Wiktoria Lipińska

Vita Saska

Katarzyna Siuzdak

Jakub Karczewski

Karol Zalęski

Emerson Coy

Anne De Poulpiquet

Ievgen Mazurenko

Elisabeth Lojou
email: lojou@imm.cnrs.fr

Karol Załęski

Interaction between bilirubin oxidase and Au nanoparticles distributed over dimpled titanium foil towards oxygen reduction reaction

Keywords: Au nanoparticles, Electrode nanostructuration, Enzymes, Catalysis, Self-Assembled-Monolayers, Bioelectrochemistry, Bilirubin oxidase

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Redox enzymes are very active and extremely selective catalysts toward specific reactions allowing their use in bioelectrochemical devices such as biosensors, bioreactors or biofuel cells [START_REF] Beaufils | From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes[END_REF][START_REF] Kano | Fundamental insight into redox enzyme-based bioelectrocatalysis[END_REF]. Multi-copper oxidases (MCO) are among the most explored ones because of their ability to oxygen reduction reaction (ORR) into water [START_REF] Mano | O2 Reduction in Enzymatic Biofuel Cells[END_REF]. Within MCO family, apart from laccases [START_REF] Aleksejeva | Electrochemistry of a high redox potential laccase obtained by computer-guided mutagenesis combined with directed evolution[END_REF][START_REF] Zhang | Application of eukaryotic and prokaryotic laccases in biosensor and biofuel cells: recent advances and electrochemical aspects[END_REF] and copper efflux oxidases [START_REF] Mazurenko | Electrochemistry of copper efflux oxidase-like multicopper oxidases involved in copper homeostasis[END_REF], bilirubin oxidases (BOD) are characterized by high activity and stability at neutral pH with low overpotential towards ORR [START_REF] Al-Lolage | Site-Directed Immobilization of Bilirubin Oxidase for Electrocatalytic Oxygen Reduction[END_REF][START_REF] De Poulpiquet | Mechanism of Chloride Inhibition of Bilirubin Oxidases and Its Dependence on Potential and pH[END_REF][START_REF] Gentil | Direct Electrochemistry of Bilirubin Oxidase from Magnaporthe orizae on Covalently-Functionalized MWCNT for the Design of High-Performance Oxygen-Reducing Biocathodes[END_REF]. BODs contain four copper centers, namely a Cu T1 center and a trinuclear center (TNC) composed of Cu T2 and T3. ORR by BOD at an electrochemical interface involves a first electron transfer from the electrode to Cu T1 site, intramolecular electron transfer from Cu T1 to TNC site and finally O2 reduction to H2O at the TNC [START_REF] Sekretareva | Electron Transfer to the Trinuclear Copper Cluster in Electrocatalysis by the Multicopper Oxidases[END_REF][START_REF] Jones | Electron transfer and reaction mechanism of laccases[END_REF]. One challenge remains the electron channeling from the electrode to the Cu T1 binding site of immobilized enzymes. Gold and carbon-based are the most popular electrode materials for such efficient immobilization [START_REF] Mazurenko | Recent advances in surface chemistry of electrodes to promote direct enzymatic bioelectrocatalysis[END_REF][START_REF] Tominaga | High electrochemical stability of hyperthermophilic archaeal multicopper enzyme adsorbed on gold electrodes compared to fungal laccase[END_REF][START_REF] Lalaoui | Nitrob enzoic acid-functionalize d gold nanoparticles: DET promoter of multicopper oxidases and electrocatalyst for NAD-dependent glucose dehydrogenase[END_REF][START_REF] Takahashi | Improved direct electron transfertype bioelectrocatalysis of bilirubin oxidase using porous gold electrodes[END_REF][START_REF] Hitaishi | Controlling Redox Enzyme Orientation at Planar Electrodes[END_REF]. Further application of nanomaterials such as carbon nanotubes, nanoporous gold and gold nanopartic les (AuNP), increases surface area, hence potential anchorage sites for enzyme immobilizatio n, leading to enhanced catalytic currents. Furthermore, depending on their shape and size, nanomaterials can impact the conformation of proteins hence their stability [START_REF] Chen | New Insights on the Size -Dependent Inhibition of Enzymes by Gold Nanoparticles[END_REF][START_REF] Kizling | Gold nanoparticles in bioelectrocatalysis -The role of nanoparticle size[END_REF]. Last, nanomaterials can be chemically modified to introduce additional functional groups on the surface and tune the interfacial properties for the grafting of enzymes in a conformation suitable for electrocatalysis. In the particular case of AuNPs, such functionalization can be efficie ntly realized through self-assembled-monolayers (SAM) of thiols [START_REF] Chumillas | Comprehensive Study of the Enzymatic Catalysis of the Electrochemical Oxygen Reduction Reaction (ORR) by Immobilized Copper Efflux Oxidase (CueO) From Escherichia coli[END_REF][START_REF] Climent | Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO[END_REF][START_REF] Gutierrez-Sanchez | Efficiency of Enzymatic O-2 Reduction by Myrothecium verrucaria Bilirubin Oxidase Probed by Surface Plasmon Resonance, PMIRRAS, and Electrochemistry[END_REF][START_REF] Dagys | Oxygen electroreduction catalysed by laccase wired to gold nanoparticles via the trinuclear copper cluster[END_REF]. Using this strategy, it was especially highlighted the role of electrostatic interactions in the oriented immobiliza tio n of the fungal Myrothecium verrucaria BOD (Mv BOD) toward a high number of molecules catalyzing O2 reduction with a high turnover [START_REF] Hitaishi | Electrostatic-Driven Activity, Loading, Dynamics, and Stability of a Redox Enzyme on Functionalized-Gold Electrodes for Bioelectrocatalysis[END_REF]. It was also shown an optimum enzyme coverage for electrocatalysis between rigid monolayer and protein crowding [START_REF] Gutierrez-Sanchez | Efficiency of Enzymatic O-2 Reduction by Myrothecium verrucaria Bilirubin Oxidase Probed by Surface Plasmon Resonance, PMIRRAS, and Electrochemistry[END_REF][START_REF] Mcardle | Optimizing the Mass-Specific Activity of Bilirubin Oxidase Adlayers through Combined Electrochemical Quartz Crystal Microbalance and Dual Polarization Interferometry Analyses[END_REF]. But the reason underlying this observation is still obscure. To get insight into this, surface patterning may allow tuning in a controlled manner the spatial distribution of enzymes on the surface [START_REF] Wu | Advances in nucleic acid architectures for electrochemical sensing[END_REF].

Toward this goal, a nanosecond laser interaction with nanometric gold films deposited on indium tin oxide (ITO) support was performed to generate in situ AuNPs with different sizes and densities [START_REF] Hitaishi | Nanosecond Laser-Fabricated Monolayer of Gold Nanoparticles on ITO for Bioelectrocatalysis[END_REF]. However, the size of the obtained nanoparticles prevented any study on the effect of the enzyme coverage on the electrocatalysis. Moreover, the ITO substrate itself was shown to allow MCO-based catalysis, complexifying the analysis of the electrochemical signal.

Another way to generate AuNPs is the furnace [START_REF] Lipinska | The optimization of enzyme immobilization at Au-Ti nanotextured platform and its impact onto the response towards glucose in neutral media[END_REF] or laser dewetting [START_REF] Lipinska | Electrochemical glucose sensor based on the glucose oxidase entrapped in chitosan immobilized onto laser-processed Au-Ti electrode[END_REF] of thin gold films sputtered over a nanostructured titanium platform received by chemical etching of previously anodized TiO2 nanotubes. The anodization process followed by chemical etching of TiO2 nanotubes leads to the formation of well-organized array of Ti dimples that reproduce the bottom part of nanotubes. Following the dewetting process, AuNPs are located in the Ti dimples, their size being less than 100 nm. Such electrodes were previously applied for glucose detection by glucose oxidase embedded in chitosan matrix [START_REF] Lipinska | Electrochemical glucose sensor based on the glucose oxidase entrapped in chitosan immobilized onto laser-processed Au-Ti electrode[END_REF]. The objective was mainly towards enhanced performances of glucose biosensing, and no tuning of the AuNPs size was researched. As far as we know, no study on other enzymes deposited on AuNPs formed on nanostructured titanium foil has been reported.

The goal of the current work is to study Mv BOD immobilization on various sizes of AuNPs deposited on nanostructured Ti platform, and evaluate the effect on enzyme activity toward ORR. The size of AuNPs was controlled by the thickness of the gold layer sputtered onto the dimpled Ti substrate. After in depth characterization of the nanomaterials, the electrocatalytic activity of the electrode material with anchored Mv BOD was evaluated via cyclic voltammetry and chronoamperometry measurements. It is proved that the thickness of the initial gold film and in consequence the size of AuNPs used for further enzyme immobilization has a significant influence on the catalytic current both in terms of rate of the interfacial electron transfer process and on the stability of the bioelectrocatalytic signal in high ionic strength electrolytes.

Experimental

Materials and Reagents

Titanium foil (99.7 %, Strem), acetone (99.5 %, Chempur), ethanol (96 %, Chempur), ammonium fluoride (98%, Chempur), ethylene glycol (99%, Chempur), oxalic acid dihydrate (99.5%, Sigma-Aldrich) were used for anodization process of Ti and further chemical etching of as-anodized Ti plates. Gold target (99.99%, Quorum Technologies) was used for thin gold layer deposition. 6-mercaptohexanoic acid (6-MHA) (Sigma Aldrich) and bilirubin oxidase from Myrothecium verrucaria (Mv BOD) (kind gift from Amano Enzymes Inc. Nagoya, Japan) were used for the AuNPs/Ti electrode modification. Disodium hydrogen phosphate and sodium dihydrogen phosphate (99.0 %, Chempur), potassium ferricyanide K3[Fe(CN)6] and potassium ferrocyanide K4[Fe(CN)6] from Sigma, hexaammineruthenium (II) chloride Ru(NH3)6Cl2 from Thermo Scientific and hexaammineruthenium (III) chloride Ru(NH3)6Cl3 from Sigma were used for electrochemical tests. All solutions were prepared with Milli-Q water.

Electrode Preparation

Firstly, Ti foil was cut into 2×3 cm 2 plates and then ultrasonically cleaned. Secondly, TiO2 nanotubes were prepared through two-step anodization process and then chemica lly etched in the oxalic acid solution forming Ti nanodimples (Ti) [START_REF] Lipinska | The optimization of enzyme immobilization at Au-Ti nanotextured platform and its impact onto the response towards glucose in neutral media[END_REF][START_REF] Lipinska | Exploring multi-step glucose oxidation kinetics at GOx-functionalized nanotextured gold surfaces with differential impedimetric technique[END_REF]. Thirdly, the nanostructured titanium foil was covered by different thicknesses of gold, namely 2 nm, 5 nm, 10 nm and 15 nm, using magnetron sputtering machine (Q150T S system, Quorum Technologies) and pure gold plate as a target. Then electrodes were annealed at 450°C for 10 minutes in muffle furnace (MidiSUN KXP3 ET, Neoterm). Depending of the initial gold thickess different morphologies of the material were provided. In the current manuscript, the electrodes are labelled as following: 2Au@Ti, 5Au@Ti, 10Au@Ti and 15Au@Ti for Ti covered by respectively 2 nm, 5 nm, 10 nm and 15 nm Au layer. The catalytic efficiency of the so-fabricated electrodes was compared to a typical disc gold electrode (labelled as Audisc) from pure gold and manufactured by Mineral with a geometrical surface area of 0.0314 cm 2 . Ti electrodes after thermal treatment of the film sputtered on or pure nanostructured Ti substrate were chemically modified by incubation during night in 5 mM 6-MHA solution dissolved in ethanol. For enzyme adsorption, electrodes were incubated for 15 min at 4°C in freshly prepared enzyme solution in 100 mM phosphate buffer (PBS) pH 6. Increasing Mv BOD concentratio ns from 2 nM, 200 nM, 500 nM, 2 µM, 20 µM, 50 µM, up to 100 µM were studied. Bioelectrodes were then gently washed with 100 mM phosphate buffer solution pH 6 to remove the loosely adsorbed enzymes before transfer to the electrochemical cell containing 100 mM phosphate buffer pH 6 for further electrocatalytic experiments. Electrodes are inserted in a special home made holder for connection to the potentiostat, and partially covered by parafilm defining a surface area in contact with the electrolyte equal to ca. 0.6 cm 2 . The final material was labelled as BOD-M-XAu@Ti, where X stands for the thickness of the initial gold film, and M for 6-MHA modification. Scheme 1 illustrates the fabrication process of the electrodes used throughout this work, and the detailed description of the various electrodes with their respective names is summarized in Table 1. Scheme 1. Schematic fabrication process of enzyme modified Au@Ti electrodes. Table 1. Description and denomination of the various electrodes.

Electrode (M: SAM, X: thickness of the initial gold layer) Description

XAu@Ti 2Au@Ti, 5Au@Ti, 10Au@Ti, 15Au@Ti Au nanoparticles deposited on Ti nanodimples

M-XAu@Ti

M-2Au@Ti, M-5Au@Ti, M-10Au@Ti T hiol modified Au NPs deposited on Ti nanodimples BOD-M-XAu@Ti BOD-M-2Au@Ti, BOD-M-5Au@Ti, BOD-M-10Au@Ti Enzyme immobilized on SAM-modified Au NPs deposited on Ti nanodimples

M-Ti

T hiol modified T i nanodimples without gold

BOD-M-Ti

Enzyme and thiol modified T i nanodimples without gold

BOD-M-Audisc

Enzyme and thiol modified Audisc electrode

Morphology characterization

The morphology of 2Au@Ti, 5Au@Ti, 10Au@Ti and 15Au@Ti was investigated by scanning electron microscope (SEM) (FEI Quanta FEG 250) equipped with Everhart-Thor nle y secondary electron detector and with the beam accelerating voltage kept at 20 kV. The topography of surfaces was inspected by atomic force microscope (AFM) (Nanosurf EasyScan

2) in the contact mode regime. The 2Au@Ti sample was measured in aberration corrected highresolution transmission electron microscope (TEM) (JEOL ARM 200F) equipped with an EDX (energy-dispersive X-ray) detector. The preparation of cross-section lamellas for TEM was carried out using a focused ion beam system (JEOL JIB-4000). During this process, the current of gallium ions and acceleration voltages were adjusted, from 23 pA to 10 nA and from 3 to 30 kV, respectively. Prior to ion milling, the region of interest was carefully coated to avoid ion damage, following the procedure described elsewhere [START_REF] Park | Use of permanent marker to deposit a protection layer against FIB damage in TEM specimen preparation[END_REF].

Electrochemical Setup

Cyclic voltammetry (CV), chronoamperometry (CA) and electrochemical impedance spectroscopy (EIS) were performed in a standard 3-electrode cell using AutoLab PGStat 302N potentiostatgalvanostat. Ag/AgCl KCl sat. and Pt mesh were used as reference and counter electrode, respectively. The electrolyte was composed of 0.1 M PBS pH 6. In the case of measurements at different pHs (Figure 7), 0.1 M Britton Robinson Solution (BRBS) was used.

Electrolytes were deaerated with N2 or Ar or saturated with oxygen before electrochemica l measurement. The impedance spectra were recorded in 5 mM K3Fe(CN)6 + K4Fe(CN)6 in the frequency range from 20 kHz to 0.1 Hz using the signal amplitude of 10 mV and at the OCP.

The fitting procedure was carried out with the use of EIS Analysis [START_REF] Bondarenko | Inverse problem in potentiodynamic electrochemical impedance, 3rd Winter School on Chemometrics (WSC-3)Pushkinskie Gory[END_REF] using Powell algorithm and on the basis of the proposed electric equivalent circuits described in supporting informa tio n (Figures S1 andS2). Values obtained for all the elements present in the used electric equiva le nt circuit together with the goodness of fitting are set in Tables S1 andS2.

In order to precisely define the electrochemical activity of all electrodes listed in Table 1, the current was calculated and reported according to 3 options. In the option (1) the current is given as the direct measure in A (Figures 5,6). In the option (2), the current is reported as the percentage between the actual current (ix) and the maximum current (imax) value registered at a particular condition. In the case of pH dependent measurements, imax was the highest for pH 6 and therefore was chosen for calculation (Figure 7). To evaluate the stability of the gold containing nanostructured electrode modified with BOD in high ionic strength conditions, CA measurements were recorded without and in the presence of successive addition of Na2SO4 to PBS electrolyte. The measured current was divided by the initial value of current (i0) recorded at the beginning of the experiment (Figure 11). In the option (3), the current is reported as a current density considering either the gold geometric area (Figures 8, 9 and 10).

CV fitting

The CVs were fitted to the steady-state models which considered the dispersion of enzyme orientations (d0 parameter) on the surface and the kinetics of the heterogeneous electron transfer (k 0 constant) (Equation ( 1)). Since the current for BOD-M-2Au@Ti demonstrated an almost straight line, it was not possible to fit it reliably to the same equation than BOD-M-5Au@Ti and BOD-M-10Au@Ti and therefore another equation considering an infinite dispersion of the orientations (𝛽𝑑 0 → ∞) was used (Equation ( 2)) [START_REF] Fourmond | Modelling the voltammetry of adsorbed enzymes and molecular catalysts[END_REF].

𝑖 = 𝑖 𝑙𝑖𝑚 𝑟𝑒𝑑 𝑎 × [1 + 1 𝛽𝑑 0 𝑙𝑛 𝑎+ 𝑏 𝑘 𝑂𝑅 0 𝑘 𝑟𝑒𝑑 ⁄ 𝑎+ 𝑏×exp (𝛽𝑑 0 ) 𝑘 𝑂𝑅 0 𝑘 𝑟𝑒𝑑 ⁄ ] (1)
𝑖 = 𝑖 𝑙𝑖𝑚 𝑟𝑒𝑑 𝛽𝑑 0 1 𝑎 × 𝑙𝑛 (1 + 𝑎 𝑏 𝑘 𝑂𝑅 0 𝑘 𝑟𝑒𝑑 ) (2)
Where:

𝑎 = 1 + 𝑒 𝑂𝑅 = 1 + exp ( 𝐹 𝑅𝑇 (𝐸 -𝐸 𝑜𝑥/𝑅 0 )) 𝑏 = 𝑒 𝑂𝑅 𝛼 = 𝑒 𝑂𝑅 1/2 = exp ( 𝐹 2𝑅𝑇 (𝐸 -𝐸 𝑜𝑥/𝑅 0 ))

Results and discussion

Optimizing the Au layer thickness to control the size of Au nanoparticles

Due to the anodization and etching procedure, nanodimpled titanium foil with an average diameter of one nanodimple reaching ca. 90 nm was received. This nanostructured titanium is serving as a substrate ensuring spatial ditribution of AuNPs formed out of the thin gold film. As can be seen from SEM images (Figure 1), the size and spatial distribution of AuNPs depend on the initial Au layer thickness. The usage of the thickest Au layer of 15 nm led to the production of agglomerates looking as gold microislands that are randomly distributed over the substrate, providing an inhomogeneous material. For the three other materials, namely 2Au@Ti, 5Au@Ti and 10Au@Ti, the distribution of AuNP is homogeneous. Magnetron sputtering of 2, 5 nm or 10 nm gold layer thickness followed by thermal treatment allows obtaining single gold nanoparticles, although some gold nanoparticles can be linked forming the shape remaining string with beads. Figure 2 shows the size distribution of AuNPs for these three materials. In the case of 2Au@Ti, a diameter of 13±6 nm is obtained (Figure 2). The thicker the Au layer is, the bigger the diameter of the nanoparticles reaching 46±12 nm for 5Au@Ti. For 10 nm gold thickness, single nanoparticles with a diameter of 81±13 nm are formed. One main objective here was to obtain AuNPs with size close to enzyme dimens io n (less than 10 nm diameter range) with the ultimate goal of fixing only one enzyme molecule per AuNP. Mv BOD for O2 redution has molecular size 4×5×6 nm, meaning that 2Au@Ti should be the most suitable for our fundametal purpose [START_REF] Pankratov | Interfacial Behavior and Activity of Laccase and Bilirubin Oxidase on Bare Gold Surfaces[END_REF]. To be noted also, the AuNP size obtained in the current work are smaller than those previously obtained by laser ablation of gold films where the average size was superior to 110 nm [START_REF] Hitaishi | Nanosecond Laser-Fabricated Monolayer of Gold Nanoparticles on ITO for Bioelectrocatalysis[END_REF].

Figure 1. SEM images of a) 2Au@Ti, b) 5Au@Ti, c) 10Au@Ti, d) 15Au@Ti. More information about the structuration of AuNPs formed on nanostructured titanium foil is provided by AFM images (Figure 3). In the case of the 2Au@Ti (Figure 3a), Ti nanodimpled platform is still visible, in contrast to larger nanoparticles (Figures 3b, 3c and3d for 5Au@Ti, 10Au@Ti, 15Au@Ti). In the cases of the 5Au@Ti (Figure 3b) and the 10Au@Ti (Figure 3c), nanoparticles are uniformly distributed over the whole sample, in agreement with previously shown SEM images (Figure 1). For the 15Au@Ti (Figure 3d), gold nanoislands are inhomogeneously distributed, also in agreement with SEM pictures. AuNPs can be recognized as elements protruding from the electrode surface.

Figure 3. AFM images of a) 2Au@Ti, b) 5Au@Ti, c) 10Au@Ti, d) 15Au@Ti.

The size of NPs from 2Au@Ti compared to the dimple size raises the question of their spatial distribution and chemical composition. TEM pictures for the 2Au@Ti electrode presented in Figure 4 provide relevant information. The nanostructured titanium substrate is clearly imaged in the form of regularly spaced dimples with a depth of ca. 25 nm and a diameter of ca. 90 nm (Figure 4a). The Ti is covered by a thin 15 nm titanium oxide layer. During annealing, not only AuNPs but also the TiO2 layer is formed in the closest surrounding of the nanoparticle as a result of exposition to oxygen containing atmosphere during thermal treatment. A large number of white spots can be obseved on Figure 4b which corresponds to AuNPs with diameters from 10 to 15 nm consistent with SEM observation. EDX maps confir m the presence of gold nanoparticles mainly at the bottom part of Ti nanodimples (Figure 4c).

Some AuNPs are also visualized on the upper edges of dimples (as marked with a red arrow in Figure 4c). Actually, the diameter of nanoparticles obtained from 2 nm thick gold film (13±6 nm) is significantly smaller than the Ti nanodimple (ca. 90 nm). Consequently, NPs can enter the dimple, covering the bottom part but also partially the edges, leaving the dimple visible as highlighted by AFM. S3) highlights that Aelec is significantly lower than Atotal for 2Au@Ti.

Also, with the significant decrease of NP diameter deposited on TiO2/Ti substrate, the electrochemical features arising on CV scans tend to semiconductor behavior. Indeed, in our experimental conditions, TiO2 layer which is present between gold nanostructures is expected to interact more significantly with the smallest NPs [START_REF] Lipinska | Influence of Annealing Atmospheres on Photoelectrochemical Activity of TiO2 Nanotubes Modified with AuCu Nanoparticles[END_REF][START_REF] Lipinska | Thermally tuneable optical and electrochemical properties of Au-Cu nanomosaic formed over the host titanium dimples[END_REF]. Not only such a configura tio n might induce an underestimated surface, but it is expected to impact the reactivity of AuNPs as further demonstrated below.

Selectivity of AuNP over Ti for enzymatic oxygen reduction

To get more insight in Ti-based surface chemical modification and further bioelectrocatalysis, CV and EIS measurements in 5 mM K3Fe(CN)6 + K4Fe(CN)6 in 0.1 M PBS solution were recorded (Figure 5). The 10Au@Ti electrode was chosen for this study since it exhibits the highest degree of AuNPs ordering. Electron transfer between the redox couple and the electrode occurs only after treatment of the semiconductor surface by gold nanostructure (Figures 5a and5c). After each modification step of AuNPs, the electron transfer is slowed down as a consequence of 6-MHA modification, then BOD immobilization [START_REF] Lipinska | Electrochemical glucose sensor based on the glucose oxidase entrapped in chitosan immobilized onto laser-processed Au-Ti electrode[END_REF][START_REF] Nidzworski | A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond[END_REF].

Accordingly, after each modification step the electron-transfer resistance R2 increases (Figure 5d, Table S1) which confirms succesful binding between AuNP and 6-MHA as well as 6-MHA and BOD.

For the Ti and Ti modified electrodes (Figures 5a and5b, Table S2), changes in Warburg parameter (W) should be considered. Indeed, because TiO2 is a semiconductor material, it is not able to carry out the reversible redox reaction (as seen in Figure 5a). It can only absorb or modify those redox species reaching the electrode surface. Therefore changes in the Warburg parameter can provide information about potential absorption layers. The Warburg parameter W increases ca. 2 times after modification of Ti by either 6-MHA or BOD (Table S2). It does not change after BOD incubation in thiol-modified Ti (BOD-M-Ti electrode). This strongly suggests that both 6-MHA or BOD can adsorb on Ti, but BOD cannot be adsorbed on the thiol layer formed on Ti, possibly because of the inappropriate positioning of functional groups. To further verify the selectivity of gold NPs over the Ti/TiO2 electrode surface for bioelectrocatalysis, the bare Ti dimpled foil was modified by 6-MHA and after incubation with Mv BOD CVs were performed in 0.1 M PBS (Figure 6). CV curves almost overlap no matter of the presence or not of O2 in the electrolyte, indicating that Ti alone is not able to undergo any enzymatic reduction of O2. Interestingly, this behavior is opposite to the case when ITO was used as a substrate for BOD anchoring [START_REF] Hitaishi | Nanosecond Laser-Fabricated Monolayer of Gold Nanoparticles on ITO for Bioelectrocatalysis[END_REF]. The lack of bioelectrocatalysis on Ti surface may result either from the abscence of BOD or from an unfavorable orientation of BOD for direct catalysis. While 6-MHA can readily form SAMs on gold [START_REF] Noh | Growth processes and control of two-dimensional structure of carboxylic acid-terminated self-assembled monolayers on Au(111)[END_REF], ITO substrate or copper selenide quantum dots [START_REF] Pacoste | Mercaptoalkanoic Acid-Induced Band Gap Attenuation of Copper Selenide Quantum Dot[END_REF], no report showing successful modification on Ti or TiO2 substrate was reported. As was indicated by Trino et al., bonding of acid containing -SH group onto the titanium dioxide surface may occur only through the grain boundaries due to the higher surface energy in those regions [START_REF] Trino | Surface Physicochemical and Structural Analysis of Functionalized Titanium Dioxide Films[END_REF]. Such a property would not favour DET orientation of BOD. One advantage is that the catalytic signals that will be recorded thereafter will be exclusively linked to the presence of AuNPs.

Enzymatic O 2 reduction on XAu@Ti electrodes

O2 reduction by Mv BOD absorbed on SAM-modified AuNPs deposited on dimple titanium foil in different pH electrolytes was first carried out. It is widely reported that the electrocatalytic activity of enzymes is strongly dependent on pH values [START_REF] Hitaishi | Electrostatic-Driven Activity, Loading, Dynamics, and Stability of a Redox Enzyme on Functionalized-Gold Electrodes for Bioelectrocatalysis[END_REF]. The aim of this preliminary study was to investigate whether main well known electroenzymatic properties are maintained on substrate containing titanium, which has never been reported so far. The BOD-M-10Au@Ti electrode was chosen as a reference electrode due to its configuration of one AuNP per one Ti dimple exhibiting high degree of ordering. Figure 7 presents pH dependence of enzymatic ORR occurring at the BOD-M-10Au@Ti electrode. Measurements were carried out in 0.1 M Britton Robinson Solution in the range pH 4 to 7. For all pHs, a catalytic response was obtained in the presence of O2. The highest current for ORR was obtained for pH 6 (Figure 7c), and the lowest one for pH 7 (Figure 7d). The pH-electrocatalysis relationship obtained is very similar to previously reported data at 6-MHA modified gold disk electrodes [START_REF] Hitaishi | Electrostatic-Driven Activity, Loading, Dynamics, and Stability of a Redox Enzyme on Functionalized-Gold Electrodes for Bioelectrocatalysis[END_REF]. Such responses have been assigned to pH-dependent changes in intrinsic activity of enzyme as well as electrostatic interactions between Mv BOD and SAMs [START_REF] Hitaishi | Electrostatic-Driven Activity, Loading, Dynamics, and Stability of a Redox Enzyme on Functionalized-Gold Electrodes for Bioelectrocatalysis[END_REF]. The onset potentials vs RHE for the four pH investigated are gathered in Table S4. Whereas a similar potential is obtained at pH The electroactive properties of the different electrodes modified with the enzyme, namely BOD-M-2Au@Ti, BOD-M-5Au@Ti, BOD-M-10Au@Ti and BOD-M-Audisc electrodes were then investigated using CV in oxygenated 0.1 M PBS solution (Figure 8). Not only different sizes of nanoparticles but also various Mv BOD concentrations were investigated.

Biocatalytic oxygen reduction occurs whatever the electrode type. For all the investigated electrodes, a bell-shape relation ship between the catalytic current and the enzyme concentration was obtained with the highest current recorded for 20 µM BOD. This tendency has been already reported on other materials, and could be explained by changes in enzyme conformation in a crowding enironment [START_REF] Mcardle | Optimizing the Mass-Specific Activity of Bilirubin Oxidase Adlayers through Combined Electrochemical Quartz Crystal Microbalance and Dual Polarization Interferometry Analyses[END_REF]. The ORR onset potential for the BOD-M-2Au@Ti, BOD-M-5Au@Ti, and BOD-M-10Au@Ti reaches ca. 1.13 V, whereas, in the case of BOD-M-Audisc electrode it is 1.10 V vs. RHE. By comparing the current densities reported to the geometric surface areas at BOD-M-Audisc (Figure S4) and at BOD-M-5Au@Ti and BOD-M-10Au@Ti for 20 µM BOD, an enhancement of 2.5 and 3 times is obtained respectively, while the current profile for BOD-M-2Au@Ti is very similar to that of BOD-M-Audisc. To be noted, in addition to weakly enhanced catalytic currents in the case of BOD-M-2Au@Ti, the CV shapes also significantly differ from those at BOD-M-5Au@Ti and BOD-M-10Au@Ti. No plateau can be reached in the cathodic regime, and the CV curve displays an almost straight line behavior. With this specific material, the shape of the CV curves and the magnitude of the catalytic current is also little affected by 6-MHA modification (Figure 9a).

This contrasts with the marked increase in current and CV shape turning to a narrower distribution of electron transfer rates when both 5Au@Ti and 10Au@Ti are modified by the thiol before incubation in BOD solution (Figures 9b and9c) [START_REF] Hitaishi | Electrostatic-Driven Activity, Loading, Dynamics, and Stability of a Redox Enzyme on Functionalized-Gold Electrodes for Bioelectrocatalysis[END_REF]. As noted above, the semiconducting properties of TiO2 alter those of the smalles t

AuNPs. It was reported that changes in local charge distribution occur on AuNPs interacting with TiO2 semiconductor [START_REF] Mcentee | Electric Field Changes on Au Nanoparticles on Semiconductor Supports -The Molecular Voltmeter and Other Methods to Observe Adsorbate-Induced Charge-Transfer Effects in Au/TiO2 Nanocatalysts[END_REF]. Such charge distribution results from electron injection from the n-type semiconductor supporting material. Moreover, decreasing nanoparticle size, the surface energy density increases owing to formation of high-energy electrons (hot electrons) localised at the NPs [START_REF] Xie | A first-principle study on the atomic-level mechanism of surface effect in nanoparticles[END_REF]. In addition, the role of quantum size effect on electronic properties based on density functional theory simulations predicts spill-out of electrons outside the nanoparticle and change of its dielectric properties at the surface. Quantum size effect can occur for nanopartic les with diameter of ~ 10 nm [START_REF] Tame | Quantum plasmonics[END_REF]. Therefore, 2Au@Ti NPs with a thickness ratio of Au to TiO2 lower than in the cases of 5Au@Ti and 10Au@Ti samples are negatively charged (Scheme 2).

This specific charge distribution tends to repel the negatively charged 6-MHA, precluding SAM formation and disfavoring favorable BOD orientation for direct bioelectrocatalysis.

Scheme 2. Schematic representation of the charge distribution over 10Au@Ti, 5Au@Ti and 2Au@Ti electrodes.

An alternative explanation may arise from prevalent semi-conducting properties of 2Au@Ti material. The electrochemical behavior of FeCN6 3-and Ru(NH3)6 3+ redox probes were run on 2Au@Ti, 5Au@Ti and 10Au@NP at different scan rates (Figure S6). Whereas FeCN6 3- behaves as a quasi-reversible electrochemical couple on 5Au@Ti and 10Au@Ti with peak potential differences about 120 mV at 100 mV/s, the shape of the CV at 2Au@Ti reveals a very slow electron transfer process. Such a modification in the electron transfer rate could be induce d by the negative charge of the 2Au@Ti NP. However, the electron transfer process is also affected in the case of Ru(NH3)6 3+ , with furthermore increase in the current magnitude on the smallest NPs as would have been expected in case of favorable attraction. These data support the prevalent effect of AuNP/TiO2 interactions which affects the electron transfer rate, the greatest in the case of 2Au@Ti NPs. A direct consequence is that the evolution of catalytic currents reported versus the electroactive area (Figure S5) which suggests that 2Au@Ti should be much more efficient than the bigger particle sizes, is instead a consequence of Au/TiO2 interaction that induced an under-estimation of the electroactive surface area.

Fitting of the CVs (Figure 10 and Table 2) obtained with BOD-M-2Au@Ti, BOD-M-5Au @Ti and BOD-M-10Au@Ti leads to additional information. In the cases of BOD-M-5Au@Ti and BOD-M-10Au@Ti, the d0 orientation parameter takes values of 9.1 and 6.5

while the ratio of k0/kcat are respectively 19 and 14. These values are in agreement with a quite narrow orientation of the enzyme on the thiol-modified materials [START_REF] Hitaishi | Electrostatic-Driven Activity, Loading, Dynamics, and Stability of a Redox Enzyme on Functionalized-Gold Electrodes for Bioelectrocatalysis[END_REF]. On the other hand, the CV curve for the BOD-M-2Au@Ti can only be fit with a model where d0 tends to infinity, and k0/kcat is decreased to 1, i.e. one order of magnitude smaller than for the higher size NPs.

Since kcat is not expected to vary since the same enzyme is used, this suggests a decrease in the interfacial electron transfer rate constant induced by the presence of TiO2 in the surrounding of the AuNPs. The increase of 𝛽𝑑 0 for the smallest NPs can be explained either by the increase of distance distribution 𝑑 0 between enzyme active centre and the gold surface because of the absence of thiol linker, or by the increase of 𝛽, an exponential decay constant dependant on the nature of the space between the active centre and the electrode. A thin isolating layer of TiO 2 in the close surrounding of the gold NPs as demonstrated by TEM images would increase the potential energy barrier that electron needs to overcome and thus would increase 𝛽.

Stability of BOD-M-10Au@Ti in the presence of high salt concentration

To explore further the capacity of 10Au@Ti to support efficient bioelectrocatalysis, we studied the impact of salt additions on the bioelectrocatalysis. Indeed, using high electrolyte concentrations is often a key in bioelectrocatalysis to overcome local pH changes [START_REF] Tassy | In Situ Fluorescence Tomography Enables a 3D Mapping of Enzymatic O-2 Reduction at the Electrochemical Interface[END_REF][START_REF] Moore | Understanding the local chemical environment of bioelectrocatalysis[END_REF]. We recently reported the complex effect of increasing concentrations of salts on ORR by Mv BOD immobilized on CNT films or on thiol-modified gold electrodes [START_REF] Saska | High electrolyte concentration effect on enzymatic oxygen reduction[END_REF]. We especially highlighted the reversible flipping of the protein on a SAM-modified gold disk electrode surface when electrostatic interactions were decreased by addition of salts at concentrations higher than 1 M.

We also highlighted that whereas the catalytic current decreases with salt addition with the enzyme adsorbed on thiol-based planar gold electrodes, it increases when the protein is covalently attached. Enzyme compaction in the presence of high salt concentrations which decreases the distance between Cu T1 and the electrode was proposed to explain this last phenomenon.

Immobilization of the enzyme on AuNPs is expected to change the nature of electrode/protein interactions, hence modifying the effect of salt addition. Chronoamperome tr y measurements were then carried at the potential of +0.43 V vs. Ag/AgCl with the successive addition of Na2SO4 solution to PBS (Figure 11). As previously reported, the current recorded for BOD-M-Audisc electrode decreases upon stepwise increase of Na2SO4 content in PBS, being 40% of the initial current after 1 M salt addition. The same experiment conducted on BOD-M-10Au@Ti induces a completely different CA response. First of all, high stability of the current with time is observed before any salt addition. For salt concentrations in the range 100 mM to 400 mM, an increase in the catalytic current is observed. This step is followed by a slight decrease of the current, the remaining current at 1 M salt concentration being similar to the initial current. This enhanced stability of the nanobioelectrode is very attractive and may be related to NP surface curvature, that offers specific surface of contact for the enzyme [START_REF] Beaufils | From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes[END_REF].

Smaller area for protein contact compared to Audisc, as well as suppression of unfavorab le protein-protein lateral interactions are most probably the reasons that can account for stabilization effect of AuNPs.

Conclusions

We investigated in this work the efficiency of enzymatic oxygen reduction once Mv BOD was immobilized on gold nanoparticles (AuNPs) spatially distributed in Ti nanodimples. The used synthesis method allows to control the size of AuNPs and regularely distribute them on the conducting surface which is not easely achieved otherwise. The advantage of such constructio n was not only to investigate AuNPs with sizes from less than 100 nm down to 10 nm, but also to take benefit of the semi-conducting properties of TiO2 allowing to restrict occurrence of biocatalysis only on AuNPs. We showed that biggest AuNPs enhanced the current density by more than 2 times. However, we also demonstrated that surrounding of the smallest AuNPs with a layer of TiO2 induced a modification in the electron transfer process most probably related to the decrease of the interfacial electron transfer rate k 0 and an increase in the constant decay parameter . This means that the use of AuNPs via the Ti nanodimple method requires a balance between efficient size for biocatalysis and impact of TiO2 layer. High stability of the enzymatic current was obtained in the presence of high concentration of salts. This finding is very interesting in view of applications which require high ionic strength to mitigate any local pH changes. In depth studies are now required to search for the reasons for such a stabilizatio n.

Figure 2 .

 2 Figure 2. Size distribution of AuNPs for a) 2Au@Ti, b) 5Au@Ti, c) 10Au@Ti electrode.

Figure 4 .

 4 Figure 4. TEM images of the 2Au@Ti electrode (a, b, d) and EDX elemental mapping c, e) gold, f) titania, g) oxygen, h) Au, Ti, O elements.

Figure 5 .

 5 Figure 5. CVs curves (scan rate 10 mV/s) of a) Ti and Ti modified electrodes, and c) 10Au@Ti and 10Au@Ti

Figure 6 .

 6 Figure 6. CVs curves of the BOD-M-Ti electrode modified by 100 µM BOD in 0.1 M PBS deareated with N2 (pink curve) and saturated with O2 (blue curve) (scan rate 10 mV/s).

6 and pH 7 ,

 7 a 20-30 mV/UpH deviation is measured at more acidic pHs. Actually, the evolutio n of onset potentials with pH is admitted in the literature to be linked to BOD CuT1 potential changes. For BOD from Magnaporthe oryzae, Al-Lolage et al. reported a two slope behavior with a pH shift in the pH 6-7.6 range corresponding to a 1 e -/1 H + process, while a sub-Nerstian response was obtained in the pH 4-6 range[START_REF] Al-Lolage | Site-Directed Immobilization of Bilirubin Oxidase for Electrocatalytic Oxygen Reduction[END_REF]. This variation agrees with the current study, and in accordance with the literature, we stated that such variation could be attributed to protonation/deprotonation of amino acid residues in the vicinity of Cu T1[START_REF] Tsujimura | Electrochemical Oxygen Reduction Catal yzed by Bilirubin Oxidase with the Aid of 2,2 '-Azinobis(3-ethylbenzothiaZolin-6-sulfonate) on a MgO-template Carbon Electrode[END_REF].

Figure 7 .

 7 Figure 7. CV curves of the BOD-M-10Au@Ti electrode modified by 20 µM BOD at different pH a) pH 4, b) pH 5, c) pH 6, d) pH 7, 0.1 M Britton Robinson Solution deareated with N2 (black curves) and saturated with O2 (orange curves) (scan rate 10 mV/s).

Figure 8 .

 8 Figure 8. CVs curves of a) BOD-M-2Au@Ti, b) BOD-M-5Au@Ti, c) BOD-M-10Au@Ti electrode as a function of increasing Mv BOD concentrations in 0.1 M PBS saturated with O2 (scan rate 10 mV/s), d) relationship between catalytic current density reported to the geometric surface area and enzyme concentration at the potential of +0.95 V vs RHE.

Figure 9 .

 9 Figure 9. CVs curves of a) 2Au@Ti, b) 5Au@Ti, c) 10Au@Ti electrodes modified by 20 µM BOD in 0.1 M PBS saturated with O2 without (blue curves) or with (orange curves) thiol modification. Black curve is assigned to the BOD-XAu@Ti electrode under N2. Scan rate 10 mV/s.

Figure 10 .

 10 Figure 10. Electrocatalytic curves of a) BOD-M-2Au@Ti, b) BOD-M-5Au@Ti, c) BOD-M-10Au@Ti electrode with subtracted blank and fitted to the equation 2 (a) or 1 (b,c).

Figure 11 .

 11 Figure 11. CA curves for the BOD-M-Audisc and BOD-M-10Au@Ti in 0.1 M PBS with addition of Na2SO4 saturated with O2.

Table 2 .

 2 Parameters derived from the fitting

Parameter BOD-M-2Au@Ti BOD-M-5Au@Ti BOD-M-10Au@Ti

		Value	Error	Value	Error	Value	Error
	𝐼 𝑙𝑖𝑚 , 𝜇𝐴/𝑐𝑚 2	-	-	-29.4	0.17	-18.2	0.1
	𝛽𝑑 0	> 9	-	9.1	0.2	6.5	0.3
	𝐼 𝑙𝑖𝑚 𝛽𝑑 0 , 𝜇𝐴/𝑐𝑚 2 ⁄	-16.6	0.15	-	-	-	-
	𝑘 𝑚𝑎𝑥 0 ⁄ 𝑘 𝑐𝑎𝑡	1.0	0.2	19	2	14	3

Acknowledgments

Research is financed by National Science Centre (Poland): Grant No. 2019/35/N/ST5/02604 and ANR (ENZYMOR-ANR-16-CE05-0024).

CRediT authorship contribution statement

Declaration of Competing Interest

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


Laurent Sagart

The phonetic nature of PAN *j

Keywords: Proto-Austronesian phonological reconstruction, Campidanese Sardinian, palatal glide fortition, prenasalized stops. PAN (Blust)

This paper brings evidence from Campidanese Sardinian and other languages to support and refine the author's earlier proposal that PAN *j was a palatal nasal whose modern reflexes arose through an episode of palatal glide fortition. It clarifies the evolutions of *j in Formosan, responds to some criticisms and details the circumstances leading to loss of the nasal component after glide fortition. It concludes that where an alveolar or palatal nasal is in correspondence with [g], [ɟ] or [d], the default historical interpretation is out of the nasal.

1. INTRODUCTION. 1 It is well known that a sound correspondence exists among Formosan languages between [n] in Siraya, Kavalan, Basay, Trobiawan and Amis, [g] in Atayal and Rukai, [d] in Favorlang, Paiwan, and Puyuma, [z] in Pazeh and Saisiyat, [ð] in Thao, [l] in Kanakanabu, and zero in Bunun and Tsou (Table 1 northwestern Basque (Egurtzegi 2013:158); from Kiput, a MP language (Blust 2002:402).

Fortition of [j] following a consonant occurs in Kirundi after b, v, r : /ja-raab-je/ [jaɾaːvɟe] 's/he looked', /ku-vjuura/ [kuvɟuːɾa] 'to wake up', /ku-ri-a/ (> rj) [kuɾɟa]~[kuɟa] 'to eat' [START_REF] Kochetov | Palatalization and glide strengthening as competing repair strategies: Evidence from Kirundi[END_REF]. For Tibetan, Li Fang-kuei's second law [START_REF] Li | Tibetan Glo-ba-'dring[END_REF][START_REF] Hill | An Inventory of Tibetan Sound Laws[END_REF] Specifically after n, j-fortition will result in a prenasalized consonant. Lai (2022) observed a correspondence in Gyalrongic, a Sino-Tibetan group, whereby Krhoskyabs, a language within the western branch, has ɟ against n or nj in the other Gyalrongic languages, whether western or eastern. Lai argued that in Khroskyabs, "*nj is fortified into ɟin initial position and the original nasality is preserved when preceded by a vowel". He posited the sequence *nj > * n ɟ > ɟ (Lai 2022: 219). Cited examples are few, however, and come from different Gyalrongic languages.

The clearest examples of palatal fortition after n come from Sardinian dialects [START_REF] Lausberg | Linguistica romanza[END_REF], Puddu 2000[START_REF] Lai | Preinitial denasalization and palatal fortition in Khroskyabs and the Gyalrongic word for 'man[END_REF]. atin *ni and *ne merge to evolve to [ɲɟ] in Campidanese 4 and to the 3 Where -j-is intrusive, from an unknown source.

variants [nz] ~ [ndz] in Logudorese (
4 Lausberg (1976:330) transcribes Campidanese -ngi-as [ñǵ], equivalent to [ɲɟ] in the current version of the IPA.

3. PAN *j AS A NASAL. Aiming for a phonetically more realistic account of the PAN consonant system and of the evolutions of PAN *j, Sagart (2004:429) proposed to reinterpret *j as a palatal nasal *n j [ɲʲ], evolving to [ɲɟ] through palatal glide fortition, Campidanese-style. This has the advantage of accounting for the nasal reflexes without the need for an unattested [g j ] > n sound change. It also makes the consonant system more balanced-as [g j ], *j lacks a voiceless counterpart.

Wolff's reconstruction of *j as [g], which has a voiceless counterpart in PAN *k, avoids the imbalance issue. He notes that PAN *g and *j as understood by other scholars are complementarily distributed: the former does not occur word-medially and is extremely rare, or inexistent, word-finally, while the latter does not occur word-initially. Wolff's PAN *g thus stands for both *g and *j of other scholars. However his account requires *g [g] to palatalize between any two vowels, whether palatal or not. This is phonetically curious. A more common evolution for an intervocalic -g-would be to lenite to [ɣ]. As it happens, *g is also in complementary distribution with *y: like *j, *y does not occur word-initially. It is possible, then, that pre-PAN intervocalic *-g-evolved to PAN -y-, leaving a gap in the distribution of *g. I find the -g-> -ɣ-> -j-explanation better supported than Wolff's: intervocalic -g-leniting to -ɣ-does not require a palatal context, and neither does further lenition of [ɣ] into the approximant -j-. The evolution -g-> -ɣ-> -j-in intervocalic position is attested in northern and southern Italian dialects: Piemontese, Tessinese, Tarento (Kümmel 2007:85). As to why PAN *j does not occur word-initially, I take PAN *j to originate in pre-PAN *n preceded by a palatal segment such as *i or *y. This explains its absence word-initially: as there was no preceding segment, evolution to *j [n j ]was not possible. Siraya, Basai, Trobiawan, Kavalan and Amis followed the second or third path, and the resulting palatal nasal then merged with *n. There is no ground for supposing that the phonetically trivial n/ ɲ merger occurred only once.

CLASH

Denasalization itself cannot be modeled as a single innovation: the denasalizing shifts often providing θ with a voiced counterpart. These episodes of denasalization must be independent.

The final denasalizing event occurred before PMP, where *j was [g j ] or similar, as proposed by

Dempwolff.

Chinese provides us with a parallel for the loss of secondary prenasalization in a language without prenasalized stops. Like all Old Chinese (OC) nonpharyngealized initial consonants, OC *n developed a medial palatal glide in late OC [START_REF] Baxter | Old Chinese: a new reconstruction[END_REF]). In the first and second centuries CE, it served to transcribe Sanskrit /ñ/ (Coblin 1983:56). It is generally thought to have been a palatal nasal in Middle Chinese: [START_REF] Baxter | A Handbook of Old Chinese phonology[END_REF] symbolizes it as ny-in Early Middle

Chinese (around 550-600 CE). Based on its modern dialect reflexes, Karlgren (1915) We can exclude that Beijing ʐfricativized directly out of an earlier palatal glide: ʐ < ʝ < j: if so, the glide would surely have merged with Middle Chinese y-[j] along the way. But MC y-and ny-are not merged in Beijing, with MC y-retaining its phonetic quality as a palatal glide:

引

  It appears, then, that PMP *ñ originates in two PAN phonemes: PAN *N and *n, when followed by a palatalizing context, here noted as a diphthong of unstressed i plus a stressed vowel: *niV and *NiV. In PMP *n and *N merged as *n at a time when *iV strings were still intact: so *niV and *NiV merged as *niV, which then palatalized to *ñV. Kanakanabu independently followed the same evolution, and further velarized [ñ] to[ŋ]. This is the reason why most of the evidence adduced in support of putative *ñ by Tsuchida and Blust comes from two languages that merge *n and *N: PAN and Kanakanabu. Outside of Kanakanabu, Formosan languages appear to have reduced the iV diphthong to V following *n and *N, so that where *niV and *NiV merge, they do so as nV, feeding into inherited *nV, without producing a reflex distinct from that of *n.As already evident inWolff (2010), PAN *n and *N (Wolff's *n and *ɬ) are sufficient to account for the range of phenomena earlier attributed to *ñ. Thus Wolff (2010) has PAN *qáɬuj opposite Blust's *qañud 'adrift' but *peniyu opposite Blust's *peñu 'sea turtle'. Putative PAN *ñ is superfluous: therefore reinterpreting *j to *n j does not entail a clash with another PAN palatal nasal. 5. EVOLUTION OF *n j IN FORMOSAN. Emending PAN *j to a palatal nasal undergoing fortition to [ɲɟ] introduces a prenasalized phoneme into a sound system lacking such consonants. The new consonant was thus poorly integrated. Different paths were available to remove the oddity: -by removing nasal component, to [ɟ]; -by removing the stop component, to [ɲ]; -via progressive nasalization and degemination [ɲɟ] > [ɲɲ] > [ɲ]. 5 Taokas, Atayal, Rukai, Favorlang, Papora, Paiwan, Puyuma chose the first path. The resulting [ɟ] lacked a voiceless counterpart and, even after denasalization, was poorly integrated. As a result [ɟ] was regularized across the board: no modern Formosan language reflects *j as [ɟ]. The main avenues to regularize [ɟ] were to [d] (Favorlang, Paiwan, Puyuma, Papora), [t] (Taokas) and [g] (Atayal, Rukai). The change ɟ > g occurs in Danish and Egyptian Arabic (Kümmel 2007: 242).

  motivated by the need to fill gaps in the consonantal system. Thus in Atayal, where PAN *g devoiced to k-, [ɲɟ] evolved to g-. In Amis, where *d shifted to r, [ɲɟ] went to d. In Paiwan, where *d changed to dj, [ɲɟ] went to d. In Thao, PAN *C went to θ, and [ɲɟ] changed to ð,

  ABBREVIATIONS

Table 1

 1 ).

		rice	how many	navel	bile	know	name
	PAN (Blust)	*pajay	*pija	*puja	*qapeju	*bajaq	*ŋajan
	Kavalan	n panay	-	-	-	-	ŋanan (<M)
	Basai	n -	-	puna	-	-	nanan
	Amis	n panay	pina	pona	-	fanaʔ	ŋaŋan (<A)
	Siraya	n -	-pina	-	parno	vana-vana	nanang
	Atayal	g pagai	piya 2	puga	-	bak !	-
	Rukai	g pagai	-	-	pagu	vaga	naganə
						'language'

. Formosan reflexes of PAN *j. Sources: Austronesian Comparative Dictionary,

Wolff (2010)

;

Adelaar (2011)

for Siraya.

Austronesianists attribute this correspondence to a PAN phoneme for which the symbol *j has become standard. This phoneme is the PAN precursor of PMP *j [gʲ], part of a series of palatovelars (hintere Palatale) in Dempwolff's system. In both MP and Formosan, d and g reflexes occur, pointing to an original palatal articulation. An important difference is that nasal reflexes occur in Formosan, while MP has none. That *j never gives nasals in MP, a group of c. 1,200 languages, while 5 out of 25 or so Formosan languages reflect it as n, in itself suggests a phonetic difference, involving nasality, between the PAN and PMP stages of this phoneme.

Like Dempwolff, authors on PAN interpret *j phonetically as a voiced obstruent in the palatal or dorsal region: [ɟ]

(Dahl 1981:152

); [g]

[START_REF] Wolff | The PAN consonant system[END_REF](Wolff , 2010);); [ɣʲ]

or

[ʝ]

[START_REF] Ross | The Sound of Proto-Austronesian: An Outsider's View of the Formosan Evidence[END_REF](Ross , 2015))

; [g j ] (Blust 1999). As already mentioned, [d] and [g] reflexes occur in both MP and Formosan. The other reflexes, such as [z], [ð], [j], [l] and zero, can be viewed as somehow derived. The

[n]

Table 2

 2

	manzanu

). The nasal component is preserved, as prenasalized voiced stops are licit word-internally. Campidanese Logudorese at. v n a 'vineyard' bíngia id. bintza, binza Lat. e trān us 'foreign' istràngiu id. istranzu at. m n a 'duties, functions' múngia 'weariness, strain' munzaɲ at. mān 'morning' + suffix -anu mangiànu id.

Table 3 :

 3 N, putative PAN *ñ occurs in two mutually exclusive sets of words: one with the reflexes e pected of *n, and another with the refle es of *N. This can be shown using Blust's own PAN reconstructions (Table3):

	WITH *ñ. Blust (2014: 359-361) argued that the reinterpretation of *j as *n j in Sagart
	(2004) clashes with *ñ, a palatal nasal he takes to be reconstructible to PAN. *ñ was first
	reconstructed by Dempwolff for the proto-language we now call PMP. Impressed by Tsuchida's
	observation (1976) that Dempwolff's *ñ is reflected as [ŋ] in Kanakanabu, Blust projected PMP

*ñ back to PAN apparently without verifying that this putative proto-phoneme has a single set of reflexes in Formosan languages. In fact, in those Formosan languages which distinguish *n and *Two sets of Formosan reflexes for

Blust's *ñ in the ACD (accessed July 7, 2015)

.

have the reflexes of *N; columns 6-9 ('sea turtle' though 'wash'), those of *n.

Table 4 :

 4

	saw it as a

reflexes of OC nonpharyngealized *n in

Beijing, Wenzhou, Meixian (tone marks omitted)

Thanks to Sander Adelaar, Eva Büchi, William Baxter, Isabelle Bril, Guillaume Jacques, John Wolff and to three anonymous reviewers for useful discussion. Any errors are mine alone.

Atayal and Kanakanabu have a special reflex after *i.

Lausberg (1976:330) states that the change of at. -NI -to [ɲ] in western Romance occurred through a [ɲɲ] interstage, still observable in southern and central Italian dialects.

Online resource:

ACD: Austronesian Comparative Dictionary, web edition. Robert Blust and Stephen Trussel: www.trussel2.com/ACD.