N Claessens

F Demoisson

T Dufour

Ali Mansour

A Felten

J Guillot

J-J Pireaux

F Reniers

Carbon nanotubes decorated with gold, platinum and rhodium clusters by injection of colloidal solutions into the post-discharge of an RF atmospheric plasma Authors

In this paper, we present a new, simple, robust and efficient technique to decorate multi-wall carbon nanotubes (MWCNT) with metal nanoparticles. As case studies, Au, Pt and Rh nanoparticles are grafted onto MWCNTs by spraying a colloidal solution into the post-discharge of an atmospheric argon or argon/oxygen RF plasma. The method that we introduce here is different from those usually described in the literature, since the treatment is operated at atmospheric pressure, allowing the realization in only one step of the surface activation and the deposition processes. We demonstrate experimentally that the addition of oxygen gas in the plasma increases significantly the amount of grafted metal nanoparticles. Moreover, TEM pictures clearly show that the grafted nanoparticles are well controlled in size.

I. Introduction

Thanks to their unique electrical properties, high chemical stability and high surface-to-volume ratios, multi-wall carbon nanotubes (MWCNTs) have been proposed as ideal metal supports for catalytic and sensing applications. Therefore, many different methods have been developed to activate and decorate carbon nanotubes with metal nanoparticles. It is well accepted that the activation of a carbon surface is based on the formation of surface defects and/or the adsorption of reactive species, such as oxygencontaining groups. In most of the studies, this activation is carried out via wet chemistry techniques, requiring the use of hot nitric acid solutions [1]. In some others, the activation and deposition processes are based on vacuum techniques requiring ion guns, low pressure plasma and/or thermal evaporators [2]. In this work, we have successfully transposed a technique recently used for the deposition of Au nanoparticles onto HOPG (highly oriented pyrolytic graphite) [3] to the deposition of metal nanoparticles (Au [4], Pt and Rh) onto MWCNTs. Besides the advantage of working at atmospheric pressure, this technique guarantees the cleaning and the activation of the substrate surface in only one step [5]. Moreover, it allows the deposition of uniform, and well controlled in size, metal nanoparticles. Another successful method that has been recently reported to decorate MWCNTs with gold, Rh and Pt nanoparticles is based on a thermal evaporation process [6][7][START_REF] Suarez-Martinez | ACS Nano at press[END_REF]. Although the density of the grafted nanoparticles is higher than those we obtained, such a technique requires us to work at (ultra) high vacuum pressure. Moreover, the yield of this process is much lower than the one obtained when using the sprayed nanoparticles.

II. Materials & Experimental setup

MWCNTs were provided by Nanocyl S.A. (ref 3100). They were grown by chemical vapour deposition (CVD) with a purity higher than 95%. Their length was as high as 50 μm and their outer and inner diameters ranged respectively from 15 to 3 nm and 7 to 2 nm.

The metal nanoparticles to graft onto these MWCNTs stemmed from colloidal solutions. The gold colloidal solution was synthesized by the citrate thermal reduction method [3]. The colloidal solutions of platinum and rhodium were supplied by the Department of Chemistry (University of California) in Berkeley; their synthesis conditions are described in [START_REF] Rioux | [END_REF] and [10]. These suspensions resulted in stable dispersions of metal nanoparticles with an average diameter of 10 nm, 8 nm and 4 nm, respectively, for gold, rhodium and platinum.

Before starting any deposition, MWCNTs were soaked in a methanol solution for up to 15 min. Then, their surface was activated by exposure to the post-discharge of an atmospheric plasma during 2 min. This plasma was generated with an RF torch (Atomflo™-250 from Surfx Technologies LLC [START_REF] Reniers | Proc´ed´e de dépôt de nanoparticules sur un support[END_REF]) using argon as the vector gas. The decoration of the carbon nanotubes surface was performed by spraying the colloidal solution into the postdischarge (during 30 s). The resulting surface was then exposed to the post-discharge for 3 min. Once this step was achieved, the treated MWCNTs were introduced into a methanol solution for up to 5 min and subjected to ultrasonication. Their surface composition was finally analyzed by X-ray photoelectron spectroscopy (XPS) and their surface morphology (dispersion and shape of the metal nanoclusters) was characterized by transmission electron microscopy (TEM).

To evaluate the chemical composition of the MWCNTs' surface, XPS analyses were performed by using two instruments. The HP 5950 A instrument from the LISE team (Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium) was dedicated to the characterization of the Au/MWCNTs samples. The ThermoVG Microlab 350 instrument from the SAM team (CRPGL, Luxembourg) was used to analyze the Pt/MWCNT and Rh/MWCNT samples. In both cases, the RX source was a monochromatized Al Kα line (hv = 1486.6 eV). The samples were pressed onto a Cu tape on a holder and introduced into the spectrometer. The base pressure in the analytical chamber was ≈ 10 -9 mbar. Survey scans were used to determine the chemical elements present at the MWCNT surface. Narrow-region electron spectra were used for the chemical study of the C 1s, Au 4f, Pt 4f and Rh 3d peaks. The elemental composition was calculated after removal of a Shirley background line and using relative sensitivity factors from [START_REF] Briggs | Practical Surface Analysis 2nd edn, vol 1 Auger and X-ray Photoelectron spectroscopy[END_REF]. The obtained compositions must be taken as indicative and are used only for comparison between the different plasma treatments (with/without O2) and the different deposited nanoparticles. They do not reflect the absolute surface composition. In our case, the multiply analyzed regions on the same sample always lead to the same relative compositions.

In order to determine the size and the distribution of the metal nanoclusters and to evaluate the effect of the plasma treatment on the MWCNTs, transmission electron microscopy (TEM) measurements were carried out on a Philips Tecnai 10 microscope operating at 80 kV from the LISE team for the characterization of the Au/MWCNT samples and on a LEO 922 OMEGA operating at 200 kV with a point-to-point resolution of 0.29 nm (TEM) from the SAM team for the characterization of the Pt/MWCNT and Rh/MWCNT samples. For this study, the MWCNT powder was dispersed in ethanol with ultrasonication for 5 min, and a drop was deposited onto a honeycomb carbon film supported by a copper grid. As a TEM image only gives local information, each sample was observed at different regions, so that each image presented in this paper is representative of the mean global observation.

III. Results & Discussion

As already mentioned in the experimental procedure, the decoration step was preceded by the exposure of the MWCNT surface to the post-discharge. During this plasma activation step, the sidewall structure of the carbon nanotubes can be modified. Figure 1 shows the XPS O 1s and C 1s core levels in three cases: no plasma activation, plasma activation (argon flow rate: 30 L.min-1) and plasma activation using both argon and oxygen (with the respective flow rates: 30 L.min -1 and 20 mL.min -1 ). The carbon composition is 96.6% when no activation is performed. For the pure argon activation, this value decreases to 91.4% since the oxygen from the air was sufficient to create local oxygen vacancies. Then, for the Ar/O2 plasma activation, the C 1s peak indicates a relative composition of only 88.1%, thus suggesting the significant impact of the oxygen during the plasma activation. TEM pictures were acquired to evaluate the impact of this Ar/O2 plasma activation (figure 2(b)) with respect to the reference case where no plasma activation was performed (figure 2(a)). The comparison shows no significant damage on the external sidewalls that could be attributed to the plasma activation. In this sense, the HRTEM picture of the plasma-treated MWCNTs (figure 2(c)) shows their integrated layer structure, their smooth surface and the parallel graphene layers. Contrary to other studies where the carbon nanotubes are treated inside low pressure plasma, our MWNTs are exposed to the post-discharge of an atmospheric plasma. The ion-induced damage is thus strongly reduced [START_REF] Bittencourt | [END_REF].

Figure 2. TEM pictures of (a) pure MWCNTs and (b) MWCNTs plasmaactivated with Ar and O2. (c) is an HRTEM picture of case (b).

After an argon plasma treatment with nanoparticles at atmospheric pressure, the XPS analysis highlighted the presence of metal on the MWCNTs' surface. The survey spectra (figure 3) indicated Au at. = 0.5%, Pt at. = 0.7% and Rh at. = 1.2%, respectively, for the Au/MWCNT, Pt/MWCNT and Rh/MWCNT samples. This surface composition was calculated with the usual formula, assuming therefore a homogeneous distribution of the elements in the sample (which is obviously not the case) and no matrix effects. With or without the ultrasonication step, the amount of metal detected was unchanged, thus suggesting a strong adhesion of the metal nanoparticles onto the MWCNTs. Note that the nitrogen detected on the carbon nanotubes decorated with Pt or Rh originates from the colloidal solution, and more specifically from its capping agent (polyvinylpyrrolidone). As according to the spectra of figure 3 the surface composition was approximately the same in any case, it is clear that gold, platinum and rhodium present a similarity of behavior This same surface composition also indicates a strong reproducibility of the plasma treatment for the metal cluster deposition. In figure 4, we show the fitted core levels of the Au 4f, Pt 4f, Rh 3d and C 1s peaks. The fitted Au 4f (Au 4f5/2-Au 4f7/2 spin-orbit doublets with a fixed 0.75:1 intensity ratio and 3.7 eV energy separation) [14], Pt 4f (Pt 4f5/2-Pt 4f7/2 spin-orbit doublets with a fixed 0.78:1 intensity ratio and 3.3 eV energy separation) [14] and Rh 3d (Rh 3d3/2-Rh 3d5/2 spin-orbit doublets with a fixed 0.64:1 intensity ratio and 4.75 eV energy separation) [14] spectra can be unambiguously assigned in the majority to the metallic state [15].

Figure 4. Fitted core levels for (a) Au 4f, (b) Pt 4f, (c) Rh 3d and (d) C 1s peaks after the atmospheric argon plasma treatment. Note that the presented fitted C 1s level corresponds to Au/MWCNT sample characterization. The fitted C 1s level is similar for the Pt/MWCNT and Rh/MWCNT samples.

As already observed on an HOPG surface [3], the high-resolution C 1s spectra highlighted the presence of carbon-oxygen species. In the present case, illustrated in figure 4(d), the C 1s spectra can be fitted to five lines with binding energies at 284.9, 286.2, 287.4, 288.8 and 290.9 eV, with a constant FWHM of 1.3 eV [16]. These peaks correspond respectively to (sp 2 C=C)/(sp 3 C-C), C-O, C=O, COOH and π-π * contributions [4]. As the process does not operate in a closed system but in an open environment, the presence of the carbon-oxygen species can reasonably be attributed to the fact that the MWCNTs' surface was exposed to an oxidizing atmosphere during the plasma treatment. Indeed, optical emission spectrometry of the post-discharge (not shown) reveals the presence of atomic oxygen lines at 777 and 852 nm. Therefore, traces of oxygen and/or water cannot be avoided because of the humidity of the air. The solvent from the colloidal solutions (which is water) can also enhance the formation of these carbon-oxygen species.

The role played by these carbon-oxygen species is quite important on the amount of metal nanoparticles grafted onto the MWCNTs' surface. Theoretical calculations predicted that the surface must be in an appropriate energetic state to allow the condensation of metals on it. In the case of a decoration with gold nanoparticles, it has been calculated that the nucleation would preferably take place on defect sites, especially on oxygenated vacancies (VO2) which would act as trapping sites for gold atoms [4].

To verify experimentally these predictions, we replaced the previous argon plasma treatment by a new one in which an oxygen flow rate of 20 mL.min In agreement with the previous XPS spectra (figure 5), we observed on the TEM images (figure 6) a higher number of gold nanoparticles grafted onto the MWCNT surface when oxygen is mixed with argon. TEM images of MWCNTs decorated with platinum or rhodium nanoparticles are respectively presented in figures 7 and 8. In both cases, we obtained a similar increase in the number of metal nanoparticles grafted when operating under an argon-oxygen plasma treatment. This behavior could be attributed to the increase of oxygen-containing groups and/or of structural defects on the MWCNTs' surface, under the assumption of a metal binding at a C-O or defect site [4]. TEM images (figures 6-8) show that most of the MWCNTs are isolated and that almost no agglomeration of carbon nanoparticles has occurred. Note that the diameters of spherical Au, Pt and Rh nanoclusters (respectively 10 nm, 4 nm and 8 nm) are like those of the preformed particles in the colloidal solution. Similar results have been obtained on HOPG substrates [3]. Finally, independent of their nature (Au, Pt or Rh), the dispersion of the nanoparticles was always better when an oxygen flow rate of 20 mL.min -1 was mixed with the argon gas; and the grafted platinum nanoparticles were particularly homogeneously distributed (figure 7).

IV. Conclusion

We have successfully achieved the deposition of gold, platinum and rhodium nanoparticles on MWCNTs by using an atmospheric plasma source. We have demonstrated the major role of oxygen during the plasma treatment. By adding an oxygen flow rate as low as 20 mL.min -1 to the argon gas of the plasma torch, we managed to increase subsequently the amount of metal nanoparticles grafted onto MWCNTs (up to 4% in the case of gold), while preserving the original size of the nanoparticles from the colloidal solution. Our results point out a very interesting new and simple method which allows the deposition of metal nanoparticles with a desired uniform and well-controlled size.

Figure 1 .

 1 Figure 1. XPS spectra core levels of O 1s and C 1s of MWCNTs: not activated, plasma-activated with an argon flow rate of 30 L.min -1 and plasma-activated with an argon flow rate of 30 L.min -1 and oxygen flow rate of 20 mL.min -1 . The RF plasma power was 80 W.

Figure 3 .

 3 Figure 3. Survey XPS spectra recorded on (a) Au/MWCNTs, (b) Pt/MWCNTs and (c) Rh/MWCNTs after atmospheric Ar plasma treatment.

  -1 is added to the argon flow rate (30 L.min -1 ). This new plasma treatment clearly modifies the surface composition of the MWCNTs, as illustrated by the XPS survey Nanotechnology, 2010, Vol. 21, Issue 38, 385603, http://dx.doi.org/10.1088/0957-4484/21/38/385603 spectra in figure5. The first spectrum (figure5(a)) highlights about 4 at.% of gold deposited on the MWCNT surface instead of the 0.5 at.% detected without oxygen in the plasma gas (figure3(a)). The potassium detected in the Au/MWCNT sample originates from the colloidal solution (potassium citrate was used in the synthesis process). In the case of platinum deposition (figure5(b)), the absolute number of grafted nanoparticles has considerably increased since the Pt 4f peak is much stronger than in figure5(b). Finally, in figure5(c) the relative surface composition of Rh is almost twice as high (2.2 at.% versus 1.2 at.%) when the postdischarge is enriched in oxygen. All these results are consistent and clearly show the benefit of mixing oxygen with argon, particularly in the case of gold deposition.

Figure 5 .

 5 Figure 5. Survey XPS spectra recorded on (a) (b) Pt/MWCNTs and (c) Rh/MWCNTs after the argon-oxygen plasma treatment. The flow rates were 30 L.min -1 for argon and 20 mL.min -1 for oxygen.

Figure 6 .

 6 Figure 6. TEM images of MWCNT samples covered with gold metallic nanoclusters (10 nm) deposited by the atmospheric plasma treatment using (a) Ar and (b) Ar/O2 mixture. Flow rates were 30 L.min -1 for Ar and 20 mL.min -1 for O2.

Figure 7 .

 7 Figure 7. TEM images of MWCNT samples covered with platinum metallic nanoclusters (4 nm in diameter) deposited by the atmospheric plasma treatment using (a) Ar and (b) Ar/O2 mixture. Flow rates were 30 L.min -1 for Ar and 20mL.min -1 for O2.

Figure 8 .

 8 Figure 8. TEM images of MWCNT samples covered with rhodium metallic nanoclusters (8 nm in diameter) deposited by the atmospheric plasma treatment using (a) Ar and (b) Ar/O2 mixture. Flow rates were 30 L.min -1for Ar and 20mL.min -1 for O2.

Nanotechnology, 2010, Vol. 21, Issue 38, 385603, http://dx.doi.org/10.1088/0957-4484/21/38/385603

Nanotechnology, 2010, Vol. 21, Issue 38, 385603, http://dx.doi.org/10.1088/0957-4484/21/38/385603

Nanotechnology, 2010, Vol. 21, Issue 38, 385603, http://dx.doi.org/10.1088/0957-4484/21/38/385603

V. Acknowledgments

The authors warmly thank Professor Gabor A Somorjai from the Department of Chemistry (University of California) in Berkeley, for supplying the colloidal solutions of rhodium and platinum. This work was financially supported under the European NMP3-CT-2006-033311 NANO2HYBRIDS project.

VI. References


R Leghrib

T Dufour

F Demoisson

N Claessens

F Reniers

E Llobet

Affiliations

Gas sensing properties of multiwall carbon nanotubes decorated with rhodium nanoparticles Authors

In the present work, multiwalled carbon nanotubes were decorated with rhodium nanoparticles using a colloidal solution in the post-discharge of an RF atmospheric plasma of argon (Ar) or argon/oxygen (Ar:O2). The properties of these hybrid materials towards the room temperature detection of NO2, C2H4, CO, C6H6 and moisture were investigated and discussed in view of compositional and morphological studies. It was found that the presence of oxygen in the plasma treatment is essential to significantly enhance the gas response of Rh-decorated multiwalled carbon nanotubes and to avoid response saturation even at low gas/vapor concentrations. These desirable effects are attributed to the presence of oxygen during the CNT plasma treatment since oxygenated vacancies act both as active adsorption sites for gases and as anchoring sites for Rh nanoparticles (the presence of Rh nanoclusters is nearly doubled in Ar:O2 treated samples as compared to Ar treated samples). The oxygen treatment also makes easier the charge transfer between Rh nanoparticles and carbon nanotubes upon gas adsorption. The method for treating and decorating multiwalled carbon nanotubes used here is simple, fast and scalable for producing gas sensitive nanohybrid materials with uniform and well-controlled properties.

I. Introduction

Carbon nanotubes (CNTs) have been widely studied in different applications of nanotechnology thanks to their interesting physico-chemical properties [START_REF] Dresselhaus | Science of Fullerenes and Carbon Nanotubes[END_REF][START_REF] Kauffman | Carbon nanotube gas and vapor sensors[END_REF][START_REF] Zhang | Recent progress in carbon nanotube-based gas sensors[END_REF]. Considering their chemical stability and large surface area to volume ratio, CNTs have been considered as new supports for metal catalysts [START_REF] Auer | Carbons as supports for industrial precious metal catalysts[END_REF][START_REF] Coq | Fullerence-based materials as new support media in heterogeneous catalysis by metals[END_REF]. Since the adsorption of a small quantity of chemical species can result in a dramatic change of their conductivity, CNTs are suited to detect gases at low concentrations (e.g. low ppb level) [START_REF] Valentini | Highly sensitive and selective sensors based on carbon nanotubes thin films for molecular detection[END_REF]. Furthermore, the possibility to operate CNT gas sensors at room temperature makes them very interesting to integrate low power consumption detectors [START_REF] Leghrib | Room-temperature, selective detection of benzene at traces levels using plasma-treated metal-decorated multiwalled carbon nanotubes[END_REF]. A gas sensor can be built using a simple transducer (comb electrodes) to monitor the electrical resistance of a CNTbased film.

In recent years, numerous theoretical and experimental studies have been focused on the functionalization of CNT surface and/or the decoration of their sidewalls with metal nanoclusters, in a quest for enhancing sensitivity and ameliorating selectivity to different toxic gases or vapors [START_REF] Leghrib | Room-temperature, selective detection of benzene at traces levels using plasma-treated metal-decorated multiwalled carbon nanotubes[END_REF][START_REF] Kong | Functionalized carbon nanotubes for molecular hydrogen sensors[END_REF][9][START_REF] Kauffman | Understanding the sensor response of metaldecorated carbon nanotubes[END_REF][START_REF] Zhao | Carbon nanotubesmetal cluster composites: a new road to chemical sensors[END_REF][START_REF] Kumar | Nanostructured Pt functionalized multiwalled carbon nanotubes based hydrogen sensor[END_REF][START_REF] Star | Gas sensor array based on metal-decorated carbon nanotubes[END_REF]. The modification of CNT surfaces with metal nanoparticles is of interest for gas sensing since metal nanoclusters have a broad range of diverse structures and can provide a wide range of reactivity with different species. Indeed, the concept of using CNT-metal cluster hybrids as the sensitive material of a device where the metal cluster surfaces act as reactive sites for the adsorption of the target molecules was theoretically introduced in [START_REF] Zhao | Carbon nanotubesmetal cluster composites: a new road to chemical sensors[END_REF]. The first researchers that experimentally exploited this concept were Kong et al. [START_REF] Kong | Functionalized carbon nanotubes for molecular hydrogen sensors[END_REF], Kumar and Ramaprabhu [START_REF] Kumar | Nanostructured Pt functionalized multiwalled carbon nanotubes based hydrogen sensor[END_REF] and Star et al. [START_REF] Star | Gas sensor array based on metal-decorated carbon nanotubes[END_REF], who used Pd-decorated CNTs or Pt decorated CNTs to detect hydrogen, and an array of single wall CNTs decorated with different metals (Rh, Ni, Au, Pd, etc.) for detecting H2S, CH4, H2, CO and NH3, respectively. Recently [START_REF] Leghrib | Room-temperature, selective detection of benzene at traces levels using plasma-treated metal-decorated multiwalled carbon nanotubes[END_REF], we have shown the selective detection of low C6H6 concentrations employing an array of Rh, Ni, Au, and Pd decorated multiwall carbon nanotube (MWCNT) gas sensors operated at room temperature.

Among the different metals one can consider for decorating carbon nanotube sidewalls, rhodium is of interest since the Rh-MWCNT catalyst has been reported to be effective in the hydrogenation of aromatic rings at room temperature. In addition, the catalyst can be reused at least six times without losing catalytic activity for arene hydrogenation and its catalytic activity is much higher than that from commercially available Rh nanocatalysts [START_REF] Pan | Sonochemical one-pot synthesis of carbon nanotubesupported rhodium nanoparticles for roomtemperature hydrogenation of arenes[END_REF][START_REF] Pan | One-step synthesis of size-tunable rhodium nanoparticles on carbon nanotubes: a study[END_REF]. Although there is a weak interaction between molecular benzene and CNTs [START_REF] Tourmus | Ab initio study of benzene adsorption on carbon nanotubes[END_REF], the presence of Rh nanoparticles on the surface of MWCNTs helps enhancing the sensor signal upon benzene adsorption (i.e., among the different metals studied, Rh led to the highest benzene response) [START_REF] Leghrib | Room-temperature, selective detection of benzene at traces levels using plasma-treated metal-decorated multiwalled carbon nanotubes[END_REF].

In this paper, the effects of using two different atmospheric plasmas (i.e., Ar or an Ar-O2 mixture) while preparing Rh decorated multiwall carbon nanotubes on the sensing properties of the resulting nanohybrid materials are studied. The performance of gas sensors employing such materials are compared in terms of their response towards NO2, CO, C2H4, C6H6 and moisture. The differences in their response are discussed in view of the data gathered by Transmission Electronic Microscopy (TEM) and X-ray Photoelectron Spectroscopy (XPS) analyses. Combined, the gas sensing, compositional and morphological studies give insight about the role of oxygen species grafted to CNTs during the plasma treatment both for ameliorating the decoration with metal nanoparticles and enhancing the gas response of the resulting nanohybrid material.

II. Experimental setup II.1. Hybrid nanomaterials: synthesis and characterization

The multiwall carbon nanotubes used were provided by Nanocyl S.A. They were grown by Chemical Vapor Deposition with purity higher than 95%. Nanotubes were up to 50 m in length and their outer and inner diameters ranged from 3 to 15 nm and 3 to 7 nm, respectively. These nanotubes were decorated with Rh nanoparticles employing a colloid solution supplied by the Department of Chemistry from the University of California at Berkeley (USA). For this solution the capping agent was poly(vinylpyrrolidone) (PVP) and the solvent was ethanol. Solution concentration was 4 mmol/L, which represented 0.31 g.L -1 of Rh. The size of Rh nanoparticles was 8.8 ± 1.7 nm [START_REF] Wang | Preparation of tractable platinum, rhodium, and ruthenium nanoclusters with small particle size in organic media[END_REF]. The plasma activation and deposition processes are further explained in [START_REF] Claessens | Carbon nanotubes decorated with gold, platinum and rhodium clusters by injection of colloidal solutions into the post-discharge of an RF atmospheric plasma[END_REF][START_REF] Reniers | Procédé de dépôt de nanoparticules sur un support[END_REF]. At first, the experimental process required the cleaning of carbon nanotubes for 15 min in a methanol solution. Surface treatment and metal decoration were conducted using plasma post-discharge at atmospheric pressure. The plasma was generated with a RF torch (Atomflo-250, from Surfx Technologies LLC), powered at 80 W and working for an argon flow of 30 L/min. For some of the samples prepared, oxygen was mixed to the argon gas at the rate of 20 mL/min. Carbon nanotubes, placed 10 mm away from the RF torch were exposed to the plasma postdischarge for 2 min to activate their surface. Then, during 30 s, the rhodium colloid solution was sprayed in the plasma postdischarge. Treated nanotubes had to be exposed 3 more minutes before switching off the RF torch. As a final step, they were introduced for 5 min into a methanol solution under ultrasonication.

The chemical composition and the surface morphology of the different hybrid materials employed as active layers of the sensors fabricated were analyzed by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM), respectively. The results of these chemical and morphological studies can be found in [START_REF] Claessens | Carbon nanotubes decorated with gold, platinum and rhodium clusters by injection of colloidal solutions into the post-discharge of an RF atmospheric plasma[END_REF].

II.2. Sensor fabrication and characterization

Sensor substrates consisted of an integrated array of four inert micro-hotplate substrates, each one comprising a polysilicon heating resistor sandwiched between two thin silicon nitride layers and a pair of interdigitated Pt electrodes on top (the electrode gap was 50 µm). The membrane thickness was about 0.6 µm and the active (hot) area of 500 µm × 500 µm [START_REF] Rickerby | Structural and dimensional control in micromachined integrated solid state gas sensors[END_REF]. Plasma-treated Rhdecorated MWCNT were dispersed in dimethyl formamide (DMF) by ultrasonication at room temperature to form uniform suspensions. The suspensions were airbrushed onto the interdigitated electrodes. After DMF evaporated (sensors are kept on a hotplate during the airbrushing process), a mat of metal decorated CNTs spanned across neighboring fingers. By carefully controlling its process parameters (e.g., nozzle to substrate distance, substrate temperature, carrier flow and amount of CNTs within a suspension), highly reproducible mats of Rh-decorated CNTs are obtained. Finally, sensors were annealed at 250°C for 4 h in an air flow to completely remove the remnant DMF and improve the nanotube-electrode contact.

The gas sensing properties at room temperature of Rh-decorated carbon nanotubes treated in Ar or Ar+O2 plasmas were tested in the presence of NO2, C2H4, CO, and C6H6. Humidity response was also investigated. To perform the measurements, the gas sensors were placed in a 2 × 10 -5 m 3 volume Teflon/stainless steel test chamber. The desired concentrations of each species were obtained using a computer-controlled measurement rig that employed mass flow meters and calibrated gas bottles. Dry air was used as carrier gas. R.H. in the measurement rig was 10% at 30°C. The concentrations tested were as follows: 50, 100, 200, 500 and 1000 ppb for benzene vapors; 50, 100, 500 and 1000 ppb for NO2; 3, 7, 15 and 30 ppm for C2H4; and 2, 5, 10 and 20 ppm for CO. Moisture responses to changes in R.H. from 10 to 50%, 50% to 80% and 10% to 80% were measured. Finally, to better study moisture effects on gas response, 50 and 100 ppb of NO2; 2 and 5 ppm of CO; and 50 and 100 ppb of benzene were measured at 50% R.H. To assess the reproducibility of results, each measurement was replicated 4 times. An Agilent 34970A multimeter was used for continuously monitoring the electrical resistance of the sensors. DC measurements were performed throughout this study. The measurement process was as follows (identical for all species tested): the air flow was set to 100 sccm and kept constant. Data acquisition started 20 min before injecting a continuous flow of the desired concentration of a given species into the measurement chamber. 20 min after the injection, the sensors were flushed with dry air and their temperature was raised to 150°C for promoting the cleaning of their surface. 20 min after heating had been switched off, a new measurement was performed.

III. Results and discussion

III.1. Effect of plasma treatment conditions on the nanohybrid materials XPS and TEM results show that the introduction of oxygen in the plasma used to treat the surface of CNTs induces the grafting of a higher amount of Rh nanoparticles onto CNT sidewalls [START_REF] Claessens | Carbon nanotubes decorated with gold, platinum and rhodium clusters by injection of colloidal solutions into the post-discharge of an RF atmospheric plasma[END_REF]. XPS confirms that adding 20 mL/min of oxygen to the Ar plasma increases the amount of Rh from 1.2% (in wt.%) in Ar treated samples to 2.2% (in wt.%) in Ar-O2 treated samples. Moreover, the oxidation state of the grafted Rh remains unchanged. TEM analysis shows that this method preserves the original size of the Rh nanoparticles from the colloidal solution. These results can be found elsewhere [START_REF] Claessens | Carbon nanotubes decorated with gold, platinum and rhodium clusters by injection of colloidal solutions into the post-discharge of an RF atmospheric plasma[END_REF]. Additionally Suarez-Martinez et al. showed in [START_REF] Suarez-Martinez | Study of the interface between rhodium and carbon nanotubes[END_REF] that the inclusion of oxygen in an RF plasma treatment grafts oxygen species to the CNT surface, which helps creating nucleation/anchoring sites for metal clusters. As a result, more Rh nanoparticles are attached to CNT sidewalls and with more uniform distribution than on pristine tubes or on tubes treated with Ar plasma only. In [START_REF] Claessens | Carbon nanotubes decorated with gold, platinum and rhodium clusters by injection of colloidal solutions into the post-discharge of an RF atmospheric plasma[END_REF], it is suggested that the introduction of oxygen during CNT plasma treatment favors the decoration with Rh nanoparticles because these are immobilized at oxygenated vacancies present in CNT sidewalls and the capping agent of colloidal particles is activated, which helps forming a bridge between C and Rh atoms. Giordano et al. [START_REF] Giordano | Preparation of rhodium catalysts supported on carbon nanotubes by a surface mediated organometallic reaction[END_REF] found that the oxidation of the outer surface of multi-walled carbon nanotubes by nitric acid leads to the formation of carboxylic groups that can be used to anchor rhodium carbonyl moieties. Thus, the pretreatment of CNTs in oxygen plasma is very necessary to get a high amount of metal particles attached onto nanotube surface with good homogeneity [START_REF] Claessens | Carbon nanotubes decorated with gold, platinum and rhodium clusters by injection of colloidal solutions into the post-discharge of an RF atmospheric plasma[END_REF], which are of paramount importance for getting high reactivity to gases of the resulting hybrid material. The amounts of Rh grafted on CNT sidewalls are highly reproducible provided that the plasma parameters are kept the same. Furthermore, the functionalization of CNT surface with oxygen species prior to metal decoration affects the density of states of valence bands [START_REF] Ago | Work functions and surface functional groups of multiwall carbon nanotubes[END_REF] and this improves the exchange of electrons at the interface between CNTs and metal nanoparticles, which in this way increases the sensitivity of the material.

III.2. Gas sensing analysis

After exposing the sensors based on Rh-CNTs treated in argon or argon/oxygen plasmas to the different gases studied, it was found that sensors showed different reactivities depending on the material treatment and the gas detected. Fig. 1 shows the typical responses, at room temperature, of our hybrid materials towards NO2, C6H6 and CO. Although sensors can recover their baseline by cleaning them with air at room temperature, heating at 150°C speeds up sensor baseline recovery. The slow recovery of baseline resistance at room temperature and the effect of heating at 150°C are shown in Fig. 2. This moderate temperature helps desorbing the species adsorbed on the surface of the hybrid materials. This is supported by the fact that their response to the different species tested decreases if the sensors are operated at 150°C. effect is confirmed by DFT calculations performed on defective CNTs [START_REF] Zanolli | Defective carbon nanotubes for single-molecule sensing[END_REF]. Saturation in the response is seen for CO concentrations higher than 5 ppm (see Fig. 1e in which if CO concentration is further increased to 10 or 20 ppm, sensor resistance variations remain below the noise level). If response time is defined as the time needed to reach 90% of the steady state resistance value after gas concentration is changed stepwise, in Rh-decorated CNT sensors response time is below 10 min for CO, C6H6 and C2H4 and near 20 min for NO2.

Fig. 2. (a) Response and slow recovery of a Rh-decorated CNT sensor at room temperature and (b) room temperature response and complete baseline recovery of a Rh-decorated CNT sensor when heating is applied during the cleaning cycle.

The subplots in Fig. 3 show the response of the two hybrid materials (i.e., Rh-MWCNT treated in Ar or in Ar:O2 plasmas) towards different concentrations of C2H4, CO, NO2 and C6H6. Response is defined as (Rg-Ra)/Ra, where Ra is the sensor resistance in clean air (i.e. baseline resistance) and Rg is the resistance in the presence of a given species. Considering the species measured (see Fig. 3a-d), for both materials and for the different concentrations tested, the absolute value of the response is higher for NO2 followed by C2H4, CO and finally C6H6. Rh-MWCNTs treated in argon plasma show a clear saturation effect in their response that appears, in some cases, even at the lowest concentrations measured. On the other hand, Rh-MWCNTs treated in argon-oxygen plasma show good linearity in their response to NO2 and C6H6. However, for C2H4 and CO their response saturates at 2 and 10 ppm, respectively.

Fig. 3. Room-temperature response and associated error bars of sensors employing Rh-CNTs either treated in argon or argon-oxygen plasmas to (a) nitrogen dioxide, (b) ethylene, (c) carbon monoxide and (d) benzene.

Fig. 3 shows that hybrid materials treated in Ar plasma show lower response to the gases tested than materials treated in Ar:O2 plasma. The increase in response achieved by the treatment under an Ar:O2 plasma is due to both the increased amount Rh nanoparticles grafted to the outer surface of CNTs and to the creation of oxygenated vacancies, which can act as active adsorption sites for gas molecules [START_REF] Ionescu | Oxygen functionalization of MWNT and their use as gas sensitive thick-film layers[END_REF]. The early saturation in response experienced with materials treated in argon plasma only could be due to their lower coverage with Rh nanoparticles and lower amount of oxygenated defects. The fact that the saturation in response is also observed in Ar:O2 plasma treated samples in the particular case of ethylene could be due to the non-suitability of Rh for detecting this species [START_REF] Leghrib | Room-temperature, selective detection of benzene at traces levels using plasma-treated metal-decorated multiwalled carbon nanotubes[END_REF]. These results determine the dominant interaction mechanism between the gases tested and the metal decorated CNTs. This mechanism is the charge transfer between adsorbed gas molecules and CNTs via Rh nanoparticles. This alters the conductivity of the hybrid material. The direction of this charge transfer depends on the oxidizing or reducing nature of the gas environment [START_REF] Lu | Room temperature methane detection using palladium loaded single-walled carbon nanotubes sensors[END_REF]. Recently, Zanolli and co-workers published a deep combined experimental and theoretical study on the sensing properties of Au decorated CNTs towards benzene, carbon monoxide and nitrogen dioxide [START_REF] Zanolli | Gas sensing with Au-decorated carbon nanotubes[END_REF].

In this study, nitrogen dioxide is predicted to accept electronic charge from the Au-CNT system, which is consistent with the electron acceptor character of that molecule. The nature of the charge transfer between NO2 and Au-CNT indicates that holes are injected in the metal decorated carbon nanotube strengthening its p-type semiconductor behavior and lowering further the Fermi level of the system. This explains the decrease in resistance measured in Au-CNT sensors and also applies to the Rh-MWCNT sensors studied here. On the other hand, the interactions of Au-CNTs with benzene are so weak that no significant charge transfer could be computed, suggesting that Au-CNTs are not suitable for detecting benzene [START_REF] Zanolli | Gas sensing with Au-decorated carbon nanotubes[END_REF]. A metal such as Rh, which shows a stronger interaction than Au with CNTs is more suitable for detecting benzene since the detection mechanism relies on the biding of the molecule to the nanoparticle. Benzene is well-known to be an electron donor. Upon benzene adsorption on Rh nanoparticles, electrons are injected into the Rh-CNT system decreasing the concentration of free carriers (i.e., holes), which explains the increase in resistance measured in Rh-MWCNT samples.

The adsorption of a CO molecule on Au-CNT results in a small fraction of electron charge transferred towards CO [START_REF] Zanolli | Gas sensing with Au-decorated carbon nanotubes[END_REF]. The interaction between CO and Au nanoclusters is quite complex [START_REF] Mpourmpakis | Identification of descriptors for the CO interaction with metal nanoparticles[END_REF] since it strongly depends on the overlap of the 5 and 2* orbitals of the CO with the d orbitals of the gold nanoparticle, resulting in a mechanism of electron donation/back-donation (i.e., CO donates electrons from its  orbitals to the d orbitals of gold and the filled d orbitals of Au donate electrons back into the empty  orbitals of CO). A similar mechanism could explain the decrease in resistance observed for Rh-MWCNT sensors in the presence of CO.

The operational principle in many ceramic humidity sensors is physisorption of water molecules on an initially chemisorbed hydroxyl layer. Water physisorption leads to protonic conduction on the surface, which increases the overall conductance of the sensor [START_REF] Traversa | Ceramic sensors for humidity detection: the state of the art and future developments[END_REF]. The process of physisorption onto a hydroxyl layer cannot explain the present results since Rh-MWCNT sensors decrease their conductance when humidity increases. These experimental results are in agreement with those previously reported by Varghese et al. in [START_REF] Varghese | Gas sensing characteristics of multi-wall carbon nanotubes[END_REF], in which DC and AC measurements ruled out water physisorption as the operational principle in MWCNTs. Instead, they suggested that water vapor directly interacts with p-type MWCNTs donating electrons into the valence band and thus, decreasing the number of holes. Additionally, in a recently published paper [START_REF] Zanolli | Defective carbon nanotubes for single-molecule sensing[END_REF], Zanolli and coworkers have reported computational studies on the interaction of water vapor with defective carbon nanotubes (e.g., CNTs with oxygenated vacancies). Water vapor is predicted to mildly interact with defective tubes in which a small charge transfer occurs between adsorbed water molecules and CNTs.

Similar studies should be performed to elucidate the interaction of ethylene molecules with metal decorated CNTs. Fig. 4 shows the response to moisture of the different sensing hybrid materials. Three different cases were considered: the responses when relative humidity changed from 10 to 50%, from 10 to 80%, and from 50 to 80% were measured. Hybrid materials treated in Ar:O2 plasma show a significantly higher humidity response than materials treated in Ar plasma only. In fact, another effect of introducing oxygen during the plasma treatment of CNTs is the modification of their surface polarity resulting in increased hydrophilicity, which enhances the response to moisture. Fig. 4 shows that there is a moderate response when relative humidity changes from 50 to 80%. Furthermore, a sudden 30% change in relative humidity is quite unlikely. Therefore, this fact suggests that the material would suffer from moderate humidity interference provided that sensors are operated in an ambient atmosphere where moisture remains within this range. Fig. 5 compares the response of an Ar:O2 plasma treated Rh-MWCNT sensor to nitrogen dioxide, carbon monoxide and benzene when relative humidity was set either to 10% or 50%. This study was performed for oxygen treated sensors because these were the most responsive to the different species tested. Moisture levels were set to 10% and 50% because sensors significantly responded to humidity changes in this range (see Fig. 4). The absolute values of the slopes of the curves shown in Fig. 5 decrease when humidity increases. In other words, the sensitivity towards these species slightly diminishes when background moisture is raised from 10% to 50%. These results seem to indicate that moisture and the different gases and vapors tested compete for adsorption sites at the surface of CNTs and Rh clusters. The use of a humidity sensor should be considered for compensating the effects of small humidity changes. According to the number of species studied, different concentrations tested and number of replicate measurements performed, every sensor characterized underwent 80 detectionrecovery cycles, which spanned for about one month. No deactivation of Rh nanoparticle activity (i.e., significant response drift or response losses) was observed.

IV. Conclusion

The introduction of oxygen during the plasma treatment of carbon nanotubes leads to the creation of active sites (i.e. oxygenated vacancies) on the surface of CNTs. Additionally, reactive oxygen activates the capping agent of colloidal nanoparticles. Combined, these two effects improve the metal nanoparticle-CNT surface interaction, which results in a higher amount of metal nanoparticles grafted to CNT sidewalls and a more homogeneous decoration of CNTs. For example, the inclusion of oxygen in the Ar plasma leads to nearly doubling the weight of Rh attached to CNTs and preserves the original size of the nanoparticles from the colloidal solution. Furthermore, oxidative treatments affect the density of states of valence bands and increase the work function of MWCNTs making it closer to that of metals such as Rh. As a result, it is easier for electrons to travel between the metal nanoparticles and the CNT, with the direction of charge transfer depending on the gaseous environment.

Therefore, the response of oxygen plasma treated, Rh-decorated MWCNTs to different gases and vapors (e.g. NO2, C2H4, CO or C6H6) is substantially increased. It is worth stressing that the saturation in the response observed with Ar plasma treated, Rh-decorated MWCNTs is solved when oxygen is included in the plasma treatment. The inclusion of oxygen during the plasma treatment turns CNTs hydrophilic and this has an impact in their response to ambient moisture. However, the response of the oxygen-argon plasma treated hybrid materials to humidity changes in the 50 to 80% R.H. range remains moderate.

The CNT treatment and metal decoration employed here uses an atmospheric plasma source. It is a simple, fast and scalable method for producing gas sensitive nanohybrid materials with uniform and well-controlled properties.

3

  Sensors and actuators B: Chemical, 2011, Vol. 160, Issue 1, 974-980, http://dx.doi.org/10.1016/j.snb.2011.09.014

Fig. 1 .

 1 Fig. 1. Typical room-temperature responses of sensors employing plasmatreated Rh-CNTs to nitrogen dioxide: (a) Ar:O2 plasma, (b) Ar plasma; benzene: (c) Ar:O2 plasma, (d) Ar plasma and carbon monoxide: (e) Ar:O2 plasma, (f) Ar plasma.

6Fig. 4 .Fig. 5 .

 45 Fig. 4. Room-temperature response to moisture changes of sensors employing Rh-CNTs either treated in argon or argon-oxygen plasmas.

Sensors and actuators B: Chemical, 2011, Vol. 160, Issue 1, 974-980, http://dx.doi.org/10.1016/j.snb.2011.09.014

Sensors and actuators B: Chemical, 2011, Vol. 160, Issue 1, 974-980, http://dx.doi.org/10.1016/j.snb.2011.09.014

V. Acknowledgments

This work was supported in part by the European Commission under the FP6-NMP grant Nano2hybrids (no. 033311 STREP), and the URV PhD fellowship program. We are thankful to Dr. Isabel Gràcia and Dr. C. Cané from the CNM (IMB-CSIC, Spain) for providing us with the microhotplate transducers and to Prof. Gabor A. Somorjai from the Department of Chemistry (University of California) in Berkeley, for supplying the colloidal solutions of rhodium.

VI. References


Adrien Bussonnière

Yannick Miron

Michaël Baudoin
email: michael.baudoin@univ-lille1.fr

Olivier Bou-Matar

Michel Grandbois

Paul Charette
email: paul.g.charette@usherbrooke.ca

Alan Renaudin
email: alan.renaudin@usherbrooke.ca

Cell detachment and label-free cell sorting using modulated surface acoustic waves (SAW) in droplet-based microfluidics †

We present a droplet-based surface acoustic wave (SAW) system designed to viably detach biological cells from a surface and sort cell types based on differences in adhesion strength (adhesion contrast), without the need to label cells with molecular markers. The system uses modulated SAW to generate pulsatile flows in the droplets and efficiently detach the cells, thereby minimizing SAW excitation power and exposure time. As a proof-of-principle, the system is shown to efficiently sort HEK 293 from A7r5 cells based on adhesion contrast. Results are obtained in minutes with sorting purity and efficiency reaching 97 % and 95 %, respectively.

Introduction

Cell sorting is critical for many biological and biomedical applications such as cell biology, biomedical engineering, diagnostics and therapeutics. Indeed, numerous biological analyses are based on the separation of different cell types harvested from a raw heterogeneous sample such as whole blood. Fluorescence-activated cell-sorting (FACS) 1 and magnetic-activated cell-sorting (MACS) 2 are well established methods for cell and particle sorting known for their high-throughput and specificity. Both methods, however, require pre-processing for cell tagging with markers, an important time and cost expense for some applications.

In contrast, by making use of differences in the intrinsic physical properties of cells (size, density, adhesion strength, stiffness, electrical and optical polarizability), label-free sorting methods do not require molecular tagging. Compared to FACS and MACS, however, the specificity of label-free methods is often limited owing to insufficient contrast in physical properties, thereby restricting their widespread use. As with tagging-based systems, label-free cell-sorting methods have been implemented in microfluidic devices 3 using techniques such as deterministic lateral displacement 4 , hydrodynamic filtration 5 , dielectrophoresis (DEP) 6,7 , optical lattices 8,9 , stiffness separation 10,11 , acoustophoresis 12,13 and adhesion-based sorting [14][15][16] . In some cases, such as for sorting based on ad-hesion to the substrate, performance has been improved with surface nanostructuring 15 and bio-functionalization 16 to enhance adhesion contrast between cell types.

In addition to sorting, the dynamics of cell detachment from solid surfaces is of interest in and of itself, either to harvest cells or to study the mechanisms of cell adhesion to surfaces. Cell dissociation and detachment from a solid substrate is normally achieved by cleaving bonding proteins with trypsin 17 . This process is quite aggressive as cells can be damaged if left exposed to trypsin for too long and a post-treatment rinsing step is required. In contrast, cell detachment based on microfluidic effects alone requires no external agents or rising. Cell detachment under constant fluid shear stresses has been demonstrated using spinning discs 18 , flow chambers 19 and more recently using surface acoustic wave (SAW)-actuated flow [START_REF] Schneider | Chemphyschem : a European journal of chemical physics and physical chemistry[END_REF][START_REF] Hartmann | [END_REF] .

The miniaturization of cell-manipulation methods has led to their integration into lab-on-chip (LOC) platforms, where cell detachment and sorting have been widely investigated in flow-based microchannel formats 19,22,23 . Comparatively few studies [24][25][26][27] , however, have explored cell separation or detachment in droplet-based microfluidics as in digital microfluidics (DMF) [START_REF] Berthier | Microdrops and Digital Microfluidics[END_REF] . Indeed, the physics of microfluidics in droplets are completely distinct from flow-through closed-channels systems. Microfluidics properties such as bulk and surface modes of vibration, which are unique to droplet-based systems, can be exploited to great effect. In general, unlike continuous flow systems which are often optimized for high-volume cellsorting, droplet-based systems are best suited to studies of cell properties in small populations of cells, such as cell adhesion modulation mechanisms which are highly complex and of wide-ranging interest [START_REF] Parsons | [END_REF]30 .

Droplet actuation in DMF is generally accomplished with surface acoustic waves (SAW) [31][32][33] , electrowetting on dielectrics (EWOD) 34 or dielectrophoresis (DEP) 35 . SAW-based DMF have been used in a range of biological applications [36][START_REF] Lyford | Ultrasonics Symposium (IUS)[END_REF][START_REF] Renaudin | [END_REF] to implement functions as diverse as mixing 39 , droplet displacement 40,41 , atomization 42 , as well as for particle and cell manipulation 43 . Though EWOD has been used successfully to manipulate cells 25 , large or strongly-adhering cells are difficult to detach and/or transport with the relatively weak electrowetting forces exerted by EWOD. In the case of DEP, the oscillatory forces applied to the cell are subject to changes in the physical composition of the membrane 44 , which may or may not be desirable depending on the nature of the experiment. The strong electrical fields involved in DEP also have the potential to alter cell membrane characteristics [START_REF] Sengupta | Microfluidic Diagnostic Systems for the Rapid Detection and Quantification of Pathogens[END_REF] .

Recently [START_REF] Bussonnière | Proceedings of Meetings on Acoustics[END_REF] , we presented preliminary results on the use of SAW-based fluid actuation in droplets to detach biological cells from a surface. We showed that under continuous SAW excitation, fully confluent cell layers could be detached en masse at sufficiently high SAW power, whereas isolated cells were very resistant to detachment, even at power levels above the threshold for cell viability [START_REF] Li | [END_REF] . In a previous publication, we showed that the acoustic power required to move or deform droplets with SAW can be significantly reduced by using modulated rather than continuous excitation 48 . Based on this work, we demonstrate here that modulated SAW can be used to viably detach cells from a surface and sort cells based on adhesion contrast, without the need for labeling. The experimental results presented show that two distinct cell types can be separated with a final purity of up to 97 % and an efficiency greater than 95 %. Results are achieved with characteristic processing times on the order of one minute without adversely affecting cell viability or requiring the cell layer to be fully confluent.

Methods and Materials

Apparatus setup and experimental procedure

Experiments were run on cells adhered to the surface of a LiNbO 3 substrate and immersed in 20 µl droplets of phosphate buffered saline solution (PBS).

Rayleigh-type SAW were generated at the surface of the LiNbO 3 substrate by applying a 17.1 MHz sinusoidal radio frequency (RF) excitation to interdigitated transducer (IDT) thin-film metal electrodes. The excitation frequency was selected to maximize the energy transmission from the substrate surface to the liquid. The IDT were designed as "electrode width controlled single-phase unidirectional transducers" (EWC-SPUDT) [START_REF] Hartmann | Proceedings IEEE Ultrasonics Symposium[END_REF] , a configuration that ensures that the acoustic energy is directed solely in the forward direction. The excitation signal was supplied by an RF generator (Agilent, model N9310a) and amplified to 30 dBm using an RF amplifier (Empower, model BBM0D3FEL).

Once transmitted to the fluid, the acoustic waves induce interface stresses and internal flow resulting in deformations of the droplet free surface due to two types of nonlinear effects: acoustic radiation pressure and acoustic streaming 40 . As explained below, cyclic droplet deformations were induced by switching the SAW excitation on and off with an appropriate period and duty cycle, resulting in large shear stresses in the fluid causing cells to detach.

An 8×8 mm2 "cell-attachment zone" was defined by markers patterned onto the LiNbO 3 substrates. The devices were mounted under a phase contrast microscope (Motic, model AE 30/31) with a 10× objective to focus on the cell layer (2 mm diameter field of view). A high-speed 10 bit CMOS camera (PCO, model pco.1200hs) was used to capture video of cell detachment. The setup is depicted in Figure 1.

Images sequences recorded by the camera were processed using ImageJ (NIH, rsbweb.nih.gov/ij) and cell populations were counted using the ImageJ cell counting tool. The two cell types used in the experiments could be easily distinguished in the images based on differences in their morphological characteristics. Following each experiment, cell short term viability assays were performed with Trypan blue exclusion.

SAW Device fabrication

The SAW devices were fabricated from 1 mm thickness Xcut Z-propagating LiNbO 3 wafers (Newlight Photonics). This particular cut was chosen for its efficient electromechanical coupling along both Y and Z perpendicular propagation directions (K z = 4.9 %, K y = 3.1 %), allowing for two-dimensional droplet actuation if required. Indeed, the K 2 values along the two directions are greater than for a typical 128

HEK A7r5

• Y-X cut LiNbO 3 crystal (K x = 5.5 %, K y = 1.2 %).
The metal IDT electrodes and cell-attachment zone markers were fabricated by photoresist (Shipley S18-13, Microchem) spin-coating on the LiNbO 3 wafers, patterning with standard photolithography processes, metal deposition (Ti/Au, 20/200nm), and lift-off.

Cells culture and surface preparation

Two cell lines were used in the experiments: adherent vascular smooth muscle cells (A7r5) and human embryonic kidney cells (HEK 293). These particular cell lines were selected because they have been shown to exhibit surface adhesion strengths comparable to cancerous [START_REF] Plouffe | [END_REF] and other normal 51 cell types. Cell adhesion strength, however, is highly dependent on surface preparation specifics. We chose to adhere cells directly to a bare lithium niobate (LiNbO 3 ), the piezoelectric material most commonly used to generate surface acoustic waves, in order to provide a recognizable point of reference. The adhesion of cells to bare LiNbO 3 has been studied by other groups [START_REF] Rodaite-Riseviciene | The 7th IEEE International Conference on Nano/Molecular Medicine and Engineering[END_REF] . As shown below, the fluid shear stresses required to detach cells from a bare LiNbO 3 surface are in the same range as that reported by others for surface preparations commonly used for cell studies. If required, LiNbO 3 can be readily functionalized for specific surface preparations [START_REF] Seeger | Preparation and Characterization of Antibody Films on Lithium Niobate Surfaces[END_REF][START_REF] Bennès | [END_REF] . Cells were adhered to the device surfaces either by growing the cells directly on the LiNbO 3 substrates in the case of single cell line reference experiments, or by incubating the LiNbO 3 substrates in a solution of pre-grown re-suspended cells in the case of single and dual cell line experiments.

A7r5 and HEK 293 cells were grown separately by seeding in 60 mm petri dishes and cultured in growth media (DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 IU/mL penicillin, 50µg/mL streptomycin, Wisent) under an atmosphere of 5% CO2 at 37 • C for 24h. For experiments requiring cells to be grown directly onto LiNbO 3 surfaces, the LiNbO 3 substrates were placed at the bottom of the petri dish for the duration of the incubation time.

For cells not grown directly onto the LiNbO 3 substrates, single cell line re-suspended cell solutions were prepared by rising the cell cultures in PBS, incubating for 5 min in 500 µl trypsin-EDTA solution at 37 • C, and re-suspending in 2 ml growth medium to stop the trypsin digestion. After 5 min centrifugation, cells were re-suspended in HEPES-buffered salt solution (HBSS) (20 mM Hepes at pH 7.4, 120 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO4, 1.8 mM CaCl2, and 11.1 mM dextrose). Dual cell line re-suspended solutions were prepared by combining HEK 293 and A7r5 re-suspended solutions in experimentally-determined proportions as required for roughly equal numbers of both cell types to adhere to the surface (Fig. 2). LiNbO 3 substrates were placed in a petri dish immersed in re-suspended cell solutions for incubation times ranging from 15 to 90 min to study various adhesion states.

Following cell adhesion by either of the two methods above, the LiNbO 3 substrates were rinsed in PBS, cells outside the attachment zone were dried and mechanically removed, leaving a uniformly distributed 8×8 mm 2 adherent cell layer covered by a thin PBS film. A 20 µl droplet of PBS was added atop the attachment zone using a calibrated micropipette prior to experiments. Note that due to adherent cell secretions, the cell-attachment zone was highly hydrophilic and the droplets spread across the squared-shaped area with a high contact angle and flat profile. The 50 ms SAW excitation bursts displaced the distal liquid-solid contact line (droplet edge furthest from the SAW electrodes) at a rate of a few microns per cycle while the proximal contact line (droplet edge closest to the SAW electrodes) remained pinned due to the high contact angle hysteresis. As a result, the spread in the direction of SAW propagation at a velocity of ∼ 0.01 mm/s. As shown in Fig. 4, this expansion elicited three distinct fluidic regimes in the droplets. Since the camera field of view (vertical column in Fig. 4) was fixed relative to the spreading droplet, the three fluidic regimes swept sequentially across the field of view and thus could be separated in time in the image sequences.

At the start of the experiments, the expansion was sufficiently small so that the droplets essentially oscillated between the two shape extrema shown schematically in Fig. 4b and in photographs of (Fig. 3b). During SAW excitation (Fig. 4b-top), most of the fluid was displaced in a "bulk" zone at the distal end of the droplet whilst leaving behind a thin film "tail", as previously observed by Rezk et al and Collins et al 55,56 . During relaxation, the droplet returned to its symmetric relaxed shape due to surface tension effects (Fig. 4b-bottom).

After a sufficient number of cycles, the distal contact line displacement became significant enough (∼ 0.5 mm) that a 150 ms relaxation interval between SAW excitation bursts was no longer sufficient to return the droplets to a symmetric relaxed shape. As a result, three distinct fluid dynamics regimes (Fig4.c) could be distinguished in the droplets. As before, the extrema were: (1) the bulk (shaded blue area), where shear stresses result from a combination of large scale vortices induced by Eckart streaming 57 and small scale vortices due to Rayleigh and Schlichting streaming 58,[START_REF] Schlichting | Boundary-Layer Theory[END_REF] near the vis-cous boundary layer, and (2) the thin film tail (shaded orange area), where Schlichting and Rayleigh streaming generated by a standing wave (as evidenced by periodic patterns in Fig. 3c) are dominant 55 . In between the two, a transient zone appeared (gray shaded area), swept by a strong oscillating "wavefront" resulting from the fluid transitions to and from the extrema.

As shown in the experimental results below, the rate of cell detachment from the surface was consistently highest in the transient zone. Eventually, as the droplets spread sufficiently, cells in the field of view of the camera were confined to the thin film tail (Fig. 4d, shaded orange area).

τ = µ ∂ v ∂ y ∼ µ V wave f ront h , ( 1
)
where y is the distance perpendicular to the surface, v is the fluid velocity in the direction parallel to the surface at height y, and µ is the dynamic viscosity of the medium (10 -3 m 2 .s -1 for PBS at 20 • C). In this formula, V wave f ront and h denote the surface velocity and the height of the thin film, respectively, estimated from a side-view image sequence (h ∼ 100 µm). Under SAW excitation, the "forward" wavefront velocity was about 100 mm/s, resulting in an estimated vertical shear stress of 1 Pa. When SAW excitation was turned off, the "backward" wavefront velocity was much lower (∼ 10 mm/s), resulting in an estimated shear stress of 0.1 Pa. For comparison, values of shear stresses reported in the literature to detach cells from treated and untreated surfaces with flow-based systems typically lie between 0.01 and 10 Pa. 16,19,[START_REF] Hartmann | [END_REF][START_REF] García | [END_REF] In addition to viscous stresses, pulsatile flow also induces so-called unsteady forces (Added mass). The relative magnitudes of viscous and unsteady forces can be quantified by the dimensionless Womersley number Wo = ωρh 2 µ . In our experiments, Wo was typically ∼ 0.5, meaning that unsteady forces likely also played an important role in cell detachment.

Cell detachment with single cell line (HEK 293)

We first investigated the dynamics of cell detachment using our proposed method in experiments with a single cell line (HEK 293). The experiments sought to compare detachment behavior with substrates prepared with the two protocols described earlier: (1) cells adhered directly to the LiNbO 3 substrates after a 24 h incubation and (2) LiNbO 3 substrates incubated in a solution of re-suspended cells (incubation period of 60 min in this case). In the experiments, ∼ 300 cells were typically adhered initially to the surface in the field of view of the camera.

Because the dynamics of cell detachment from a surface will vary greatly depending on particular conditions, experimental results should be compared in terms of normalized parameters. At a constant rate of detachment, the number of adhered cells will decrease by the same fraction over a time interval of fixed length at any point during the detachment process. As a result, the number of cells adhered to the surface as a function of time, N(t), will follow a decaying exponential profile, N(t) = N 0 e -σt , where N 0 is the initial number of attached cells and σ is a detachment rate parameter. Cell detachment dynamics are characterized here in terms of their normalized rate of detachment from the surface, R(t) : The calculation of R(t) is therefore equivalent to estimating the instantaneous value of the detachment rate parameter, σ , at time, t. Fig. 5 shows measurements of the fraction of adhered cells over time and the calculated normalized detachment rates under cyclic SAW actuation for experiments with HEK 293 cells prepared with the two adhesion protocols the transition times was on the order of ±5 s. The purpose of these experiments was to explore the relative differences in detachment kinetics between the three fluidic regimes and to extract "order of magnitude" information if possible about the dynamics. Indeed, a much broader range of experimental conditions would be required to form any kind of quantitative conclusion specific to these particular cell lines.

R(t) = - 1 N(t) dN(t) dt . (2)
The results in Fig. 5 clearly show that the cell detachment rate is highest in the transient zone. The two graphs show similar maximum rates of detachment, indicating that both cell adhesion methods can yield similar adhesion strengths to the surface (assuming detachment rate is an indication of adhesion strength). The inverse of the maximum normalized detachment rate, 1/max(R(t)), can be considered as the "characteristic time" of the system under maximum efficiency, that is to say, the time taken for the number of adhered cells to fall to (1/e)N 0 at the maximum rate of detachment. From Fig. 5, this value is ∼ 35 s in both cases. The effect of the duration of the transient regime is also interesting to consider: in Fig5-top, the transient regime is longer (∼ 63 s) resulting in a higher detachment efficacy (> 10% residual adhered cells), whereas in Fig. 5-bottom due to a faster spreading of the droplet, the transient regime is shorter (∼ 46 s) resulting in a lower detachment efficacy (∼ 25% residual adhered cells). In both cases, the majority of cells is detached in minutes, which compares very favorably with results from others methods [START_REF] Hartmann | [END_REF]22 .

Cell detachment with dual cel lines (sorting)

We next investigated the selective detachment (sorting) of cell types based on adhesion contrast. LiNbO 3 substrates were prepared by incubation in a dual cell-solution of re-suspended cells line (A7r5 and HEK293) for a range of incubation periods: 15, 25 and 60 min. Cell sorting performance was characterized by 2 parameters, "purity" and "efficiency", calculated once the system had reached equilibrium, i.e. when the number of adhered cells no longer changed (∼ 2 min typically):

Purity = % HEK 293 detached % HEK 293 detached + % A7r5 detached . (3)
Efficiency = % HEK 293 detached .

Fig. 6a shows calculations of cell sorting purity for the three different incubation times. Interestingly, results indicate that purity increases with incubation time. We speculate that this behavior arises because: (1) cells require a certain time to achieve complete adhesion and thereby maximizing adhesion contrast between cell types and (2) it is possible that excretion of extracellular matrix proteins by A7r5 cells negatively modulates HEK 293 adhesion. Indeed, adhesion modulation by competing species has been observed by other groups, such as the improvement of cancer cell (MCF7) adhesion in the presence of human breast epithelial cells (MCF10A) 15 . In all cases, sorting efficiency was greater than 90 % (Fig. 6b). Once detached, cells remained re-suspended in the droplets. Short-term viability assays were performed after experiments both with SAW excitation and without SAW as a negative control. Results indicated that SAW excitation only slightly affected viability (apoptosis rate below 5 %).

Conclusions

In this paper, we purpose a method to selectively and viably detach cells from a solid substrate in fluid droplets using mod-

6 | 1-8
ulated surface acoustic waves (SAW). Experiments were designed to study the effects of different fluid dynamics regimes by using a fixed imaging field of view with respect to droplet expansion under SAW actuation. Results show that cell detachment rate is highest in the middle regime, termed the "transient regime", where viscous shear stresses are estimated to be of the order of 1 Pa. Under the chosen SAW modulation protocol, HEK 293 and A7R5 cells adhered to bare LiNbO 3 surfaces were successfully sorted based on adhesion contrast. Results show that cells were detached in the order of minutes and the contrast in adhesion strength varies with incubation time. This method could be generalized to other cell lines exhibiting either intrinsic or controlled (via surface bio-functionalization) adhesion contrasts. Importantly, cell adhesion strength is highly dependent on surface preparation specifics and adhesion modulation between competing species. Therefore, SAW excitation parameters required to viably detach and sort cell types under different experimental conditions can be expected to vary according to the characteristics of cell lines, surface preparation, and adhesion modulation between competing cell species. Similarly, sorting purity and efficiency are expected to be highly dependent on particular experimental conditions.

Interestingly, modulated SAW could be combined with EWOD to detach strongly adhered cells and enhanced EWOD cell manipulation and sorting performance. Further investigations with a view to optimizing unsteady forces would also be of interest, for example with bi-lateral SAW excitation.

Acknowledgements

Fig. 1

 1 Fig. 1 Schematic diagram of the setup showing the cell layer (red dashed line) immersed in a 20 µl PBS droplet atop a LiNbO 3 piezoelectric substrate. The diagram also shows the interdigitated transducers (IDT), RF amplifier and signal generator used for Rayleigh type SAW generation. Cell visualization is achieved using a phase contrast phase microscope, 10× objective, and CMOS camera.

Fig. 2

 2 Fig. 2 Photograph of vascular smooth muscle cells (A7r5) and human embryonic kidney cells (HEK 293) adhere to the surface of a LiNbO 3 substrate after incubation in a re-suspended cell solution at 37 • C. The arrows indicate typical specimens of HEK 293 and A7r5 cells which exhibit significantly different morphological features.

Fig. 3

 3 Fig. 3 (a) Schematic diagram of the fluid droplet in its relaxed position spread across the 8×8 mm 2 droplet positioning zone atop the LiNbO 3 substrate, in relation to the SAW electrodes (EWC-SPUDT); (b) side-view camera images showing the two shape extrema of the droplet during one SAW excitation modulation cycle (ON: 50 ms, OFF: 150 ms). (c) Zoom on the tail section of the drop showing a standing wave pattern. (d) Flow profile in the tail section and characteristic lengths.

1- 8 | 3 3and discussion 3 . 1

 8331 Results Droplet dynamics and cell-fluid interactionAs stated earlier, the purpose of this work was to investigate the potential of modulated SAW fluid actuation to selectively detach and sort cells in a droplet. Fig.3ashows a schematic diagram of PBS the fluid droplet atop the 8×8 mm 2 droplet positioning zone on the LiNbO 3 substrate, in relation to the SAW electrodes (EWC-SPUDT). Cyclic deformations of the droplets between a relaxed state and a deformed state (sideview camera images shown in Fig.3b-top and Fig.3b-bottom, respectively) were induced by switching the SAW signal on (excitation) and off (relaxation) with an appropriate period and duty cycle. During excitation, internal flow and surface deformation are induced by nonlinear acoustic forces. During relaxation, the potential energy stored as capillary surface energy produces a restoring flow in the opposite direction. Optimal values for the period (200 ms), duty cycle (25 %), and power (30 dBm) were based on the droplet intrinsic relaxation time, measured experimentally.

Fig. 4

 4 Fig.4Schematic diagrams of the different droplet fluid dynamics regimes under modulated SAW actuation (ON: 50 ms, OFF: 150 ms). The vertical bar shows the location of the circular area (2 mm diameter) imaged by the microscope; (a) Relaxed shape of the droplet mainly determined by its wetting properties on the cell-covered LiNbO 3 substrate; (b) Initial shape extrema pair in each excitation/relaxation cycle. (c) After a sufficient number of cycles, a 150 ms relaxation interval between SAW excitation bursts is no longer sufficient to return the droplets to a symmetric relaxed shape of (b). As a result, three distinct fluid dynamics zones can be distinguished: bulk (blue), transient (gray), and thin-film "tail" (orange); (d) Eventually, as the droplet spreads sufficiently, the field of view of the camera is confined to the thin-film tail.

3. 2

 2 Magnitude of the shear stresses and unsteady forces in the transient zone Shear stresses in the fluid acting on the cells were estimated from the image sequences in the transient zone. Assuming noslip boundary condition (null velocity at the liquid-solid interface), the vertical shear stress, τ, in the fluid can be linearly 4 | 1-8 approximated (Fig3.d) by:

Fig. 5

 5 Fig.5Normalized detachment rate and percentage of adhered cells over time under cyclic SAW actuation; TOP: result from a typical experiment with HEK 293 cells adhered directly to a LiNbO 3 substrate after a 24 h incubation; BOTTOM: result from a typical experiment with HEK 293 cells adhered to the substrate after incubation in a solution of re-suspended cells for 60 min.

  (top: direct to LiNbO 3 ; bottom: incubation in re-suspended cells). The three fluid dynamics regimes (bulk, transient and tail) are highlighted by different background colors. Transitions between successive regimes were determined by observation in the image sequence where the top of the wavefront in the transient zone could be clearly seen. The uncertainty on estimations of 1-8 | 5

Fig. 6

 6 Fig. 6 Cell sorting purity (a) and efficiency (b) for LiNbO 3 substrates prepared by incubation in a dual cell-line re-suspended solutions (A7r5 and HEK 293) for a range of incubation periods: 15, 25 and 60 min.

  This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), Créneau Biotech Sant du Projet ACCORD du Ministère des Finances et de l' Économie (MFEQ) du Québec (Canada), Agence Nationale pour la Recherche ANR-12-BS09-021-02 et 01 (France), Direction Générale de l'Armement (France) and Région Nord-Pas-de-Calais (France). The authors would like to thank the Institut Interdisciplinaire d'Innovation Technologique (3IT) at the Université de Sherbrooke (Canada) for technical support.

| 1-8