Rabih Khoder

Hafsa Korri-Youssoufi

E-DNA biosensors of M. tuberculosis based on Nanostructured Polypyrrole

Keywords: Polypyrrole, Nanowire, DNA, Electrochemical, Detection, M. tuberculosis

The objective of this paper is to demonstrate the potential of nanostructured polypyrrole formed by template free as platform for amperometric detection of DNA. The nanowires of polypyrrole (nw-PPy) are formed through electrochemical polymerization and chemically modified by electrochemical oxidation of ethylene diamine or dendrimers PAMAM to obtain aminated surface. The DNA probe and ferrocenyl group, as redox reporter, were covalently linked to the surface of nw-PPy. The chemical structure of nanostructured platform was characterized through SEM, FT-IR and XPS and the electrochemical properties through cyclic voltammetry and electrochemical impedance spectroscopy (EIS). We show that the properties of nw-PPy such as, hydrophilic character and large surface area have large effect on the electronic properties. Thus, the electrochemical performance is increased compared to others nanomaterials considering the obtained value of the rate of electron transfer of 18 s -1 . These properties allow enhanced DNA sensing where detection limit of 0.36 atomolar without any amplification step. The biosensor can be applied in detection of genomic DNA of Mycobacterium tuberculosis and the mutated one which present the resistance to rifampicin and large selectivity was demonstrated. We believe that nw-PPy modified with redox marker is a promising platform for electrochemical biosensors and can be applied for various diagnosis prospects.

Introduction

A rapid, accurate, cost-effective of diagnostics of pathogenic and viral DNA biomarkers stills remains a challenge. The development of extremely sensitive, highly selective, simple, robust and yet inexpensive biosensing platforms appears thus essential for a wide range of applications, including not only clinical diagnostics [START_REF] Gubala | Point of Care Diagnostics: Status and Future[END_REF] but also environmental monitoring [START_REF] Jokerst | Advances in microfluidics for environmental analysis[END_REF] and food safety testing [START_REF] Atalay | Microfluidic analytical systems for food analysis[END_REF]. The detection and identification of multi-resistant strains are raising therapeutic problems. Tuberculosis remains one of major global health problem worldwide. Despite the advent of antibiotics which have widely reduced mortality, antibioticresistant strains have now emerged. The ability to obtain sequence-specific genetic information about infectious pathogens and resistant stand remains of great importance. Most methods used for DNA detection for diagnosis are PCR [START_REF] Mengoli | Use of PCR for diagnosis of invasive aspergillosis: systematic review and meta-analysis[END_REF] and Q-PCR [START_REF] Bustin | Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis[END_REF]. Recently various strategies were developed for label free DNA detection, such as fluorescence [START_REF] Wang | DNA-templated Au nanoclusters and MnO2 sheets: a label-free and universal fluorescence biosensing platform[END_REF], [START_REF] Wang | DNA-templated Au nanoclusters coupled with proximity-dependent hybridization and guanine-rich DNA induced quenching: a sensitive fluorescent biosensing platform for DNA detection[END_REF]. Even these methods are sensitive they are not adapted to point of care analysis system. Thus, electrochemical DNA (E-DNA) biosensors have recently found extensive applications in diagnosis as well as analysis. E-DNA biosensors usually composed of two basic components, a DNA recognition system and a transducer which is a device that converts the chemical response into electrical signals that can be detected by various detection approaches [START_REF] Ferrier | Micro-and nano-structure based oligonucleotide sensors[END_REF] [START_REF] Odenthal | An introduction to electrochemical DNAbiosensors[END_REF].

Several E-DNA biosensors using transducers have been developed for real time detection without amplification step. They include conducting materials [START_REF] Peng | Conducting polymers for electrochemical DNA sensing[END_REF] able to change their electrical properties after such hybridization reaction on their surface.

Conducting polymers (CPs) are widely used as transducers for biological interactions. CPs are π conjugated polymers with an electronic structure giving them intrinsic characteristics such as electrical conductivity, which can be controlled by a doping/de-doping process, low ionization potential, high electron transfer ability and optical properties. Polypyrrole (CPPy) is one of the conducting polymers extensively applied for the design of bioanalytical sensors [START_REF] Cosnier | Biosensors based on electropolymerized films[END_REF] [START_REF] Ateh | Polypyrrole-based conducting polymers and interactions with biological tissues[END_REF] including DNA detection [START_REF] Korri-Youssoufi | Electrochemical Probing of DNA Based on Oligonucleotide-Functionalized Polypyrrole[END_REF]. This is related to their electrical, electrochemical properties as well as their ability of immobilization onto micro size surface by electropolymerisation [START_REF] Lê | Effect of the size of electrode on electrochemical properties of ferrocene-functionalized polypyrrole towards DNA sensing[END_REF]. One of their potential powerful is their ability for chemical modification and introduction of functional group that has a great's importance in the construction of biosensors devices [START_REF] Korri-Youssoufi | New method of polypyrrole functionalization toward molecular recognition[END_REF]. E-DNA hybridization sensing based on CPPy has been achieved by measuring their electrical properties through impedance [START_REF] Tlili | Electrochemical impedance probing of DNA hybridisation on oligonucleotide-functionalised polypyrrole[END_REF], amperometric [START_REF] Ramanaviciene | Pulsed amperometric detection of DNA with an ssDNA/polypyrrole-modified electrode[END_REF] or redox markers [START_REF] Korri-Youssoufi | Electrochemical biosensing of DNA hybridization by ferrocenyl groups functionalized polypyrrole[END_REF]. Numerous publications concerning E-DNA biosensors based on CPPy with various approaches were developed. For example, molecular imprinted PPy [START_REF] Ratautaite | Molecularly Imprinted Polypyrrole for DNA Determination[END_REF], association of PPy with graphene [START_REF] Khodadadi | A new epirubicin biosensor based on amplifying DNA interactions with polypyrrole and nitrogendoped reduced graphene: Experimental and docking theoretical investigations[END_REF] or with carbon nanotube [START_REF] Cheraghi | A nanostructure label-free DNA biosensor for ciprofloxacin analysis as a chemotherapeutic agent: an experimental and theoretical investigation[END_REF] were shown to improve the detection performance. However, CPPy nanostructures present a greats interest in biosensors devices as these nanomaterials present high surface area comparable to carbon nanotubes, with tunable conductivity, flexibility, chemical diversity and easy processing [START_REF] Rajesh | Recent progress in the development of nano-structured conducting polymers/nanocomposites for sensor applications[END_REF].

Current research showed that vertically aligned PPy nanowires exhibit a unique combination of morphology and intrinsic properties, such as high surface to volume ratio, fast oxidation/reduction kinetic and electrical conductivity [START_REF] Debiemme-Chouvy | Electrosynthesis of polypyrrole nano/micro structures using an electrogenerated oriented polypyrrole nanowire array as framework[END_REF].

nw-PPy can be obtained by a number of ways including growing on templates of inorganic materials such as anodic aluminium oxide [START_REF] Nazemi | Aluminium oxide nanowires synthesis from high purity aluminium films via two-step anodization[END_REF] (AAO) membranes, zinc oxide [START_REF] Sameera | Temperature dependent current-voltage characteristics of zinc oxide nanowire/polypyrrole nanocomposite[END_REF] or template with organic materials such as, gelatin as soft mold [START_REF] Tran | In-channel-grown Polypyrrole Nanowire for the Detection of DNA Hybridization in an Electrochemical Microfluidic Biosensor[END_REF]. The nanostructures of polypyrrole could also be formed through the nanotechnology approaches, through nanocontact printing and controlled polymerization [START_REF] Errachid | Electropolymerization of nano-dimensioned polypyrrole micro-ring arrays on gold substrates prepared using submerged micro-contact printing[END_REF]. Template free method of synthesizing nanostructures of CPPy has several advantages, like simple synthesis with no template removing steps needed. In addition, uniform and reproducible nanostructures are formed [START_REF] Zhang | Bulk Synthesis of Polypyrrole Nanofibers by a Seeding Approach[END_REF]. Various nanostructures could be formed within direct electropolymerization of pyrrole on electrode, depending on chemical and electrochemical conditions of use. For example, spherical PPy nanoparticles could be formed using potential pulses [START_REF] Plausinaitis | Electrochemical polypyrrole formation from pyrrole 'adlayer[END_REF] where the size could be controlled [START_REF] Plausinaitis | Quartz Crystal Microbalance-Based Evaluation of the Electrochemical Formation of an Aggregated Polypyrrole Particle-Based Layer[END_REF]. nw-PPy without template has been demonstrated in the presence of non-acidic and week-acidic anions by the electropolymerization at fixed potential [START_REF] Qu | Preparation of polypyrrole microstructures by direct electrochemical oxidation of pyrrole in an aqueous solution of camphorsulfonic acid[END_REF],

where the length of the wire could be controlled [START_REF] Debiemme-Chouvy | Template-free one-step electrochemical formation of polypyrrole nanowire array[END_REF]. Various electrochemical sensors based on nw-PPy have been presented for detection of small molecules such as NO 3

-ions [START_REF] Aravamudhan | Development of micro-fluidic nitrate-selective sensor based on doped-polypyrrole nanowires[END_REF], Cu 2+

[34], ammonia gas [START_REF] Zhang | A novel ammonia sensor based on high density, small diameter polypyrrole nanowire arrays[END_REF], glutamic acid [START_REF] Yang | Surface Molecularly Imprinted Nanowires for Biorecognition[END_REF], spermidine [START_REF] Ghanbari | Electrochemically fabricated polypyrrole nanofibermodified electrode as a new electrochemical DNA biosensor[END_REF], glucose [START_REF] Liu | Electrochemically fabricated nanoelectrode ensembles for glucose biosensors[END_REF] [39] and pH sensor [START_REF] Sulka | pH sensors based on polypyrrole nanowire arrays[END_REF]. Several groups have demonstrated E-DNA biosensors based on nw-PPy formed with template. The detection methods are based on the measure of resistance properties of polypyrrole layers using field effect transistors [START_REF] Bangar | Single Conducting Polymer Nanowire Based Sequence-Specific, Base-Pair-Length Dependant Label-free DNA Sensor[END_REF] or through the conductivity variation [START_REF] Mai | Highly sensitive DNA sensor based on polypyrrole nanowire[END_REF].

E-DNA based on amperometric measurements, with nw-PPy is still challenging, as the most demonstrated in literature are not conducting enough to allow high electron transfer and to provide amperometric measurement of attached redox marker.

In this work, we would like to prove that aligned nw-PPy, formed by electropolymerisation with template free, could be employed as transducer and provide chemical and electronic properties which improve the DNA immobilization and detection. We demonstrate that modification of nw-PPy could be achieved through electrochemical deposition of groups bearing amine such as ethylene diamine (EDA) and polyamidoamine dendrimers (PAMAM).

Redox molecules such as ferrocene could be attached to PPy nanostructure to follow their electrical properties and their electron transfer ability. Such nanostructure could be applied for M. tuberculosis detection and discrimination of resistant stand with single nucleotide polymorphism. The results will be compared to other materials based on polypyrrole. the methods described previously [START_REF] Miodek | E-DNA Sensor of Mycobacterium tuberculosis Based on Electrochemical Assembly of Nanomaterials (MWCNTs/PPy/PAMAM)[END_REF].

Pyrrole, ethylene diamine (EDA), dendrimer polyamidoamine of second generation (PAMAM G2) and lithium perchlorate (LiClO 4 ), dibasic potassium phosphate (K 2 HPO 4 ) were purchased from Sigma-Aldrich. The pyrrole is distilled before use. The (1,1'-(phtalimidebutanoate) ferrocene is bearing two acid activated by phtalimido groups and is obtained following the procedure described previously [START_REF] Godillot | Direct chemical functionalization of as-grown electroactive polypyrrole film containing leaving groups[END_REF].

Biosensors formation

Electrodeposition of polypyrrole nanowire:

Films of nw-PPy were grown on the gold surface electrode by potentiostatic method during 2 minutes in a solution containing 0.15M pyrrole, 1 mM LiClO 4 and 0.2 M K 2 HPO 4 in 5 mL of distilled water. During the electropolymerisation, the working and counter electrode were separated in a small volume cell from the reference electrode. After electrodeposition, the electrode was rinsed several times with double distilled water. Various applied potentials were studied 0.8V, 0.84 and 0.88 vs Ag/AgCl to underline their effect on polypyrrole morphology.

Functionalization of PPY with EDA and PAMAM G2 Dendrimers

The functionalization of nw-PPy by PAMAM was performed by CV in a solution of 1 µM of PAMAM and 0.5 M LiClO 4 by scanning the potential from 0 to 1.2 V at 50 mVs -1 during three cycles. This leads to a deposition of PAMAM on nw-PPy layer by covalent bonding and affords amine functions on the surface of PPy. The functionalization of nw-PPy with EDA was performed with the same method. The modified electrode with nw-PPy was placed in water solution containing 0.5 M LiClO 4 and 1M of EDA and the deposition was performed by scanning the potential from 0 to 0.7 V vs. Ag/AgCl during 10 cycles at scan rate of 50 mVs -1 .

Then electrode was washed three times with double distilled water.

Covalent attachment of ferrocene and DNA Probe

These steps were carried out according the optimized procedures described in previous work [START_REF] Miodek | Electrochemical functionalization of polypyrrole through amine oxidation of poly(amidoamine) dendrimers: Application to DNA biosensor[END_REF].

Methods

The electrochemical measurements are based on a potensiostat biologic controlled with Eclab software. All electrochemistry measured was performed with a three-electrode cell with a reference Ag/AgCl electrode, a platinum stem as counter electrode and a working electrode of gold with area of 0.03 cm 2 . The working electrodes are polished several times with different thickness disc (6, 3, 1 µm) and diamond paste with respectively various thickness and subsequently activated in a sulfuric acid solution of 0.5 M by cyclic voltammetry by sweeping the potential between 0 to 1.8 V at 100 mV/S for 15 cycles.

X-ray photoelectron spectroscopy (XPS) was performed on a K Alpha spectrometer from Thermofisher, using monochromated X-ray source (Al Kα 1486, 6ev). The spectra obtained were treated by Advantage software provided by Thermo-Fisher. A Shirley-type background subtraction was used. align and form a well-ordered bundle structure on the surface where the grown of the PPy leads to aligned polypyrrole nanotubes on gold surface [START_REF] Qu | Preparation of polypyrrole microstructures by direct electrochemical oxidation of pyrrole in an aqueous solution of camphorsulfonic acid[END_REF]. Another explanation was also provided, where the mechanism supposes the formation of self-assembled gas bubbles on the working electrode which could act as the chemical template for the growth of nw-PPy [START_REF] Fakhry | Mechanism of formation of templateless electrogenerated polypyrrole nanostructures[END_REF].

To confirm such mechanism, electropolymerisation of N-methyl pyrrole (Py-N-CH 3 ) has been performed in the same condition. SEM image shows that the polymerization process leads to the formation of nanowire (See SI.1, Figure S1). This demonstrates that the formation of nanowire polypyrrole could be obtained even for N-protected pyrrole and favors the mechanism of nanobubble of oxygen as template [START_REF] Debiemme-Chouvy | Template-free one-step electrochemical formation of polypyrrole nanowire array[END_REF].

The electrical properties of the nw-PPy were analyzed through the electron transfer ability of redox process of Fe(CN) 6 4-/Fe(CN) 6 3-system (Figure 3A). CVs show the response obtained with non-modified gold electrode and after the nw-PPy deposition. The CV shows high redox current obtained when gold coated nw-PPy. This demonstrates the formation of conducting polypyrrole layer with high surface area. S3), where one charge-transfer step is obtained according to bode and phase diagram. Various circuit models could be used to fit the PPy layer properties when EIS was

C

performed in the absence of redox markers. Some of them separate the ionic diffusion related to charge compensation and doping process from the electron transfer [START_REF] Ramanavicius | Electrochemical impedance spectroscopy of polypyrrole based electrochemical immunosensor[END_REF]. In this work where low potential range is applied, the circuit model with one interface fits well with the equivalent circuit presented. The measured charge transfer resistance could be related to the electrical properties of nw-PPy and its conductivity. Thus Rct informs on both the electron transfer occurred at the PPy as well as the anion transfer for charge compensation due to doping/undoping process. Result shown that nw-PPy has a characteristic of conducting surface where Rct value around 100 Ω which is in same order of magnitude of PPy compact film (See SI.3; table S3) and in the same order of magnitude of data described in literature [START_REF] Ramanavicius | Electrochemical impedance spectroscopy of polypyrrole based electrochemical immunosensor[END_REF]. The analytical treatment of EIS shows the presence of CPE behavior with n value of 0.8 instead of capacitance which demonstrates that the distribution of charge is not the same in all the thickness of the nanostructure. This behavior is generally observed in the case of nanostructured PPy [START_REF] Ghanbari | Electrochemically fabricated polypyrrole nanofibermodified electrode as a new electrochemical DNA biosensor[END_REF].

Functionalization of nw-PPy and structural characterization

In order to functionalize nw-PPy with functional groups bearing amine in terminal position, electrochemical deposition of molecules containing amine groups were obtained through amine oxidation [47][48]. EDA could be attached to nw-PPy surface through formation of carbon-nitrogen link on the film layer, where the others terminal amine groups could serve for additional functionalization. To increase the number of functional groups dendrimers PAMAM of various generation could be attached with same way as demonstrated previously in the case of compacts film of PPy [START_REF] Miodek | Electrochemical functionalization of polypyrrole through amine oxidation of poly(amidoamine) dendrimers: Application to DNA biosensor[END_REF]. The surface of nw-PPy film was modified with EDA and dendrimers PAMAM of second generation (PAMAM G2). The modification was performed by scanning the potential and during oxidation, amine groups forms radical cations -NH •+ which interact with carbon of PPy leading to covalent grafting.

FT-IR and XPS were performed for the characterization of such reaction. FT-IR spectroscopy allowed analyzing the structural properties of nw-PPy before and after electrochemical deposition of amines. The spectra of nw-PPy and nw-PPy-PAMAM give significant information (See SI.2.1, Figure S2). The fundamental bands of nw-PPy related to the asymmetric (C C) and symmetric (C N) ring stretching vibration of pyrrole ring are observed at ∼1545 and 1305 cm -1 respectively. The bands at 1175 and 903 cm -1 are characteristic of doped nw-PPy chains as demonstrated previously [START_REF] Wu | Synthesis and characterization of conductive polypyrrole with improved conductivity and processability[END_REF]. Covalent attachment of dendrimers PAMAM leads to new bands at 1709 cm -1 attributed to the amide bond C=O of PAMAM.

XPS analysis, in carbon and nitrogen regions for nw-PPy and nw-PPy-PAMAM layers was also achieved (Fig. 4, table S1). The XPS spectrum in carbon region of nw-PPy showed five bands after deconvolution and peaks separations with maximum at 283.92, 284.82, 285.81, 287.65 and 290.94 (Fig. 4A). The two peaks at lowest energy correspond to α and β carbon atoms of nw-PPy. The peak (c) observed at 285.81 eV corresponds to imine C=N or C-N + structures and does at 287.65 eV could be associated to C=N + band. The peak (e) at 290.94

eV is much weaker and could be attributed to π-π* satellite commonly found in aromatic system. The spectrum obtained after PAMAM G2 electrodeposition (Fig. 4B) shows a significantly increase of the peaks ratio of 285.79 eV and 287.62 eV corresponding respectively to C-N and C=O bonds, which could be related to immobilized PAMAM G2.

In specific nitrogen region, nw-PPy layer was characterized with component of the peaks at 401 eV which could be attributed respectively to N-H bonds and to the positively charged nitrogen (Fig. 4C). Small contribution at 398.1 eV could be associated to aromatic amines structure. After modification of the surface with PAMAM G2, significant modifications of nw-PPy-PAMAM spectrum were observed (Fig. 4D). The nitrogen peaks showed two maximums at 399.80 and 401.36 eV which could be attributed to amine and amide bonds respectively. The relative intensity at 399.80 eV is much higher than for nw-PPy before modification and could correspond to C-N and N-H bonds of PAMAM G2 linked to the surface. The component at binding energy region of 401-403 eV corresponds usually to positively charged nitrogen, and the energy band obtained at 399.16 eV could be attributed to amines of PAMAM G2. These results confirm the association of PAMAM G2 dendrimers through covalent attachment to polymer. To demonstrate that the covalent attachment of PAMAM G2 to nw-PPy is through carbonnitrogen link on pyrrole ring and not nitrogen-nitrogen between the amine of polypyrrole and PAMAM, electrodeposition of PAMAM was realized also on nw-PPy-N-CH 3 modified in nitrogen by methyl group. XPS analysis was then used for further investigation on PPy-N-CH 3 and PPy-N-CH 3 -PAMAM (see SI.2.2, Figure S3, Table S2). The deconvolution of N 1S in the case of PPy-N-CH 3 gives one peak 399,9 eV related to carbon-nitrogen bond. After grafted of PAMAM in PPy-N-CH 3 the nitrogen region shows after deconvolution a peak related to carbon-nitrogen bond and new peak at 402.4 which could be attributed to nitrogen positively charged in PAMAM. This result proves that the PAMAM groups are well grafted on the surface of PPy-N-CH 3 and this link is between the nitrogen of PAMAM and carbon β of pyrrole.

Electrochemical characterization of modified nw-PPy

Electrochemical characterization through EIS measurements allowed the characterization of electrical properties of the nw-PPy modified with PAMAM and EDA and the comparison with non-structured PPy (Figure 5). Nyquist plots were performed in PBS solution free of redox markers. The EIS data were successfully fitted by the same equivalent circuit model as shown previously (Figure 3B inset). For the two PPy and nw-PPy, an important increase in the resistance was observed after PAMAM immobilization (Figure 5 A, B and Table S3). The enlargement in high frequency region demonstrates well the small semicircle obtained in the case of the non-modified PPy and nw-PPy compared to those obtained after modification (See SI.3, Figure S4). This is due to the saturation of the surface with larges molecules such as PAMAM which has generally positive charge causing blocking effect. This behavior is more important in the case of nw-PPy-PAMAM than PPy-PAMAM (Figure 5C). This could be attributed to larger amount of the dendrimers covalently bonded to nw-PPy which present higher surface to volume ratio compared to PPy. In the case of surface nw-PPy-EDA (Figure 5D) smaller variation of the charge transfer resistance is obtained due the small size of EDA molecules. PAMAM-FC and nw-PPy-PAMAM-FC. The symbols are the experimental data, and the solid lines are the fitted curves using the equivalent circuit showed in Figure 3.

Association of ferrocene redox marker to the nw-PPy-PAMAM and nw-PPy-EDA was realized to enhance the kinetic of charge transfer within the nw-PPy layer and to study the electron transfer ability. Ferrocene bearing two activated acid groups (N-Hydroxyphtalimide) could react on both the surface of the aminated surface of PPy and the DNA probe bearing amine group in terminal 5'position. After the attachment of ferrocene, the nw-PPy/EDA/Fc and nw-PPy/PAMAMG2/Fc films were analyzed by CV and square wave voltammetry (SWV) (Figure 6). Redox signal of ferrocene with oxidation peak at 0.09 V (vs. Ag/AgCl) and reduction peak at 0.04 V (vs. Ag/AgCl) are obtained after ferrocene attachment on modified nw-PPy-PAMAM and nw-PPy-EDA. The variation of anodic and cathodic peak potential gives a value of 50mV which demonstrated non-ideality reversible redox system.

The formal potential measured is 0.05 V vs Ag/AgCl which is lower compared to ferrocene attached to others surface and nanomaterials (see Table 1). The shift toward lower potentials of ferrocene compared to others materials may be explained by the polar properties of nw-PPy which is known as super hydrophilic structures [START_REF] Fakhry | Templateless electrogeneration of polypyrrole nanostructures: impact of the anionic composition and pH of the monomer solution[END_REF]. SWVs (Figure 6 B) show well-defined peaks of oxidation of ferrocene where the current is much higher for the nw-PPy-PAMAM than that modified with EDA. This result demonstrates clearly that nw-PPy-PAMAM allows to bind a large numbers of redox markers compared to nw-PPy-EDA. The full-widths at halfmaximum (fwhm) of the oxidation peaks could give information about the homogeneity and the distribution of redox molecules on the surface. The theoretical value of fwhm of redox reversible redox center immobilized on the surface is expected at 91mV at 25°C [START_REF] Laviron | General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems[END_REF].

Analysis of electrochemical characteristic of ferrocene attached to nw-PPy through EDA or PAMAM demonstrate that fwhm is 146 mV in both case which is higher that the ideally a one-electron transfer and also higher of monolayer of ferrocene calculated at 114mV [START_REF] Smalley | Heterogeneous Electron-Transfer Kinetics for Ruthenium and Ferrocene Redox Moieties through Alkanethiol Monolayers on Gold[END_REF]. The increased value of fwhm could be related to chemical inhomogeneity of the surface or the interaction between neighboring ferrocene. In the last case, dilutions of redox site by spacing their attachment leads to decrease fwhm. Concerning nw-PPy the same value is obtained with both nw-PPy modified with EDA and PAMAM G2. This underlines that the higher value of fwhm is related to the inhomogeneity of the surface of nw-PPy. Similar report has been demonstrated in the case of ferrocene anchored on nanofiber of carbon where large value has been attributed to the presence of different local environments of the redox couple [54] [55].

The charge exchanged during redox process allows calculation of the surface coverage of ferrocene groups, bonded to the modified nw-PPy, following the equation:

= Q/ nFA (1)
 is surface coverage, Q is the charge under the cathodic or anodic waves, n is number of electrons involved in the redox process, F is the Faraday constant, and A is the area of the electrode.

Based on equation (1) we calculated the average coverage of the surface for nw-PPy nanowires modified with EDA and with PAMAM G2 (see table 1). Immobilization of ferrocene on nw-PPy-EDA leads to surface coverage of 49.10 -9 mole.cm -2 , where surface coverage of 64.10 -9 mole.cm -2 is obtained in the case of nw-PPy-PAMAM. This confirms that the formation of the nano-array of polypyrrole improves the surface area and then the number of attached molecules. EIS were performed after covalent attachment of electroactive ferrocene for both modified electrodes nw-PPy-PAMAM-Fc and nw-PPy-EDA-Fc. Nyquist plots show a decrease in semi-circle after ferrocene attachment which underlines lower charge transfer resistance (Fig. 6C, Table S3). This proves that the bonding of the electroactive species to the modified electrode surface increase the charge transfer ability. ESI shows a more significant decrease of charge transfer resistance in case of nw-PPy-PAMAM-Fc compared nw-PPy-EDA-Fc.

This confirm the previously result demonstrating that the number of ferrocene grafted on the surface of modified electrode is higher in the case of PAMAM immobilization increasing the charge transfer ability.

Kinetic characterizations of attached ferrocene on nw-PPy

To understand how the structure of polypyrrole, impact the electron transfer process, the electron-transfer rate constants are calculated from the peak-to-peak separation of CVs using Laviron method [START_REF] Laviron | General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems[END_REF]. The method uses the numerical integration to correlate the standard electron-transfer rate constant ks with the separation between anodic peaks ∆Ep at different scan rates (equation 2).

1 𝑚 = 𝑛𝑣𝐹 𝑅𝑇 1 𝐾 𝑆 (2)
Where n is number of electrons exchanged in the redox reaction (n=1), v is the scan rate m is the dimensionless rate constant, R is universal gas constant, T temperature, F faraday constant. The value of m was determined by adjustment of the curve Ep = f(m -1 ).

The k s calculated for ferrocene attached on nw-PPy-PAMAM-Fc was measured and compared to PPy-PAMAM-Fc layer obtained by binding of PAMAM to compact PPy film [START_REF] Miodek | Electrochemical functionalization of polypyrrole through amine oxidation of poly(amidoamine) dendrimers: Application to DNA biosensor[END_REF]. An increase of k s from (1.67±0.01) s -1 to (7.44±0.03) s -1 was obtained in the case of nw-PPy-PAMAM-Fc compared to compact layer. This demonstrates that the nanowires improve the electron transfer between neighboring ferrocene. This improvement of the ks could be related to the large surface area of nanowire PPy and their chemical properties which favor the electron hopping process. To test how the molecular length affects the electron-transfer process, the CV data obtained with nw-PPy-EDA-Fc with attached EDA, smaller molecule compared to PAMAM, were analyzed. The data showed that there are significant change in the electron transfer rate constant as the length of the carbon chain is varied from PAMAM to EDA with increase of the k s from (7.44±0.03) to (18.77±0.06) respectively.

Eox (V)

(mole.cm super-hydrophilic character of nw-PPy as their polar and hydrated environment surface which disfavors the interaction of the relatively hydrophobic nucleobase of DNA [START_REF] Elder | Effect of Hydrophobic and Hydrophilic Surfaces on the Stability of Double-Stranded DNA[END_REF].

The reproducibility was tested by measuring the response with 3 fresh biosensors and standard deviation of 10% is measured. This reproducibility could be improved by controlling the surface properties of the gold and the geometry of the cell.

Detection in real sample of M. Tuberculosis and resistant strand

The biosensor was used also for detection of DNA pathogen agent of M. This biosensors present high sensitivity and easy to used compared to those demonstrated in the case of DNA biosensors were amplification steps is generally needed after the DNA hybridization step (see table 2).

2. Material and methods 2 . 1

 21 Reagents: DNA sequences were obtained by Eurogentec Company. The DNA probes are with a sequence of 25 bases with a chain of six carbons and an amino group at its 5 ' phosphate position: NH 2 -C6-5'TCA -ATC-GCT -GGA-TCA -ATC-ATG -TTA -G3'. The complementary DNA has a sequence of 25 bases: 5'CTA -ACA -TTG -AGA -TTC -GCC -AGA-TTG -A3 '. A noncomplementary sequence of DNA also has been used: 5'CTA -ACA -TTG -AGA -TTC -TGA -CGA -GAT -GAT -CTT-C3 ' to study the selectivity of the biosensor. The DNA probe for M. tuberculosis is strand from rpob gene and is contained 18 bases with the sequences NH 2 -C12-5'CCG-ACT-GTT-GGC-GCT-GGG3' and the DNA probe resistant drug with the mutation TTG in position 531 of rpob gene (TCG/TTG) NH 2 -C12-5'CCG-ACT-GTC-GGC-GCT-GGG3'. The DNA of M. Tuberculosis is obtained by amplification of a fragment of 411 bp length of the rpob gene and the mutated one following

Figure 1 : 7 SEM

 17 Figure 1: Schematic presentation of DNA biosensor based on modified nw-PPy: a) electro polymerization of nw-PPy film; b) electrodeposition of PAMAM G2; c) covalent grafting of ferrocene; d) anchoring of ssDNA probe; e) detection of specific DNA target

Figure 2 shows

 2 Figure 2 shows SEMs images of the electrode surface modified with nw-PPy formed using various potentials of electrodeposition. nw-PPy formed with applied potential of 0.88V underlines low organization of the nanowire and large width around 400 nm (Fig 2, image a). Decreasing the applied potential to 0.86V and 0.84V leads to lower width of nanowire and formation of array (Fig 2, images b et c). This proves the relationship between the potential applied for the polymerization and the structure of nanowire where applied potential has a large effect on the structure. The optimized condition retained thereafter is, applied potential of 0.84V during 120s. The wide of nanofibers are around 100 nm.

Figure 2 :

 2 Figure 2: SEM images of gold surface covered by nw-PPy with different scale (20µm and 2µm) the films were formed with various applied potential a) E=0.84V; b) E=0.88V; c) E=0.80V.

Figure 3 :

 3 Figure 3: (A) Cyclic Voltammetry of the gold electrode analyzed in solution of Fe(CN) 6 4-/Fe(CN) 6 3-before and after polymerization with nw-PPy; (B) Nyquist plots obtained after modifications of gold electrode with PPy and nw-PPy the measurements were obtained in PBS buffer, in frequency range from 0.1Hz to 100 kHz at 0.10 V vs. Ag/AgCl by applying DC potential of 10 mV. The symbols are the experimental data, and the solid lines are the fitted curves using the equivalent circuit showed in inset. (C) Same Nyquist plot as (B) with enlargement in high frequency range.

Figure 4 :

 4 Figure 4: Deconvolution of XPS spectra in carbon and nitrogen for nw-PPy layer (A,C) and nw-PPy-PAMAM (B, D) after modification with PAMAM G2 dendrimers.

Figure 5 :

 5 Figure 5: Nyquist plots obtained after attachment of dendrimers PAMAM G2 on PPy(A) and nw-PPy( B) and (C) comparison of response obtained between structured and non structured

Figure 6 :

 6 Figure 6: (A) CV (B) SWV of electrochemical signal of ferrocenyl group (Fc) linked to nw-PPy modified with dendrimers PAMAM and EDA, (C) Nyquist plots obtained after covalent attachment of ferrocene to the nw-PPy-PAMAM and nw-PPy-EDA modified electrode. The measurements were obtained in PBS buffer, in frequency range from 0.1Hz to 100 kHz at 0.10 V vs. Ag/AgCl by applying DC potential of 10 mV. The symbols are the experimental data, and the solid are the fitted curves using the equivalent circuit.

1 :

 1 Electrochemical parameters obtained for various modified polypyrrole structures3.5 Properties of biosensors towards DNA detectionTo study the effects of nanostructured polypyrrole in sensing properties, the covalent attachment of DNA and then hybridization reaction between ssDNA probe immobilized and DNA target in solution has been realized. The concentration range of DNA target studied was from 1aM to 1 pM. This was measured by the changes of current corresponding to redox signal of ferrocene attached to the nw-PPy. SWVs show the variation of the redox current after DNA hybridization within the various concentrations (Figure7A). The decrease of redox current is related to the low electron transfer happened after the hybridization of complementary DNA and the formation of double strand on the surface. This current variation is depending on the concentration of DNA target in solution. Calibration curve was plotted by presenting the average current variation versus the logarithmic scale of the concentration (Figure7B). We can see that large dynamic range of DNA detection is obtained with this biosensor from 1 aM to 100fM where the saturation is started at 100fM. The calculated detection limit of 0.36 aM is obtained, based on signal-to-noise ratio of 3 and by measuring the standard deviation of the blank test. This sensitivity could be related to various parameters provided by polypyrrole nanostructure and their functionalization such as the high surface area of nw-PPy, that allows loading a large amount of DNA probe, the conductivity of the nw-PPy layer and hydrophilic character, which enhances the electron transfer ability. This result demonstrates the potential of nanowire polypyrrole formed by electrodeposition as platform for DNA detection.

Figure 7 : 3 . 6

 736 Figure 7: (A) SWV recorded in PBS solution after incubation of biosensor with various DNA concentrations (1aM-100 fM). (B) Plot of the relative changes of the current peak vs. concentration of DNA target ΔI=(I 0 -I)/I 0 , where I 0 is the peak current of biosensor and I the current measured after incubation of the biosensor with certain concentration of DNA target. (C) SWV obtained after incubation of biosensors the 1µM of ncDNA and 1fM of c DNA target.

  tuberculosis, and was applied to discriminate the single nucleotide polymorphism (SNP) (TCG/TTG) which gives the resistance of M.tuberculosis to rifampicin drug. The PCR sample were composed of 411 bases of DNA fragment from rpoB gene called WT, and fragment with mutated DNA called Mut. The DNA probe specific to WT and Mut was composed of 18 bases modified with amine group and was attached to the nw-PPY surface. The hybridization reaction with WT and Mut DNA was followed by SWV of redox signal of ferrocene (Figure8).

Figure 8 :

 8 Figure 8: (A) SWV response obtained with biosensors modified with WT probe (solid line) and after detection with PCR sample with mutated DNA (dash line) and after detection with PCR sample with

work 4 .

 4 ConclusionNanowire polypyrrole formed by template free with simple electropolymerization could be used as sensitive E-DNA biosensors through amperometric measurement. We demonstrated that nw-PPy could be formed by template free and highlight the mechanism of their formation as well as high electrical properties. The obtained nw-PPy could be modified through EDA or PAMAM electrodeposition through carbon-nitrogen coupling reaction which leads to introduction of aminated surface. Covalent attachment of ferrocene as redox marker on the modified nw-PPy could be performed and the electrochemical properties exhibits lower redox potential and high rate of electron transfer (18s -1 ). This is obtained thanks to high hydrophilic chemical structure character, good conductivity and high surface area of nw-PPy. E-DNA biosensor based on nw-PPy modified with ferrocene as redox marker shows a detection limit of 0.36aM without any amplification procedure. Improvement of the sensitivity to DNA hybridization was demonstrated compared to compact PPy layer and others nanostructured materials. Their selectivity was proven by their ability to discriminate rpob gene of M.Tuberculosis from mutated with single polymorphism. The biosensors are able to measure DNA from complex sample such as PCR. These analytical performances properties were obtained by dint of the chemical and electronic properties and their soft functionalization through amine oxidation. Thus, high surface area of nw-PPy allows loading a large amount of DNA probe, the conductivity of the nw-PPy layer enhances electron transfer ability, where hydrophilic character of nw-PPy prevents non-specific interaction. The developed electrochemical DNA based on nw-PPy could be used as platform for detection of various biomolecules in complex sample involving specific bioreceptor.

  ; (D) comparison of ESI obtained after the attachment of EDA or PAMAM to nw-PPy. The measurements were obtained in PBS buffer, in frequency range from 0.1Hz to 100 kHz at 0.10 V vs. Ag/AgCl by applying DC potential of 10 mV. (C) Comparison between PPy-

	A	8.0x10 4			PPy PPy-PAMAM	B		8.0x10 4					nw-PPy nw-PPy-PAMAM
		6.0x10 4
									6.0x10 4
	-Z''()	4.0x10 4					-Z''()	4.0x10 4
		2.0x10 4							2.0x10 4
		0.0
									0.0
		0.0	2.0x10 4	4.0x10 4	6.0x10 4	8.0x10 4				0.0	2.0x10 4	4.0x10 4	6.0x10 4	8.0x10 4
				Z'()
													Z'
	C	8.0x10 4		nw-PPy-PAMAM PPy-PAMAM		D	8.0x10	4					nw-PPy-PAMAM nw-PPy-EDA
		6.0x10 4						6.0x10	4
	-z''	4.0x10 4					-Z''	4.0x10	4
		2.0x10 4						2.0x10	4
		0.0							0.0
		0.0	2.0x10 4	4.0x10 4	6.0x10 4	8.0x10 4				0.0	2.0x10	4	4.0x10	4	6.0x10	4	8.0x10	4
				z'									Z

surface

Table 2 :

 2 Comparison of the present method for M. tuberculosis detection with other reported works based on electrochemical biosensors. (AuNPs Gold nanoparticles; RGO reduced graphene oxide; MPs magnetic nanoparticles, PANi polyaniline; ALP Alkaline phosphatase,

	MB methylene blue)
	Biosensor	Method	of	Detection	Dynamic range	Reference
		detection	or	limit		s
		amplification
	Dextrin/AuNPs	Amplification		0.01ng/µl	0.01-10 ng/µl	[59]
		MPs/AuNPs
	CNT/ZrO 2	impedimetric		0.065 ng/µl 1-150ng/µl	[60]

Acknowledgments:

The authors' acknowledge University Paris-Saclay and CNRS for finding this project and thanks the Lebanese government for the PhD fellowship. The project PHC N° 39382RE is acknowledged for supporting a part of this project.


domain: phys chim

text:
Meryem Bouaziz

Wei Zhang

Yongfeng Tong

Hamid Oughaddou

Hanna Enriquez

Rym Mlika

Hafsa Korri-Youssoufi

Zhesheng Chen

Heqi Xiong

Yingchun Cheng
email: iamyccheng@njtech.edu.cn

Azzedine Bendounan
email: azzedine.bendounan@synchrotron-soleil.fr

Phase transition from Au-Te surface alloy towards tellurene-like monolayer

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

energy electron diffraction (LEED) supported by scanning tunneling microscopy (STM) shows the existence of several surface reconstructions depending on the Te film thickness in the sub-monolayer regime. We observed that the well-known spin-split Shockley state of the Au(111) surface survives the Te deposition and is even shifted to higher binding energy, suggesting a charge transfer at the interface. For a coverage of 0.33ML of Te, new dispersive bands are observed by angle-resolved photoemission (ARPES), which arise from a strong hybridization between the electronic states of Te and Au. With a substantially low intensity and a back-folding at the boundaries of the reduced surface Brillouin zone, these electronic bands represent a proof of the existence of a naturel 2D electron gas, strongly disturbed by the surface reconstruction. It is therefore possible that an Au 2 Te alloy forms on the surface. By increasing the coverage to 0.5ML, a rich, thickness-dependent transition develops from the surface alloy to tellurene-like structure and completely excludes the growth of AuTe 2 monolayer on the surface. Both the surface alloy and the tellurene-type monolayer have a semiconductor character with a gap in the occupied states of 0.65 eV.

gap gap TOC (3x3)R30° (3x3)

1. Introduction

In the recent years, two dimensional (2D) materials have attracted enormous scientific and technological interests. In particular, the transition metal dichalcogenides (TMDs) in the monolayer or multilayer regimes have received increasing and impressive attention due to the remarkable success in combining their unique optoelectronic properties with their reduced dimensions at the nanometer scale [START_REF] Wang | Electronics and Optoelectronics of Two-Dimensional Transition Metal Dichalcogenides[END_REF] . In fact, their intrinsic band gap is thickness dependent, thus makes them as a promising alternative to the most studied 2D materials, i.e. graphene, which lacks an intrinsic band gap. For example, MoS 2 monolayer exhibits a direct energy gap of 1.8 eV and pronounced photoluminescence [START_REF] Mark | Atomically Thin MoS 2 : A New Direct-Gap Semiconductor[END_REF][START_REF] Splendiani | Emerging Photoluminescence in Monolayer MoS 2[END_REF] , while bulky MoS 2 has a trivial photo-response and an indirect band-gap. In addition, size reduction of the TMDs systems can induce highly sought-after exotic properties, such as superconductivity [START_REF] Xi | Ising Pairing in Superconducting NbSe 2 Atomic Layers[END_REF][START_REF] Saito | Superconductivity Protected by Spin-Valley Locking in Ion-gated MoS 2[END_REF] , charge density waves [START_REF] Barja | Charge Density Wave Order in 1D Mirror Twin Boundaries of Single-Layer MoSe2[END_REF] , Quantum spin Hall effect [START_REF] Qian | Quantum Spin Hall Effect in Two-Dimensional Transition Metal Dichalcogenides[END_REF] , and valley-spin splitting [START_REF] Cao | Valley-Selective Circular Dichroism of Monolayer Molybdenum Disulphide[END_REF][START_REF] Wang | Spin-Orbit Enginnering in Transition Metal Dichalcogenide Alloy Monolayers[END_REF] .

Tellurium (Te)-based TMDs constitute ideal model systems for fundamental and technological researches. Being the heaviest non-radioactive element of the chalcogen group, Te is characterized by high air stability, strong spin-orbit coupling and a p-type semiconductor nature, which are valuable parameters for modern high-level technological applications. Te is also a monotonically tuned bandgap semiconductor with decreasing thickness from 0.33eV in a

Te solid crystal to 1.0eV in the 2D monolayer system. On the other hand, noble metal surfaces, especially Au(111), can provide a platform for hosting an epitaxial TMD layer or binary surface alloy, both of which may accommodate fascinating 2D electron gas. Indeed, the in-situ deposition of Te on metallic surfaces (M) does not necessarily form TMD monolayers of MTe 2 composition, but can also generate a monolayer of surface alloy as M 2 Te. In recent study, we have identified Cu 2 Te alloy monolayer as an interesting new 2D material [START_REF] Tong | Evidence of New 2D Material: Cu 2 Te. 2[END_REF] , which was also confirmed by another group [START_REF] Qian | Epitaxial Growth and Air-Stability of Monolayer Cu 2 Te[END_REF] . This behavior seems to depend on the metal substrate itself because epitaxial growth of Se on Pt(111) leads to the formation of PtSe 2 having identical properties to MoS 2 sheets [START_REF] Wang | Monolayer PtSe 2 , a New Semiconducting Transition-Metal-Dichalcogenide, Epitaxially Grown by Direct Selenization of Pt[END_REF][START_REF] Tong | Phase Transition and Thermal Stability of Epitaxial PtSe 2 Nanolayer on Pt(111)[END_REF] . Also, it was shown that co-deposition of Te and Pt on 6H-SiC(0001) yields to the formation of PtTe 2 film [START_REF] Lin | Dimensionality-Mediated Semimetal-Semiconductor Transition in Ultrathin PtTe 2 Films[END_REF] . For practical purpose, the monolayer of Te proved to be an ideal buffer layer to decouple blue phosphorus film from the Au substrate [START_REF] Gu | Growth of Quasi-Free-Standing Single-Layer Blue Phosphorus on Tellurium Monolayer Functionalized Au(111)[END_REF] .

More interesting, it has been reported that Te doping boosts the transport performances and the stability to air of black phosphorus based Field Effect Transistors [START_REF] Yang | Te-Doped Black Phosphorus Field-Effect Transistors[END_REF] . In the herein study, we characterized the atomic and electronic structures of low amount of Te (in the submonolayer regime) deposited by UHV-MBE on Au(111). One of the expected scenarios is the formation of one monolayer of AuTe 2 . This composite called also Calaverite, is the most common gold mineral and is the only compound in nature from which one can extract gold on an industrial scale. Moreover, it develops a superconductivity phenomenon when doped with Pt 17 (Pd [START_REF] Kudo | Composition-Induced Structural Instability and Strong-Coupling Superconductivity in Au 1-x Pd x Te 2[END_REF] ) or submitted to high pressure [START_REF] Kitagawa | Pressure-Induced Superconductivity in Mineral Calaverite AuTe2[END_REF] . To our limited knowledge, there is to date no evidence of an epitaxial or isolated monolayer of AuTe 2 . The second scenario is the formation of an Au 2 Te surface alloy, where Te atoms are embedded in the topmost Au layer, as has been effectively demonstrated for the other two noble metals Cu and Ag. A third scenario is also plausible, which consists of the emergence of the so-called "tellurene" monolayer floating on the Au(111) surface, as in the case of flat stanene on Cu(111) [START_REF] Deng | Epitaxial Growth of Ultraflat Stanene with Topological Band Inversion[END_REF][START_REF] Ahmed | Structure Determination of Ultra-Flat Stanene on Cu(111) Using Low Energy Electron Diffraction[END_REF] . In fact, tellurene, an unaltered monolayer of Te with a honeycomb structure, has been successfully synthesized and employed in hightech applications, due to its large on/off ratios, excellent ambient condition stability, and high carrier mobility [START_REF] Wu | Tellurene: its Physical Properties, Scalable Nanomanufacturing, and Device Applications[END_REF][START_REF] Wang | Field-Effect Transistors Made from Solution-Grown Two-Dimensional Tellurene[END_REF][START_REF] Shen | Tellurene Photodetector with High Gain and Wide Bandwidth[END_REF] .

Although the atomic structure of Te/Au(111) was the subject of long discussion [START_REF] Guan | Superstructures at Te/Au(111) Interface Evolving upon Increasing Te Coverage[END_REF][START_REF] Schouteden | Adsorption of Te Atoms on Au(111) and the Emergence of an Adatom-Induced Bound State[END_REF][START_REF] Sorenson | Formation of and Phase Transitions in Electrodeposited Tellurium Atomic Layers on Au(111)[END_REF][START_REF] Nagashima | Growth Modes and Surface Structures of Te Overlayers Deposited on Au(111) Surfaces[END_REF][START_REF] Hayden | In-Situ STM Study of Te UPD Layers on Low Index Planes of Gold[END_REF][START_REF] Ikemiya | Atomic Structures and Growth Morphologies of Electrodeposited Te Film on Au(100) and Au(111) Observed by In-Situ Atomic Force Microscopy[END_REF] , its electronic structure is still unexplored and no literature referring to the behavior of the valence electrons is reported up to day. After deposition of Te, the well-known 23x3 herringbone reconstruction is lifted, which suggests strong Te-Au interaction at the surface. Thereby, we present systematic investigation using complementary techniques to analyze the interplay between structural and electronic properties. Through LEED and STM, we have exanimated the crystal structure and atomic arrangement of the surface. To learn about the interaction inducing a chemical shift, XPS measurements were performed on the Au4f and Te4d core levels.

The effect of the surface reconstruction depending on the Te coverage was systematically analyzed. Interestingly, by ARPES we have studied in detail the characteristics of the Shockley state, as well as new spectroscopic bands that result from hybridization between Te and Au electronic states. A structural phase transition depending on the Te coverage is observed and the formation of tellurene-type monolayer showing a honeycomb-like structure is suggested.

Experiment

Sample preparation: the Au(111) surface was prepared in-situ by standard procedure of multiple cycles consisting of Ar + sputtering followed by annealing at 500°C in ultra-high vacuum (UHV). The surface cleanliness was monitored by high-resolution XPS on the carbon and oxygen core levels, while the crystalline structure of the surface was characterized by LEED. For the present work, we have used a home-made Knudsen cell for the evaporation of Te in UHV. The evaporation rate was monitored by quartz microbalance and calibrated with respect to reference data from the literature [START_REF] Guan | Superstructures at Te/Au(111) Interface Evolving upon Increasing Te Coverage[END_REF] . We have also employed an alternative method based on chemical deposition that proved to give the same results and be successfully utilized for elaboration of other systems [START_REF] Tong | Evidence of New 2D Material: Cu 2 Te. 2[END_REF][START_REF] Tong | Phase Transition and Thermal Stability of Epitaxial PtSe 2 Nanolayer on Pt(111)[END_REF] . It consists in immersion of the Au(111) substrate in 0.1 ML NaTe 2 solution for few minutes. The resulting surface is then well rinsed with Milli-Q water and dried under N 2 flow before reintroducing into the UHV analysis chamber for LEED, STM and XPS analysis.

Characterization: to carry out the high-resolution photoemission experiments we used the synchrotron radiation at TEMPO Beamline of Synchrotron SOLEIL, France. For probing the Au and Te core levels it was necessary to utilize photon energy of 260eV, which represents a compromise for obtaining reasonable photoionization cross section and high energy resolution

(estimated of about 50meV). The ARPES measurements were made with photon energy of 60eV in excellent conditions of high energy and angle resolutions. The energy scale was also calibrated with the Au4f 7/2 on a clean Au(111) surface at the corresponding photon energy. The experimental binding energy calibration error for the reported core level binding energies (CLBE) is estimated to be of ±50 meV. LEED and Low temperature STM measurements were conducted on an Omicron STM station at ISMO-CNRS laboratory, Paris-Saclay University. The temperature reached 77K with liquid N 2 cooling during measurements. No further treatment of the STM images was applied.

Results description

LEED and STM investigations

First, we review here how the atomic structure of the Au(111) surface evolves after deposition of Te. Fig. 1 displays large size STM topographies obtained on clean and covered Au(111) surface by Te sub-monolayer amounting to 0.33ML and 0.5ML, respectively. As expected, the pristine Au(111) surface shows the well-known herringbone reconstruction which originates from an uniaxial contraction of about 4.4% of the surface topmost layer with respect to the bulk, resulting in a reduction of the lateral Au-Au interatomic separation at the surface [START_REF] Barth | Scanning Tunneling Microscopy Observations on the Reconstructed Au(111) surface: Atomic Structure, Long-Range Superstructure, Rotational Domains, and Surface Defects[END_REF][START_REF] Wang | Simulation of the Au(111)-(22x3) Surface Reconstruction[END_REF] . Thus, due to this constraint, the Au surface atoms occupy fcc or hcp sites of the underlying bulk lattice and 23 surface atoms occupy 22 substrate positions along the close-packed [1-10] direction [START_REF] Barth | Scanning Tunneling Microscopy Observations on the Reconstructed Au(111) surface: Atomic Structure, Long-Range Superstructure, Rotational Domains, and Surface Defects[END_REF] .

The regions with high density of Au atoms appears as bright stripes in the STM image and correspond to the so-called domain walls or soliton walls which separate the wide fcc and narrow hcp regions [START_REF] Barth | Scanning Tunneling Microscopy Observations on the Reconstructed Au(111) surface: Atomic Structure, Long-Range Superstructure, Rotational Domains, and Surface Defects[END_REF] . We observe that this peculiar reconstruction of Au(111) is lifted when Te atoms are deposited on the surface. Such an effect is attributed to the reduction of the surface stress via charge redistribution induced by the Te electronegative adsorbates. Thus, this lifting of reconstruction is accompanied by the release of gold atoms from the herringbone structure.

Note that when the adsorbate binds to the Au(111) surface by a simple van der Waals-like (a)

7.7Å

9.4nm

(b) (c)
interaction, the herringbone reconstruction remains imperturbable, as has been observed for organic molecular films [START_REF] Kelly | An Investigation into the Interactions Between Self-Assembled Adenine Molecules and a Au(111) Surface[END_REF][START_REF] Kezilebieke | Steric and Electronic Selectivity in the Synthesis of Fe-TCNB Complexes on Au(111): from Topological Confinement to Bond Formation[END_REF] and also surprisingly after the deposition of Sn on Au(111), which induces a SnAu 2 alloy on the surface [START_REF] Maniraj | Structure and Electronic Properties of the (3x3)R30° SnAu 2 /Au(111) Surface[END_REF] . For the present system, even a slight variation in Te coverage causes a radical change of the atomic arrangement, as shown in Fig. 1. Note however that the STM topographies display surfaces completely covered with very ordered superstructures at great distances. According to literature results, the formation of isolated and large islands of pure Te is excluded and, instead, the Te atoms are statistically distributed over the entire surface even at a very low coverage [START_REF] Schouteden | Adsorption of Te Atoms on Au(111) and the Emergence of an Adatom-Induced Bound State[END_REF] and substitute Au atoms of the topmost surface layer when the thickness exceeds 0.24ML [START_REF] Guan | Superstructures at Te/Au(111) Interface Evolving upon Increasing Te Coverage[END_REF] . For Te coverage of about 1/3 ML, the surface exhibits a (3x3)R30° super-lattice, with Te atoms in the hollow site positions. Such a reconstruction has also been observed for Te/Cu(111) [START_REF] Tong | Evidence of New 2D Material: Cu 2 Te. 2[END_REF] and Te/Ag(111) [START_REF] Shah | Two-Dimensional Binary Honeycomb Layer Formed by Ag and Te on Ag(111)[END_REF] , and for many other systems [START_REF] Ast | Giant Spin Splitting through Surface Alloying[END_REF][START_REF] Moreschini | Influence of the Substrate on the Spin-Orbit Splitting in Surface Alloys on (111) Noble Metal Surfaces[END_REF][START_REF] Moreschini | Assessing the Atomic Contribution to the Rashba Spin-Orbit Splitting in Surface Alloys: Sb/Ag(111)[END_REF] . In addition, there is the coexistence of another reconstruction of a (321x321)R10° super-periodicity, as previously shown [START_REF] Guan | Superstructures at Te/Au(111) Interface Evolving upon Increasing Te Coverage[END_REF] . At coverage of ~ 0.5 ML, the morphology changes significantly and new reconstruction occurs at the surface, which consists of highly ordered honeycomb-like structure at large distance, as demonstrated in Fig. 1c. Moreover, on-top of the reconstructed surface upper-layer, self-organization phenomenon of adatoms or clusters is observed. In fact, the adatoms occurring as bright dots in the STM image form well-defined and oriented atomic chain structures.

In the following, more detail is given on the atomic structure and the distribution of the Te atoms within the topmost surface layer. LEED patterns together with the corresponding atomically resolved STM images, as well as the profile line details are reported in Fig. 2. At Te coverage of about 0.25ML, it was shown that (√3x√3)R30° superstructure starts to develop on the surface [START_REF] Guan | Superstructures at Te/Au(111) Interface Evolving upon Increasing Te Coverage[END_REF] . In addition, it was demonstrated that a large scale reconstruction type (√111x√111)R4.7° is also present [START_REF] Guan | Superstructures at Te/Au(111) Interface Evolving upon Increasing Te Coverage[END_REF] . Here, we restrict ourselves to two characteristic Te thicknesses: 0.33 ML and 0.5ML.

(d) (e) (f)

(b) (a)

4.0nm (c)

Indeed, by increasing the Te coverage to 0.33ML, the surface evolves sensively at the atomic scale. As shown in Fig. 2a, the STM image reveals a highly structured surface and the LEED pattern (in inset) indicates the coexistence of two types of superstructure. The (√3x√3)R30° reconstruction is preserved whereas the 2D rhombus phase changes to another reconstruction with larger supercell (3√21x3√21)R10°. Thereby, the supercell size of √111 2D is converted to 3√21 by adding Te atoms. There is also a change in the orientation of the rhombus phase from 4.7° to 10°. From the STM image, we can destinguish a periodic flower-shaped pattern characterized by ordered arrows forming diamond-like patches that are abutted by two triangles. There are dark and bright patterned Te atoms, which are all sitting on (√3x√3)R30° superlattice with inter-distance of 0.5nm (Fig. 2b). The distance between the centers of the flower feature is approximately 4nm, as obtained from the profile line shown in Fig. 2c. This value is consistent with the moiré superstructure of (3√21x3√21)R10° periodicity. Because of the large radius of Te atoms, there is no sufficient space for them to always sit on the exact same 3-fold sites of Au(111). This leads to the formation of dark furrows along the edge of the diamond structure, where the Te atoms on each side are in different 3-fold sites and slightly further apart. On one side of a furrow, the atoms are on face-centered cubic (fcc) sites whereas the atoms on the other side are on hexagonal close-packed (hcp) sites.

When the Te coverage reaches 0.5 ML, one observes a honeycomb-like structure characterized by holes forming super-cell with hexagonal symmetry, as shown in Fig. 2d,e. Careful examination of a line profile (Fig. 2f) yields an average hole spacing (between two successful depletion regions) of approximately 8.7 Å, which corresponds to 3x the Au(111) lattice parameter. This is the signature of a (3x3) reconstruction, which is confirmed in the LEED pattern reported in the inset of Fig. 2d and occurs in perfect accord with the literature [START_REF] Guan | Superstructures at Te/Au(111) Interface Evolving upon Increasing Te Coverage[END_REF] . Additionally, small clusters of Te adatoms nucleate on the holes of honeycomb-like structure and tend to align in quasi-1D chains following the symmetry direction of the honeycomb-like superstructure underneath.

There are two types of adatom rows. On the one hand, relatively long rows composed of separate adatoms and, on the other hand, very dense and short rows. Indeed, the dominant distance between neighboring adatoms is 8.70 Å, while its value within the dense rows averages about 4.35 Å. The spacing between chains varies between 8.7 Å and 34.5 Å.

Photoemission analysis

Fig. 3 shows the XPS spectra measured on Au(111) surface covered by 0.5ML of Te, recorded at normal and grazing emission angles, respectively. The spectra display an intense doublet due to electrons photo-emitted from the Au 4f 7/2 (84eV) and Au4f 5/2 (87.6eV) core levels and very weak structures attributed to the Au5p levels. Deposition of Te film provokes the occurrence of spectroscopic features at binding energy of around 41 eV, which are assigned to the Te4d core levels. Note that the low intensity of the Te4d peaks compared to the Au4f is related to the effect of the photoionization cross section, which is significantly larger for Au4f than for Te4d at the selected photon energy (260eV) [START_REF] Yeh | Atomic Subshell Photoionization Cross Sections and Asymmetry Parameters: 1  Z  103[END_REF] . The spectral weight of the Te4d photoelectrons increases as we move from the normal to the grazing emission geometry, while the opposite effect is observed for Au4f. This means that the Te atoms are mostly on the surface of the sample and indicates the absence of any segregation process of Au atoms to the surface.

Fig.3: XPS spectra measured with h=260 on Au(111) covered by 0.5ML of Te at normal and grazing emission geometries, respectively. Zooms are given on the Au4f and Te4d core levels.

Fig. 4 illustrates a comparison between the Au4f spectra of the clean Au substrate and that after deposition of Te. The main component of the Au 4f 7/2 peak at 84.0eV is associated with bulk Au (Au B ), whereas the shoulder at 83.66eV is associated with the reconstruction of the Au surface atoms (Au S ), since its intensity increases at the grazing emission geometry at which the surface contribution becomes high [START_REF] Jia | Selenium Adsorption on Au(111) and Ag(111) Surfaces: Adsorbed Selenium and Selenide Films[END_REF] . After substation of a Shirley background, the experimental spectrum was deconvoluted into two components Au B and Au S as shown in Fig. 4, using a Voigt line profile with essentially the same parameters. After Te adsorption, the Au S component at 83.66eV vanishes as a result of interaction with Te. By analogy with gold oxidation, one could expect the appearance of a higher binding energy component if a gold telluride is formed, but this is not the case here. Indeed, we obtain an effect comparable to the case of Se/Au(111) [START_REF] Jia | Selenium Adsorption on Au(111) and Ag(111) Surfaces: Adsorbed Selenium and Selenide Films[END_REF] system and one expects a weakly-chemisorbed Te on the Au(111) surface.

Fig.4: Core level Au4f XPS spectra recorded at normal emission before and after deposition of 0.5ML of Te on Au(111). As shown, the deconvolution of these spectra yields to two contributions for the clean substrate, Au B stands the bulk and Au S the surface, while only the Au B one is present after growing Te on the surface.

The Te4d core level spectra obtained on Te covering Au(111) at thicknesses ranging from 0.25ML to a thick layer (greater than 20ML) are shown in Fig. 5. Two main spectroscopic structures associated with 4d 3/2 and 4d 5/2 core levels are observed. For a detailed analysis, we limit ourselves to the 4d 5/2 characteristics in the rest of the discussion. Starting from thick-layer of Te on Au(111), the fit using voigt-like peaks of the 4d 5/2 feature indicates the presence of one contribution positioned at 40.44eV and assigned to the Te bulk. This value is consistent with the binding energy obtained on Te/Cu(111) elaborated by chemical deposition method [START_REF] Tong | Evidence of New 2D Material: Cu 2 Te. 2[END_REF] . When the Te film is much thinner around 0.5ML, the shape of the photoemission spectrum remains almost unchanged, however, the peak position of the 4d 5/2 level occurs shifted to 40.1eV. The peak line-width is almost identical and is about 0.54 eV. At this thickness, the surface displays (3X3) honeycomb-like reconstruction on which self-organized adatom chains are formed, as discussed above. Surprisingly, we observed only one chemical environment, whereas one can expect at least two contributions; one related to adatom chains and the other to the (3x3) reconstruction behind. One possible explanation of this result is that both contributions have the same binding energy. This means that the atoms from chains and those of the underlying layer have comparable interactions with the Au substrate, which are in this case significantly weak. Thus, it can be suggested that the (3x3) reconstructed layer is decoupled from the Au(111) substrate and can be described as a floating monolayer of Tellurene-type, since it also has a honeycomb-like structure.

Fig.5: XPS spectra of the Te4d core levels obtained for different thicknesses of Te on

Au(111). Each spectrum is deconvoluted using fit with voigt-like peaks.

When the Te thickness is about 0.33ML, the photoemission spectrum changes and exhibits features significantly asymmetric due to the presence of a shoulder at the high energy side.

After a Shirley background subtraction the spectrum was deconvoluted (Fig. 5) by choosing the same peak width as that used for the other thicknesses. An accurate fit required utilization of two voigt-type contributions; one is associated with the shoulder structure at binding energy of about 40.13eV and second contribution characterized by high intensity and located at 39.77eV, as seen in Fig. 5. Thus, two chemical environments of Te exist in the reconstruction shown in As seen in Fig. 6, the Shockley state of pristine Au(111) corresponds to two parabolic electron dispersion shifted in the right and in the left from the -point. This net offset in the k-direction

+ /
represents to the Rashba spin-orbit (RSO) splitting, which is well described in the literature [START_REF] Hoesch | Spin Structure of the Shockley Surface State on Au(111)[END_REF][START_REF] Henk | Spin Polarization of the L-Gap Surface States on Au(111)[END_REF] , also for other systems [START_REF] Forster | The Shockley-type Surface State on Ar Covered Au(111): High Resolution Photoemission Results and the Description by Slab-Layer DFT Calculations[END_REF][START_REF] Bendounan | Role of the Shockley State in Doping of Organic Molecule Monolayer[END_REF] .

After deposition of 0.33ML of Te, we observe a significant downward energy shift of the Shockley state band, which is attributed to an electron transfer process from the Te atoms to the Au(111) surface. Also, there is a significant change in the effective mass, whereas it is not obvious to estimate the variation in the RSO splitting due to the broadening of the peak, which is attributed to the effect of the high anisotropy of the surface reconstruction causing a loss of momentum conservation of the emitted photoelectron and hence leading to a significant decay of the Shockley state intensity and to a high background. The contribution of undesirable adsorbates such as oxygen, carbon or water is totally excluded since the XPS indicated an Au(111) surface free of such molecules. To obtain the Shockley state parameters for Te/Au(111), we used the dispersion formula of a nearly free electron behavior, in which we assumed the same RSO amplitude as for pristine Au(111). Thereby, its energy dispersion can be described within the Bychkov-Rashba (BR) [START_REF] Bychkov | Properties of a 2D Electron Gas with Lifted Spectral Degeneracy[END_REF] superstructure, which is a typical behavior of a 2D electron system. As seen in Fig. 7, these hybridization bands are characterized by low intensity, as the photoelectrons are probably strongly scattered by the profound corrugation of the long range moiré structure. On the other hand, along Γ-M direction, it is seen that the Shockley state band in second SBZ is back-folded due also to the effect of the moiré superstructure of the surface alloy observed by STM. This finding suggests the formation of 2D like electrons system localized at the Au 2 Te surface alloy.

The ARPES data reveal also a semiconductor character of the Au 2 Te monolayer with a gap limit of about 0.65 eV, which in agreement with the STS measurements given in ref. [START_REF] Guan | Superstructures at Te/Au(111) Interface Evolving upon Increasing Te Coverage[END_REF] .

By increasing the Te coverage to 0.5ML, the surface reconstruction changes radically and as discussed above the upper layer has a honeycomb-like network similar to that of graphene. The band structure also changes, as shown in Fig. 8, where we report the ARPES mapping along the ΓM direction of the SBZ. First, we observe persistence of the Shockley state band, although it appears much broadened and shifted back towards its original position of clean Au(111). This is a signature of a weakening of the interaction between the Te layer and the Au substrate, in good agreement with the XPS data. Furthermore, a new band that we label S 3 occurs at a bottom binding energy of 0.65eV and is characterized by a peculiar invert U shape, i.e almost (c)

Fig. 1 :

 1 Fig.1: (a) Large-scale STM image of clean Au(111) surface, showing the herringbone reconstruction (U=-0.7V, I=1.0nA) and insert displays the associated image with atomic resolution. (b,c) Large-scale images measured on Au(111) covered by 0.33 ML and 0.5 ML of Te, respectively.

Fig. 2 :

 2 Fig.2: Quantitative analysis of STM images with atomic resolution obtained on Te/Au(111) system: (a) image at 0.33ML of Te, where the surface shows the coexistence of moiré and (3x3)R30° reconstructions, which are also highlighted in the LEED pattern given in the insert. (b,c) Profiles taken respectively along lines A and B indicated in image (a). (d) STM image at 0.5ML of Te displaying a honeycomb-like superstructure on which selforganization mechanism of Te atoms is observed. (e) Close STM inspection of the honeycomb-like network characterized by (3x3) super-lattice, as demonstrated by LEED (insert) and by the profile shown in (f) taken along line C in figure (d).

Fig. 2d .

 2d Fig.2d. In fact, upon decreasing the Te coverage, the Te contribution associated with (3x3) becomes weaker and new structure develops. The shape of the XPS spectrum does not change too much when the Te coverage is about 0.25ML and exhibits two spectroscopic features at the same energies as for 0.33ML. Indeed, this indicates the existence of two different chemical vacancies, one at 40.13eV corresponds to Te weakly bond to the substrate and the second at 39.77eV is supposed to be related to the Te-Au chemical bonding which suggests the presence of alloy monolayer giving rise to the (3x3)R30° reconstruction. Thus, the XPS data indicate a phase transition as a function on the Te coverage; the formation of Surface alloy which transforms progressively into decoupled Te monolayer. This result is consistent with the STM analysis, suggesting the existence of a tellurene-type monolayer.

Fig. 6 :

 6 Fig.6: Shockley state bands measured by ARPES on pristine Au(111) (left panel) and on Au(111) covered by 0.33 ML of Te (right panel). The ARPES data have been recorded at normal emission along -M direction and at T=100K with a photon energy of 60eV.

2 N

 2 model as: E± (k ¯˙) = E0 ℎ 2 k 2 * ± αR |k|, where ± refers to the two spin states,  R is the Rashba parameter, E 0 is the bottom band energy and m* represents the effective mass. The binding energy of the bottom band lies at 0.68 eV, while its value is found to be 0.46 eV on clean Au(111). Likewise, the effective mass increases to (0.36±0.01)m 0 (m 0 denotes the free electron mass) compared to its value (0.28±0.01)m 0 on free Au(111) surface. This can be ascribed to the Te-induced significant structural and chemical change of the in-plane gradient of the surface potential, besides the presence of reconstructions which can also have an effect. By performing scans throughout the SBZ, we were able map the overall band structure of Te/Au(111), to gain insight on the behavior of valence electrons. Fig.7shows a wide energy scan along the ΓK and ΓM directions of the SBZ recorded on Au(111) surface covered by 0.33ML of Te. Besides the Shockley states around the -point, we observe highly dispersive bands crossing the Fermi level, which correspond to the sp-states of the Au(111) surface. Additionally, we distinguish broad bands having a parabolic-type dispersion with negative effective mass, labeled S 1 and S 2 . By analogy with a sister system Cu 2 Te/Cu(111), those bands can originate from the Te adsorbates inducing an Au 2 Te alloy at the surface and result from strong hybridization between the Au 5d/6s and Te 5p orbitals. Moreover, we observe a back-folding behavior of these bands in the adjacent reduced SBZ (R-SBZ) of the (√3 x √3)R30°

Fig. 7 :

 7 Fig.7: (a) LEED pattern and (b) the corresponding schematic of the SBZs of the (3x3)R30° reconstruction obtained after deposition of 0.33ML of Te on Au(111) surface. (c) ARPES mapping of band structure along K--M- directions measured also on Au(111) covered by 0.33 ML of Te. The ARPES data have been recorded at T=100K with a photon energy of 60eV and the LEED pattern measured at energy of 80 eV.

Fig. 8 :

 8 Fig.8: (a) LEED pattern obtained on Au(111) surface covered by 0.5ML of Te, showing (3x3) reconstruction and (b) the corresponding surface Brillouin zone. (c) ARPES results obtained on the same surface along the M directions of the Au(111) SBZ.

  have studied here the changes in the structural and electronic properties of Au(111) surface induced after deposition of Te. At large distance, LEED and STM analyses indicated the formation of well-ordered and long-range reconstructions, however, details at the atomic scale demonstrated rich and complicated structures depending on the Te coverage. The XPS data indicated the formation of an Au 2 Te surface alloy at Te thickness below 0.33ML and a transition to a so-called tellurene monolayer with a unique chemical environment when the Te coverage reaches 0.5ML. The existence of a single Au chemical environment ruled out the possible formation of AuTe2, which is one of the scenarios discussed here. By ARPES, new dispersive bands were revealed at 0.33ML, which originate from a strong hybridization between Te and Au electronic states. These bands are strongly influenced by the surface reconstructions and undergo folding at the boundaries of the reduced surface Brillouin zone. This finding clearly highlights the presence of 2D-type electron system within the AuTe alloy layer. More interesting, the band structure changes when the tellurene-like monolayer is formed at 0.5ML and a peculiar band is observed. The ARPES data reveal the existence of a band gap and suggest a semiconductor character of the Au 2 Te surface alloy and also of the tellurene monolayer.

Acknowledgments M. B. and A. B. acknowledges for Synchrotron SOLEIL direction for supporting the project. We thank also the TEMPO beamline staff for the technical support. W. Zhang and Y. Tong are grateful to the China Scholarship Council (CSC) for the financial support (scholarship) for their Ph.D. works in France.