Stella K Papadopoulou
email: papadopoulou.styliani@gmail.com

Nicolas Papaiconomou

Stéphane Baup

Cristina Iojoiu

Lenka Svecova

Pierre-Xavier Thivel

Surface characterization of 1-butyl-1-ethylpiperidinium bromide by inverse gas chromatography

Keywords: Ionic liquids, Inverse gas chromatography, 1-butyl-1-ethylpiperidinium bromide, Surface energy, Acid-base interactions

This work addresses the surface characterization of an organic salt based on a piperidinium cation and a halide anion, similar to a first generation ionic liquid, using the Inverse Gas Chromatography (IGC) technique. IGC was employed in order to assess the dispersive surface energy and the acid/base character of 1-butyl-1-ethylpiperidinium bromide, [C 2 C 4 PIP]Br at a temperature range (313.15-343.15 K) well below its melting point, where the retention mechanism is governed by the surface adsorption of the probes. This type of characterization was possible due to the high melting point of [C 2 C 4 PIP]Br, namely 413.15 K. The dispersive component of the surface energy was estimated with the aid of the Schultz method and the Dorris-Gray method. Results obtained using the first method were higher than the ones obtained by the latter. The discrepancy between the two methods was found to increase with the increase of temperature. The acid/base characterization was implemented by using the Flour and Papirer approach as well as the Brookman and Sawyer method. The acidity and basicity constants of the surface of [C 2 C 4 PIP]Br revealed that it is amphoteric with a predominantly basic character.

Introduction

Ionic liquids (ILs) are salts exhibiting low melting points, typically below 100 °C, composed of anions and large organic cations, such as imidazolium, pyridinium, pyrrolidinium and less commonly piperidinium [START_REF] Genand-Pinaz | Removal of platinum from water by precipitation or liquid-liquid extraction and separation from gold using ionic liquids[END_REF][START_REF] Koller | A Simple Prediction Method for the Surface Tension of Ionic Liquids as a Function of Temperature[END_REF][START_REF] Mandai | Criteria for solvate ionic liquids[END_REF][START_REF] Mazan | Experimental connections between aqueousaqueous and aqueous-ionic liquid biphasic systems[END_REF][START_REF] Pollet | Solvents for sustainable chemical processes[END_REF][START_REF] Pena-Pereira | Perspectives on the replacement of harmful organic solvents in analytical methodologies: a framework toward the implementation of a generation of eco-friendly alternatives[END_REF]. ILs have a number of interesting properties such as nonvolatility, non-flammability, high thermal and ionic conductivity and an enhanced solvation ability for a wide range of materials [START_REF] Genand-Pinaz | Removal of platinum from water by precipitation or liquid-liquid extraction and separation from gold using ionic liquids[END_REF][START_REF] Mazan | Experimental connections between aqueousaqueous and aqueous-ionic liquid biphasic systems[END_REF][START_REF] Pollet | Solvents for sustainable chemical processes[END_REF][START_REF] Almeida | Influence of the anion on the surface tension of 1-ethyl-3-methylimidazolium-based ionic liquids[END_REF]. On these grounds, ILs have been widely explored as 'green' media in chemical and catalytic reactions, distillations, liquid-liquid extractions and solvent-aided crystallizations [START_REF] Genand-Pinaz | Removal of platinum from water by precipitation or liquid-liquid extraction and separation from gold using ionic liquids[END_REF][START_REF] Mazan | Experimental connections between aqueousaqueous and aqueous-ionic liquid biphasic systems[END_REF][START_REF] Pollet | Solvents for sustainable chemical processes[END_REF]. Other studies report the utilization of ILs as lubricants, in gas separation processes and as gas storage media [START_REF] Almeida | Influence of the anion on the surface tension of 1-ethyl-3-methylimidazolium-based ionic liquids[END_REF]. ILs were also employed as electrolytes in batteries, electrochemical capacitors [START_REF] Mandai | Criteria for solvate ionic liquids[END_REF] and as catalysts [START_REF] Deng | Determination of the solubility parameter of ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate by inverse gas chromatography[END_REF].

Piperidinium based ILs are of special interest for electrochemical applications due to their water immiscibility, high conductivities and wide electrochemical windows [START_REF] Shukla | A Comparative Study of Piperidinium and Imidazolium Based Ionic Liquids: Thermal, Spectroscopic and Theoretical Studies[END_REF][START_REF] Shukla | Importance of weak interactions and conformational equilibrium in N-butyl-N-methylpiperidinium bis (trifluromethanesulfonyl) imide room temperature ionic liquids: Vibrational and theoretical studies[END_REF]. As piperidinium based ionic liquids contain only saturated carbons, Shukla demonstrated that for a given anion, these ILs exhibit higher melting points and are thermally more stable than the respective imidazolium ILs having the same alkyl chains appended on the cation [START_REF] Shukla | A Comparative Study of Piperidinium and Imidazolium Based Ionic Liquids: Thermal, Spectroscopic and Theoretical Studies[END_REF][START_REF] Shukla | Importance of weak interactions and conformational equilibrium in N-butyl-N-methylpiperidinium bis (trifluromethanesulfonyl) imide room temperature ionic liquids: Vibrational and theoretical studies[END_REF]. In particular, piperidinium bromide salts exhibit very high melting points (in the vicinity of 200°C) whereas their homologous imidazolium bromide salts are either liquids or present low melting points [START_REF] Shukla | A Comparative Study of Piperidinium and Imidazolium Based Ionic Liquids: Thermal, Spectroscopic and Theoretical Studies[END_REF][START_REF] Shukla | Importance of weak interactions and conformational equilibrium in N-butyl-N-methylpiperidinium bis (trifluromethanesulfonyl) imide room temperature ionic liquids: Vibrational and theoretical studies[END_REF]. When ionic liquids are involved in applications such as extraction and catalysis, the presence of a second phase (liquid of gas) other than the ionic liquid is required.

Reactions, interactions and chemical exchanges that take place at the interface of the twophase systems strongly depend on the surface properties of each fluid [START_REF] Járvás | A novel method for the surface tension estimation of ionic liquids based on COSMO-RS theory[END_REF]. Therefore, the understanding of surface related properties of ionic liquids, such as their surface energy and acid-base character is necessary for the elucidation of the mechanisms that govern these reactions [START_REF] Almeida | Influence of the anion on the surface tension of 1-ethyl-3-methylimidazolium-based ionic liquids[END_REF][START_REF] Koller | Surface tension of tricyanomethanide-and tetracyanoborate-based imidazolium ionic liquids by using the pendant drop method[END_REF][START_REF] Law | Surface tension measurements of N-alkylimidazolium ionic liquids[END_REF][START_REF] Mirkhani | Prediction of surface tension of ionic liquids by molecular approach[END_REF][START_REF] Restolho | On the interfacial behavior of ionic liquids: Surface tensions and contact angles[END_REF].

Although there is a plethora of experimental studies investigating the surface tension of ionic liquids with various techniques [START_REF] Tariq | Surface tension of ionic liquids and ionic liquid solutions[END_REF], only a restricted number of publications address the estimation of the dispersive surface energy of ionic liquids with the use of inverse gas chromatography (IGC). Over the past years, inverse gas chromatography has proved to be a valuable tool towards the characterization of the surface and thermodynamic properties of various inorganic and organic materials such as polymers, fillers and pharmaceuticals [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF]. It should be outlined that the choice of the measurement temperature range is crucial in IGC as it determines the state (melt or solid) of the material under study, governing thus the retention mechanism of the probes (surface adsorption in the case of solids or mixing with bulk of the melt) [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF].

Although during the last decade IGC appears to be very popular for the thermodynamic characterization of ionic liquids [START_REF] Deng | Determination of the solubility parameter of ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate by inverse gas chromatography[END_REF][START_REF] Chen | Determination of the Solubility Parameter of Ionic Liquid 1-Hexyl-3-methylimidazolium Hexafluorophosphate by Inverse Gas Chromatography[END_REF][START_REF] Domańska | Measurements of activity coefficients at infinite dilution of aromatic and aliphatic hydrocarbons, alcohols, and water in the new ionic liquid [EMIM][SCNusing GLC[END_REF][START_REF] Heydar | Determination of Activity Coefficients at Infinite Dilution of Solutes in New Dicationic Ionic Liquids Based on Morpholine Using Gas-Liquid Chromatography[END_REF][START_REF] Królikowski | The study of activity coefficients at infinite dilution for organic solutes and water in 1-butyl-4-methylpyridinium dicyanamide[END_REF][START_REF] Marciniak | Activity Coefficients at Infinite Dilution Measurements for Organic Solutes and Water in the Ionic Liquid 1-(3-Hydroxypropyl)pyridinium Trifluorotris(perfluoroethyl)phosphate[END_REF][START_REF] Marciniak | Activity coefficients at infinite dilution and physicochemical properties for organic solutes and water in the ionic liquid 1-(2-methoxyethyl)-1methylpiperidinium bis(trifluoromethylsulfonyl)-amide[END_REF][START_REF] Mutelet | Accurate measurements of thermodynamic properties of solutes in ionic liquids using inverse gas chromatography[END_REF][START_REF] Mutelet | Measurement of activity coefficients at infinite dilution in 1hexadecyl-3-methylimidazolium tetrafluoroborate ionic liquid[END_REF][START_REF] Wang | Determination of the solubility parameter of ionic liquid 1-allyl-3-methylimidazolium chloride by inverse gas chromatography[END_REF], it has rarely been utilized for their surface characterization. This is due to the fact that such characterization should be conducted at a temperature range below the melting point of the IL in order to ensure that the interaction mechanism between the probes and IL is surface adsorption [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF]. On these grounds, as ILs exhibit low melting points, IGC is rather appropriate for the surface analysis of high melting point salts or for the thermodynamic characterization of ionic liquids.

The group of Wang has published a series of papers on the surface [START_REF] Wang | Determination of Surface Characteristics of Ionic Liquid [1-Hexyl-3-methylimidazolium Hexafluorophosphate] by Inverse Gas Chromatography[END_REF][START_REF] Chen | Characterization of surface properties of 1-allyl-3-methylimidazolium chloride ionic liquid by inverse gas chromatography[END_REF][START_REF] Ban | Surface Characterization of a Series of 1-Alkyl-3methylimidazolium-Based Ionic Liquids by Inverse Gas Chromatography[END_REF] and thermodynamic characterization [START_REF] Deng | Determination of the solubility parameter of ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate by inverse gas chromatography[END_REF][START_REF] Chen | Determination of the Solubility Parameter of Ionic Liquid 1-Hexyl-3-methylimidazolium Hexafluorophosphate by Inverse Gas Chromatography[END_REF][START_REF] Li | Determination of the thermodynamic parameters of ionic liquid 1-hexyl-3•methylimidazolium tetrafluoroborate by inverse gas chromatography[END_REF][START_REF] Li | Determination of the thermodynamic parameters of ionic liquid 1-hexyl-3-methylimidazolium chloride by inverse gas chromatography[END_REF][START_REF] Deng | Determination of thermodynamic parameters for ionic liquid 1-hexyl-3-methylimidazolium trifluoromethansulfonate by inverse gas chromatography[END_REF][33] of methyl imidazolium ILs by means of IGC.

To our knowledge, these are the only IGC studies addressing the surface characterization of ionic liquids [START_REF] Wang | Determination of Surface Characteristics of Ionic Liquid [1-Hexyl-3-methylimidazolium Hexafluorophosphate] by Inverse Gas Chromatography[END_REF][START_REF] Chen | Characterization of surface properties of 1-allyl-3-methylimidazolium chloride ionic liquid by inverse gas chromatography[END_REF][START_REF] Ban | Surface Characterization of a Series of 1-Alkyl-3methylimidazolium-Based Ionic Liquids by Inverse Gas Chromatography[END_REF]. It has to be stressed that the authors conducted both the surface and the thermodynamic characterizations at the same experimental temperature range, which is well above the melting point of the studied ILs [START_REF] Zhang | Ionic Liquids: Physicochemical Properties[END_REF]. Given the fact that the studied substances were in all cases in the liquid state and thus the probes' interaction mechanism was bulk absorption, the measurement temperatures used in these three studies were not appropriate for the surface characterization of the methyl imidazolium ILs.

In the current contribution, we present the results of the IGC surface characterization of 1butyl-1-ethylpiperidinium bromide [C 2 C 4 PIP]Br. Piperidinium based ILs is a family of ionic liquids much less studied than those based on imidazolium or pyridinium cations for example [START_REF] Domańska | Physico-chemical properties and phase behaviour of piperidinium-based ionic liquids[END_REF]. In this article, after the theoretical background section and a section dedicated to detailing the procedure used for carrying out the IGC analysis, results will be presented and discussed.

Theoretical background

Contrary to the conventional gas chromatography, in IGC, the material under study is packed into the column where injections of carefully selected probes with known physicochemical properties are made [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF][START_REF] Belgacem | Surface characterization of cellulose fibres by XPS and inverse gas chromatography[END_REF][START_REF] Kolodziejek | Characterization of hybrid materials by means of inverse gas chromatography and chemometrics[END_REF]. The probes can be injected onto the column either at infinite dilution or at finite concentration. In the case of infinite dilution, the injection of minor amounts of the tested solvent makes sure that the retention is governed by the stationary phase-probe interactions and the probe-probe interactions are avoided [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF].

The key measurement in IGC experiments is the net retention volume of the probes, . It is expressed as the volume of the carrier gas necessary to elute the solute from the column and is calculated by the following equation [START_REF] Conder | Physicochemical Measurement by Gas Chromatography[END_REF]:

= ( -) (1 -) (1)
where , are the probe's and marker's retention times, respectively, is the carrier gas flow rate measured at the column outlet at ambient pressure, and at room temperature, .

Also, is the column temperature, is the vapor pressure of water at and is the James and Martin factor used to correct the gas carrier compressibility, defined as [START_REF] Conder | Physicochemical Measurement by Gas Chromatography[END_REF]:

= !" # !" $ (2)
where % and are the column inlet and outlet pressures, respectively.

Dispersive interactions

The dispersive component of the surface energy of a solid, & ' ( , can be determined, using the Dorris-Gray method [START_REF] Domańska | Measurements of activity coefficients at infinite dilution of organic solutes and water in 1-propyl-1-methylpiperidinium bis {(trifluoromethyl) sulfonyl}imide ionic liquid using g.l.c[END_REF], as described by the following equation:

& ' ( = ) " *+ ,- . ) !/0 ,- 1 ,- . (3)
where Δ3 45

Δ3 45 = -> ln ) A B(CDE) A B(C) . (4)
where (FG") and (F) are the retention volumes of n-alkanes with n + 1 and n carbon atoms, respectively, is the column temperature and > is the ideal gas constant. The slope of the straight line obtained by a plot of the surface free energy of adsorption of n-alkanes > ln versus the number of their carbon atoms is equal to the free energy of adsorption of a methylene group, Δ3 45 .

Another method used for the calculation of the dispersive component of the surface free energy, is the one proposed by Schultz et al. [START_REF] Schultz | The role of the interface in carbon fibre-epoxy composites[END_REF] and is based on the following equation:

-Δ3 H = > ln = 267J& ' ( & K ( + M ( 5
)
where 7 is the surface area of the probe molecules, & ' ( and & K ( are the dispersive components of the surface energy of the solid and the liquid probe, respectively and M is a constant.

& '

( can be extracted from the slope of the linear plot of the > ln values of n-alkanes versus the term 7J& K ( .

Specific interactions

The non-dispersive character of the stationary phase is studied by the employment of probes that can also interact through specific intermolecular forces with the stationary phase. When a polar probe is injected in the column, it interacts with the material under study by both dispersive and specific interactions. The term specific includes all types of interactions, i.e. polar, hydrogen bonding, metallic or magnetic, apart from the dispersive ones (London forces) [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF]. Hence, the contribution of specific interactions to the free energy of adsorption, Δ3 ' , can be considered as the difference between the total free energy of adsorption, Δ3 H(' , and the dispersive contribution to the free energy of adsorption Δ3 ( . Several methods have been proposed for the determination of the Δ3 ' [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF]. In the present study, Δ3 ' was calculated through the use of the Flour and Papirer approach [START_REF] Saint Flour | Gas-solid chromatography: a quick method of estimating surface free energy variations induced by the treatment of short glass fibers[END_REF] and the Brookman and

Sawyer method [START_REF] Brookman | Specific Interactions Affecting Gas Chromatographic Retention for Modified Alumina Columns[END_REF]. The estimation of Δ3 ' is based on a plot of Δ3 or simply > ln F values of both polar and apolar probes versus their boiling temperatures ( N , in °C) according to the Brookman and Sawyer method, or the log , logarithm of their saturated vapor pressure, according to the Flour and Papirer method. The n-alkane series leads to a linear plot, which constitutes a reference straight line for the dispersive interactions. The contribution of specific interactions to the free energy of adsorption, Δ3 ' , corresponds to the vertical distance between the -Δ3 H(' value and the reference line according to the equation:

(-Δ3 ' ) = (-Δ3 H(' ) -(-Δ3 QRS ) = > lnT ,QRS ⁄ V (6)
where is the net retention volume of the polar probe and ,QRS the net retention volume of a hypothetical alkane with the same boiling point or saturated vapor pressure as that of the polar probe.

The specific enthalpy, ΔW ' , can be determined after the calculation of the specific component of the free energy of adsorption at various temperatures. When plotting the Δ3 '

values as a function of the reciprocal temperature, !" , ΔW ' is calculated from the slope of the straight line corresponding to the following relation:

Δ3 ' ⁄ = ΔW ' • 1 ⁄ -ΔY ' (7)
The enthalpies of specific interactions between the examined surface and the polar probe can be used for the estimation of the Z [ and Z \ indices reflecting the acidity (electron acceptor)

and basicity (electron donor) of a solid surface, respectively [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF][START_REF] Gutmann | The Donor-Acceptor Approach to Molecular Interactions[END_REF][START_REF] Kamdem | Inverse Gas-Chromatography Characterization of Birch Wood Meal[END_REF][START_REF] Riddle | Spectral Shifts in Acid-Base Chemistry .1. Vanderwaals Contributions to Acceptor Numbers[END_REF] and are related to the acidic or basic character through the equation:

-ΔW ' ]6 * ⁄ = Z [ • _6 ]6 * ⁄ + Z \ (8)
where _6 [START_REF] Gutmann | The Donor-Acceptor Approach to Molecular Interactions[END_REF] the surface is considered to be acidic [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF].

Experimental

Materials

For the synthesis of the ionic liquid, 1-bromobutane (99%), 1-ethylpiperidine (99%) and acetonitrile were used and were purchased from Sigma Aldrich.

For the IGC analysis, the following solvents were used as probes: n-octane, n-nonane, ndecane, n-undecane, n-dodecane, acetonitrile, n-butanol, pyridine and chloroform. The characteristics of the solvents used for the IGC analysis are presented in Table 1. All solvents were of the highest available purity and were purchased from Sigma Aldrich. Chromosorb W HP (100/120) mesh was used as the solid support (Antelia, France). All the gases used were purchased from Air Liquide and were of high purity.

Synthesis and characterization of 1-butyl-1-ethyl piperidinium bromide

1-butyl-1-ethylpiperidinium bromide (Figure 1) was synthesized as previously reported [START_REF] Papaiconomou | Synthesis, Physicochemical Properties, and Toxicity Data of New Hydrophobic Ionic Liquids Containing Dimethylpyridinium and Trimethylpyridinium Cations †[END_REF].

Briefly, 1-bromobutane was mixed with 1-ethylpiperidine in 100 mL acetonitrile at reflux for 2 days. The solvent was then evaporated using a rotary evaporator. The resulting product appearing as a white solid was washed several times with ethyl acetate. After filtering, the product was initially dried on a rotary evaporator to remove the remaining solvent and then dried under vacuum (~0.1 mbar) for 24 hours. The final product was obtained as a white solid.

Purity was checked by Nuclear Magnetic Resonance Spectroscopy ( 1 H NMR). The 1 H NMR spectrum was collected on a Bruker WM 400 MHz spectrometer in Dimethysulfoxide (DMSO-d6) as solvent. The spectrum is given in the Supplementary Material section (Figure S1). Material section (Figure S2). The melting point of the [C 2 C 4 PIP]Br was found to be 140 °C.

Column preparation and IGC setup

The IGC experiments were performed using a Perkin Elmer Clarus 480 gas chromatograph, equipped with a flame ionization detector (FID). The data acquisition was made with the AZUR software. The retention times of the probes were determined after the calculation of the first-order moment of the concentration distribution. This was necessary due to the slight "tailing" observed at the elution profile of the probes [START_REF] Conder | Physicochemical Measurement by Gas Chromatography[END_REF]. High purity helium was used as the carrier gas and methane was used as a marker. The flow rate of helium was measured with a soap bubble flowmeter connected to the end of the column, at room temperature. The experiments were performed at infinite dilution, by injecting manually 0.1 µL of each probe with a 1 µL Hamilton syringe. At least three injections of each probe were made and the retention time was taken as the average of the three measurements. The standard deviation was less than 2% in all cases.

The IGC experimental conditions and the column characteristics are presented in Table 2. A stainless steel column was used for the measurements and was washed with acetone prior to use. The stationary phase of the column was prepared via the coating method proposed by Al-Saigh and Munk in order to better control the amount of the salt coated on the solid support [START_REF] Al-Saigh | Study of polymer-polymer interaction coefficients in polymer blends using inverse gas chromatography[END_REF]. [C 2 C 4 PIP]Br was coated onto Chromosorb W HP at a loading of 20.7% w/w. The column was preconditioned overnight to the working conditions (temperature and helium flow rate) in order to remove any possible contaminants that could be eluted during measurements.

The protocol regarding the preparation of the column was described in detail elsewhere [START_REF] Papadopoulou | Surface characterization of poly (2, 2, 3, 3, 3-pentafluoropropyl methacrylate) by inverse gas chromatography and contact angle measurements[END_REF][START_REF] Papadopoulou | Assessment of the thermodynamic properties of poly(2,2,2-trifluoroethyl methacrylate) by inverse gas chromatography[END_REF][START_REF] Papadopoulou | Thermodynamic characterization of poly(2,2,3,3,3-pentafluoropropyl methacrylate)[END_REF].

Dispersive component of the surface energy

The dispersive component of the surface energy of 1-butyl-1-ethylpiperidinium bromide was calculated at the temperature range 313.15-343.15 K, using the Dorris-Gray method and the approach proposed by Schultz.

Dorris-Gray method

The slope of the line obtained when plotting the > ln values of n-alkanes versus their number of carbon atoms yields the free energy of adsorption of a methylene group, Δ3 45 as indicated by Eq. ( 4). The corresponding plot is illustrated in Figure 2. The dispersive component of the surface energy of [C 2 C 4 PIP]Br was calculated via Eq. ( 3) according to the Dorris-Gray method. The values of & ' ( together with their maximum errors are presented in Table 3. 1 in combination with Eq. ( 5) were used for this purpose. Results are listed in Table 3. plotted as a function of temperature (Figure 4) and the values are given in Table 3. For all studied temperatures, high correlation coefficients R² values were found. It appears that the dispersive component of the surface energy of [C 2 C 4 PIP]Br presents a decreasing trend with the increase of temperature [START_REF] Ramanaiah | Effect of temperature on the surface free energy and acid-base properties of Gabapentin and Pregabalin drugs -a comparative study[END_REF]. Moreover, based on Figure 4 we notice that the dependence of & ' ( with temperature is almost linear, and when comparing the slopes corresponding at the descending trend obtained by both methods, we observe that they are similar.

In addition, we notice that the & ' ( values calculated with the aid of the Dorris-Gray method are higher with respect to those obtained with the Schultz method. The discrepancy between the results calculated by these methods increases with temperature. This trend is in line with the results presented in pertinent literature [START_REF] Voelkel | Determination of Hansen solubility parameters by means of gas-solid inverse gas chromatography[END_REF][START_REF] Van Asten | Surface characterization of industrial fibers with inverse gas chromatography[END_REF][START_REF] Shi | Comparison of Dorris-Gray and Schultz methods for the calculation of surface dispersive free energy by inverse gas chromatography[END_REF][START_REF] Segeren | Surface energy characteristics of toner particles by automated inverse gas chromatography[END_REF][62][START_REF] Mohammad | Chromatographic adhesion law to simplify surface energy calculation[END_REF][START_REF] Lapčík | Surface heterogeneity: Information from inverse gas chromatography and application to model pharmaceutical substances[END_REF][START_REF] Gamelas | An insight into the surface properties of calcined kaolinitic clays: The grinding effect[END_REF][START_REF] Bilgiç | Determination of the acid/base properties of MgY and NH4Y molecular sieves by inverse gas chromatography[END_REF].

As it was mentioned in the introduction, there is no study in literature addressing the estimation of the dispersive surface energy of piperidinium or bromide based ILs. However, [69] / 35.3 [68] 34.2 -303.2 34.5 [67] / 35.1 [68] 34.0 -313.2 34.5 [68] 33.5 34.0 -37.5

323.2 34.0 [68] 33.0 31.3 -35.2 333.2 33.5 [68] 32.5 29.7 -34.0 343.2 33.0 [68] 32.1 26.3 -30.6

Osada et al. [START_REF] Osada | Surface tension of room temperature ionic liquids measured by dynamic light scattering[END_REF] reported the surface energy of 1-methyl- 4) with literature data is not straightforward for several reasons; firstly, the latter concern surface energy values contrary to the former which concern values of dispersive surface energy. Furthermore, as it has been shown, the measuring techniques of the surface energy as well as the sample purity have an impact on the values of & [START_REF] Tariq | Surface tension of ionic liquids and ionic liquid solutions[END_REF][START_REF] Bhattacharjee | The effect of the cation aromaticity upon the thermophysical properties of piperidinium-and pyridinium-based ionic liquids[END_REF]. Moreover, the anion [NTf 2 ] -((bistrifluoromethanesulfonyl)imide) reported in the literature is a polyatomic, bulky and very weakly coordinating anion. It is thus very different from the small and coordinating bromide ion studied here. On these grounds, only a rough comparison between literature data and the results of this study can be made. As a first remark, the same temperature dependence of & and is & ' ( observed for all ILs, all values decrease with increasing temperature. It is well known that the surface tension depends on the contribution of the groups which are exposed at the surface [START_REF] Tariq | Surface tension of ionic liquids and ionic liquid solutions[END_REF][START_REF] Bhattacharjee | The effect of the cation aromaticity upon the thermophysical properties of piperidinium-and pyridinium-based ionic liquids[END_REF]. In the case of ionic liquids, when the cation bears long alkyl side chains and is thus bulkier than the anion, the surface tension is affected primarily by the nature of the cation [START_REF] Tariq | Surface tension of ionic liquids and ionic liquid solutions[END_REF][START_REF] Bhattacharjee | The effect of the cation aromaticity upon the thermophysical properties of piperidinium-and pyridinium-based ionic liquids[END_REF].

Acid-base interactions

The ability of the ionic liquid to intermolecular specific interactions with other substances is strongly dependent on the presence of acidic and basic sites on its surface.

The assessment of the acid-base interactions of a surface includes the calculation of the specific component of the free energy of adsorption of a probe, Δ3 ' , the specific component of the free enthalpy of adsorption of a probe, ΔW ' and the ratio Z \ Z [ ⁄ of the examined surface [START_REF] Voelkel | Inverse gas chromatography as a source of physiochemical data[END_REF]. As it has been mentioned in Section 2.2, several methods have been developed

for the acid-base characterisation of a material through the use of the IGC technique. The Schultz method allows the estimation of Δ3 ' through the use of the term J& K ( , whereas the Papirer method and the Brookman and Sawyer method are based on the saturated vapor pressures and the boiling points of the probes, respectively. The latter two properties are readily available in literature, contrary to the molecular area and the dispersive component of the surface tension of polar probes. The discrepancy between the values of & ' ( and b of polar probes appearing in literature [START_REF] Gamelas | An insight into the surface properties of calcined kaolinitic clays: The grinding effect[END_REF][START_REF] Belgacem | Interfacial Phenomena in Chromatography[END_REF] is due to the presence of one or more functional groups [START_REF] Van Asten | Surface characterization of industrial fibers with inverse gas chromatography[END_REF]. For the above reasons, although in the present study the Schultz method was employed for the dispersive surface energy calculation of [C 2 C 4 PIP]Br, the acid-base interactions were studied with the aid of the Flour and Papirer method and the Brookman and Sawyer method.

Flour and Papirer method

Brookman and Sawyer method

The contribution of specific interactions to the free energy of adsorption Δ3 ' of polar probes, was calculated by the aid of the Brookman and Sawyer method, as shown in Figure 6. values versus the reciprocal temperature, !" . The results obtained by both methods employed are reported in Table 5. ' values, indicating that the surface is amphoteric. Moreover, we notice that all ΔY ' values are negative, with the exception of acetonitrile. The adsorption of a probe from a surface is usually accompanied by an entropy loss as the probe passes from the less ordered vapor phase to the more ordered adsorbed phase. In the case of acetonitrile, the entropy increase implies an increase in the degrees of freedom which could be attributed to the rearrangement of the surface [START_REF] Santos | Characterisation of the surface Lewis acid-base properties of the components of pigmented, impact-modified, bisphenol A polycarbonate-poly (butylene terephthalate) blends by inverse gas chromatographyphase separation and phase preferences[END_REF].

Acidity and basicity constants

The values for the specific enthalpies of polar probes were employed in order to evaluate the Bearing in mind the structure of 1-butyl-1-ethylpiperidinium bromide, we notice that both acidic and basic moieties are present in the molecule. As no comparable work has been carried out previously for solid organic salts or ionic liquids below their melting point, neither the comparison with literature nor the interpretation of our results are straightforward. Nevertheless, a tentative explanation can be given as following. Since in our case, the ions of the salt are relatively small and bound together, the overall basicity of [C 2 C 4 PIP]Br is due to an average of all of its parts, i.e. the alkyl groups, the positive charge on the nitrogen atom and the Br -anion. As reported previously in IGC studies dealing with polymers, CH 2 and CH 3 groups are known to be weak Lewis acidic sites [START_REF] Papadopoulou | Surface characterization of poly (2, 2, 3, 3, 3-pentafluoropropyl methacrylate) by inverse gas chromatography and contact angle measurements[END_REF][START_REF] Santos | Characterisation of the surface Lewis acid-base properties of the components of pigmented, impact-modified, bisphenol A polycarbonate-poly (butylene terephthalate) blends by inverse gas chromatographyphase separation and phase preferences[END_REF][START_REF] Santos | Study of a core-shell type impact modifier by inverse gas chromatography[END_REF]. On the other hand, Br -, as F -or Cl -anions, is a dense, monoatomic and relatively electronegative anion, and is known to be basic [START_REF] Papadopoulou | Surface characterization of poly (2, 2, 3, 3, 3-pentafluoropropyl methacrylate) by inverse gas chromatography and contact angle measurements[END_REF][START_REF] Abel | Adsorption of poly(methyl methacrylate) and poly(vinyl chloride) blends onto polypyrrole: Study by X-ray photoelectron spectroscopy, time-of-flight static secondary ion mass spectroscopy, and inverse gas chromatography[END_REF]. The positive charge on the nitrogen is acidic, but is expected to be hidden and to be sterically hindered by the alkyl groups bound to it. Therefore, we can surmise that the general basic character of [C 2 C 4 PIP]Br is due to the relatively dense and monoatomic bromide anion.

Conclusions

This contribution reports for the first time on the surface characterization of a piperidinium based organic salt, 1-butyl-1-ethylpiperidinium bromide, by means of IGC. Measurements

  Concerning their IGC characterizations, only a limited number of studies have reported the estimation of the thermodynamic properties of piperidinium-based ILs bearing anions other than the bromide anion, or with different alkyl chain appended onto the cation, as those present in [C 2 C 4 PIP]Br [36-38]. 1-butyl-1-ethylpiperidinium bromide is an organic salt being by all aspects similar to halide-based ionic liquid, with the exception that its melting point is above 373 K. Hence, IGC measurements were performed at 313.15-343.15 K, a temperature range which is below the melting point of [C 2 C 4 PIP]Br. The energy, enthalpy and entropy of adsorption of polar probes on the surface of the salt, the dispersive component of the surface energy and the Lewis acidity and basicity constants of [C 2 C 4 PIP]Br were determined. The dispersive interactions were investigated with the aid of well-known methods proposed by Dorris and Gray and by Schultz, whereas the specific interactions of the polar probes were calculated using the Brookman and Sawyer approach and the Flour and Papirer method.

Figure 1 .

 1 Figure 1. Structure of [C 2 C 4 PIP]Br. The protons observed in the NMR spectrum were identified.

Figure 2 .Figure 3

 23 Figure 2. Surface free energy of adsorption of n-alkanes > ln on [C 2 C 4 PIP]Br versus their number of carbon atoms, measured at the temperature range 313.15-343.15 K.

Figure 3 .

 3 Figure 3. Surface free energy of adsorption of n-alkanes > ln , on [C 2 C 4 PIP]Br versus bJ& K ( measured at the temperature range 313.15-343.15 K.

Figure 4 .

 4 Figure 4. Variation of the dispersive component of the surface energy of [C 2 C 4 PIP]Br with temperature, at the temperature range 313.15-343.15 K, obtained by the Dorris-Gray and the Schultz methods

  of piperidinium ILs has been assessed by techniques other than IGC (see

  1-propylpiperidinium bis(trifluoromethanesulfonyl)imide ([C 3 C 1 PIP][NTf 2 ]) measured with the Dynamic Light Scattering (DLS) technique. Bhattacharjee et al. [68] assessed the surface energy of [C 3 C 1 PIP][NTf 2 ] and [C 4 C 1 PIP][NTf 2 ] (1-Butyl-1-methylpiperidinium bis (trifluoromethyl sulfonyl)imide) by using the contact angle method. The comparison between our results (last column in Table

Figure 5 Figure 5 .

 55 Figure 5 presents the results of the energies of adsorption of n-alkanes and polar probes on the surface of [C 2 C 4 PIP]Br versus the logarithm of their saturated vapour pressure, according to the Flour and Papirer method. The straight reference line defines the London dispersive interactions, while the energies of adsorption polar probes lie above this line.

Figure 6 .

 6 Figure 6. Energy of adsorption of n-alkanes and polar probes on the surface of [C 2 C 4 PIP]Br versus their boiling temperatures, at 343.15 K.

Figure 7 .

 7 Figure 7. Determination of the Z [ and Z \ values of the of [C 2 C 4 PIP]Br based on the Flour and Papirer approach.

Figure 8 .

 8 Figure 8. Determination of the Z [ and Z \ values of the of [C 2 C 4 PIP]Br based on the Brookman and Sawyer method.

  were conducted at a temperature range (313.[START_REF] Restolho | On the interfacial behavior of ionic liquids: Surface tensions and contact angles[END_REF]-343.15 K), well below the melting point of [C 2 C 4 PIP]Br (413.15 K) to ensure that the dominant retention mechanism of the probes is adsorption onto the surface. The Schultz and the Dorris-Gray methods were used for the calculation of the dispersive component of the surface energy. Results showed that & ' ( decreases with temperature. Moreover, the comparison of the dispersive component of the surface energy of [C 2 C 4 PIP]Br with that of two piperidinium based ILs revealed that the results of this study are comparable with literature values. The anion does therefore not appear to play a crucial role in the surface energy of the piperidnium-based organic salts studied here and in the literature. The acid/base characterization based on the Flour and Papirer approach and the Brookman and Sawyer method showed that the surface of [C 2 C 4 PIP]Br is amphoteric with a predominantely basic character, a conclusion which is further supported by taking into account the molecular structure of [C 2 C 4 PIP]Br bearing, both, weak Lewis acidic and basic sites.

Table 1 .

 1 Physical and chemical properties of the probes used for the IGC analysis[START_REF] Wang | Determination of Surface Characteristics of Ionic Liquid [1-Hexyl-3-methylimidazolium Hexafluorophosphate] by Inverse Gas Chromatography[END_REF][START_REF] Riddle | Spectral Shifts in Acid-Base Chemistry .1. Vanderwaals Contributions to Acceptor Numbers[END_REF][START_REF] Cava | Surface characterization of poly (lactic acid) and polycaprolactone by inverse gas chromatography[END_REF][START_REF] Daubert | Physical and Thermodynamic Properties of Pure Chemicals: Data Compilation[END_REF][START_REF] Papadopoulou | Surface characterization of poly (2, 2, 3, 3, 3-pentafluoropropyl methacrylate) by inverse gas chromatography and contact angle measurements[END_REF][START_REF] Santos | Lewis acid/base character and crystallisation properties of poly(butylene terephthalate)[END_REF][START_REF] Vasanth Kumar | On the effect of a non-ionic surfactant on the surface of sucrose crystals and on the crystal growth process by inverse gas chromatography[END_REF]

	Probe	Boiling point	Saturated vapor	7 (10 -19 m 2 )	& K ( (mJ/m 2 )	_6 (kJ mol -1 )	(kJ mol -1 ) ]6 *	Character
		(°C)	pressure
			(kPa)
	n-Octane	125.7	1.871	6.29	21.3	n/a	n/a	Neutral
	n-Nonane	150.8	0.580	6.89	22.7	n/a	n/a	Neutral
	n-Decane	174.2	0.181	7.5	23.4	n/a	n/a	Neutral
	n-Undecane 195.9	0.056	8.1	24.6	n/a	n/a	Neutral
	n-Dodecane 216.3	0.018	8.7	25.4	n/a	n/a	Neutral
	n-Butanol	117.7	0.930	-	-	0	38.1	Acidic
	Pyridine	115.3	2.772	-	-	138.6	0.6	Basic
	Chloroform	61.2	26.165	-	-	0	22.6	Acidic
	Acetonitrile	81.6	12.147	-	-	59	19.7	Amphoteric

Table 2 .

 2 Chromatographic conditions and column specifications

		1-butyl-1-ethylpiperidinium bromide
	Injector temperature (K)	473.15
	Detector temperature (K)	473.15
	Column temperatures (K)	313.15, 323.15, 333.15, 343.15
	Column type of material	SS 316 ASTM A-269
	Column length (cm)	70
	Column O.D (inch)	1/8
	[C 2 C 4 PIP]Br loading (%)	20.7
	Flow rate (mL/min)	10
	4	Results

Table 3 .

 3 Dispersive component of the surface energy of [C 2 C 4 PIP]Br at the temperature range

		12	313.15 K
			323.15 K
		10	333.15 K 343.15 K
	N (kJ/mol)	6 8
	RTlnV	4
		2
		0
		-2
			3.00E-018	3.50E-018	4.00E-018	4.50E-018
				α(γ d l ) 0.5 [m 2 (mJ/m 2 ) 0.5 ]
	313.15-343.15 K, obtained by the Dorris-Gray and the Schultz methods
	T (K)		Dorris-Gray method		Schultz method
			& ' ( (mJ/m 2 )	>	& ' ( (mJ/m 2 )	>
	313.15		37.5 ± 1.7	0.998	34.0 ± 1.7	0.998
	323.15		35.2 ± 0.8	0.999	31.3 ± 1.2	0.999
	333.15		33.9 ± 1.3	0.999	29.7 ± 1.4	0.998
	343.15		30.6 ± 0.7	0.999	26.3 ± 1.0	0.999

Table 4 ) . Table 4 .

 4.4 Surface tension values of piperidinium based ILs taken from literature and dispersive

	component of the surface energy results of [C 2 C 4 PIP]Br of the present study
		& (mJ/m 2 )	& ' ( (mJ/m 2 )
	T (K)	[C 3 C 1 PIP][NTf 2 ]	[C 4 C 1 PIP][NTf 2 ] [68]	[C 2 C 4 PIP]Br
				(this work)
	298	35.2

Table 5 .

 5 Specific components of the enthalpy and entropy of adsorption of polar probes on the surface of [C 2 C 4 PIP]Br at the temperature range 313.15-343.15 K The enthalpies and entropies of adsorption of polar probes on the surface of [C 2 C 4 PIP]Br estimated with the Flour and the Brookman methods are comparable, with the exception of nbutanol. The enthalpy of adsorption of all probes was found to be exothermic in all cases. Acetonitrile which is an amphoteric probe presents the highest enthalpy of adsorption whereas chloroform which is acidic has the lowest ΔW ' implying that [C 2 C 4 PIP]Br is amphoteric with a basic character. The values of the specific components of the entropy of adsorption of polar probes on the surface of [C 2 C 4 PIP]Br follow the same trend as discussed above. Acetonitrile has the highest ΔY

		Flour and Papirer method	Brookman and Sawyer method
	Probe	-ΔW '	-ΔY '	-ΔW '	-ΔY '
		(kJ/mol)	(J/mol K)	(kJ/mol)	(kJ/mol K)
	n-Butanol	5.59	6.48	11.31	15.56
	Pyridine	7.74	6.51	9.30	8.99
	Chloroform	24.07	59.58	28.73	66.41
	Acetonitrile	0.63	-18.48	3.55	-13.84

  based on the high values of the regression coefficients. Similar plots for the estimation of the Z [ and Z \ constants appear in literature[START_REF] Wang | Determination of Surface Characteristics of Ionic Liquid [1-Hexyl-3-methylimidazolium Hexafluorophosphate] by Inverse Gas Chromatography[END_REF][START_REF] Santos | Lewis acid/base character and crystallisation properties of poly(butylene terephthalate)[END_REF] 62,[START_REF] Bhattacharjee | The effect of the cation aromaticity upon the thermophysical properties of piperidinium-and pyridinium-based ionic liquids[END_REF]. The results of the acidity and basicity constants of the surface of [C 2 C 4 PIP]Br are reported in Table6.

	acidity and basicity constants of [C 2 C 4 PIP]Br. According to Eq. (8) the Z [ and Z \ constants can be assessed by the slope and the intercept, respectively, of a plot of ΔW ' ]6 * ⁄ versus _6 ]6 deduce that, in both cases, linear dependencies of ΔW ' ]6 * ⁄ versus _6 ]6 * ⁄ were achieved,

* ⁄ . Based on the values of the Gutmann's donor and modified acceptor numbers of the polar probes presented in Table 1 as well as the values of ΔW ' obtained by the two methods used, Z [ and Z \ constants were evaluated (Figures 7 and 8). Examining the two plots we can

Table 6 .

 6 Acidity and basicity constants of the surface of [C 2 C 4 PIP]Br ratios calculated by the Flour and Papirer method and the Brookman and Sawyer method suggest that the surface of 1-butyl-1-ethylpiperidinium bromide has a predominantly Lewis basic character. This conclusion is in line with the findings of the analysis based on the values of the energy and enthalpy of polar probes, presented in paragraphs 4.2.1 and 4.2.2. Despite the fact that the Z \ Z [ ⁄ ratios are not similar, we can safely conclude that [C 2 C 4 PIP]Br is a Lewis base, since in both cases the Z \ Z [ ⁄ ratio is greater than 1.

	Method	Z [	Z \	Z \ Z [ ⁄
	Flour and Papirer	0.054	0.36	6.67
	Brookman and Sawyer	0.065	0.52	8.00
	The Z \ Z [ ⁄

Acknowledgments

The authors acknowledge Grenoble INP for post-doctoral fellowship for S.P. and financial support for this project.


Abir Swaidan

Priyakshree Borthakur

Purna K Boruah

Manash R Das

Alexandre Barras

Salah Hamieh

Joumana Toufaily

Tayssir Hamieh

Sabine Szunerits

Rabah Boukherroub
email: rabah.boukherroub@univ-lille.fr

A facile preparation of CuS-BSA nanocomposite as enzyme mimics: Application for selective and sensitive sensing of Cr(VI) ions

Keywords: CuS-BSA, Peroxidase mimic, Cr(VI) ions, Colorimetric, Detection

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

Introduction

Contaminated water by heavy metal ions represents a major risk to human health. Although most of heavy metal ions are essential to human body, when their concentration exceeds the normal level, they cause chronic toxicities. Thus, the detection of metal ions in water is a persistent need for environmental protection.

Chromium generally occurs in the environment in two main forms: Cr(VI) and Cr(III).

Cr(III) is considered as an essential element for carbohydrate, protein and lipid metabolism, as well as insulin efficiency [START_REF] Anderson | Chromium, glucose intolerance and diabetes[END_REF] due to its enhancement in transporting glucose through the cell membrane. Indeed, supplemental Cr(III) as chromium picolinate enhances the blood glucose, insulin, hemoglobin and cholesterol in a concentration dependent way in Type 2 diabetes mellitus. In contrast, deficiency in Cr(III) can result in nerve and brain damage. The first report to highlight chromium deficiency was in 1977 [START_REF] Jeejeebhoy | Chromium deficiency, glucose intolerance, and neuropathy reversed by chromium supplementation, in a patient receiving long-term total parenteral nutrition[END_REF], when a patient suffered from low insulin levels due to glucose intolerance, where it has been observed that Cr requirement is closely related to the level of glucose metabolism. Unlike Cr(III), Cr(VI) is highly toxic and considered to be carcinogen [START_REF] Li | Non-aggregation based label free colorimetric sensor for the detection of Cr(VI) based on selective etching of gold nanorods[END_REF]. This form of chromium compound can be found in the earth's crust and used in many industrial processes such as chrome plating, manufacturing of pigments and dyes, paints, plastics, wood preservation, and leather tanning [START_REF] Blade | Hexavalent chromium exposures and exposure-control technologies in American enterprise: results of a NIOSH field research study[END_REF]. Therefore, the risk of Cr(VI) disposal to water, air, and soil is restricted to means of industrial facilities. The high solubility and mobility of anionic forms of Cr(VI) (HCrO4 -or CrO4 2-) raised concern on the hazardous effect of these species. Indeed, Cr(VI) ions can be also transported through cellular membranes by sulphate/phosphate anion channels due to their similarity in structure, since at physiological pH, hexavalent chromium exists as oxyanion with an overall -2 charge [START_REF] Epa | Toxicological review of hexavalent chromium[END_REF]; once inside the cells, it can interact with biological species (ex. DNA) [START_REF] Shi | Reaction of Cr (VI) with ascorbate and hydrogen peroxide generates hydroxyl radicals and causes DNA damage: role of a Cr(IV)-mediated Fenton-like reaction[END_REF], causing extensive damage and leading to serious deficiencies.

On the other hand, H2O2, a naturally occurring by-product of oxygen metabolism, is of biological interest, due to its ability to transform into harmful free radicals that can readily interact with biological molecules. Recently, it has been found that H2O2 can form an intermediate with thiol-group containing species in the cellular membrane for the activation of Cr(VI) ions to enhance their toxicity and mutagenicity [START_REF] Perez-Benito | A Kinetic Study of the Chromium (VI)-Hydrogen Peroxide Reaction. Role of the Diperoxochromate (VI) Intermediates[END_REF]. The interaction between H2O2 and Cr(VI) ions is demonstrated to be pH-dependent. Joaquin et al. [START_REF] Perez-Benito | A Kinetic Study of the Chromium (VI)-Hydrogen Peroxide Reaction. Role of the Diperoxochromate (VI) Intermediates[END_REF] reported that at low pH, Cr(VI) acts as an oxidizing agent towards the generation of hydroxyl radicals.

The U.S. EPA (Environmental Protection Agency) Guidelines for Carcinogen Risk Assessment [START_REF] Glasser | Kinetics and Mechanisms of Cr 6+ Reduction by Structural Fe(II) in Clay Minerals[END_REF][START_REF] Epa | Guidelines for Carcinogen Risk Assessment US Environmental Protection Agency[END_REF], stated that Cr(VI) concentration in drinking water exceeding 0.1 mg/L is considered to be carcinogen through mutagenic mode of action. Thus, people residing near sources of industrial waste of Cr(VI) compounds and consuming Cr(VI) in drinking water are more susceptible to cancer. Therefore, it is mandatory to develop a highly efficient technique for Cr(VI) detection.

Many methods have been reported for efficient sensing of heavy metal ions, such as atomic absorption [START_REF] Trindade | Multivariate optimization of ultrasound-assisted extraction for determination of Cu, Fe, Ni and Zn in vegetable oils by high-resolution continuum source atomic absorption spectrometry[END_REF], emission [START_REF] Losev | Silica sequentially modified with polyhexamethylene guanidine and Arsenazo I for preconcentration and ICP-OES determination of metals in natural waters[END_REF], and mass spectroscopies [START_REF] Wang | Chip-based array magnetic solid phase microextraction on-line coupled with inductively coupled plasma mass spectrometry for the determination of trace heavy metals in cells[END_REF]. Although these methods are highly selective and sensitive for detecting heavy metal ions, they are time-consuming, require specialized equipment and cannot be applied for real-time or field analysis. Thereby, the colorimetric detection holds interest due to its simplicity, low cost, fast response, and low detection limit.

With the emergence of nanotechnology, the development of enzyme mimics as colorimetric biosensors offers great advantages in the environmental and biomedical fields. In 2007, Gao et al. [START_REF] Gao | Intrinsic peroxidase-like activity of ferromagnetic nanoparticles[END_REF] demonstrated the use of ferromagnetic Fe3O4 nanoparticles (NPs) that possess enzymelike activity similar to the naturally occurring horseradish peroxidase enzyme (HRP). Since then, a tremendous amount of efforts has been devoted to the preparation of a plethora of enzyme-like nanomaterials (commonly designed as nanoenzymes) for sensing of H2O2, glucose and other biologically relevant molecules [START_REF] Cormode | Emerging biomedical applications of enzyme-like catalytic nanomaterials[END_REF].

In recent years, semiconductor chalcogenides and particularly copper sulfide (CuS) nanostructures have received tremendous interest, owing to their high abundance, low cost, and unique electronic and optical properties. These materials have found widespread use in various fields like photovoltaics, photocatalysis, energy conversion devices, biosensors, etc. [START_REF] Coughlan | Compound copper chalcogenide nanocrystals[END_REF]. However, in absence of appropriate surface coating, these nanostructures tend to agglomerate in aqueous media, hampering their applications [START_REF] Zhao | Hollow micro/nanomaterials with multilevel interior structures[END_REF].

Many studies reported that biomolecules such as proteins can be used to prevent nanomaterial's aggregation [START_REF] Huang | Synthesis and characterization of bovine serum albuminconjugated copper sulfide nanocomposites[END_REF]. Therefore, great efforts have been made to develop peroxidase mimics with excellent stability. The high water solubility and stability of bovine serum albumin (BSA) protein, as well the presence of various binding sites in its structure, makes it a key factor to stabilize CuS NPs and prevent their aggregation to enhance their sensing performance.

Herein, BSA was used as a template to synthesize water dispersible copper sulfide nanoparticles (CuS NPs) via a facile method. The obtained CuS-BSA nanocomposite was applied for selective Cr(VI) colorimetric sensing. The as-synthesized CuS-BSA nanocomposite exhibited good stability and dispersibility in various aqueous media and displayed intrinsic peroxidase-mimic for efficient catalytic oxidization of 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) substrate in the presence of H2O2. This property was further exploited for the detection of Cr(VI) in acidic aqueous medium, as confirmed by a naked eye detection of the color change of the solution from transparent to blue. Additionally, UV/vis absorbance spectroscopy was used for the quantitative determination of the oxidized TMB (oxTMB) at 654 nm. The mechanism of TMB oxidation was enhanced by H2O2 decomposition by Cr(VI) to generate HO • in the presence of CuS-BSA. The experimental parameters for colorimetric Cr(VI) detection including temperature, pH and incubation time were also assessed. A detection limit of Cr(VI) as low as 50 nM was achieved under optimized conditions.

Experimental section

Materials and Methods

Bovine serum albumin (BSA, ≥ 96%), copper(II) acetate [Cu(Ac)2, 98.0%], sodium sulfide hydrate (Na2S.xH2O, ≥ 97.0%), sodium hydroxide pellets (NaOH, ≥ 97.0%), 3,3',5,5'tetramethylbenzidine (TMB, ≥ 95.0%), sodium acetate (anhydrous) [CH3COONa, ≥ 99.0%], chromium(III) chloride hexahydrate (CrCl3•6H2O, 96.0%), chromium trioxide (CrO3, ≥ 99.0%), nickel(II) chloride hexahydrate (NiCl2.6H2O, ≥ 98.0%), cobalt(II) nitrate hexahydrate (CoN2O₆.6H₂O, ≥ 98.0%), mercury(II) nitrate monohydrate (HgN2O₆.H₂O, ≥ 99.99%), sodium sulfite (Na2SO3, ≥ 98.0%), zinc chloride (ZnCl2, ≥ 98.0%), iron(III) chloride (FeCl3, ≥ 99.99%), and terephthalic acid (C6H4-1,4-(CO2H)2, 98.0 %) were purchased from Sigma-Aldrich. Ethanol (95% v/v), dimethyl sulfoxide (DMSO, ≥99.9%), glacial acetic acid (99.0 %), and hydrogen peroxide (30 wt%, H2O2) were purchased from fisher scientific. Milli-Q (MQ) water was used throughout the whole procedure.

Sample preparation via mineralization approach

The CuS-BSA nanocomposite was prepared via mineralization process according to a previous procedure [START_REF] Yang | Protein-Nanoreactor-Assisted Synthesis of Semiconductor Nanocrystals for Efficient Cancer Theranostics[END_REF] with some modifications. In short, an aqueous solution of BSA was prepared by dissolving 250 mg of BSA in 9 mL MQ-water. Then 1.0 mL of Cu(Ac)2 (0.1 M) was added dropwise to the above solution under magnetic stirring, resulting in a blue cloudy mixture, after which a light green color was observed. 0.5 mL of an aqueous NaOH (2.0 M) solution was used to adjust the pH at 12, after which the mixture turned directly to purple. Then 52.0 mg of Na2S (0.4 M) were added under stirring; the color of the solution turned to brown immediately. The mixture was further processed at 55 °C for 5 h to give a green solution. The resulting CuS-BSA nanocomposite was purified by dialysis against a 12-14 kDa molecular mass cut off dialysis membrane in MQ-water for 24 h to remove excess copper ions (to exclude their contribution in the catalytic process), then stored at 4 °C for later use. A fluffy-like dark green powder of CuS-BSA was collected by lyophilization for further characterization, which can be redispersed easily in MQ-water.

Peroxidase-mimic based on CuS-BSA nanocomposite

The catalytic activity of CuS-BSA as a peroxidase-mimic was assessed via the oxidization of TMB substrate in presence of H2O2. In brief, the reaction was carried out first by mixing 150 µL of each TMB (1.6 mM dissolved in 96% ethanol), H2O2 (200 mM), and the as-prepared CuS-BSA nanocomposite (1 mg/mL) at a temperature of 25 °C for 25 min. 0.1 M acetate buffer (pH=3.9) was used to adjust the reaction volume to 1.5 mL. Absorption spectra were recorded by UV/vis absorption spectrophotometer in the 350-850 nm range to follow the changes of the oxidized TMB (oxTMB) peak at 654 nm.

The effect of CuS-BSA concentration over the range 0.03-0.20 mg/mL, pH (2.5-10.0), temperature (25-50 °C), and incubation time (0-30 min) were performed to investigate their influence on the catalytic oxidation reaction.

A set of control experiments were also conducted to highlight the catalytic activity of CuS-BSA towards the oxidation of TMB.

Kinetic analysis of CuS-BSA peroxidase-like activity and determination of the

Michaelis-Menten constant and maximum velocity

Enzyme affinity to its substrate can be assessed by the determination of the Michaelis-Menten constant (Km), where smaller Km value indicates a high affinity.

A Lineweaver-Burk double reciprocal plot is used to determine the enzyme affinity using the following relation (1):

1/V = (Km/Vmax) (1/[S]) + 1/Vmax (1)
Where V corresponds to the initial rate of the reaction, Vmax represents the maximum rate, Km is the Michaelis-Menten constant, and [S] is the concentration of the substrate.

The kinetic measurements were recorded at 654 nm in a time-scan mode at different concentrations of one substrate (TMB or H2O2) and at a fixed concentration of the other. The experiments were performed by mixing different concentrations of TMB (0.01-0.16 mM) with H2O2 (20 mM) and CuS-BSA (0.1 mg/mL) catalyst in 1.5 mL acetate buffer (0.1 M) at pH 3.9.

Similarly, using H2O2 as a substrate, the reaction was executed by mixing different concentrations of H2O2 (5-20 mM) with TMB (0.16 mM) and CuS-BSA (0.1 mg/mL) in 1.5 mL of 0.1 M acetate buffer (pH=3.9).

Colorimetric sensing of Cr(VI) in aqueous medium

The colorimetric detection of Cr(VI) ion was carried out by adding various concentrations of CrO3 (dissolved in water) to a mixture of CuS-BSA (0.1 mg/mL), TMB (0.16 mM), and H2O2

(20 mM). The reaction volume was adjusted by 0.1 M acetate buffer (pH = 3.9) to 1.5 mL. The resulting mixture was incubated for 25 min at 25 °C, and the absorption spectra were measured by monitoring the absorbance at 654 nm.

Cr(VI) detection was also recorded in the absence of H2O2 under the same conditions mentioned above.

Selectivity and reproducibility

The selectivity of CuS-BSA was investigated for speciation of Cr(VI). The credibility of this assay was studied by determining the absorbance at 654 nm in presence of different potentially co-existing ions. Thus, 20 µM and 100 µM of different ions (Na + , Hg 2+ , Zn 2+ , Co 2+ , Ni 2+ , Cr 3+ )

were mixed with CuS-BSA (0.1 mg/mL), TMB (0.16 mM), and H2O2 (20 mM) in 0.1 M acetate buffer (pH = 3.9) to examine the response of the proposed sensor for the colorimetric detection of Cr(VI) ion using other ions at same and five-times higher than the concentration of Cr(VI).

The reusability of the nanocomposite was also determined for several repeated cycles under the optimized experimental conditions.

Mechanism of CuS-BSA catalytic activity in the formation of hydroxyl radicals (HO • )

In order to confirm the catalytic activity of CuS-BSA towards TMB oxidation through the formation of hydroxyl radicals from H2O2 decomposition in acidic medium, terephthalic acid

Sensing of Cr(VI) ion in real environmental water samples

To investigate the practical application of CuS-BSA as a colorimetric sensor, concentration of The sensing experiments were conducted as follows: 150 µL of the as-prepared CuS-BSA suspension (1 mg/mL), 150 µL of TMB (1.6 mM in ethanol) and 150 µL of H2O2 (200 mM) were added successively to 150 µL of each water sample containing different concentrations of Cr(VI) (10, 50, 100 nM), and the total volume was adjusted to 1.5 mL by adding acetate buffer (pH = 3.9). The reaction mixture was kept at 25 °C for 25 min, and the absorbance was monitored at 654 nm by UV/vis absorbance spectrophotometry.

Results and discussion

Characterization of CuS-BSA nanocomposite

The synthesis of CuS-BSA nanocomposite was carried out in two steps, first by forming Cu 2+ -BSA complex through the addition of Cu(Ac)2 into BSA solution, where Cu 2+ cations can bind with BSA through exposing the binding sites of BSA to Copper ions in alkaline pH medium (pH 12), followed by the release of S 2-by adding Na2S. The presence of free -SH in cysteine amino acid of BSA [START_REF] Yang | Fabrication of proteinconjugated silver sulfide nanorods in the bovine serum albumin solution[END_REF] enhances the conjugation of metal sulfides to BSA, and acts as a nucleating agent for the formation of CuS-BSA. The formation of CuS-BSA can also be easily visualized by color change, as clearly observed in Fig. S1.

The chemical composition of CuS-BSA nanocomposite and BSA precursor was evaluated by X-ray photoelectron spectroscopy (XPS) analysis. The XPS survey spectrum of BSA (Fig. S2a)

clearly indicates the presence of C1s (285 eV), O1s (532 eV), N1s (400 eV) corresponding to peptide nitrogen, and S2p at 163 eV due to the presence of free thiol (SH). The atomic concentrations of C1s, N1s, O1s, and S2p are 66.43%, 14.53%, 18.52% and 0.52%, respectively.

The deconvolution of C1s high resolution spectrum (Fig. S2b) showed the contribution of various functional groups at 286.0 eV (C-O/C-N), and 287.7 eV due to carbonyl carbon (C=O/C=N), a peak at 284.7 eV ascribed to the aliphatic carbon [START_REF] Pradier | Adsorption of Bovine Serum Albumin on Chromium and Molybdenum Surfaces Investigated by Fourier-Transform Infrared Reflection-Absorption Spectroscopy (FT-IRRAS) and X-ray Photoelectron Spectroscopy[END_REF][START_REF] Iucci | XPS and FT-IR spectroscopy study of albumin adsorption on the surface of a π-conjugated polymer film[END_REF], and the C-S bond of cysteine [START_REF] Gong | Graphene oxide-based electrochemical sensor: a platform for ultrasensitive detection of heavy metal ions[END_REF] in BSA. The high resolution N1s spectrum can be fitted with two bands attributed to C=N [START_REF] Belatik | Locating the binding sites of Pb (II) ion with human and bovine serum albumins[END_REF] and C-N [START_REF] Mahmood | Two-dimensional polyaniline (C3N) from carbonized organic single crystals in solid state[END_REF] at around 397.5 and 400.0 eV, respectively (Fig. S2c). The O1s spectrum (Fig. S2d) can be fitted with one symmetrical peak at 531.0 eV due N-C=O and OH in BSA [START_REF] Pradier | Adsorption of Bovine Serum Albumin on Chromium and Molybdenum Surfaces Investigated by Fourier-Transform Infrared Reflection-Absorption Spectroscopy (FT-IRRAS) and X-ray Photoelectron Spectroscopy[END_REF]. In the S2p high resolution XPS spectrum (Fig. S2e), the peaks at 163 and 168 eV are attributed to SH [START_REF] Escudeiro | Adsorption of bovine serum albumin on Zr cosputtered aC (: H) films: implication on wear behaviour[END_REF] and SO4 2-, respectively. SO4 2-originates most likely from partial sulfur oxidation under ambient conditions. (398.84 eV) and (C)3-N (400.82 eV) of BSA [START_REF] Luo | Pyridinic N doped graphene: synthesis, electronic structure, and electrocatalytic property[END_REF][START_REF] Liu | Hydrothermal treatment of grass: a low-cost, green route to nitrogen-doped, carbon-rich, photoluminescent polymer nanodots as an effective fluorescent sensing platform for label-free detection of Cu (II) ions[END_REF]. This is not surprising since nitrogen contribution originates from BSA only. Fig. 1d represents the deconvoluted spectrum of Cu2p with peaks at 932.06 and 951.82 eV assigned to Cu2p3/2 and Cu2p1/2, respectively. A characteristic satellite peak is observed at around 943 eV, indicating the presence of paramagnetic Cu 2+ ions of CuS [START_REF] Zhang | Visible light photocatalytic H2-production activity of CuS/ZnS porous nanosheets based on photoinduced interfacial charge transfer[END_REF].

In the S2p high resolution XPS spectrum of CuS-BSA (Fig. 1e), the peaks at 164.64 and 167.88 eV are attributed to CuS and CuSO4, respectively [START_REF] Qian | Enhanced wet hydrogen peroxide catalytic oxidation performances based on CuS nanocrystals/reduced graphene oxide composites[END_REF][START_REF] Ancutiene | Structural, chemical and optical properties of the polyethylene-copper sulfide composite thin films synthesized using polythionic acid as sulfur source[END_REF][START_REF] Borthakur | Microwave assisted synthesis of CuS-reduced graphene oxide nanocomposite with efficient photocatalytic activity towards azo dye degradation[END_REF] even though one cannot rule out a contribution of oxidized sulfur in BSA. The peak at ~163 eV originates from S2p in BSA.

The morphology, structure and particle size of CuS-BSA nanocomposite were characterized by TEM analysis (Fig. 2). TEM images (Fig. 2a,b) indicate that CuS-BSA nanocomposite consists CuS-BSA aqueous solution was stable for five months at a concentration ~10 mg/mL with no noticeable precipitates, which indicates the long-term colloidal stability and dispersibility of our nanocomposite. The long-term stability of CuS-BSA nanocomposite was also examined in different solvents (phosphate buffer saline (PBS), acetate buffer, DMSO, Dulbecco's modified Eagle's medium (DMEM)), where no apparent precipitation was observed at a concentration of 1 mg/mL (Fig. 3). Therefore, BSA proved to be an excellent capping agent to prevent aggregation of CuS NPs. at 1655 and 1539 cm -1 , respectively, OH at 3445-3296 cm -1 and amide A (-NH) at 3061 cm -1 [START_REF] Huang | Synthesis and characterization of bovine serum albuminconjugated copper sulfide nanocomposites[END_REF]. The same bands appear in the FTIR spectrum of CuS-BSA, suggesting the presence of BSA in the nanocomposite [START_REF] Liang | Biocompatible tumor-targeting nanocomposites based on CuS for tumor imaging and photothermal therapy[END_REF]. However, by comparing both spectra, one can notice a slight decrease of the intensity of the characteristic -NH peak in CuS-BSA most likely due to the coupling between NH in BSA and Cu 2+ . Additionally, the OH peak in the FTIR spectrum of CuS-BSA is shifted by 49 cm -1 [START_REF] Chen | Synthesis of protein-assisted aqueous Ag2S quantum dots in the bovine serum albumin solution[END_REF], confirming the interaction between BSA and CuS, as BSA can provide various binding sites such as -NH, -OH, COOH, and -SH functionalities. The UV-Vis absorption spectrum of BSA depicts an optical absorption peak at 280 nm [START_REF] Huang | Synthesis and characterization of bovine serum albuminconjugated copper sulfide nanocomposites[END_REF].

The same peak appears in the absorption spectrum of CuS-BSA nanocomposite, indicating the growth of CuS on BSA surface. Furthermore, the absorption spectrum of CuS-BSA shows an increase in absorption tail in the near-infrared (NIR) region [START_REF] Yadav | Synthesis of copper sulfide nanoparticles: pH dependent phase stabilization[END_REF], which is a fundamental characteristic of CuS nanoparticles that absorb light in the NIR range (Fig S6).

Thermogravimetric analysis (TGA) and derivative thermogravemetric (DTG) were conducted to confirm the interaction between CuS NPs and BSA and to study their thermal behavior. DTG is performed to detect the slight changes in weight that cannot be observed by TGA. The TGA curve of CuS-BSA exhibited a total weight loss ca. 78.88% from room temperature (RT) to 980 °C (Fig. S7), with 21.12 % of residual mass left at the end of heating, while the TGA curve of pure BSA showed a total weight loss ca 82.43% from RT to 980 °C (Fig. S8) with 17.57% of residual mass. This decrease in weight loss for CuS-BSA is attributed to the conjugation between BSA and CuS NPs. Pristine BSA showed 3 stages of weight loss observed by DTG, from 30 to 100 °C, 170 to 250 °C, and from 250 to 565 °C. The first stage of weight loss is due to evaporation of water molecules, while the second mass loss is assigned to BSA degradation (most likely oxygen containing groups). The last step corresponds to the decomposition of amino acid residues, which occurs at higher temperature range [START_REF] Chen | Synthesis of protein-assisted aqueous Ag2S quantum dots in the bovine serum albumin solution[END_REF]. However, after conjugation of BSA with CuS NPs, the thermal curve displayed only two stages of weight loss, from RT to 100 °C, and from 160 to 480 °C. The decrease in decomposition temperature is attributed to the reduction of thermal stability of BSA after hybridization with the nanoparticles. BSA exhibited a steep curve at 320 °C, which is also displayed in CuS-BSA with a slight decrease in temperature.

These results indicated the decrease in thermal stability of BSA could be ascribed to the physical adsorption of CuS on BSA rather than chemical interaction.

Peroxidase-like catalytic activity of CuS-BSA NPs

The peroxidase-like catalytic activity of CuS-BSA in mimicking the naturally occurring horse-radish peroxidase (HRP) enzyme was examined by studying the oxidation of the TMB molecule. As shown in Fig. S9, the oxidation of TMB occurred within 30 s when H2O2 was added to CuS-BSA catalyst in acidic medium, confirmed by a visual color change from transparent to blue, and by dynamic increase of the absorbance peak at 654 nm. In order to confirm the catalytic nature of the process, control experiments were carried out in the absence of CuS-BSA and H2O2, respectively. Surprisingly, the experimental systems without H2O2

showed almost a negligible color change, while in absence of CuS-BSA, a slight blue color was observed (Fig. S10), indicating that both CuS-BSA and H2O2 are responsible for the generation of blue colored oxTMB.

It is worth to notice that the oxidation of TMB resulted from the hydroxyl radicals ( • OH) generated from H2O2 decomposition in an acidic medium [START_REF] Borthakur | Metal free MoS 2 2D sheets as a peroxidase enzyme and visible-light-induced photocatalyst towards detection and reduction of Cr (VI) ions[END_REF], which are catalyzed by Cu 2+ ions on the CuS-BSA surface, similar to the Fenton reaction [START_REF] He | Understanding the formation of CuS concave superstructures with peroxidase-like activity[END_REF]. Basically, H2O2 degradation is catalytically mediated by Cu 2+ , in which a reactive intermediate (Cu 3+ ) is formed through the process. Thereby, copper is being progressively cycled between two different oxidation forms (+2 and +3) (Fig. 5).

The mechanism of • OH formation can be summarized as follows:

Experimental condition optimization

To attain the optimal conditions for the catalytic reaction, TMB oxidation was performed by varying different parameters like the catalyst concentration, solution pH, and temperature of the reaction. The absorbance at 654 nm versus concentration of CuS-BSA response curve was plotted in Fig. S11. It is clear that the absorbance gradually increases with the increase of CuS-BSA concentration and saturates at almost 0.1 mg/mL, which is selected for the following experiments. Notably, the rate of the catalytic reaction is highly dependent on catalyst concentration due to the excessive amount of TMB [START_REF] Liu | Au@CuxOS yolk-shell nanomaterials with porous shells act as a new peroxidase mimic for the colorimetric detection of H2O2[END_REF].

The catalytic activity of CuS-BSA NPs, like natural enzymes, depends on pH and temperature. Changes of pH (2.5-10) and temperature (25-50 °C) were recorded to demonstrate their influence on the oxidation of TMB. As revealed in Fig. S12, the relative activity of CuS-BSA was plotted as a function of temperature, whereby the maximum absorbance intensity at 654 nm was set as 100 %.

Relative activities were determined by the following equation (Eq. 2):

R.A = (A654 nm/A654max) × 100 %

Where R.A represents the relative activity, A654 is to the absorbance of oxTMB at 654 nm, and A654max is the maximum absorbance at 654 nm.

The catalytic oxidation of TMB was stable over a wide temperature range and reaches an optimum intensity at 40 °C, close to the physiological environment, due to the increase in the rate of the enzyme-like reaction at this value. A slight decrease in enzymatic activity takes place at 50 °C, most probably due to partial decomposition of H2O2. This result indicates that CuS-BSA displayed better catalytic performance than natural enzymes, which lose their activity completely at high temperature. Similarly, CuS-BSA revealed higher activity at pH 3.9 (Fig. S13) due to the enhanced solubility of TMB in acidic environment [START_REF] Liu | Au@CuxOS yolk-shell nanomaterials with porous shells act as a new peroxidase mimic for the colorimetric detection of H2O2[END_REF][START_REF] Zhang | Fe3O4 Nanoparticles Loaded on Lignin Nanoparticles Applied as a Peroxidase Mimic for the Sensitively Colorimetric Detection of H2O2[END_REF]. Therefore, we performed the following assays in acidic medium and we selected 25 °C for the remaining experiments.

In order to evaluate the rate of the catalytic process, TMB oxidation was monitored at different incubation times in the presence of H2O2 (20 mM), CuS-BSA (0.1 mg/mL), and TMB (0.16 mM) in 1.5 mL acetate buffer (pH=3.9). As observed in Fig. S14, the catalytic activity of CuS-BSA increased upon increasing the incubation time to reach a maximum value after 25 min;

this incubation time was adopted to complete the oxidation reaction in the following experiments.

Steady-state kinetic mechanism of CuS-BSA as a peroxidase-mimic and determining the Michaelis-Menten constant

The kinetic assay of CuS-BSA as a peroxidase mimic was carried out by determining the initial rate of each substrate, i.e. the slope of ΔA654 per unit time (min). In order to determine the reaction rate of each substrate (TMB and H2O2), the UV/vis absorption spectra were recorded at 654 nm in a time-scan mode for different concentrations of substrate (Fig. S15 andS17). Then the Beer-Lambert law (Eq. 3) was applied to convert the absorbance values to the corresponding concentrations:

A = × c × l ( 3
)
Where A is the absorbance at 654 nm, ε is the molar absorptivity (ε = 3.9 ×10 4 for oxTMB at 654 nm in M -1 .cm -1 ), c represents the concentration of one substrate, and l corresponds to the optical path length (l=1 cm). Fig. S16 and S18 depict respectively typical Michaelis-Menten curves for TMB and H2O2, where the data are obtained by plotting the initial reaction rate versus concentration of one substrate and changing the concentration of the other. The catalytic parameters (Km and Vmax), determined from a Lineweaver-Burk plot, are listed in Table 1,

where the y-intercept allows to determine the Vmax and the x-intercept gives the Km value. Cu-rGO [START_REF] Darabdhara | Cu-Ag bimetallic nanoparticles on reduced graphene oxide nanosheets as peroxidase mimic for glucose and ascorbic acid detection[END_REF] 0.0263 10.5×10 -4 5.385×10 -8 3.289×10 -8 It was found that the catalytic oxidation of TMB by CuS-BSA follows a typical Michaelis-Menten kinetics, and the low Km value of CuS-BSA indicates a high affinity towards TMB. The Km value of CuS-BSA with TMB as the substrate was calculated to be 2×10 -4 M, which is lower than that of HRP enzyme [START_REF] Lu | A highly selective and sensitive colorimetric uric acid biosensor based on Cu (II)-catalyzed oxidation of 3,3′,5,5′tetramethylbenzidine[END_REF]. However, the Km value of CuS-BSA with H2O2 as substrate is 0.014 M, suggesting a better binding affinity of CuS-BSA to TMB along with a good peroxidase-enzyme mimic behavior, compared to other previously reported nanomaterials (Table 1).

Colorimetric sensing of Cr(VI)

To further widen the scope of the CuS-BSA nanoenzyme, we have investigated the direct sensing of Cr(VI) using the optimized experimental conditions of TMB oxidation by H2O2.

Indeed, the catalytic activity of CuS-BSA towards TMB oxidation was enhanced in the presence of Cr(VI), whereby in a low pH medium (pH=3.9), Cr(VI) induces the decomposition of H2O2 into H2O and O2 [START_REF] Borthakur | Metal free MoS 2 2D sheets as a peroxidase enzyme and visible-light-induced photocatalyst towards detection and reduction of Cr (VI) ions[END_REF], which can readily generate HO • radicals when combined with Cu 2+ on the surface of CuS-BSA nanocomposite; the reaction of Cr(VI) with H2O2 can also generate HO • radicals (Haber Weiss-type reaction), which is favored in acidic medium [START_REF] Bokare | Chromate-induced activation of hydrogen peroxide for oxidative degradation of aqueous organic pollutants[END_REF][START_REF] Shi | Reduction of chromium (VI) and its relationship to carcinogenesis[END_REF]. Cr(VI) sensing was recorded under various experimental conditions, i.e. in the absence (Fig. S19) and presence of H2O2 (Fig. S20) under the optimized reaction conditions. A good linear relationship (R 2 = 0.99372) was obtained for Cr(VI) in the 80-200 µM concentration range in the absence of H2O2

(inset of Fig. 6); the limit of detection (LOD) was determined using the following formula (Eq.

4):

LOD = (3× standard deviation of the blank)/slope of linear regression line (4)

The LOD for Cr(VI) was determined to be 18 µM at a signal-to-noise ratio of 3.

Based on this result, the detection of Cr(VI) was examined in the presence of H2O2. Noticeably, the LOD of Cr(VI) was enhanced in the presence of H2O2 (Fig. S21), and a good linear relation (R 2 = 0.9984) between the absorbance A654 nm and the concentration of Cr(VI) within the 0-100 nM and 1-20 µM ranges was observed (Fig. 7 and Inset of Fig. S21). The LOD was found to be 50 nM at a signal-to-noise ratio of 3 using the lower linear range, below the detectable level determined by the U.S EPA (1.9 µM) [START_REF] Dong | Selective colorimetric detection of Cr (III) and Cr (VI) using gallic acid capped gold nanoparticles[END_REF], demonstrating the sensitive detection of Cr(VI) using CuS-BSA nanocomposite.

The performance of the developed sensor is better than that reported for the same metal ion using colorimetric detection scheme (

Detection of Cr(VI) in real environmental water samples

To validate the practical application of our proposed enzyme mimic, Cr(VI) detection was tested in different environmental samples using the optimized conditions (CuS-BSA (0. In order to evaluate the selectivity of CuS-BSA, the absorbance (A=654 nm) was monitored in presence of different interfering ions (Na + , Hg 2+ , Zn 2+ , Co 2+ , Ni 2+ , Cr 3+ ) under the same conditions using 5-times higher concentration than the optimized Cr(VI) concentration. The specificity of our sensor was tested with the same experimental procedure as mentioned above.

As can be seen from Fig. 9, Cr(VI) showed the highest remarkable response, indicating the good selectivity of the developed sensor. Interestingly, the absorbance was not enhanced much, even when the concentration of the interfering ions was 5 times higher (100 µM) than that of Cr(VI) (Fig. S22). Also, in the absence of H2O2, the interfering ions did not induce blue color change.

Notably, due to the higher redox potential of Cr 6+ /Cr 3+ (E= +1.33 V) [START_REF] Dehghani | Reduction of noxious Cr (VI) ion to Cr (III) ion in aqueous solutions using H2O2 and UV/H2O2 systems[END_REF] among other interfering metal ions, Na + /Na (-2.71 V), Co 2+ /Co (-0.28 V), Zn 2+ /Zn (-0.76 V), Ni + /Ni (-0.36 V), Hg 2+ /Hg (E= +0.92 V), Cr 3+ /Cr (-0.74 V), it acts as a strong oxidizing agent in an acidic environment for the dismutation of H2O2 [START_REF] Perez-Benito | A Kinetic Study of the Chromium (VI)-Hydrogen Peroxide Reaction. Role of the Diperoxochromate (VI) Intermediates[END_REF]. Therefore, the results demonstrated high specificity, sensitivity, selectivity, and colorimetric assay can be achieved using CuS-BSA nanocomposite for Cr(VI) detection.

Conclusion

In summary, this work presented a simple and cost-effective method for the synthesis of CuS-BSA nanocomposite with peroxidase-like activity towards the catalytic oxidation of TMB. The enhanced activity of the nanoenzyme was further applied for the sensitive and selective detection of hexavalent chromium ions. The proposed sensor showed that the catalytic reaction follows typical Michaelis-Menten kinetics in a pH-dependent manner. Under optimized experimental conditions, a good linear calibration plot was obtained in the concentration range of 0-100 nM and 1-20 µM of Cr(VI) with a detection limit of 50 nM using the lower linear range. Using terephthalic acid, it was possible to confirm the generation of HO • radicals during the TMB catalytic oxidation by H2O2 in the presence of CuS-BSA, a process that has been enhanced in the presence of Cr(VI). The method was effectively tested for the determination of Cr(VI) content in spiked real environmental water samples. Based on these observations, the CuS-BSA nanocomposite showed better performance over natural enzymes which opened new promising applications in many fields.

(

  TA) was used as a probe molecule that can readily react with • OH radicals to produce 2hydroxyterephthalic acid (HTA). The HTA exhibits a fluorescence emission peak at 430 nm under excitation wavelength of 315 nm. In brief, TA (0.15 mM in DMSO) was subsequently treated with (a) H2O2 (20 mM), (b) Cr (VI) (20 µM), (c) CuS-BSA (0.1 mg/mL), (d) H2O2 (20 mM) + CuS-BSA (0.1 mg/mL), (e) H2O2 (20 mM) + Cr(VI) (20 µM), (f) Cr(VI) (20 µM) + CuS-BSA (0.1 mg/mL), (g) H2O2 (20 mM) + Cr(VI) (20 µM) + CuS-BSA (0.1 mg/mL) in 1.5 mL acetate buffer (pH=3.9) and incubated for 25 min at 25 °C. The fluorescence emission spectra were then collected in the range of 325-550 nm using a Safas Xenius XC fluorescence spectrophotometer (Monaco) under excitation wavelength of 315 nm.

  Cr(VI) ions were determined in various real environmental water samples (sea, pond, tap and tube well water) spiked with different concentrations of Cr(VI) ions. The tube well water samples were taken from local area of Lille city, Rihour, France. The pond water samples were collected from the Parc du Héron, Villeneuve d'Ascq (France). The tap water samples were obtained from our laboratory (IEMN, Villeneuve d'Ascq), and the sea water samples were from the North Sea (marginal sea of Atlantic Ocean). The collected water samples were purified by centrifugation at 13,500 rpm for 10 min and the supernatant was then collected and filtered through 0.2 µm pore size membrane, before standard solutions of Cr(VI) ions (10, 50, 100 nM) were added separately.

Figure 1 :

 1 Figure 1: XPS survey spectrum (a), and high resolution XPS spectra of C1s (b), N1s (c), Cu2p (d) and S2p (e) of CuS-BSA nanocomposite.

Figure 2 :

 2 Figure 2: (a, b) TEM images, (c) HRTEM image and SAED pattern (inset), (d) particle size distribution, (e) HAADF-STEM image, elemental mapping for (f) carbon, (g) nitrogen, (h) oxygen, (i) copper, (j) sulfur of CuS-BSA nanocomposite.

Figure 3 :

 3 Figure 3: The long-term stability of CuS-BSA (1 mg/mL) dispersed in different solvents (left to right: PBS (a), sodium acetate buffer (b), DMSO (c), and DMEM (d) medium) for one month.

Figure 4 :

 4 Figure 4: FTIR spectra of native BSA and CuS-BSA nanocomposite.

Figure 5 :

 5 Figure 5: Schematic illustration of the oxidation of TMB.

  Fig.S20depicts the absorbance of oxTMB (A654 nm) at various H2O2 concentrations, where above 20 mM the catalytic activity of CuS-BSA was not much improved. Therefore, we fixed the concentration of H2O2 to 20 mM to determine the LOD of Cr(VI) within the 0.01-

Figure 6 :

 6 Figure 6: A dose-responsive curve of Cr(VI) using CuS-BSA as a catalyst. Absorbance of oxTMB versus Cr(VI) concentration at 654 nm in the presence of CuS-BSA (0.1 mg/mL), TMB (0.16 mM), pH=3.9, T=25 °C, incubation time=25 min; inset: The linear calibration plot for the determination of Cr(VI).

Figure 7 :

 7 Figure 7: A dose-responsive curve of Cr(VI) using CuS-BSA as a catalyst. Absorbance of oxTMB versus Cr(VI) concentration at 654 nm in the presence of CuS-BSA (0.1 mg/mL), TMB (0.16 mM), H2O2 (20 mM), pH=3.9, T=25 °C, incubation time=25 min; inset: The calibration curve in the lower concentration range.

Figure 8 :

 8 Figure 8: Fluorescence spectra to identify hydroxyl radicals' formation using terephthalic acid (TA) as a fluorescent probe. Reaction conditions: CuS-BSA (0.1 mg/mL), H2O2 (20 mM), Cr(VI) (20 µM), TA (0.15 mM) and 0.1 M acetate buffer (pH = 3.9) for 25 min at 25 °C.

Figure 9 :Figure 10 :

 910 Figure 9: UV-vis absorption at 654 nm of oxTMB in the presence of different interfering ions of concentration 20 µM. Reaction conditions: CuS-BSA (0.1 mg/mL), H2O2 (20 mM), TMB (0.16 mM), Cr(VI) (20 µM) and 0.1 M acetate buffer (pH = 3.9), T=25 °C, incubation time=25 min.

Table 1 :

 1 Michaelis-Menten constant and maximum velocity for typical TMB and H2O2 substrates in comparison with previously reported materials.

	Catalyst		Km (M)		Vmax (M.s -1 )
		H2O2	TMB	H2O2	TMB
	CuS-BSA (our work) 0.014	2 ×10 -4 2×10 -8	3.3×10 -8
	HRP [13]	0.0037	4.34×10 -4 8.71×10 -8	10×10 -8
	MoS2 [38]	1.16×10 -4	3.87×10 -4	2.42×10 -8	7.23×10 -8

Table 2 ).

 2

		0.64
		0.62		0.590		y= 0.28835x+ 0.5622
	A654 nm	0.60	A654 nm	0.575 0.580 0.585		R2= 0.99846
				0.570
		0.58		0.565
				0.560
				0.00	0.02	0.04	0.06	0.08	0.10
		0.56				[Cr(VI)] (µM)
			0 5 10 15 20 25 30 35 40 45 50 55
				[Cr(VI)] (µM)

Table 2 :

 2 Comparison of different nanomaterials for colorimetric sensing of Cr(VI).

	Materials	LOD	Linear range	Ref.
	GA-Au NPs	2 µM	1-20 µM	[46]
	BSA-Au NPs/STCP	280 nM	0.5-50.0 μM	[47]
	frGO-Cu	67.13 nM	0-200 nM	[48]
	CuS-BSA	50 nM and 6.8 µM	0-100 nM and 1-20	Our work
			μM

Table 3 ,

 3 the CuS-BSA nanocomposite displayed good recovery percentage for Cr(VI) sensing in different environmental water samples.

	1

Table 3 :

 3 Detection of Cr(VI) in real environmental water samples (n=3).

	Samples	Cr(VI) added	Cr(VI) found	Recovery	RSD
		(nM)	(nM)	(%)	(%)
		10	9.95	99.5	0.71
	Tap water	50	49.5	99	1.42
		100	96	96	2.94
		10	9.6	96.3	5.79
	Pond water	50	49.3	98.6	1.24
		100	97.6	97.6	0.59
		10	9	90	15.7
	Sea water	50	49.5	99	1.4
		100	92	92	3.07
		10	9.2	96.5	5.12
	Tube-well water	50	49	98	5.7
		100	98.5	98.5	0.71

. Selectivity and reproducibility of CuS-BSA enzyme-mimic for sensing heavy metal Cr(VI) ions

The XPS survey spectrum of CuS-BSA (Fig. 1a) comprises bands attributed to C1s (285 eV), N1s (400 eV), O1s (532 eV), Cu2p (932 eV) and S2p (163 eV) with atomic concentrations of 62.97%, 15.44%, 19.69%, 0.77% and 1.12%, respectively. The atomic ratio of Cu/S is calculated to be 0.68:1; the deviation from 1 is due to the presence of S groups in BSA.

Acknowledgement

AS, AB, SS and RB acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), the University of Lille, the Hauts-de-France region and the CPER "Photonics for Society".