Karine Reybier
email: reybier@cict.fr

Clotilde Ribaut

Agnès Coste

Jérôme Launay

Paul Louis Fabre

Franc ¸oise Nepveu

Characterization of oxidative stress in Leishmaniasis-infected or LPS-stimulated macrophages using electrochemical impedance spectroscopy

The physiological changes caused by external stimuli can be employed as parameters to study pathogen infection in cells and the effect of drugs. Among analytical methods, impedance is potentially useful to give insight into cellular behavior by studying morphological changes, alterations in the physiological state, production of charged or redox species without interfering with in vitro cellular metabolism and labeling. The present work describes the use of electrochemical impedance spectroscopy to simply monitor by modeling impedance plots (Nyquist diagram) in appropriate equivalent circuit, the changes affecting murine macrophage cell line (RAW 264.7) in response to parasite infection by Leishmania amazonensis or to lipopolysaccharide (LPS) treatment. These results demonstrate the ability of electrochemical impedance spectroscopy to discriminate between two opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. Indeed, the study has allowed the characterization, from an electrical point of view, of the extra-cellular NO radical produced endogenously and in great quantities by the inducible form of NOsynthase in the case of LPS-stimulated macrophages. This production was not observed in the case of Leishmania-infected macrophages for which to survive and multiply, the parasite itself possesses mechanisms which may interfere with NO production. In this latest case, only the intracellular production of ROS was observed. To confirm these interpretations confocal microscopy analysis using the ROS (reactive oxygen species) fluorescent probe 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate and electron paramagnetic resonance experiments using Fe(DETC) 2 as NO radical spin trap were carried out.

Introduction

Cells represent the minimum functional and integrating communicable units of living systems. During their life cycle they act both as transducers and transmitters of various chemical and physical signals through the production of specific molecules. The physiological changes caused by external stimuli might be employed as parameters to study pathogen infection in cells, and the effect of drugs. Cell-based sensors are new hybrid systems using the abilities of the cell to detect, transduce and amplify small changes originating from stimuli (McFadden, 2002;Stenger et al., 2001). These assays offer a significant range of opportunities based on the ability of cells to recognize a broad range of biologically active substances affecting their response. Cell-based biosensors have been implemented for a number of applications ranging from pharmaceutical screening (Asphahani and Zhang, 2007), to detection of environmental pollutants (Pinsino et al., 2008) or pathogens (bacteria and viruses) (Rider et al., 2008). Applications are very dependent on the transduction mode which can be electric, optic or even piezoelectric or a combination of different types (De Blasio et al., 2004), and to cell types including macrophages (Kowolenko et al., 1990), fibroblasts [START_REF] Tlili | [END_REF]Xiao and Luong, 2005), epithelial and endothelial (Marx et al., 2005) cells. Among cellular biosensors, impedance cell-based sensor arrays which were first described by [START_REF] Giaever | Proc. Natl. Acad. Sci[END_REF], are potentially useful to give insight into cellular behavior, to detect morphological changes (Arndt et al., 2004), to study cell adhesion (De Blasio et al., 2004), attachment and spreading (Luong et al., 2001;Xiao et al., 2002), alterations in the physiological state or to test the efficiency of drugs (Otto et al., 2004) or effectors (Nguyen et al., 2004;[START_REF] Tiruppathi | Proc. Natl. Acad. Sci[END_REF][START_REF] Tlili | [END_REF]. The physical background to the technique is based on the electrically insulating effect of cell membranes at low frequencies. The cellular sensing method allows real-time monitoring of cells, avoids the use of labeled molecules and does not interfere with in vitro cellular metabolism. Even if cellular biosensors have been applied in many different fields, the literature on cell-based devices involving two living entities, such as a parasite developing in its host cell, is very rare.

Leishmaniasis is an infectious disease caused by intracellular protozoa from the genus Leishmania affecting over 12 million people worldwide. After entry into its mammalian host, Leishmania is phagocytosed by macrophages and confined to a lysosomelike compartment, known as the parasitophorous vacuole (Russell, 1995), where it develops and proliferates. When parasites are numerous enough, i.e. after 20-30 h, the macrophage disrupts and releases amastigotes internalized by new naive macrophages. Hence, the macrophage constitutes both the host and the effector cell against Leishmania infections (Ritting and Bogdan, 2000). Furthe macrophage possesses a number of primary defence mechanisms against microbial pathogens, including the production of reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs). Leishmania promastigotes have been shown to be susceptible to both ROIs and RNIs.

The aim of the present work is to use electrochemical impedance spectroscopy (EIS) to simply monitor changes from an electrical point of view, and in particular oxidative stress generated by murine macrophage cell line RAW 264.7 in response to parasite infection by Leishmania amazonensis (L. amazonensis) or to lipopolysaccharide (LPS) (Stuehr and Marletta, 1987) treatment. Identification of morphological changes and oxidative events taking place in macrophages during activation by Leishmania could help in the comprehension of infectious mechanisms to fight this neglected disease which has only a few efficient treatment drugs, most of them being very toxic.

Materials and methods

RAW 264.7 cell culture

RAW 264.7 cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with sodium bicarbonate (1.5 g/L; Lonza), d-(+)-glucose (4.5 g/L; Sigma-Aldrich), l-glutamine (4 mM; Lonza) and 10% decomplemented foetal bovine serum (Lonza). The cells were maintained at 37 • C in a 5% CO 2 atmosphere.

Parasites and in vitro cultures

Promastigote cultures were derived from axenically grown amastigote stages by subpassage at 25 ± 1 • C in medium RPMI 1640 (Roswell Park Memorial Institute medium, Lonza) buffered with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Lonza) and 2 mM NaHCO 3 (Sigma-Aldrich), pH 7.2, supplemented with 20% heat-inactivated foetal bovine serum (FBS, Lonza), 2 mM l-glutamine, 100 U/mL penicillin and 100 g/mL streptomycin (Gibco, BRL). Promastigote cultures were maintained at 25 ± 1 • C in RMPI 1640 supplemented with 10% of decomplemented FBS.

In vitro RAW 264.7 cell infection or activation

The macrophage murine cell line RAW 264.7 was maintained in an exponential growth phase by subsequent splitting in DMEM complemented with 10% of FBS. RAW 264.7 cells were infected with promastigote at a parasites: macrophage ratio of 5:1 for 1 h at 37 • C with 5% CO 2 . Non-internalized parasites were removed by gently washing in pre-warmed PBS.

RAW 264.7 were activated with LPS at 1 or 10 g/mL (stock solution at 1 mg/mL in sterile EBSS (Earle's Balanced Salt Solution)) depending on experiments, for 1 h at 37 • C with 5% CO 2 . The culture was then rinsed with pre-warmed PBS.

Confocal microscopy

The macrophage murine cell line RAW 264.7 was cultured on a Lab-Tek chamber slide and was infected or stimulated as describe above. Non-adhesive cells were removed by washing in pre-warmed PBS. The fixed cells were incubated with 2 ′ ,7 ′dichlorofluorescein diacetate (20 M; Sigma-Aldrich), for 20 min at 37 • C and 5% CO 2 (Kondo et al., 2001). A confocal laser-scanning microscope (LSM 510, Zeiss) was used to visualize the production of radical species such as RO • , ROO • , • NO 2 , CO • 3 , • OH and NO.

EPR experiments

Electron paramagnetic resonance spectroscopy (EPR) experiments were carried out for RAW cells activated with LPS and infected with L. amazonensis, as described above. The cells were then incubated in DMEM containing 3% (w/w) bovine albumin, NaDETC (sodium diethyldithiocarbamate, 2 mM; Sigma-Aldrich), and FeSO 4 (1 mM; Sigma-Aldrich) at 37 • C and 5% CO 2 . After 2 h of incubation, 300 L of supernatant was placed in a quartz EPR tube (inner diameter 4 mm) for EPR analysis. X-band EPR spectra were obtained at 112 K on a Bruker EMX-8/2.7 (9.86 GHz) equipped with a high-sensitivity cavity (4119/HS 0205) (Bruker, Wissembourg, France) and a teslameter (Bruker ER 0.35). Processing of EPR data was performed using WINEPR software (Bruker). Typical scanning parameters were: scan number, 5; modulation amplitude, 5 G; modulation frequency, 100 kHz; microwave power, 20.12 mW; time constant, 10.24 ms; receiver gain: 2 × 10 4 .

Electrode manufacturing process and electrode modification

Firstly, P-type (3-5 cm -1 ) silicon substrates were thermally oxidized to grow an 800 nm thick silicon oxide SiO 2 layer. A titanium/gold deposit (30 nm/300 nm) was then performed by evaporation at low deposit rates (1 nm/min).

The gold electrodes were firstly washed in a piranha mixture (70% H 2 SO 4 /30% H 2 O 2 ; Sigma-Aldrich) for 3 min. Electrode modification by cells was carried out by re-suspending adherent RAW cells in a flask whose bottom had been covered by the electrodes (gold faces up) at 37 • C in a 5% CO 2 atmosphere for 24 h.

Electrochemical impedance spectroscopy

Electrochemical experiments were performed at 25 • C using a Voltalab 80 PGZ 402 with a three-electrode cell including a calomel electrode as the reference electrode, a gold electrode (0.255 cm 2 ) as the counter electrode and a modified gold electrode (0.053 cm 2 ) as the working electrode. EIS with a chemical probe [Fe(CN) 6 ] 3-/4- (1:1) (5 mM, Sigma-Aldrich) was carried out in PBS solution (pH 7.4), while EIS without chemical probe was performed in the culture medium. Impedance spectra were recorded in a free-potential frequency range from 50 kHz to 100 mHz The amplitude of the alternating voltage was 10 mV. Impedance experiments were done in quadruplate for a same culture.

Results and discussion

Impedance biosensors are based on electrochemical impedance spectroscopy (EIS) which measures the electrical impedance of an interface by imposing or not (free potential) a small sinusoidal voltage and measuring the resulting current at different frequencies. Impedance Z is given by the ratio voltage-current. From the Nyquist plots, i.e. -Z imaginary = f(Z real ) recorded for different frequencies it is possible, by modeling, to convert the interface in equivalent circuits (composed of resistances and capacities) taking into account electronic transfer and mass diffusion which takes place at the cell/solution interface. Among EIS experiments, two methods exist. EIS using a redox couple as a probe (Fe(CN) 6

3-/4-, for example), to characterize the electrode surface by determining kinetic parameters and the surface coverage after modification, and EIS carried out probe-free, which gives information about species produced by the covering layer. In our case EIS was applied to gold electrodes modified by infected or stimulated macrophages.

Characterization of the cellular layer by EIS using a redox probe

Impedance measurements were carried out at the free potential of the electrode with an equimolar mixture of potassium ferrocyanide/ferricyanide as redox probe. The Nyquist diagrams, corresponding to the gold electrode before and after modification with RAW cells, are presented in Fig. 1. In all cases, the curves highlight a semicircle immediately followed by the Warburg line, whose slope is equal to unity. The semicircle characterizes the electron transfer and Warburg line mass transfer. It can be noted that the presence of RAW cells at the gold surface induces an increase in the semicircle diameter resulting from the insulating properties of the cells.

In order to obtain kinetic parameters of the film, Nyquist plots were fitted according to circuit model I presented in Fig. 1 (Ribaut et al., 2009). The electrochemical system is described by the standard Randles circuit: as usual, R s is the electrolyte resistance, Q dl the double-layer capacity (here a constant phase element (CPE)), R ct the charge transfer resistance and Z w the Warburg impedance under non-stationary conditions, characterized by the Warburg coefficient with Z w = /ω 1/2 -j/ω 1/2 and ω = 2f.

The cellular layer is taken into account by to the typical Randles circuit, of R f , film resistance and Q f , dielectric capacitance corresponding to the film.

Electric-component values deduced from modeling are presented in Table 1. The increase in charge transfer resistance is related to electrode coverage by the relationship 1 -= R 0 ct /R ct where is the apparent electrode coverage, assuming that all the current passes through bared spots on the electrode, R 0 ct the charge transfer resistance measured at the bare Au electrode and R ct the charge transfer resistance measured under the same conditions at the modified electrode. From R 0 ct and R ct given in Table 1, the electrode coverage is equal to 93 ± 2%. The presence of the film implies the addition of an imperfect capacitance Q f for which n, which characterizes inhomogeneities (surface roughness, 0 < n < 1) of the analyzed layer, equals to 0.7. This value is relatively weak revealing a rough surface due to the cell monolayer.

As shown in Table 1, the macrophages layer on the gold surface leads to a 80-fold increase in the double-layer capacity. This increase may result from the fact that cellular plasmatic membranes are coated with an external layer containing negatively charged polysaccharides (Mutsaers and Papadimitrou, 1988). The presence of negative charges on the surface, induces with positive charges available in the electrolyte, a large charge separation responsible for the high value recorded for Q dl .

Characterization by EIS of the cellular layer after infection by Leishmania or stimulation with LPS

Impedance spectroscopy measurements, with or without use of a probe, were carried out on healthy, Leishmania-infected and LPS-stimulated RAW macrophages. The corresponding plots are presented in Fig. 2. The corresponding plots clearly highlight great differences between the three cell states. In order to obtain information on the electrical properties of parasitized or stimulated versus normal macrophages, impedance plots corresponding to measurement completed with a probe were fitted according to the model circuit I presented in Fig. 1. However, in the case of EIS carried out without probe, the modeling did not provide satisfactory fitting with experimental data. This difficulty may come from reactivespecies production within the macrophage itself, thus rendering the equivalent circuit inappropriate; this production is masked when experiments are carried out with a redox probe. Indeed, to kill intra-and extra-cellular parasites, various oxidant-generating enzymes are activated among which NADPH oxidase and xanthine oxidase, leading to the production of superoxide anion and inducible nitric oxide synthase (iNOS), which produces nitric oxide (Gantt et al., 2001;Lemesre et al., 1997;Mehta and Shaha, 2006). For this reason, circuit model II presented in Fig. 2 was preferred to circuit model I given in Fig. 1. This circuit includes in series a charge transfer resistance R ads and a capacitance C ads which take into account redox species production in the cellular layer. R ads corresponds to a charge transfer resistance whereas C ads represents the concentration of adsorbed electroactive species.

The value of the components deduced from modeling with circuit models I or II, respectively, for EIS performed with or without probe, are detailed in Table 1. The comparison between values deduced from experiments carried with or without redox probe facilitates interpretation of the events induced by pathogen infection or LPS stimulation. It is important to mention that the control values from non-infected and non-stimulated macrophages change with each experiment because the same RAW cell line, but not in the same passage number, was used. With an increasing passage number, the cells can change morphologically, and phenotypic variations can appear leading to great variations in component values deduced from modeling as an equivalent circuit.

Electrolyte resistance is logically constant independently of the cellular state with a value equal to 330 for experiments carried out with [Fe(CN) 6 ] 3-/4 . In the case of experiments performed in the culture medium, R s is almost constant (450 ) with a very weak increase in the case of LPS-stimulated macrophages.

Concerning film characteristics, i.e. Q f and R f , it can be noted that the evolution of the value depends highly on the electrolyte used. Film capacity Q f significantly decreases in the presence of [Fe(CN) 6 ] 3-/4-from 8.2 F s n-1 for non-infected RAW to 3 F s n-1 for parasitized cells, whereas it increases from 6.3 to 11.7 F s n-1 for LPS-stimulated macrophages. Conversely, this value is constant in the absence of probe. It can also be noted that R f is constant with the probe, whereas it strongly decreases with infection or stimulation in the culture medium. All these comparisons suggest that interferences take place between the probe and the cell. The effect of ferricyanide [Fe(CN) 6 ] 3-on macrophages has already been mentioned in the literature. Baoutina et al. (2001), for example, have demonstrated the capacity of macrophages to reduce in vitro transition metal such as Fe(III) by measuring the formation of ferrocyanide [Fe(CN) 6 ] 4-from ferricyanide [Fe(CN) 6 ] 3-. The reduced transition metal ions generated (Fe(II)) might facil-itate production of oxidative species via a Fenton-type reaction with the H 2 O 2 produced. In the same way, Kaul et al. (1997) have demonstrated that ferricyanide, while this is not the case for ferrocyanide, is able to stimulate NF-kB via the redox reaction at the plasma membrane, NF-kB being the transcription factor that controls the inducible expression of a variety of genes, particularly those involved in inflammatory, immune, and acute-phase responses. The high value of Q f recorded for healthy RAW with [Fe(CN) 6 ] 3-/4-(8.2 F s n-1 ) could originate from morphological changes affecting the [Fe(CN) 6 ] 3-stimulated cells as described in the literature for LPS-stimulated macrophages. Williams and Ridley (2000) have demonstrated on glass slides that LPS induced distinct changes in cell morphology and actin organization in monocytes and Bac1 mouse macrophage cells. These morphological changes are characterized by an increase in cell adhesion and spreading. As Q f is proportional to the surface and inversely proportional to the film thickness, the high value obtained in the case of [Fe(CN) 6 ] 3- stimulated RAW cells, could originate from an increase in film surface (cellular spreading) and/or to a decrease in cellular thickness, as illustrated in Scheme 1.

Taking into account modeling results, the ferricyanide effect on macrophages seems higher in healthy RAW cells than in parasitized ones. This result could be due to the fact, as described in the literature for LPS-activated cells, that infected cells take 40% longer to activate (Camacho et al., 2008). To summarize, in Scheme 1. Morphological changes that could affect RAW in the presence of [Fe(CN)6] 3-.

Table 1

Modeling of EIS measurements performed on infected or stimulated macrophages using or not a redox probe. For EIS performed with the redox probe values of electrical components are deduced from the modeling of Nyquist diagrams (Figs.

1, 2a and b) according to the circuit model I. For EIS carried out without redox probe in the culture medium values of electrical components are deduced from the modeling of Nyquist diagrams (Fig. 2c andd)

according to the circuit model II. The errors were obtained from the standard deviation.

Rs

() the case of Leishmania-infected cells, morphological changes are delayed by infection, and film capacity decreases with the infection, whereas in the case of LPS-stimulated macrophages the effects of [Fe(CN) 6 ] 3-and LPS are combined inducing a great change in cell morphology, and thus an increase in film capacity. These opposed and cumulative morphological changes described for infected or LPS-stimulated macrophages, respectively, could also explain the different behaviors recorded for R ct . R ct decreases with the infection from 4500 to 3170 , while it increases from 2633 to 4066 in the case of LPS-stimulated RAW cells. The more swollen are the cells, the more insulating is the film, and the higher is the charge transfer resistance.

Q f (F s n-1 ) n R f () Q dl (F s n-1 ) n Rct () w ( s -1/2 ) EIS
Q f (F s n-1 ) n R f (k) Q dl (F s n-1 ) n R ads (k) C ads (F) Rct (k) w ( s -1/2 ) EIS
The stability of the film resistance in the case of experiments performed with a redox couple could also originate from interference between probe and cell. An activation of the macrophage by ferricyanide means that ions bind to the membrane as described for LPS in the case of LPS-activated RAW cells. Indeed, LPS binds to CD14 at the macrophage surface, then interacts with signaling receptor TLR4, toll-like receptors (TLRs) which recognize pathogenassociated molecular patterns and evoke various cell-signaling pathways (Moon and Pyo, 2007). Binding between the redox probe and the cell results in ion-intercalation in the cell membrane, i.e. in the cellular film itself, rendering the insulating properties independent of the cellular state, whether native or infected.

As far as the interface film/solution is concerned, in the case of impedance carried out with the redox probe, the imperfect capacity of double layer Q dl increases for infected cells from 225 to 555 F s n-1 , whereas it is stable for LPS-stimulated macrophages (∼350 F s n-1 ). The increase in Q dl recorded for parasitized macrophages toward normal ones could result from hyperpolarization of the cell membrane induced by the infection, as described by Forero et al. (1999).

Taking into account the huge effect of the redox probe on the healthy or even activated macrophages, electrochemical impedance spectroscopy using a probe is not adapted to the study of such cells.

For measurements performed in the culture medium, i.e. probefree (Table 1), the first important change concerns film resistance R f which decreases for infected and stimulated cells compared to healthy ones. These changes could originate from the Leishmania infection or LPS-activation mechanism leading to the release of a great number of reactive species including not only transient radical species such as O 2

•-, • OH and NO (Baoutina et al., 2001;Bhattacharya et al., 2008;Lemesre et al., 1997), but also calcium or potassium ions (Alexander et al., 1999;Bogdan et al., 1996) into the cellular layer. Several reports have indicated that LPS treatment can cause an increase in intracellular-free Ca 2+ concentration which is associated with TNF-␣ production in alveolar macrophages and Kuffer cells (Chen et al., 1999;Zandi et al., 1997). It has also been shown that K + channels are involved in LPS-induced activation of macrophages [START_REF] Haslberger | [END_REF]Lowry et al., 1998) and that, in murine macrophage J774.1 cells, LPS treatment changes the density of inwardly rectifying K + channels (McKinney and Gallin, 1990).

If most of the electrical components of the equivalent circuit vary in the same way for infected or LPS-stimulated cells, this is not the case for R ads and R ct which, respectively, characterize the production of electroactive species in the cellular layer or in the culture medium. These opposite variations result from the fact that these two stimuli induce diametrically contrary responses in RAW cells. Standard LPS macrophage activation leads to the production of inflammatory cytokines, reactive oxygen and nitrogen intermediates (ROIs and RNIs) among which nitric oxide produced endogenously and in great quantities by the inducible form of NO-synthase (iNOS), significantly expressed in LPS-activated macrophages. This highly diffusible radical is rapidly released from the cell. Moreover, the high concentration of nitric oxide in the cul- ture medium is responsible for the strong decrease of R ct from 4000 to 775 k following stimulation.

In the case of Leishmania infection, both in vitro and in vivo experimental models have demonstrated that leishmanicidal activity in murine macrophages is also mediated by reactive oxygen and nitrogen intermediates, especially NO (Jorens et al., 1995;Solbach and Laskay, 2000). However, to survive and multiply within the macrophage the parasite itself possesses mechanisms which may interfere with NO production (Perrella Balestieri et al., 2002). The decrease in NO produced in the case of Leishmania-infected cells compared to LPS-activated cells may explain the slighter decrease in charge transfer resistance R ct from 650 to 250 k. Conversely, the high decrease in R ads from 725 to 81 k recorded in this case could come from the production of lethal oxygen radicals by the cell in its cytosol (non-diffusible radicals) to eliminate invading pathogens (Mehta and Shaha, 2006).

In order to confirm the production of reactive oxygen species in the cell cytosol following infection by Leishmania, the production of oxygen-derived free radicals was measured with ROS fluorescent probe 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (DCF) using confocal microscopy. Non-ionized DCF is membrane-permeable and, therefore, diffuses readily into cells (Nasr-Esfahani et al., 1990). Confocal microscopic images of LPS-activated or Leishmaniainfected macrophages carried out in the presence of DCF are presented in Fig 3a andb, respectively. These images clearly show a high fluorescent intensity in the infected cell (Fig. 3b) whereas this is not the case for LPS-stimulated macrophages (Fig. 3a), confirming that Leishmania induces a huge production of reactive oxygen species in RAW cells. In the same way, to confirm nitric oxide production in the case of LPS-stimulated macrophages and its diffusion outside the cell, electron paramagnetic resonance (EPR) was carried out using water-insoluble complex N,N-diethyldithiocarbamate [Fe(II)(DETC) 2 ] as a spin trap. Fig. 3c andd complex (a N = 13.5 G, g = 2.041) was detected in the culture medium of LPS-stimulated RAW cells, confirming the presence in great quantities of nitric oxide radicals. Conversely, no triplet was detected for Leishmania-infected RAW cells, either in the supernatant (Fig. 3d), or in the cells (data not shown), confirming the ability of the parasite to interfere with NO production.

These results confirm our hypothesis of explaining variations in electric-component values (R ads , R ct ), i.e. (i) a high production of diffusible NO radicals in the case of LPS-stimulated RAW, an NO production inhibited for Leishmania-infected cells and (ii) a high production of oxygen-reactive species within infected cells, whereas this production is not observed in LPS-stimulated ones.

Conclusions

These results are very promising since they demonstrate the ability of EIS to simply characterize in real time the oxidative stress generate by an infection. In particular it demonstrates its ability to discriminate between two diametrically opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. For further insight into the redox mechanisms discriminating cellular stress states, our aim now is to associate this device to complementary selective electrodes based on amperometric transduction for the determination of NO or O 2 •-, for example.

Fig. 1 .

 1 Fig. 1. Influence of cellular layer on impedance response. Nyquist impedance plots in the frequency range 50 kHz to 100 mHz obtained in the presence of 5 mM Fe(CN)6 3-/4- (1:1) in PBS with ( ) a bare gold electrode and (᭹) a gold electrode modified with macrophages, the symbols correspond to experimental data and the line represents the simulated spectra with the parameters calculated by FRA software from the equivalent circuit model I.

Fig. 2 .

 2 Fig. 2. Influence of the macrophage state, Leishmania-infected or LPS-stimulated, on the EIS measurements. Nyquist impedance plots in the frequency range 50 kHz to 100 mHz obtained in the presence of 5 mM Fe(CN)6 3-/4-(1:1) in PBS (a and b) or in the culture medium (c and d) with a gold electrode modified with ( ) healthy, ( ) Leishmania-infected (a and c) or LPS-stimulated macrophages (b and d). The symbols correspond to experimental data and the line represents simulated spectra with parameters calculated by FRA software from equivalent circuit model I (for a and b) or II (for c and d).

Fig. 3 .

 3 Fig. 3. Production of reactive species by Leishmania-infected or LPS-stimulated RAW cells. Image (63×) by confocal microscopy of RAW 264.7 cells after incubation with H2DCFD recorded at 530 nm for (a) RAW 264.7 cells activated with LPS (1 g/L) and (b) RAW 264.7 cells infected with Leishmania amazonensis. EPR spectra of the culture medium of RAW 264.7 cells recorded after 2 h of incubation with Fe(DETC)2 for (c) macrophages activated with LPS (1 g/L) and (d) macrophages infected with Leishmania amazonensis.

  depicts X-band EPR culture-medium spectra of LPS-activated or Leishmania-infected RAW cells. Typical triple-line EPR spectra of [Fe(II)(NO)(DETC) 2 ]

Acknowledgments

Séverine Maurel-Chevalley deserves our deep gratitude for her technical assistance and knowledge of parasitology. We also thank Université Paul Sabatier and the Institut de Recherche pour le Développement for financial support.


Lu Ouyang

David M Dotzauer

Seth R Hogg

Jorge Macanás

Jean-Francois Lahitte

Merlin L Bruening
email: bruening@chemistry.msu.edu

Catalytic hollow fiber membranes prepared using layer-by-layer adsorption of polyelectrolytes and metal nanoparticles

Immobilization of metal nanoparticles in hollow fiber membranes via alternating adsorption of polyelectrolytes and negatively charged Au nanoparticles yields catalytic reactors with high surface areas. SEM images show that this technique deposits a high density of unaggregated metal nanoparticles both on the surfaces and in the pores of the hollow fibers. Catalytic reduction of 4-nitrophenol with NaBH 4 , which can be easily monitored by UV-vis spectrophotometry, demonstrates that the nanoparticles in the hollow fiber membrane are highly catalytically active. In a single pass through the membrane, >99% of the 4-nitrophenol is reduced to 4-aminophenol, but this conversion decreases over time. The conversion decline may stem from catalyst fouling caused by by-products of 4-aminophenol oxidation.

Introduction

Hollow fiber membrane reactors, which initially appeared in the 1980s [1][2][3][4], are attractive because their geometry provides a high surface area to volume ratio [5][6][7] and should facilitate gas-liquid reactions [8][9][10]. Moreover, the hollow fiber module is the membrane configuration with the highest packing density. Remarkably, the ratio of membrane surface area to module volume can reach values as high as 30,000 m 2 /m 3 [START_REF] Mulder | Basic Principles of Membrane Technology[END_REF]. Thus, a number of recent studies examine hollow fibers as catalyst supports in membrane reactors [8,[START_REF] Israni | [END_REF][13][14][15].

Metal nanoparticles are especially effective catalysts because of their relatively large percentage of surface atoms [16,17]. Additionally, these materials often possess unusual electronic properties due to their unique size, which is between the bulk and molecular regimes [16][17][18]. For most practical catalytic applications, however, the nanoparticles must be immobilized on solid supports to prevent aggregation and facilitate catalyst recovery [16,[19][20][21][22][23][24][25]. Common methods for making supported noble metal catalysts include impregnation, coprecipitation (CP) and deposition-precipitation (DP) [26]. In impregnation, the support is typically immersed into a metal salt solution and heated in air, and the adsorbed metal species are reduced in hydrogen [27][28][29]. For the CP [30,31] or DP methods [30,32], the noble metal precursor is either coprecipitated with support precursor or directly deposited on the support sur-face prior to precipitation with another reagent. These methods usually yield metal particles of non-uniform size and shape, and the degree of catalyst dispersion depends on various factors such as the type of support, the deposition pH and the concentration of precursors in the solutions [26,27,33]. To make heterogeneous catalysts with more control over particle size and shape, metal nanoparticles can be synthesized in solution and then loaded on the support [20,34].

Layer-by-layer (LBL) deposition of polycations and polyanions provides a particularly simple way to deposit a high density of well-defined metal nanoparticles on a wide range of substrates [35][36][37][38][39][40]. Alternating adsorption of charged nanoparticles and an oppositely charged polyelectrolyte affords control over the amount of nanoparticles deposited through variation of the number of adsorbed layers, and a wide range of particle sizes and compositions are available through tuning of the nanoparticle synthesis [41][42][43][44][45]. The LBL technique is effective for membrane modification because it can occur on a wide variety of substrate geometries [46], including flat surfaces, cylindrical particles, and the pores of flat sheet membranes [35,41,42,45].

This paper describes modification of hollow fiber microfiltration (MF) membranes through LBL adsorption of polyelectrolytes and citrate-stabilized metal nanoparticles (NPs) to create reactors that catalyze the reduction of 4-nitrophenol with NaBH 4 (Fig. 1). An article by Macanás et al. (in this issue) focuses mostly on coating the surfaces of fibers with nanoparticles [47], whereas this study aims to modify the interior and exterior of the membrane to increase nanoparticle loading.

Experimental

Materials

Poly(sodium 4-styrenesulfonate) (PSS, M w = 70,000 Da), poly (allylamine hydrochloride) (PAH, M w = 56,000 Da), HAuCl 4 •3H 2 O, sodium citrate, NaBH 4 and 4-nitrophenol were used as received from Sigma-Aldrich. Deionized water (Milli-Q purification system, 18.2 M cm) was employed for membrane rinsing and preparation of polyelectrolyte or reaction solutions, and the pH of the PAH solutions was adjusted with dilute HCl or NaOH.

Polyethersulfone (PES) MF hollow fiber membranes (MicroPES Capillary membrane Type TF10) were manufactured by MEM-BRANA (Wuppertal, Germany). These membranes have a specified maximum pore size in the membrane skin of 0.5 ± 0.1 m, an inner diameter of 300 ± 40 m and a wall thickness of 100 ± 25 m.

A module containing MF polysulfone (PS) hollow fiber membranes was purchased from GE Healthcare (Model #: CFP-6-D-6A), and individual fibers were removed from the module and repotted. The fibers have an average skin pore size of 0.65 m, an inner diameter of 750 m and a wall thickness of 225 m. The supplementary material contains SEM images of these fibers (Supplementary data, Fig. S1).

Preparation of citrate-stabilized Au nanoparticles

Au nanoparticles were prepared by the citrate reduction method [18,48,49], where citrate serves as both the reducing agent and the nanoparticle stabilizer. In a 250 mL round-bottom flask equipped with a condenser, 100 mL of 1 mM HAuCl 4 solution was heated to a rolling boil while stirring. Ten mL of 38.8 mM sodium citrate solution was also heated to a rolling boil and then rapidly added to the Au solution. After about 15 s, the solution changed from colorless to blue and then burgundy. The mixture was subsequently stirred with heating for another 10 min, and then without heating for 15 min. The nanoparticle solution was kept in an amber glass bottle and stored in a refrigerator until use. Prior to particle deposition, the nanoparticle solution was diluted 9:1 with deionized water. TEM images show that the particle diameter is 12 ± 1 nm [42].

Modification of hollow fiber membranes

Following a literature procedure, the hollow fibers were potted in a PVC tube with epoxy glue purchased from Axson Technologies (Cat. Number: Adekit A 130) [50]. All the modules in this study contained one fiber, with an effective fiber length (distance between the potting glue on the two ends) of about 22 cm for PES fibers and 15 cm for PS fibers. Prior to modification, the potted PES fiber was rinsed with water at 0.7 bar for 30 min. In the modification steps, all the solutions were passed through the membrane in the lumen to shell flow configuration with cross-flow (see Fig. 2) using a pressure of 0.7 bar. Cross-flow was used during modification in an effort to minimize any pore blockage caused by the polyelectrolytes. A prefilter (2.5 m Ahlstrom qualitative filter paper) was used to remove small particles that can potentially block the fiber, and this filter paper was replaced after deposition of each layer. Polyelectrolyte adsorption solutions contained 2 mM PSS or PAH and 50 mM NaCl (polyelectrolyte concentrations are always given with respect to the repeating unit). The pH of the PAH solution was adjusted to 4.5. The modification started with passage of 100 mL of PSS solution through the fiber (volume includes both transmembrane and cross-flow). This initial adsorption of PSS is likely driven by hydrophobic interactions between PSS and the PES fiber [51,52]. The fiber was then rinsed with 300 mL of water to remove any extra polyelectrolytes. Subsequently, 100 mL of PAH solution was passed through the hollow fiber membrane followed by another 300 mL water rinse. Nanoparticles were loaded by passing 100 mL of citrate-stabilized Au colloid solution (∼0.1 mM in Au atoms) through the membrane prior to rinsing with 300 mL of water. The permeate of the nanoparticle solution was initially colorless and then became light pink when the fiber was nearly saturated with Au nanoparticles. Modification of the PS fiber occurred in a similar fashion with slight modifications, and the supplementary material contains the detailed procedure.

Membrane characterization

SEM images of bare and coated hollow fiber membranes were taken using a Hitachi S-4700 II field-emission scanning electron microscope (FESEM). For shell and lumen surface characterization, 5 nm of Au was sputter-coated (Pelco SC-7 auto sputter coater) on the surface before imaging. For cross-sectional images, the fiber was dipped in ethanol for 2 min, fractured in liquid nitrogen, and the cross-section was sputter-coated with 5 nm of Au.

To determine the amount of Au loaded in the fiber, the membrane was immersed in aqua regia (3 parts HCl and 1 part HNO 3 ) for 5 min, and this solution was then diluted and analyzed for Au by atomic absorption spectroscopy (AAS) (Varian Spectra Atomic Absorption-200 Spectrometer).

The water permeability of the PES fiber was tested in a shell to lumen flow configuration (no cross-flow in the shell) at room temperature with pressures ranging from 0.7 to 2.1 bars. All fluxes reported in this work are calculated with respect to the surface area of the outside of the fiber. Hydraulic permeability values, which were obtained from the slopes of plots of flux versus pressure, were determined before and after each modification step. For the PS fiber, water permeability was determined only at 0.7 bar because the membrane manufacturer does not recommend using the fibers under pressures higher than 1 bar.

Catalytic reactions and leaching studies

Reduction of aqueous 4-nitrophenol with NaBH 4 served as a model reaction for examining the catalytic activity of the modified membranes. A feed tank was filled with 0.5-1 L of a freshly prepared solution containing 0.5 mM 4-nitrophenol and 25 mM NaBH 4 , and this solution was forced through the hollow fiber membrane under pressure. Permeate samples were collected every 5 or 10 min, and the concentration of 4-nitrophenol in the feed and permeate was determined from the solution absorbance at 400 nm [42,53] (UV-vis spectra were acquired with a Perkin-Elmer Lambda 40 spectrophotomer.) To examine the extent of catalyst leaching during the reaction, the amount of Au in the feed and permeate samples was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) (Varian 710-ES ICP Optical Emission Spectrometer). Residence time is calculated with an assumption of 30% porosity.

Results and discussion

Membrane characterization

The first sign of successful modification of the hollow fiber membranes with Au nanoparticles is a change in the fiber color from white to red. SEM images, elemental analysis by AAS, and water permeability measurements afford a more quantitative picture of the fiber modification.

SEM characterization of immobilized nanoparticles

Fig. 3 shows SEM images of bare PES hollow fiber membranes. These fibers have a layered, porous structure on both lumen and shell sides, but pore sizes are smallest at the shell surface and largest in the interior of the fiber. The extensive internal surface area of the membrane pores should allow a high loading of Au nanoparticles, and the relatively large pores (maximum pore size around 2 m) facilitates film adsorption without pore blockage. However, pore blockage sometimes occurs, especially in fiber sections that contain somewhat smaller pores (see below). Fig. 4 shows SEM images of the shell, lumen and wall surfaces of PES hollow fiber membranes modified with PSS/PAH/AuNP films. Adsorption of the films occurs by flowing solution along the lumen side of the fiber and also through the fiber wall as described in Section 2.3. Consequently, nanoparticles appear on all surfaces of the membranes. The nanoparticles are well separated with no apparent aggregation, and the particle size is about 12 nm, which is consistent with TEM images of the as-prepared particles [41,42]. Fig. 5 and Supplementary data, Fig. S2 show that modification of PS fibers with PSS/PAH/AuNP films results in a similar, uniform Au nanoparticle distribution.

Catalyst loading

SEM provides information on nanoparticle deposition in small spots with image areas of about 10 m 2 , but such images may not provide a good indication of Au loading on a macroscopic scale. To determine the overall Au content and whether the nanoparticles are evenly distributed along the length of the fiber, coated PES fibers were cut into several pieces, and the nanoparticles in each piece were dissolved in aqua regia for subsequent Au analysis by AAS. Table 1 summarizes these analyses and shows that the Au deposition in the PES fiber is very uniform. Remarkably, the amount of Au in each piece of the fiber differs by less than 8% from the average loading. The average amount of Au in a 5 cm-long PES fiber is 350 nmol, which corresponds to 0.3 mg of Au in a fiber with a length of 22 cm. Gold loading in a PS fiber is 1.2 mol in a 5 cm segment or about 1 mg in a fiber with a length of 22 cm. The higher loading in the PS membranes likely occurs because the cross-sectional area of the walls of these fibers is 5.5 times that in PES fibers. The loading in both types of membranes is comparable to the loading in a chitosan catalytic fiber that was modified by the in situ reduction method. In that case, palladium chloride was absorbed on the chitosan and subsequently reduced by hydrogen gas to give a Pd loading of 0.25-2.5 mg in a 50 cm fiber [13].

Adsorption of additional PAH/Au nanoparticle bilayers should further increase the Au loading in the film [42]. However, due to challenges with blocking of some membranes by the polyelectrolyte films (see below), the catalytic studies described here focus on modification with only one PAH/AuNP layer.

Water permeability

Although a previous study showed no detectable plugging of membrane pores during deposition of poly(acrylic acid)/PAH/AuNP films in porous alumina (pore size of 0.2 m) [42], the hollow fiber flow configuration and the spongy membrane structure in the present case may lead to pore blockage. Fig. 6 shows the water permeabilities of PES fibers after each step in the deposition of PSS/PAH/AuNP films. (Permeabilities are determined by measuring the pressure dependence of pure water flux in the shell to lumen flow mode). The bare PES fiber exhibits a water permeability of 41 m 3 /(m 2 day bar), which is in the high range of fluxes through MF membranes [START_REF] Mulder | Basic Principles of Membrane Technology[END_REF]. After PSS deposition, water permeability decreases by less than 10%, but adsorption of one bilayer of PSS/PAH reduces permeability by 30% compared to a bare fiber. Moreover, adsorption of Au nanoparticles on the PSS/PAH film results in a water permeability of only 22 m 3 /(m 2 day bar), about We should note that in some cases adsorption of PSS/PAH/AuNP films in PES fibers decreases flow to around 7% of that before modification. SEM images of different bare fibers indicate that the pore size varies greatly from roll to roll or perhaps with time (see Supplementary data, Fig. S3). As expected, smaller pores are more prone to plugging during film adsorption. To overcome this problem, we employ PS fibers, which have a permeability of 360 m 3 /(m 2 day bar). Adsorption of PSS/PAH/AuNP films in these fibers decreases permeability by less than 10%.

Examination of the catalytic activity of nanoparticle-coated fibers

The reduction of 4-nitrophenol with NaBH 4 (Fig. 1) serves as a test reaction for examining the catalytic activity of hollow fibers loaded with Au nanoparticles. This reaction is attractive because it does not proceed in the absence of catalyst, and yet it rapidly occurs in the presence of Au nanoparticles and can be easily monitored by UV-vis spectrophotometry [53,56,57]. Some previous studies examined nitrophenol degradation with chitosan hollow fibers [13,15,58]. In a typical reaction, the feed solution contains 0.5 mM 4-nitrophenol and 25 mM NaBH 4 so the amount of the reducing agent is in large excess throughout the reaction.

Catalytic reduction using PES fibers modified with PSS/PAH/AuNP films

We first reduced 4-nitrophenol in the lumen-shell flow configuration with the lumen outlet plugged. These reactions initially result in more than 99% reduction of 4-nitrophenol during flow Fig. 6. Water permeability of PES hollow fiber membranes before (bare) and after adsorption of different polyelectrolyte films.

through PES membranes modified with PSS/PAH/AuNP films [47]. Control experiments with fibers coated with PSS/PAH show less than 1% reduction of 4-nitrophenol under similar conditions. However, in this flow mode the accumulation of H 2 in the fiber can potentially cause variations in flux and effective membrane area. (Periodic opening of the fiber outlet causes increases in the transmembrane flux). Thus, all further reactions employ the shell to lumen configuration without cross-flow (Fig. 7). The fiber is perpendicular to the ground, and any trapped hydrogen should pass through the lumen outlet along with the permeate. The red plug on the shell (Fig. 7) can be removed periodically to release H 2 that accumulates in the shell. With this flow mode, the permeate flux is 0.7 ± 0.1 mL/(cm 2 min) at an applied pressure of 1.4 bar, and the residence time in the membrane is 210 ± 30 ms.

Fig. 8 shows the extent of 4-nitrophenol reduction during flow (shell to lumen) through a hollow fiber membrane modified with a PSS/PAH/AuNP film. The 4-nitrophenol reduction is more than 99% for 30 min and then decreases to 83% after 1 h. One possible reason for the decline in conversion is that the amount of reducing agent in the feed tank decreases with time due to continuous NaBH 4 degradation. A previous study reports that NaBH 4 decomposes completely to NaBO 2 and hydrogen gas in 20 min at 75 • C [59]. Although the membrane reactions occur at room temperature, an equation developed by Kreevoy suggests that 50% of the NaBH 4 should decompose at this temperature in approximately 1 h [60]. The effective fiber length is 22 cm, the fiber shell surface area is 3.45 cm 2 , the Au loading is 0.3 mg, the volume passed through the module is about 500 mL, the residence time in the membrane is 210 ± 30 ms and the permeate flux is 0.7 ± 0.1 mL/(cm 2 min). The dashed, vertical lines represent the addition of a fresh 4-nitrophenol/NaBH4 solution.

To minimize the effect of NaBH 4 degradation on conversion, the feed solution is replaced every 60 min. Upon replacing the feed solution after 1 h of reaction, 4-nitrophenol conversion recovers from 83% to values as high as 96%, but then begins decreasing again, so conversion is only 70% after 2 h (Fig. 8). When the feed is replaced a second time (after 2 h), the conversion only recovers to 77% and finally drops to 60% after 3 h. The incomplete recovery of conversion after replacing the feed shows that degradation of NaBH 4 is not the sole cause for the conversion decline.

A second possible reason for the conversion decline is that reaction by-products [58] block the catalytic sites of the nanoparticles. Comparison of SEM images of the lumen surfaces of modified PES fibers before (Fig. 4(b)) and after the NaBH 4 /4-nitrophenol reaction (Fig. 9) indicates that deposits form during the reaction. Interestingly, no deposits appear in the fiber wall where most of the catalyst is located. Another possible cause of the conversion decline, catalyst leaching, is discussed in more detail below.

Catalytic reduction using PS fibers modified with PSS/PAH/AuNP films

Reduction of 4-nitrophenol also occurs rapidly when using PSS/PAH/AuNP-modified PS hollow fiber membranes. Fig. 10 shows 4-nitrophenol conversion over a 4 h period in which the feed is replaced after 80 and 180 min. Because of the high permeability of these membranes, the permeate flux is 1.4 ± 0.2 mL/(cm 2 min) under a pressure <0.2 bar. (This flux corresponds to a membrane Fig. 9. SEM image of the lumen surface of a catalytic hollow fiber that was employed for reduction of 4-nitrophenol for 3 h. This is the same membrane that was used to obtain the data in Fig. 8. residence time of 240 ± 30 ms.) Conversion is more than 99.9% for the first 30 min and decreases to 92% after 80 min. Once the feed solution is replaced, however, conversion recovers to around 99.9% and remains more than 99% for another 40 min. Conversion drops to 90% after 180 min, and replacing the feed solution a second time does not result in recovery of the catalytic activity.

To determine whether the conversion decline stems from catalyst leaching, the Au concentration in the permeate samples was analyzed by ICP-OES. The permeate contains less than 5 ppb Au throughout the reaction period, which corresponds to <3% cumulative leaching of the Au in the membrane. Hence, Au leaching is not a likely cause for conversion decline in this experiment.

To minimize the effect of NaBH 4 degradation on conversion decline, another 4 h 4-nitrophenol reduction was performed where the feed solution (500 mL of 0.5 mM 4-nitrophenol and 25 mM NaBH 4 ) was replaced every 30 min. Fig. 11 shows the plot of conversion vs. reaction time at a flux of 1.4 mL/(cm 2 min). 4-Nitrophenol conversion is at least 99.9% for 90 min and >99.7% for another 65 min. Even after 3 h, more than 99% of the 4-nitrophenol is reduced. However, during the 4 h of reaction, conversion drops to between 95 and 98%. The Au concentration in the permeate samples is again <5 ppb, showing that conversion declines are not due to leaching of Au. The maximum 5% conversion decline is significantly less than the conversion drop of 12% in the experiment where the feed solution is only replaced every 80-100 min (Fig. 10). Conversion declines are less severe when the feed is replaced more often, and when the feed is not replaced, the NaBH 4 solution becomes less reducing. Thus, the fouling mechanism may include oxidation of aminophenol [58] in the less reducing solution and formation of polymer products that foul the catalyst.

Conclusions

Layer-by-layer adsorption of polyelectrolytes and Au nanoparticles provides a simple and effective way to modify the pores of hollow fibers and prepare catalytic membrane reactors. This procedure results in a dense layer of unaggregated nanoparticles in the membrane pores. Catalytic reduction of 4-nitrophenol with NaBH 4 shows that the immobilized nanoparticles are highly active, but their activity declines over time, presumably because of catalyst fouling. The shell-lumen flow configuration is probably better suited for this type of reaction than lumen-shell flow because in the former case gas is less likely to build up in the fiber. Current work focuses on improving catalyst stability, understanding the reaction kinetics, and exploring other catalytic reactions.

Fig. 1 .

 1 Fig. 1. Schematic diagram of a porous hollow fiber membrane modified with a polyelectrolyte/AuNP film. The fibers serve as catalytic reactors for the reduction of 4nitrophenol by NaBH4.

Fig. 2 .

 2 Fig. 2. Schematic diagram of the flow configuration for modifying a hollow fiber membrane with a PSS/PAH/AuNP film. Flow occurs both axially along the membrane and from the inside to the outside of the fiber.

Fig. 3 .

 3 Fig. 3. SEM images of a bare PES hollow fiber membrane. (a) Cross-section of the entire hollow fiber, (b) shell surface, (c) lumen surface and (d) the interior of the membrane.

Fig. 4 .

 4 Fig. 4. SEM images of PES hollow fiber membranes coated with PSS/PAH/AuNP films. (a) Shell surface, (b) lumen surface and (c) the interior of the membrane.

Fig. 5 .

 5 Fig. 5. SEM images of the interior surfaces of (a) a bare PS hollow fiber membrane and (b) a similar membrane coated with a PSS/PAH/AuNP film.

  half of the value for the bare fiber. This decrease in water permeability is somewhat surprising because typical PSS/PAH films are only 1 nm thick[54,55], and the pores sizes in these membranes are in the range of several hundred nanometers. Comparison of Figs. 4(b) and 3(c) suggests that film deposition partially occludes the lumen surface, which may account for the 50% drop in permeability after deposition of the PSS/PAH/AuNP film.

Fig. 7 .

 7 Fig. 7. Schematic diagram of the shell to lumen solution flow configuration used in most catalytic reactions in this study. (The two red cylinders represent plugged outlets.)

Fig. 8 .

 8 Fig.8. Percent reduction of 4-nitrophenol during passage of a 0.5 mM 4nitrophenol, 25 mM NaBH4 aqueous solution through a PES hollow fiber membrane coated with a PSS/PAH/AuNP film. Flow through the membrane occurs in the shell-lumen configuration without cross-flow. The effective fiber length is 22 cm, the fiber shell surface area is 3.45 cm 2 , the Au loading is 0.3 mg, the volume passed through the module is about 500 mL, the residence time in the membrane is 210 ± 30 ms and the permeate flux is 0.7 ± 0.1 mL/(cm 2 min). The dashed, vertical lines represent the addition of a fresh 4-nitrophenol/NaBH4 solution.

Fig. 10 .

 10 Fig. 10. Percent reduction of 4-nitrophenol during passage of a 0.5 mM 4nitrophenol, 25 mM NaBH4 aqueous solution through a PS hollow fiber membrane coated with a PSS/PAH/AuNP film. The reaction takes place in the shell-lumen flow configuration without cross-flow using an effective fiber length of 15.5 cm.The fiber shell surface area is 5.84 cm 2 , the Au loading is 0.7 mg, the volume passed through the module is about 2000 mL, and the flux through the membrane is 1.4 ± 0.2 mL/(cm 2 min), which corresponds to a membrane residence time of 240 ± 30 ms. The dashed, vertical lines represent the addition of a fresh 4nitrophenol/NaBH4 solution.

Fig. 11 .

 11 Fig. 11. Percent reduction of 4-nitrophenol during passage of a 0.5 mM 4nitrophenol, 25 mM NaBH4 aqueous solution through a PS hollow fiber membrane coated with a PSS/PAH/AuNP film. The reaction takes place in the shell-lumen flow configuration without cross-flow using an effective fiber length of 15.5 cm.The fiber shell surface area is 5.84 cm 2 , the Au loading is 0.7 mg, the volume passed through the module is about 2000 mL, and the flux through the membrane is 1.4 ± 0.2 mL/(cm 2 min), which corresponds to a membrane residence time of 240 ± 30 ms. The dashed, vertical lines represent the addition of a fresh 4nitrophenol/NaBH4 solution.

Table 1

 1 Au loading along PES fibers coated with PSS/PAH/AuNP. The length of each segment is approximately 5 cm.

		Segment A (nmol)	Segment B (nmol)	Segment C (nmol)	Segment D (nmol)	Average (nmol)
	Fiber 1 a	357	371	347	375	363 ± 13
	Fiber 2 a	345	339	326	370	345 ± 18

a The reported values are normalized to a fiber length of 5 cm.

Acknowledgement

We thank the U.S. National Science Foundation (OIS-0530174) for supporting this work.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cattod.2010.02.040.


M Asif Inam
email: asifinam1976@yahoo.com

Christine Frances

Effects of impurities on particle sizing by acoustic attenuation spectroscopy

It is important to have correct information regarding particle size in order to interpret, control, and optimize many industrial processes. Prior to the recent advent of acoustic attenuation spectroscopy, it was difficult to study particle size distribution online and under real process conditions in processes involving concentrated dispersions (suspensions or emulsions). The technique still needs improvement because it is less known how and under which conditions to employ the technique when dispersions involve impurities that could be soluble, insoluble, in the form of additives, and so on. This lack of understanding has almost halted the advancement in applications of the technique to various processes that essentially involve dispersions with impurities. This study investigates aqueous suspensions of CaCO 3 at different concentrations (i.e., 5%, 10% and 20% mass/mass) with added impurities of MgCO 3 (insoluble impurity), NaNO 3 (soluble impurity) and sodium polyacrylate (soluble additive) at varying proportions (5%, 10%, 20% and 30% of the weight of CaCO 3 ). The study characterizes and compares dispersion with and without impurity in order to demonstrate the possible ways in which addition of an impurity change the original acoustic attenuation spectrum of a dispersion. The study brings the conditions in which acoustic attenuation spectroscopy is capable of explaining that addition of an impurity will not change original particle size of the disperse medium.

Introduction

It is important to have correct information regarding particle size in many industries such as chemicals, paints, dyes, pharmaceuticals and cosmetics in order to interpret, control, and optimize many processes. In last few decades, many techniques of particle sizing have been developed based on different principles such as laser diffraction, dynamic light scattering, image analysis and acoustic attenuation spectroscopy. Each technique has its advantages and limitations depending upon the application. In above techniques, acoustic attenuation spectroscopy is quite prominent because of many advantages over other techniques of particle sizing. For example, it is non-destructive and non-invasive; it can study dispersions (suspensions and emulsions) without the need for diluting them; it can be employed online, under real process conditions, over a wide range of particle size (10 nm to 1000 μm) and over a wide concentration range (0.5 vol% to 50 vol%), depending upon application [START_REF] Dukhin | Acoustic and electroacoustic spectroscopy for characterizing concentrated dispersions and emulsions[END_REF][START_REF] Dukhin | New developments in acoustic and electro acoustic spectroscopy for characterizing concentrated dispersions[END_REF][START_REF] Alba | Acoustic spectroscopy as a technique for particle sizing of high concentration colloids, emulsions and suspensions[END_REF][START_REF] Challis | Ultrasound techniques for characterizing colloidal dispersions[END_REF][START_REF] Mcclements | Ultrasonic Measurements in Particle Size Analysis[END_REF]. Many studies have verified suitability of acoustic attenuation spectroscopy for simple dispersions (i.e. dispersion involving one disperse phase and one continuous phase). However, there is still lack of understanding how and under which conditions to employ the technique when dispersions are not simple. For example, in many industrial applications, dispersion of CaCO 3 are prepared from raw CaCO 3 that may contain impurities such as silica or MgCO 3 making the dispersion of CaCO 3 complex for particle sizing by means of acoustic attenuation spectroscopy. Similarly, during wet grinding processes, the addition of additives in dispersions makes them complex. The lack of understanding has almost halted the advancement in applications of the technique to various processes that essentially involve dispersions with impurities etc. Pointing out this lack, Franck Babick et al. write that only little experience has been gained for the performance under more real conditions, e.g. for the presence of contamination, for multi-component-systems or for the case that the knowledge of the involved phases is incomplete [START_REF] Babick | Dependence of ultrasonic attenuation on the material properties[END_REF]. Similarly, Povey has emphasized the importance of characterizing the surfactants in the solvent for successful particle sizing by means of acoustic attenuation spectroscopy [START_REF] Povey | Acoustic methods for particle characterization[END_REF]. This study is an attempt to bridge the gap between particle sizing of the dispersion with and without impurities by means of acoustic attenuation spectroscopy. In particular, the study aims to characterize and compare dispersions with and without impurities at different conditions in order to find possible conditions in which dispersions with impurities may be studied by means of acoustic attenuation spectroscopy.

Theory

Particle sizing by means of acoustic attenuation spectroscopy is based mainly upon ECAH theory, which is presented in terms of differential waves equations derived from fundamental laws of conservation of mass, energy, momentum, thermodynamic equations of state and stress-strain relations for isotropic elastic solids (or viscous fluids). It considers case of simple dispersions (one disperse medium in one continuous phase). The details of ECAH theory may be studied in the papers of Epstein and Carhart [START_REF] Epstein | The absorption of sound in suspensions and emulsions[END_REF] and Allegra and Hawley [START_REF] Allegra | Attenuation in sound in suspensions and emulsions: theory and experiments[END_REF] who contributed to development of this theory. With the help of ECAH theory, acoustic attenuation spectrum of a dispersion can be determined if particle size distribution and certain physical properties of the dispersion are known. The Fig. 1(a) illustrates a simple schematic of ECAH in terms of input and output. A commercial acoustic attenuation spectrometer for particle sizing (such as Malvern Ultrasizer) makes use of ECAH theory in a manner as illustrated in Fig. 1(b). It measures acoustic attenuation spectrum with the help of transducers; use information of physical properties (mentioned in Table 1) and finally determines particle size distribution by inverse calculations. The process of inverse calculations is carried out with suitable algorithms employed generally with the help of computer program.

When a sound wave of certain frequency is passed through a particulate system, it interacts with the particles (transmission, reflection, absorption and scattering etc.) and as a result it undergoes attenuation (decrease in amplitude) that can be measured in terms of attenuation coefficient [START_REF] Dukhin | Acoustic and electroacoustic spectroscopy for characterizing concentrated dispersions and emulsions[END_REF][START_REF] Dukhin | New developments in acoustic and electro acoustic spectroscopy for characterizing concentrated dispersions[END_REF][START_REF] Alba | Acoustic spectroscopy as a technique for particle sizing of high concentration colloids, emulsions and suspensions[END_REF][START_REF] Challis | Ultrasound techniques for characterizing colloidal dispersions[END_REF][START_REF] Mcclements | Ultrasonic Measurements in Particle Size Analysis[END_REF]. It is defined as the decrease in the amplitude of the sound wave per unit distance travelled and may be expressed as [START_REF] Mcclements | Ultrasonic Measurements in Particle Size Analysis[END_REF]:

α = - 1 x ln A x A 0 ð1Þ
where A 0 is the initial amplitude of the sound wave (x = 0) and A x is the amplitude after it has travelled a distance x as illustrated in Fig. 2. If a band of sound waves of different frequencies is passed through a dispersion from a certain distance (x), the attenuations in each sound wave of certain frequency may be calculated using Eq. ( 1). An acoustic attenuation spectrum describes acoustic attenuation constants expressed as a function of respective frequencies of sound waves. In a commercial acoustic attenuation spectrometer such as Malvern Ultrasizer, different values of x and range of frequencies may be selected for determining an acoustic attenuation spectrum. Alba discussed the relationship between frequency, attenuation constant and particle size distribution by means of acoustic attenuation spectroscopy [START_REF] Alba | Acoustic spectroscopy as a technique for particle sizing of high concentration colloids, emulsions and suspensions[END_REF]. If the attenuation is measured at a single frequency, there would be four potential monosize distributions, which could have produced that measured attenuation: two in the region of viscous attenuation, one in the Rayleigh scattering region, and a fourth in the diffraction zone. In the absence of either measurement or modeling errors, two frequencies would suffice to identify the correct size from the four possible sizes for each frequency.

Materials

CaCO 3

CaCO 3 , procured through Merck KGaA Germany, served as the disperse medium in the study. It was prepared by precipitation and had the following properties: density at 20 °C = 2.93 g/cm 3 , bulk density ∼400-700 kg/m 3 , solubility in water at 20 °C = 0.014 g/l, and thermal decomposition N825 °C. A Mastersizer2000 of Malvern Instruments Limited, U.K., which is based on laser diffraction technique, was used to find the mean particle size of CaCO 3 (in dry form). It was found to be 1.6 µm.

Magnesium carbonate

Magnesium Carbonate (anhydrous) was purchased from Fisher Scientific, U.K. It was insoluble in water and its density was 2.958 g/cm 3 at 25 °C. Its mean particle size (in dry form) was determined by using the laser diffraction technique with a Mastersizer2000 of Malvern Instruments Limited, U.K. It was found to be 7.3 µm.

Sodium nitrate

Sodium nitrate was purchased from Fisher Scientific, U.K. Its density was 2.26 g/cm 3 . It was highly soluble in water with a solubility 921 g/l at 25 °C.

Sodium polyacrylate

Sodium polyacrylate procured through Sigma-Aldrich Co. was used in the study. It had a density of 0.55 g/cm 3 at 25 °C and average M w ∼ 5100 (determined by gel permeation chromatography (GPC)).

H 2 O

Ultrapure water produced from "Purelab Ultra" of VWS Ltd. (U.K.) was used in the study. The density of water was found to be 0.9981 g/ cm 3 at 25 °C.

Equipment and software

An acoustic attenuation spectrometer (Ultrasizer) of Malvern Instruments Limited U.K. was employed in the study. The system has four main units: (i) instrument, (ii) electronics enclosure, (iii) heater/ chiller, and (iv) computer system. The instrument uses transducers to

Table 1

Physical properties required for particle sizing by acoustic attenuation spectroscopy.

Disperse medium (particles)

Dispersing or continuous medium 1 -Density (g/cm 3 ) 1 -Density (g/cm 3 ) 2 -Sound velocity (cm/s) 2 -Sound velocity (cm/s) 3 -Thermal dilation (°C -1 ) 3 -Thermal dilation (°C -1 ) 4 -Thermal conductivity (erg cm -1 s -1 °C-1 )

4 -Thermal conductivity (erg cm -1 s -1 °C-1 ) 5 -Heat capacity (erg g -1 °C-1 ) 5 -Heat Capacity (erg g -1 °C-1 ) 6 -Shear rigidity (dynes cm -2 ) 6 -Viscosity (Poise) Fig. 2. Passage of a sound wave through a dispersion.

pass ultrasonic waves through a sample of dispersion. The sample may be measured in batch or continuous mode. The electronic enclosure contains the microwave generator and various electronic systems for amplification, for signal processing, for control of movement of transducers and for control of temperature. The heater/chiller was used to control the temperature. The computer system is a standalone computer to run the Malvern software (Ultrasizer MSV) to control the equipment. In Malvern Ultrasizer, selection of an appropriate measurement strategy (selected value of x and frequency range) is an important step. In our study, we wanted to calculate the acoustic contribution of the impurities for which it was important to measure all the combinations of dispersions and impurities with a single measurement strategy. Repeated efforts, finally, led us to a desirable measurement strategy with a frequency range of 1.5-10.0 MHz, transducer position 1 = 0.10 and position 2 = 0.40. In each measurement, a band of 50 different sound waves, as mentioned in Table 2, was passed through the dispersions.

Fitness of the measurements

Fig. 1(b) explains that process of determination of particle size depends upon: (i) physical properties of dispersion and (ii) acoustic attenuation spectrum. For right particle sizing, the acoustic attenuation spectrum must be measured with reasonable accuracy. The fitness of an acoustic attenuation spectrum (i.e. attenuation constants vs. frequencies) depends upon accuracy of measurement of attenuation constants. In Malvern Ultrasizer, the fitness of an acoustic attenuation spectrum is measured by a parameter "spectral quality" which depends upon measurement strategy (i.e. selected value of x and frequency range). A spectral quality less than 5 indicates that the attenuation constants have been measured with reasonable accuracy. All measurements reported in this study had a spectral quality less than 5.

Experiments

The experiments were designed to sequentially study the effects of three different impurities (magnesium carbonate (insoluble), sodium nitrate (soluble) and sodium polyacrylate (additive)) added to different aqueous dispersions of CaCO 3 (5% m/m, 10% m/m and 20% m/m) in the proportions 5%, 10%, 20% and 30% of the weight (mass) of the disperse medium (CaCO 3 ). The notation m/m represents mass of CaCO 3 divided by sum of masses of CaCO 3 and water.

In all experiments, dispersions of CaCO 3 in water were prepared directly in the batch measurement cell of the acoustic equipment (Malvern Ultrasizer) at 25 °C and at an agitator speed of 1800 rpm. Mixtures of impurities in dispersions were evaluated at the same conditions of temperature and agitation speed.

The following is a detailed description of one case for elaboration of the procedure. Consider the case of 5% m/m dispersion of CaCO 3 in water that was contaminated with different concentrations of magnesium carbonate. First, 500.0 ± 0.1 g water was added to the batch measurement cell of the acoustic instrument (Malvern Ultrasizer), followed by 26.32 ± 0.1 g of CaCO 3 . Both were mixed for 20 min in the measurement cell at 25 °C and at 1800 rpm, followed by the first acoustic measurement. After 10 min, another acoustic measurement was taken under the same conditions. The second acoustic measurement was compared with the previous one. The similarity in the results of the two acoustic measurements convinced us of the homogeneity of the dispersion. In the cases where two measurements were not found to be similar, we considered the system to be inhomogeneous. It was further mixed until achievement of homogeneity. After confirmation of homogeneity of the dispersion of CaCO 3 , 1.32 ± 0.1 g of magnesium carbonate (i.e., 5% of the weight of the disperse medium) was added to the dispersion. The system was further mixed until it again became stable and homogeneous. The stability and homogeneity were confirmed by the procedure described above. The concentration of magnesium carbonate was increased to 10% of the weight of the disperse medium by a further addition of 1.32 ± 0.1 g of magnesium carbonate into the system. Acoustic measurement was repeated upon achievement of stable and homogenized state. Similarly, the concentration of magnesium carbonate was increased to 20% of the weight of the disperse medium by adding 2.63 ± 0.1 g magnesium carbonate into the system. Acoustic measurement was repeated during the stable and homogenized phase as mentioned above. The process was repeated by increasing the concentration of magnesium carbonate to 30% of the weight of the disperse medium. The same procedure was adopted in all other experiments.

Results and discussions

Effects of concentration of dispersions on acoustic attenuation spectra

A comparative approach was used to study the effects of concentration of the dispersion of CaCO 3 on acoustic spectra in the absence and in the presence of certain proportions of impurities.

Dispersions without impurities, contaminations or additives

Our experiments revealed that in this case, acoustic attenuation spectra rise with increase in concentration of dispersion, as shown in an example in Fig. 3(a). In order to confirm whether the two factors (i.e., attenuation and concentration of dispersion) are directly proportional, the attenuation values were divided by the respective values of concentrations. All three attenuation spectra were almost superimposed, indicating a proportional relationship between attenuation and concentration of dispersion, as shown in Fig. 3(b).

A slight irregularity in the first portion of the graphs in Fig. 3(a) and (b) may be due to operational incapability (higher signal to noise ratio) of the equipment that usually occurs at the beginning of an acoustic measurement. A possible explanation of the phenomenon of superimposition, as observed in Fig. 3(b), could be that at a given concentration of the dispersion, each particle (or aggregate) had a certain average size and a certain acoustic attenuation corresponding to that size. Total acoustic attenuation of the dispersion was the sum of acoustic attenuation of all particles (or aggregates). Every increase in the concentration of the dispersion added more particles (or aggregates) of same average size. These particles (or aggregates) retained their size; in other words, they did not interact to form bigger particles (flocs or aggregates). Therefore, the total acoustic attenuation increased proportionally to the number of particles (or aggregates), or in other words, proportionally to the concentration of the dispersion. The situation leads to the following conclusions:

• the particles of the same average size are added to the dispersion,

• the addition of particles (i.e. increase in concentration) does not lead to change in average particle size of the dispersion (absence of beginning of aggregation or de-aggregation etc.) and • the addition of particles does not cause to start multiple scattering.

Dispersion with impurities, contaminations or additives

As in Section 7.1.1, the effects of concentration of dispersions of CaCO 3 (5%, 10% and 20% m/m) on acoustic spectra were studied by adding the impurity MgCO 3 in the amounts of 5%, 10%, 20% and 30% of the CaCO 3 used. Similar experiments were performed using the impurity sodium polyacrylate, and later on impurity NaNO 3 . Overall, three trends are found: The conditions leading to the three trends are summarized as follows.

Trend 1 The dispersions of CaCO 3 follow trend 1 in the presence of the impurity magnesium carbonate (in all proportions: 5%, 10% 20% and 30% by weight of the disperse medium). Similarly, the dispersions of CaCO 3 follow the trend 1 in the presence of impurity NaNO 3 (only in proportions of 5% and 10%). Trend 1 is similar to the trend observed in Section 7.1.1; hence, the conclusions drawn in Section 7.1.1 may be equally applied to all combinations of the dispersions and impurities falling under this trend. The approach opens a way to study dispersions with impurities using acoustic attenuation spectroscopy. Trend 2 The dispersions of CaCO 3 follow trend 2 in the presence of the impurity NaNO 3 (in proportions of 20% and 30%). Similarly, trend 2 is observed in case of the impurity sodium polyacrylate (in proportions of 5%, 10% and 20%). Trend 2 is characterized by a non-proportional increase in acoustic spectra with concentration. There are many possible explanations for this tendency, such as the beginning of formation of aggregates or flocs between particles, between particles and the impurity or between the impurity, or the beginning of multiple scattering, etc. Trend 3 This was observed in presence of the impurity sodium polyacrylate in the proportion of 30%. The trend was found to be complex in nature. The likely causes could be deaggregation or multiple scattering that appear at a certain combination of concentrations of dispersion and dispersant.

Effects of concentration of impurities on acoustic attenuation spectra

This section analyzes the effect of an increase in concentration of an impurity on attenuation spectra of a dispersion at a given concentration.

Several combinations were studied. It was found that under some conditions, the rise of attenuation spectra was proportional to the amount of the added impurity. Fig. 7(a) shows the effects of different concentrations (proportions) of the impurity NaNO 3 on a 10% (m/m) dispersion of CaCO 3 . In order to study the relationship between the change in spectra and the concentration of the impurity, the following method was adopted. In all experiments, attenuation values of a pure dispersion were subtracted from attenuation values of the relevant impure dispersion to obtain the attenuation change due to the total quantity (%age) of impurity. This value was divided by the total quantity (%age) of impurity, and the results were plotted against corresponding frequencies to get attenuation spectra based on unit impurity. If attenuation spectra per unit impurity superimpose, then we can quantify the effects contributed by that impurity to the total acoustic attenuation spectra under given conditions. Fig. 7(b) presents an example.

The method was applied to the entire series of the experiments in order to determine the conditions at which the effects of impurities can be predicted. Table 3 presents the results.

Conclusions

The addition of an impurity into a dispersion may or may not change original particle (or aggregate) size of the disperse medium, depending on the conditions. With the help of acoustic attenuation spectroscopy, it is possible to predict the conditions (iconcentration of dispersion; iiproportion of impurity) at which the particle (or aggregate) size of the disperse medium is not influenced by the addition of the impurity. In such cases, we can calculate the acoustic contribution of the impurity in the total acoustic attenuation spectrum of the dispersion. This contribution may be subtracted from the total attenuation spectrum of the dispersion in order to get more realistic information of the particle size of the disperse medium. The cases in which the size of the disperse medium seems to be influenced by the addition of certain quantities of impurities may be explained based on the existence of multiple scattering (a complex phenomenon that disturbs attenuation in acoustic measurement), interaction between particles of the disperse medium or interaction between particles of the impurity and those of the disperse medium. In such cases, it becomes more difficult to get exact particle size information of the disperse medium by using acoustic attenuation spectroscopy. In the study, higher quantities of impurities were considered in order to emphasize their effect on acoustic response. However, under usual conditions, when the proportion of an impurity is less than 20% of the weight of the disperse medium, it may be assumed that presence of the impurities in a dispersion may lead to predictable effects, except for the impurities (such as sodium polyacrylate) that may influence electrostatic and steric stabilization of the dispersions.

Fig. 1 .

 1 Fig. 1. (a): Schematic of ECAH theory in terms of input-output. (b): Schematic of an acoustic attenuation spectrometer in terms of input-output.

  (i) Acoustic attenuation spectra rise proportionally with increasing concentrations of the dispersion (the same trend as in Section 7.1.1). Please see Fig. 4(a) and (b) as an example. (ii) Acoustic attenuation spectra rise, but non-proportionally, with increasing concentrations of the dispersion. Fig. 5(a) and (b) demonstrate this. (iii) Acoustic attenuation spectra do not show a definable relationship with concentrations of the dispersion. Fig. 6(a) and (b) demonstrate this.

Fig. 3 .

 3 Fig. 3. (a): Attenuation spectra of dispersions of calcium carbonate without addition of impurities. (b): Attenuation spectra of dispersions of calcium carbonate (without impurity) divided by respective weight concentrations.

Fig. 4 .

 4 Fig. 4. (a): Acoustic attenuation spectra of dispersions of CaCO 3 at different concentrations. In each case, 10% MgCO 3 of the weight of CaCO 3 is present as impurity. (b): The graph shows attenuation per unit concentration vs. frequency for dispersions of CaCO 3 at different concentrations. In each case, 10% MgCO 3 of the weight of CaCO 3 is present as impurity.

Fig. 5 .

 5 Fig. 5. (a): Acoustic attenuation spectra of dispersions of CaCO 3 at different concentrations. In each case, 30% NaNO 3 of the weight of CaCO 3 is present as impurity. (b): The graph shows attenuation per unit concentration vs. frequency for dispersions of CaCO 3 at different concentrations. In each case, 30% NaNO 3 of the weight of CaCO 3 is present as impurity.

Fig. 6 .

 6 Fig. 6. (a): Acoustic attenuation spectra of dispersions of CaCO 3 at different concentrations. In each case, 30% sodium polyacrylate of the weight of CaCO 3 is present as impurity. (b): The graph shows attenuation per unit concentration vs. frequency for dispersions of CaCO 3 at different concentrations. In each case, 30% sodium polyacrylate of the weight of CaCO 3 is present as impurity.

Fig. 7 .

 7 Fig. 7. (a): Effects of concentration of impurity of NaNO 3 on 10% (m/m) dispersion of CaCO 3 . (b): Effects of concentration of impurity of NaNO 3 on 10% (m/m) dispersion of CaCO 3 .

Table 2

 2 Frequency band employed in the measurements.

	No.	Frequency (MHz)	No.	Frequency (MHz)	No.	Frequency (MHz)	No.	Frequency (MHz)	No.	Frequency (MHz)
	1	1.5	11	2.2092	21	3.2537	31	4.7921	41	7.0578
	2	1.5592	12	2.2964	22	3.3822	32	4.9813	42	7.3364
	3	1.6208	13	2.3871	23	3.5157	33	5.1779	43	7.626
	4	1.6847	14	2.4813	24	3.6545	34	5.3823	44	7.9271
	5	1.7513	15	2.5793	25	3.7987	35	5.5948	45	8.24
	6	1.8204	16	2.6811	26	3.9487	36	5.8156	46	8.5653
	7	1.8922	17	2.7869	27	4.1046	37	6.0452	47	8.9034
	8	1.9669	18	2.8969	28	4.2666	38	6.2839	48	9.2549
	9	2.0446	19	3.0113	29	4.435	39	6.5319	49	9.6202
	10	2.1253	20	3.1302	30	4.6101	40	6.7898	50	10

Table 3

 3 Conditions in which the effect of an impurity may be predicted.

	Impurity	5% (m/m) dispersion of CaCO 3	10% (m/m) dispersion of CaCO 3	20% (m/m) dispersion of CaCO 3
	MaCO 3	Predictable effect	Predictable effect	Predictable effect
	NaNO 3	Predictable effect	Predictable effect	Unpredictable effect
	Sodium polyacrylate	Unpredictable effect	Unpredictable effect	Unpredictable effect


Fekher Khelifi
email: fekher.khelifi@univ-nantes.fr

Benoit Parrein
email: benoit.parrein@univ-nantes.fr

An Experimental Testbed for Underwater Acoustic Sensor Network Protocol Dedicated to Offshore Wind Turbines

Keywords: Medium Access Control, Sea Trials, TDA-MAC, Underwater Acoustic Network, Wireless Sensing, MATS-LT

Offshore wind turbine monitoring is crucial to reduce maintenance and operating costs of safety-critical components and systems, and to optimize the design of future wind turbines. Monitoring has four objectives: the acquisition of structural response data, local interrogation of collected measurement data, and wireless transmission of that data or analysis results to a underwater acoustic sensor networks (UASN). These sensing units require the design of dedicated UASN networking protocols. In this context, we implement the TDA-MAC protocol for data collection in UASN which needs any clock synchronization. The preliminary experimental results have successfully demonstrated the effectiveness of this MAC protocol on very low speed and long-range transmission devices.

INTRODUCTION

Wireless sensor networks (WSNs) have tremendous potential in monitoring aquatic environments by detecting, collecting and transferring wireless data to users in real time [START_REF] Fekher | Internet of Underwater Things to monitor offshore wind turbines fields[END_REF] [START_REF] Trevathan | An IoT General-Purpose Sensor Board for Enabling Remote Aquatic Envi-ronmental Monitoring[END_REF]. In this context, the "Blue IoT Eolia R&D" project has also addressed such an issue, in particular through the development of a solution capable of both monitoring the fatigue of the power cable and a precise analysis of the complex dynamic behavior induced by offshore wind turbines as illustrated in an example in Figure 1. The main objective of the system is to develop a solution for the networking of underwater sensors whose purpose is to ensure a continuous monitoring of environmental and operational parameters, maintenance as well as supervision of the underwater infrastructure, [START_REF] Cruciani | L'éolien offshore flottant dans sa dimension industrielle et technologique[END_REF]. for the proper functioning of offshore wind turbines.

Therefore, this paper explores the performance of the experimentation of the underwater TDA-MAC protocols [START_REF] Nils | Robust TDA-MAC for practical underwater sensor network deployment: Lessons from USMART sea trials[END_REF] implemented on the MATS-LT acoustic micro-modem [START_REF] Pascal Coince | Acoustic MATS-LT Modems User's Manual[END_REF]. The experiments were conducted in July 2021 at Port de la Grimaudière, Nantes, France. Various network scenarios and packet delivery ratio (PDR), energy consumption were used to study the performance of the TDA-MAC protocol in different contexts. one objective of this work is to produce and compare experiments from [START_REF] Nils | Robust TDA-MAC for practical underwater sensor network deployment: Lessons from USMART sea trials[END_REF] in the context of a wind turbine farm.

Recently, underwater MAC protocols have been tested in real sea experiments [START_REF] Pascal Coince | Acoustic MATS-LT Modems User's Manual[END_REF] [6] [START_REF] Petroccia | Performance evaluation of underwater medium access control protocols: At-sea experiments[END_REF]. In this study the TDA-MAC Protocol was tested in real-world offshore experiments in Fort William, UK. They focused on practical challenges and mitigation strategies related to TDA-MAC to increase its robustness in real-world deployments. Its main advantage over other MAC protocols found in the literature is that it can achieve network throughputs close to the channel capacity without clock synchronization or any other advanced functionality at the sensor nodes. Therefore, it has great potential as a practical solution for efficient data collection in UASN. The main draw-back of TDA-MAC is that it requires a centralized single-hop topology, which cannot accommodate nodes outside the gateway's cover-age. In this paper we propose to reproduce an experiments in another context and scenarios with different commercial equipment's.

EXPERIMENTAL SETUP

This section describes how we took the initial hardware concepts of the Blue-Iot-Eolia project and modified/enhanced them with the goal of creating an affordable, low-power, adaptable remote aquatic monitoring system capable of providing data in near real-time, for our testbed, we use the underwater acoustic micro-modem from the French company Sercel. Each of the beacons and gateways in the testbed is equipped with a MATS-LT acoustic micro-modem [START_REF] Pascal Coince | Acoustic MATS-LT Modems User's Manual[END_REF]. The specifications show an acoustic bit rate up to 40bps using multiple frequency shift keying (FSK). This MATS-LT acoustic micro-modem is controlled by a stm32 type microcontroller (Cortex-M0) for the beacons and a raspberry pi 3 for the Gateway.

EXPERIMENTS RESULTS

This section describes the experimentation of the underwater acoustic network in a natural environment, the river Erdre, beginning of July 2021. In this experimentation, we perform an experimental evaluation of the TDA-MAC protocol with 3 nodes and a Gateway.

To evaluate the mixed and maximum communication range and energy consumption, we placed the beacons in the river Erdre at a distance of 30m, 100m, 200m and 300m. From these points, we establish links with Gateway and variable distances. We use a reference point for Gateway, as illustrated in an example in Figure 2 Table 1 shows the PDR obtained by Gateway from each beacon. In real experience, the PDR is constant (100%); this occurs because the distance is short. For first and second test the throughput is 5.08bps and 6.93 packets/min. For the third scenario (200m). It is possible to observe that beacon 0 and beacon1 keep a similar behavior, with a PDR is higher than 93% and beacon2 at a loss of 15%. The PDR of B2 is 85%. We explain simply this by the fact that B2 is farthest from the Gateway. This leads to minimizing the throughput to 4.35bps. A fourth scenario to check the maximum range. In just this scenario the majority of the packets were also In Figure 3, we see that the battery level of the beacons decreases during the test. This is due to the number of acoustic communications. We also notice that the amount of energy consumed by beacon 0 is more than beacon 1. This is due to the number of data trans-missions made by the beacons, and it ran for 190 min, as shown in table.1.

CONCLUSIONS AND FURTHER WORK

In this paper, we have studied the results of experiments collected during a river trial. The objective is to analyze the performance of the TDA-MAC protocol using the MATS-LT acoustic micro-modem and its capability for real communication. We analyze three scenarios to study the impact of distance on communication, collecting data for real experiments to analyze and compare the PDR level and the battery level. Nevertheless, these results provided good information for future research. The next experiments will be conducted at the aquatic environment that has fewer particles, and improve flight time calculators, reduced packet loss and transmission delays. Additionally, we intend to deploy in multi-hop and other topology.

This research is supported by Pays de la Loire Region under the project Blue IoT Eolia. The partners of the project are the company Kopadia, LS2N laboratory (RIO) and SEM-REV. We would like to special thanks to François Babin and Gatien Jost from Kopadia for active participation to the experiments.

Figure 1 :

 1 Figure 1: Monitoring of a floating wind turbine with possible acoustic modems locations along the dynamic cable [3].

WUWNet' 21 ,

 21 November 22-24, 2021, Shenzhen, Guangdong, China Fekher Khelifi and Benoit Parrein

Figure 2 :

 2 Figure 2: Deployment in Port de la Grimaudière

Figure 3 :

 3 Figure 3: Battery level evolution

Table 1 :

 1 Experimental results at the Testbed

	Distance