been tuned easily. Here in a typical synthesis, the pH has been fixed close to 6.5 and the temperature controlled by the doublejacketed glass cell at 70 °C, with an aqueous gold solution (25 mM) and an aqueous NH 2 OH solution (20 mM). Different amounts of gold solution have been studied by varying the injection from 50 to 500 μL.

As expected, the density of AuNFs on the substrates increases as a function of the Au salt amount injected into the growth solution. This parameter allows a more precise control of the density of AuNFs on the samples, but it does not avoid the aggregation of flowers. However, concentration boundaries could have been observed where formation of NFs is more important. Indeed, our results show that the more relevant gold salt solution amount to avoid structure aggregation while ensuring a high AuNFs coverage rate on the surface is in the interval of 100-200 μL of HAuCl 4. This result is reinforced by the fact that, with a too high quantity of metallic salts, the shape of the structures starts to be affected, too (Figure 9f), and aggregation increased as well.

Electrocatalytic Activity of Au Dendrites. The optical properties of AuNF structures deposited onto the sensor surface have been investigated. The morphology of dendritic nanostructures, and especially the presence of their neighboring branched blocks and numerous tips at the branch apex, allows one to induce a local electromagnetic field under irradiation of a laser at a perfect wavelength. In such conditions, the appearance of a large amount of hot-spots at the apex of the flower branches or in between closed branches contributes to the enhancement of the induced localized plasmon field. In order to amplify the electrocatalytic activity of the nanostructured surface with both the required structure and cover density, optimized amounts of reactants and particular reaction parameters have been determined for the preparation of prepatterned samples [pH 6.5, T = 70 °C, 100 μL of HAuCl 4 aqueous solution (25 mM)]. This specific electrocatalytic activity could have been followed by optical spectroscopy and also voltammetry techniques.

ADECA Method. The ADECA colorimetric method allows one to quantify the number of active sites on a surface and evaluate the detection performance of AuNF structured surfaces. Three absorption times of CBB molecules on these surfaces have been recorded with UV-visible spectroscopy. Figure 10 represents SEM images of AuNFs and the variation of the absorbance of CBB on these structures as a function of three different absorption times. UV-visible spectra display the presence of a broad peak at around 650 nm, corresponding to the basic form of CBB. The absorbance values of the maximum peaks increase with increasing absorption time, ranging from 0.03 and 0.06 au. The corresponding number of active sites (AS) calculated from eq 5 are 1.27 × 10 15 , 1.35 × 10 15 , and 2.16 × 10 15 AS cm -2 for 5, 30, and 300 min of CBB absorption, respectively. As a result, the number of active sites increases as a function of absorption times, as expected. Nonetheless, an average of 10 15 AS cm -2 has been estimated for flower-like structures depicted in Figure 10a.

SERS Activity of Au Flower-like Structures. Anisotropic Au nanostructures have been usually characterized by Raman spectroscopy to evaluate their SERS effects, depending on their size and morphology.

Figure 11a displays the UV-visible spectrum of MB and Figure 11b shows the Raman spectra of MB molecules at different molar concentrations adsorbed onto AuNFs structures. The UV-visible adsorption band of MB leads to the appearance of a main intense peak at 649 nm, as displayed in Figure 11a. Investigation of the plasmon resonance adsorption of AuNFs has been done by integrating sphere spectroscopy. Results of the evolution of the reflectance of AuNFs structured surfaces vs wavelength display the appearance of a minimum peak at 500 nm (Figure S2, Supporting Information). As a result, neither the plasmon band of the AuNFs nor the adsorption wavelength of MB are in accordance; the analysis of the SERS effect of MB adsorbed onto AuNFs will be with regard to SERS conditions (and not SERRS ones). [START_REF] Xie | The Synthesis of SERSactive Gold Nanoflowers Tags for in Vivo Applications[END_REF][START_REF] Xiao | Surfactant-assisted, Shape-controlled Synthesis of Gold Nanocrystals[END_REF] The analysis of the Raman spectra of MB adsorbed onto AuNFs displays several main characteristic peaks, which are of interest for a simple study of the enhancement factor (EF). The two main intense peaks correspond to the vibration of the C-N-C skeleton bonding at 445 cm -1 and C-C bonding at 1626 cm -1 . In addition, results show that the SERS signal increases greatly when the concentration of MB increases from 10 -9 to 10 -5 M.

The enhancement factor per molecule (G) has been calculated using the formula

G I I N N SERS ref ref SERS = (8)
where I SERS is the intensity of the SERS spectrum, I ref is the intensity of the Raman spectrum of the same peak, N ref is the number of molecules excited in the reference sample, and N SERS is the number of molecules covering the structured sample within the laser spot area. Consequently, I SERS /N SERS is the SERS integrated intensity of some typical bands reduced to a single adsorbed molecule, and I ref /N ref is the corresponding spontaneous Raman intensity of the same band. To determine the values of those four parameters, a similar procedure to that described in the literature has been followed. [START_REF] Xu | Interparticle Coupling Effects in SERS[END_REF] Using an 100× objective lens, the area of the laser spot size has been estimated as 80 μm 2 . Moreover, the area of a single molecule of MB has been calculated to be approximatively 2 nm 2 . Thus, the number of MB molecules adsorbed on the 80 μm 2 sample (under the laser spot) has been evaluated as N SERS = 4 × 10 7 . In such conditions, the scattering volume of the laser containing the MB molecules for the reference sample was evaluated as 79 000 μm 3 . As a result, the number of MB molecules included in this scattered area, for a 10 -3 M solution of MB in water, has been estimated to be N ref = 4.75 × 10 10 . Therefore, the enhancement per molecule G was thus evaluated using the following equation:

G I I 1.2 10 3 SERS ref = × (9)
The enhancement factors per molecule (G) have been calculated for the different main Raman peaks characteristic of the MB molecules and are displayed in Table 1. Results show that the lower the solution concentration is, the lower the enhancement factor G is, as expected. However, those enhancement factor data indicate the high efficiency of the asprepared flower-like gold nanostructures as SERS supports. Indeed, the literature suggests that the dendritic nanostructures provide high enhancement due to their sharp corners, edges, high surface-to-volume ratio, and nanoscale junctions, which can be considered as "hot-spots". Hence, the morphology of gold structures is able to induce dramatically different enhancement of the Raman signal and could dif ferentiate chiral molecules, as observed in the literature. [START_REF] Wang | A Chiral Signal Amplified Sensor for Enantioselective Discrimination of Amino Acids Based on Charge Transfer-Induced SERS[END_REF][START_REF] Mensch | The influence of the amino acid side-chains on the Raman optical activity spectra of proteins[END_REF] Catalytic ActivityGlutathione/Hexadecanethiol. The electrocatalytic activities of that gold dendritic structure have also been investigated by cyclic voltammetry. The first "global" electrochemical measurements have been performed in an electrolytic bath (0.1 M Na 2 SO 4 ) at a scan rate of 20 mV/s. Figure 12 displays the different voltammograms corresponding to the reference and nanostructured samples: stainless steel plates (SS, black curve) or stainless steel plates covered by PS-b-P4VP (blue curve), PS-b-P4VP + gold seeds (red curve), and PS-b-P4VP + gold seeds + AuNFs (green curve).

The cyclic voltammograms displayed in Figure 12a reveal a broad large peak between 0.6 and 0.9 V. It may correspond to the oxidation of gold into gold salts. Moreover, another oxidation reaction takes place after 1.2 V, which corresponds to the oxidation of water into oxygen in an acidic electrolyte or the oxygen evolution reaction (OER). This simple experiment allows one to show the difference observed in the electrochemical response of the working electrode varying from a sample just coated with copolymer and seeds or alone and a sample covered with AuNFs. [START_REF] Zhang | Novel potential and current type chiral amino acids biosensor based on L/D-T handed double helix carbon nanotubes@polypyrrole@Au nanoparticles@L/ D-cysteine[END_REF] The overall intensity strongly increases after growth of AuNFs on the surface. The corresponding oxidation reactions are the following:

Au 4Cl AuCl 3e 0 4 + ↔ + - - - (10)
2H O O 4H 4e 2 2 ↔ + + + - (11)
These preliminary results show that the electrochemical activity is significantly modified by the growth of the AuNFs onto the substrate. Dendrites might appear to be good candidates for enhancing the electrocatalytic properties of the active surface and allowing a significant detection of molecules.

A second "local" electrochemical investigation has been performed on samples coated by PS-b-P4VP and Au-flower-like structures. Three different samples have been immersed in a 10 -9 M aqueous solution of gluthatione (GSH) and hexadecanethiol (HDT). Figure 12b displays the evolution of the electrochemical peaks as a function of the adsorbed molecule compared to a reference (substrate without adsorption).

The distribution of AuNFs onto the substrates is quite similar on all the investigated samples. In the entire voltammogram, four peaks appear. Two main oxidation peaks can be observed at 1.2 and 0.5 V and two other reduction peaks are seen at 0 and -0.5 V. The black curve, corresponding to the reference sample, displays four peaks with a very low intensity. This might be explained by the fact that no molecules have been adsorbed on it due to the absence of structuration. The two other curves (blue and red) represent the voltammograms of AuNFs after immersion into a GSH or HDT solution, respectively. A strong increase of the oxidation hysteresis has been observed on both voltammograms after adsorption of the different molecules onto the AuNFs. The slope of the HDT voltammogram is higher than that of the GSH-coated AuNFs, showing a difference in the kinetics of the oxidation reaction at the electrode. Investigation has been focused on the oxidation of gold reaction (OGR) peak appearing at a potential superior or equal to 1.2 V. Indeed, the AuNFs structuration is able to discriminate the OGR peak as a function of the adsorbed molecule. [START_REF] Zhang | Selective recognition of Histidine enantiomers using novel molecularly T imprinted organic transistor sensor[END_REF][START_REF] Arnaboldi | Review Article Enantioselective selectors for chiral electrochemistry and electroanalysis: Stereogenic elements and enantioselection performance[END_REF] The maximum of the peak for the OGR appears at 1.2 V for GSH and has been shifted to higher potential for HDT. Results shows that this OGR can occur at the same potential as the oxidation of GSH and HDT molecules. Indeed, the current intensity has reached 5.2 × 10 -5 A for GSH and 1.1 × 10 -4 A for HDT. Additional investigations should be done to show the influence of the structuration on the electrochemical response, especially the tip effect, and the limit of detection. However, these preliminary results are quite encouraging and show the sensitivity and selectivity of AuNFs through their electrochemical response after adsorption of a specific simple organic molecule.

■ CONCLUSION

In summary, a simple and controlled templated method has been developed for the synthesis of Au dendritic nanostructures.

This technique, contrary to many others, remains cheap and allows one to grow nanostructures on large areas. Beside the stainless steel plates, which could be replaced by other substrates (Si wafers, ITO, 306L stainless steel), the template fabrication can offer the possibility to deposit a soft template based on the phase separation of the mixture PS-b-P4VP/HABA at low cost, with an easy-to-make process and significant reproducibility. This nanoporous template enables one to have a better control of the distribution of gold nanostructures on the surface of samples initially by the insertion of gold seeds as growing points into the nanopores.

The resolution of physicochemical parameters (pH, temperature) and the amounts of reactants (HAuCl 4 , NH 2 OH) has enabled as better control the distribution of gold flower-like structures by the hydroxylamine approach. Both morphology and coverage rate have been first characterized by the monofractal and multifractal analysis method, respectively, and then optimized on the overall surface of samples. Particularly, the multifractal analysis has provided a tool to gain a better understanding of the microstructure growth and distribution.

The as-prepared Au nanoflowers displayed excellent electrocatalytic properties as highly Raman-active structure surfaces and could be used as microsensors, allowing optical detection of low concentrations of adsorbed organic molecules (methylene blue). Furthermore, the electrochemical experiments suggested that the sensitivity of the surface is dependent on the nature of both adsorbed molecules. A perspective of this work would be to measure the photocatalytic activity at the apex of the tip simultaneously with an electrocatalytic reaction. [START_REF] Hentschel | Chiral plasmonics[END_REF] In the long-term, a microelectromechanical systems (MEMs) generic platform could be developed to allow detection of very low traces of targeted molecules (amino acids) or biological bodies (cell, virus). The final objectives would be to obtain qualitative and quantitative data on the adsorbed molecules on the nanostructured surfaces incorporated in a microship. The physicochemical properties of the Au nanoflowers have shown excellent sensitivity (SERS) and durability (gold), which could be expanded by functionalization of the tips. MEMs are gaining notoriety for biosensing applications, due to their small size, low power consumption, and high integrability into microelectronic devices for implantable sensing applications.

Figure 1 .

 1 Figure1. Generalized dimensions D q versus moment q for a multifractal and a monofractal object.

Figure 2 .

 2 Figure 2. (a) UV-visible spectra of a mixed solution of CBB and T2HCl buffer (25/2) with different amounts of CBB and (b) plot of the maximum absorbance of CBB at 650 nm vs CBB concentration according to Beer-Lambert law (i.e., calibration curve).

Figure 3 .

 3 Figure 3. Schematic representation of AuNFs growth onto a nanopatterned surface by a template-mediated method.

Figure 4 .

 4 Figure 4. SEM images of the as-prepared Au flower-like structures obtained on stainless steel plates. (a) Scale bar: 2 μm. (b) Scale bar: 500 nm.

Figure 5 .

 5 Figure 5. (a) HRTEM image of a trunk with symmetric leaves with the corresponding SAED pattern. (b) HRTEM of the area of a leaf. (c) HRSEM images of as-prepared Au flower-like structures. (d) EBSD analysis of one flower-like structure with corresponding colors. (e) Associated XRD pattern of gold flower-like structures.

Figure 6 .

 6 Figure 6. SEM images of different gold nanostructures grown onto prepatterned substrates by the wet chemical method as a function of the solution pH: (a) pH 4, (b) pH 5, (c) pH 6, (d) pH 7, and (e) pH 8. The pH has been controlled with an ammoniac aqueous solution (500 mM). Scale bar: 500 nm. Monofractal dimensions D have been calculated for each structure.

Figure 7 .

 7 Figure 7. Typical SEM images of the Au flower-like structures grown onto prepatterned silicon wafers at different temperatures: (a) T = 20 °C, (b) T = 40 °C, (c) T = 60 °C, and (d) T = 80 °C. Scale bar: 200 μm. (e) Graph representing the average percentage coverage rate of gold structures (extracted from SEM images) as a function of reaction temperature.

Figure 8 .

 8 Figure 8. Multifractal analysis of SEM images of AuNFs structures. (a) Generalized dimension spectrum over the [-5; 5] moment q range for different reaction temperatures varying from 20 to 80 °C and (b) generalized multifractal dimensions D q over the [0; 2] q range with Y error bars of substrate nanostructured surface at different temperatures from 20 to 80 °C.

Figure 9 .

 9 Figure 9. SEM images of Au flower-like structures grown onto prepatterned stainless steel substrates and synthesized at different amount of gold salt solution: (a) 50 μL, (b) zoom in of panel a, (c) 200 μL, (d) zoom in of panel c, (e) 500 μL, and (f) zoom in of panel e.

Figure 10 .

 10 Figure 10. (a) SEM image of AuNFs deposited by a chemical pH-dependent method onto prepatterned stainless steel substrates. (b) Corresponding UV-visible spectra recorded at three adsorption times in CBB solution: 5, 30, and 300 min.

Figure 11 .

 11 Figure 11. (a) UV-vis spectrum of MB and (b) SERS spectra of MB adsorbed on gold flower-like structures at different concentrations. Reference = 50 nm gold coating deposited on a stainless steel substrate.

Figure 12 .

 12 Figure 12. (a) Global cyclic voltammograms (0.1 M Na 2 SO 4 , pH 5, 20 mV/s, cycle 10) of stainless steel substrates alone (black curve) and those prepatterned with PS-b-P4VP (blue curve), coated with gold seeds (red curve), and finally covered with gold flower-like structures (green curve). (b) Local cyclic voltammograms of stainless steel samples covered by gold flower-like structures at a scan rate of 20 mV/s in a solution of 0.1 M Na 2 SO 4 reference sample (black curve), sample immersed in a hexadecanethiol solution at a concentration of 10 -9 M (red curve), and sample immersed in a glutathione solution at a concentration of 10 -9 M (blue curve).

Table 1 .

 1 Band Assignments and Enhancement Factors (G) Calculated from SERS Spectra Displayed in Figure11b

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https://dx.doi.org/10.1021/acs.langmuir.0c01857Langmuir XXXX, XXX, XXX-XXX B

https://dx.doi.org/10.1021/acs.langmuir.0c01857Langmuir XXXX, XXX, XXX-XXX D

https://dx.doi.org/10.1021/acs.langmuir.0c01857Langmuir XXXX, XXX, XXX-XXX

https://dx.doi.org/10.1021/acs.langmuir.0c01857Langmuir XXXX, XXX, XXX-XXX H

https://dx.doi.org/10.1021/acs.langmuir.0c01857Langmuir XXXX, XXX, XXX-XXX I

We gratefully acknowledge financial support from the Centre Nationale d'Etudes Spatiales, the Fondation de l'Ecole de Chimie de Mulhouse, and the Centre National de


J Cenes

G Teyssedre

S Le Roy

L Berquez Laplace

C Moreau

P Hondaâ

V Eriksson

W Loyens

Assessment of Ageing in the Insulation of HVDC Model Cables under Thermo-electric Stress

Keywords: XLPE, HVDC, Ageing, Space charge, cables, Conductivity, Polarity reversal

The assessment of insulating materials regarding space charge features is one important aspect of HVDC cable development. Whereas most of space charge characterization is achieved under relatively short stressing time, it is important to understand if there is an evolution of materials behavior during long time under stresses, including service stresses such as combination of electrical and thermal stresses along with polarity reversal. Our purpose in this communication is to present the implementation of long term ageing tests on crosslinked polyethylene materials in such a way that relevant and valuable information can be gained as a function of stressing time. The designed stress cycle on model cables encompasses maximum fields up to 90 kV/mm at the inner semiconductor and temperature up to 100°C with repetitive grounding or polarity reversal. The stressing time is up to 18 months. The electrical and physicochemical characterizations techniques as well as the first ageing results are presented.

I. INTRODUCTION

One important property in the development of polymerinsulated cables for HVDC energy transmission is space charge build-up in the material that can potentially induce substantial field redistribution into the insulation and alter long-term performance or reliability [1]. Accordingly, one of the main properties probed when developing insulating materials and related processes is the propensity of materials to accumulate charges. However, most of published space charge studies on insulating materials involve relatively short stressing times. The approach does not necessarily account for the possible evolution of material properties during long time stressing, especially if thermal and electrical stresses are combined. Besides space charge accumulation, it is also important that the electrical conductivity of the material remains at a low and controlled level during service. In addition, the polymer structure should also remain stable in service conditions. The purpose of this paper is to present the long term ageing tests performed on crosslinked polyethylene (XLPE) materials in such a way that relevant and valuable information can be gained as a function of time and repetitive variations of the DC stress.

II. SAMPLES AND PROTOCOLS

The samples used for ageing are model cables (Fig. 1), produced by triple extrusion using crosslinked polyethylene insulation and semiconductive shield using grades developed for the currently most demanding commercial HVDC applications. The main advantage of this type of samples compared to plaques is the cylindrical geometry. Accordingly, the tests to be applied have to be adapted to this geometry. The thermal and electrical ageing conditions were designed in order to study the effect of isothermal ageing up to 100°C, thermal gradient, and the effects of grounding and polarity reversal. Ageing is planned for up to 18 months and at electrical stresses up to 90 kV/mm at the interface between the insulation and the inner semiconductive shield. Given the geometry of the cable, when applying 90 kV to the conductor, the field at the inner semiconductive shield is 90 kV/mm, at the outer semiconductive shield 45 kV/mm and the average field E av is 60 kV/mm. For each condition, electro-thermal ageing is compared to thermal ageing alone in order to distinguish the effects. As electrical characterization, space charge measurements, either on-line during ageing or off-line on aged samples, and DC conductivity are performed. Physical and chemical analyses are planned on samples taken at intermediate times in order to monitor the potential change of the properties of the XLPE during ageing.

A. Ageing protocol

After extrusion, model cables were degassed for 10 days at 70°C to remove XLPE crosslinking decomposition products [2]. Two ageing bench set-ups are run in parallel. In bench 1, relatively short ageing times are used, typically 42 days, in which a thermal gradient (TG) is applied to the cables and either grounding (G) or polarity inversion (IP), are applied periodically. Space charge profiles are measured periodically during the ageing. Samples are aged under different stresses with grounding or polarity reversal at Eav = 60 kV/mm or 45 kV/mm. The temperature Tin at the conductor is 90°C or 70°C, the corresponding calculated temperature difference between inner and outer semiconductive shields are ΔT = 5.7°C and 4.3°C, respectively. Fig. 2 shows an example of polarity inversion procedure. Fig. 3 shows the part for the PEA measurement on test bench 1. The voltage is applied using two high voltage power supplies of inverse polarity, both connected to a high voltage switch for polarity reversal. The temperature gradient is applied using Joule effect in the cable conductor through a current transformer. Three cables are installed in parallel: i/ cable 1 for on-line space charge measurement during electro-thermal ageing; ii/ cable 2 for sampling for offline electrical and physicochemical characterization during electro-thermal ageing; iii/ cable 3 same as cable 2 for thermal aging only.

In parallel to the ageing performed in bench 1, a cable is aged for 18 months in bench 2 at a constant average stress of 60 kV/mm and under isothermal condition (T=100°C) in an oven. A reference cable is also aged in parallel but only under thermal stress at T=100°C. A sampling is made every 42 days for space charge, conductivity, and physicochemical off-line measurements.

B. Space charge measurements

Space charge measurements are realized by means of pulsed electro-acoustic (PEA) method. The average applied field is 60 kV/mm in the first results presented. Measurements are recorded under thermal gradient. It is also important to note that the cable 1 in the PEA test cell has a substantially lower temperature compared to the cable part outside the cell due to the fact that it acts as a thermal sink. Therefore 'online' PEA profiles for cable 1 are measured at the same electrical stress and same ΔT = 5.7°C but at different average temperature (Tav = 36°C) compared to cable 2 (Tav = 83°C). Each space charge measurement sequence lasts for 24 h in which records are realized every 100 s under constant voltage. For off-line measurements (on samples aged in bench 2), the average field is 45 kV/mm, with a temperature gradient of 4.3°C. Each polarization and depolarization step lasts for 4 h. The pulse is generated with a frequency of 5 kHz and an amplitude of 1.5 kV on a separated cable (pulse cable), cf. Fig. 3.

C. Current measurement

Current measurement is carried out on cable sample of 90 cm long. The outer semiconductive material is stripped at both ends of the cable over 30 cm. Guard electrodes of 1 cm width are set close to the section on which the current is measured, which is about 30 cm. Measurements are performed under average DC electric field from 5 to 20 kV/mm in isothermal conditions at T = 70°C in an oven. The voltage is applied to the conductor. Current values are recorded every 2 s during the polarization/depolarization steps which last for 4 h each.

D. Physicochemical measurement

Besides electrical characteristics, different techniques are implemented to evaluate the physical and mechanical properties of the insulation material under aging. Dynamic Mechanical Thermal Analysis (DMTA) is used to follow the evolution of the mechanical properties of XLPE (real E', and imaginary E" moduli and damping tanδ [START_REF] Parpal | Proc. IEEE. Internat. Symp. Electr. Insul. (ISEI)[END_REF]). Specimens of dimension 50 7 0.5 mm³ are cut parallel to the axis of the cable (see Fig. 1) using a slicing machine. DMTA measurements are made in traction mode at a constant frequency of 1 Hz, in the temperature range from 25 to 120°C. Thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC) are used respectively to detect potential changes in the degradation kinetics and in the crystallinity degree and melting point as a function of ageing conditions (temperature ramp=10°C/min from 25°C to 180°C). Finally Fourier Transform Infrared Spectroscopy (FTIR) is used to evaluate the effects of aging on the macromolecular structure of the studied insulation. The main chemical modification is the formation of carbonyl groups by oxidation of the polymer [START_REF] Gulmine | [END_REF]. The samples for FTIR measurements are the same as for DMTA. Those for TGA and DSC are prepared in the same way as for DMTA, i.e. in the form of specimens and then cut again into adequate shape.

III. BENCHMARK

The properties of XLPE, before ageing, on cables have been carefully determined as they are the baseline for the entire study. From DSC data, the degree of crystallinity is 44% and the melting temperature is 107.8 °C.

A. Space charge results

The measurement is carried out over a period of 4 h of polarization and 4 h of depolarization for each average field of 30, 50, and 60 kV/mm. The charging and discharging steps at the different fields were applied in series on the same sample. The space charge patterns are shown in Fig. 4. For an average field of 30 kV/mm (Fig. 4a), charges are difficult to observe during polarization. However, positive charges are visible during short-circuit in the first half of the insulator. This indicates that these charges have been generated during polarization at the inner semiconductive shield and have moved towards the outer electrode. The absence of negative charges in the vicinity of the outer semiconductive shield is also noticeable. The density of injected charges is greater as the electric field is increased. Under 50 kV/mm, the positive space charge region occupies nearly all the insulation. No negative charges are observed. The resorption of positive charges during the depolarization phase is clear in Fig. 4. For stresses of 50 and 60 kV/mm, the charge amount appears higher close to the outer semiconductive shield.

Under thermal gradient, the gradient in electrical conductivity produces a field redistribution given under steady state conditions by equation 1 [5]:

( ) ( ) r r r E r E c c c σ σ = (1)
where Ec and σc are the electric field and the conductivity at the reference position rc. The corresponding 'geometrical' space charge distribution is of the form:

( ) ( ) ( ) dr r r r E E div r g σ σ ε ε ρ ∂ - = ⋅ = ) ( ) ( (2)
where ε is the dielectric permittivity. For a positive voltage applied to the conductor, and supposing that σ(r) decreases with r, the space charge is positive as is actually measured. However the accumulation of charges close to the outer semiconductive shield is not predicted by the expression.

B. Experimental results on conductivity

Fig. 5 shows experimental results of polarization current vs. time at 70°C for different stresses. The current has been normalized based on the current value at 70°C and 5 kV/mm, after 4 h of polarization. The steady state conduction is approached after 4 h of charging for the highest constraints. The transient current for 5 kV/mm has a different shape presumably because of the thermal history. The conductivity was estimated from current taken after 4 h of polarization. At 70°C, the DC conductivity is relatively low and increases substantially with the field. So the field in the cable will be redistributed according to the temperature and field dependencies of the conductivity, besides potential space charge accumulation.

IV. AGEING RESULTS

A. Space charge results after ageing

These results arise from the test bench 2, where the ageing is performed under isothermal conditions (100°C, and an average field of 60 kV/mm). During the space charge characterization, i.e. 4 h polarization at 45 kV/mm and 70°C, we observed in Fig. 6a and6b, for the thermal cable, positive charges in the bulk. The charge density intensifies near the outer semiconductive shield with ageing time. For the electrothermal cable (Fig. 6c and6d), a small positive heterocharge is detected at the vicinity of the outer SC. This density seems to increase with ageing time, but remains very low compared to the thermal cable.

B. Experimental results on conductivity after ageing

Fig. 7 shows the relative conductivity as a function of applied average field for different ageing times with a thermal gradient or under isothermal conditions. The relative conductivity is calculated based on the conductivity of the benchmark measured at 70°C and 5 kV/mm. Data from the benchmark and obtained for sample aged under thermal stress alone are added for comparison. We observe that the trend for the conductivity is a decrease with ageing time for the 4 cases tested. The effect is more important with the thermal stress than with electro-thermal stresses. Cables aged under thermal stress alone, being under thermal gradient or isothermal conditions, have a conductivity of a decade higher compared to the ones aged under electrothermal stress. So the material acts as if the electric field was driving charges outside the sample during stressing. It would be consistent with the relative in charge amount obtained in Fig. 6

V. CONCLUSION

The methodology settled for evaluating thermoelectric ageing in model cables has been described. On-line space charge measurements are achieved during ageing but not in conditions of nominal thermal stress. The implemented means provide information on space charge behavior, conductivity and physicochemical parameters. Preliminary results with stressing under relatively severe conditions in terms of field indicate a substantial decrease in conductivity under thermoelectric stress compared to thermal stresses. The space charge patterns are different considering thermal and electro thermal stress. In the forthcoming steps the impact of polarity inversion ageing on space charge will be investigated comparatively to grounding ageing.

Fig. 1 .

 1 Fig. 1.Model cable geometry and sampling for physicochemical analyses.

Fig. 2 .

 2 Fig. 2. Schematic of polarity inversion cycle (+U0 during 24h and 2 IP/hour during 24h = 48IP)

Fig. 3 .

 3 Fig. 3. Schematic of PEA test bench for space charge measurements on model cables.

Fig. 5 .

 5 Fig. 5.Polarization currents under different stresses at 70°C.

Fig. 4 .

 4 Fig. 4. Space charge mapping for a 4h / 4h polarization / depolarization protocol of the insulator under thermal gradient of ΔT=4.3°C for average fields of a) 30 kV/mm, b) 50 kV/mm; c) 60 kV/mm.

  compared to Fig 4c (though the temperature was not 90°C in space charge data).

  Relative conductivity vs. applied average field for different stresses and ageing times: a) ageing with thermal gradient; b) ageing in isothermal conditions.

TABLE I .

 I RELATIVE CONDUCTIVITY AS FONCTION OF THE AVERAGE

		FIELD AND TEMPERATURE
	Field (kV/mm)	5	10	15	20
			Conductivity (relative units) a
	T=25°C	0.122	0.139	0.134	0.135
	T=70°C	1	2.08	3.83	5.43

a Normalized based on conductivity value at 70°C and 5 kV/mm.