Melissa K Stanfield

Daniel J Eyckens

Jérôme Médard

Philippe Decorse

Jean Pinson

Using redox active

Keywords: Aryldiazonium, Surface Modification, Composite, Anthraquinone, Carbon Fibers

Introduction

Carbon fibers are well known for their desirable properties, for example, high strength, light weight, corrosion resistance, conductivity, and negligible thermal expansion, and as such have been used in a myriad of applications. [1] In another guise, carbon fibers are also receiving a great deal of attention for their suitability as capacitors, structural batteries, and as a material to remove pollutants for environmental remediation. [1][2][3] As a form of carbon, these fibers have a relatively low surface area compared to porous carbon black, graphene, carbon nanotubes and carbon nanofibers, and these latter materials have been used extensively for energy storage applications. [2] Though this is counteracted to an extent by their ability to be easily woven and processed via well-established textile processes such as weaving and braiding into complex shapes. The manufacture of carbon fibers, the modification of their surface, and the corresponding effect on macroscopic properties, has been an area of intense investigation for decades. [3,4] This has included the use of nanomaterial deposition, [5] ceramic precursor materials, [6] the use of various matrices, [7,8] and the targeted application of composites to specific environments. [9,10] Of the resins commonly used, and that which is used within this report, are thermoset polymers usually highly cross-linked and derived from epoxide containing monomers. Due to their common presence within composites, large research efforts have been put towards optimising their adhesion to various materials, including carbon fibers, [11] metals, [12,13] and naturally occurring fibres to generate green composites. [14] Moreover, the use of other additives, such as nanomaterials (graphene, nanoparticles, etc.) these resins can be made to possess interesting functional properties. [15][16][17] The choice to employ carbon fibers as the material of focus within this study was based on our historical experience. Also, the ability to fabricate carbon fibers with multi-functionality would have immediate impact on modern structures due to the widespread acceptance and use of carbon fiber reinforced composites. There have been numerous reports using the surface modification of carbon fibers with conducting polymers, [4][5][6][7][8][9] and electrolytes for use in structural batteries, though these are not typically accompanied by the evaluation of how these coatings influence fiber-to-matrix adhesion. This latter point is a critical aspect of all composites, and while it has been extensively researched for structural composites, the interfacial adhesion effected by conductive or redox active species on the fibre surface remains poorly understood. Recent work by Asp et al. showed that the interfacial adhesion for structural battery interfaces was reduced compared to commercial sizings in a vinyl ester resin, but was still viable for application in the field. [2] The area of structural batteries faces a myriad of challenges, which are being addressed, though balancing the trade-offs between efficiency, performance, and process-ability remain significant.

The ability of a material to undergo multiple electron processes in reduction-oxidation (redox) cycles has been used as a means to enhance the electrical performance of carbon materials for energy applications. Commonly multi-carbonyl compounds such as anthraquinone, [10][11][12] oxalate, [13] and organometallic species such as ferrocene [14,15] are used to functionalise the carbon surface. Work from our group has focused intently on the use of aryldiazonium salts as a facile surface manipulation strategy, largely centred on carbon fibers. [16][17][18][START_REF] Servinis | A novel approach to functionalise pristine unsized carbon fibre using in situ generated diazonium species to enhance interfacial shear strength[END_REF][START_REF] Servinis | Tailoring the fibre-to-matrix interface using click chemistry on carbon fibre surfaces[END_REF][START_REF] Servinis | A Novel Approach to the Functionalisation of Pristine Carbon Fibre Using Azomethine 1,3-Dipolar Cycloaddition[END_REF] The electrografting of these aryldiazonium salts occurs via the irreversible reductive cleavage of the Aryl-N 2 + bond to give the corresponding aryl radical and nitrogen gas. This highly reactive aryl radical is generated in close proximity to the carbon surface and thus is tethered to the surface via strong covalent C-C bonds. [22] This study examines the use of anthraquinone (AQ) films as surface modifications for carbon fiber and determination of the corresponding effect on interfacial adhesion (Figure 1). Also examined within this study is the possibility to use the conductive AQ film as a means to initiate and grow a second polymeric layer on the first layer from the fiber surface. The desire to grow a second layer of polymer to the carbon fiber surface required a species within the initial layer able to reduce aryl diazonium salts to initiate radical polymerization. Thus, AQ served as an ideal candidate for the initial conducting layer as it possesses a reduction peak on carbon fiber (vide infra) at approximately -1.0 V (vs. Ag/AgCl in ACN). Thus, it is able to easily reduce aryl diazonium salts, which typically possess a broad irreversible reduction peak centred at -0.3 V (vs. Ag/AgCl). [18] The ability to selectively install multiple surface chemistries as desired on the surface of carbon fibers will make significant gains towards the generation of gradient interface/interphases for optimal performance and the development of functional composites.

Figure 1 A schematic representation of the investigation undertaken within this work. The use of a redox active layer present on carbon fibers as a mediator to develop multilayered functionality in a composite.

Experimental Design

Reagents

See supporting information (SI) for carbon fiber and gold sputtered silica wafer preparation. The modified surfaces will be referred as Au-AQ or CF-AQ for a gold or carbon fiber surface modified by anthraquinone groups, respectively. Similarly, Au-AQ 20 -COOH for the bilayered film on gold and CF-AQ 20 -COOH for carbon fibers, with the subscript indicates the number of scans used for grafting, for example: CF-(AQ) 5 .

All chemicals were purchased from Sigma Aldrich (Australia) or Oakwood Chemical (USA), unless synthesised as outlined below.

Synthesis of Aryl-diazonium tetrafluoroborate (Aryl-N 2 )

The diazonium salts were prepared from corresponding aniline according to procedures in the literature. [START_REF] Allongue | Covalent Modification of Carbon Surfaces by Aryl Radicals Generated from the Electrochemical Reduction of Diazonium Salts[END_REF] Nitrosonium tetrafluoroborate (504 mg, 4.34 mmoL) was added to MeCN in a 2-neck RBF under inert atmosphere and the solution was cooled to -40 °C. 4-Nitroaniline (500 mg, 3.62 mmol) or 2-aminoanthraquinone (500 mg, 2.23 mmol) was added and stirred for 30 minutes. The mixture was warmed to room temperature over the following hour. Precipitation and collection of the desired products from diethyl ether (0 °C), gave the desired compound, determined to be analytically pure by 1 H NMR and 19 F NMR.

Electrochemical Grafting

The electrochemical measurements and grafting procedures were carried out on an Autolab PGSTAT204. The working electrode was either a carbon fiber tow or gold coated silica wafer fixed in the cell using copper tape and an alligator clip. A leak-less silver/silver chloride reference electrode (ET069-1, eDaq) and a platinum mesh (area = 2.5 cm 2 ) counter electrode were also used.

Measurements were recorded through NOVA software. Electrochemical functionalisation was carried out as reported in the literature. [START_REF] Ceccato | Using a Mediating Effect in the Electroreduction of Aryldiazonium Salts To Prepare Conducting Organic Films of High Thickness[END_REF] The grafting solution consisted of 2 millimolar AQ-N 2 in dry MeCN with 0.1 molar TBAPF 6 as the supporting electrolyte. Once the electrografting was finished, the gold electrodes were washed and sonicated in ethanol for 60 seconds and dried under a stream of nitrogen. The modified carbon fibre tow was washed with acetone, dichloromethane, ethanol, and allowed to air dry.

Fourier Transform Infrared Spectroscopy

A Bruker LUMOS FTIR using ATR mode was used to obtained infrared measurements, using 256 scans and a scan resolution of 4 cm -1 . To process the data, Bruker OPUS 7.0 software was used.

Atomic Force Microscopy (AFM)

AFM data was obtained using a Bruker Multimode 8 AFM instrument with a Bruker FMV-A tip model (tip radius 8 nm, spring constant nominally 2.8 N/m). Contact tapping mode was used to perform the experiments. NanoScope Analysis (Bruker, version 1.5) was used to process the acquired data.

Ellipsometry

The film thickness of each sample was calculated using a M-2000XI Spectroscopic Ellipsometer (J.A.

Woollam Co., Inc., Lincoln, NE, USA). Three measurements were taken from each sample at angles of incidence of 60°, 65°, 70° and 75° at wavelengths of 210-1685 nm. Film thicknesses were calculated by fitting a B-Spline model on top of a silicon substrate with native oxide layer using CompleteEASE software version 4.92 (J.A. Woollam Co., Inc., USA).

X-ray Photoelectron Spectroscopy (XPS)

XPS measurements were performed using a K-Alpha + spectrometer (Thermo Fisher Scientific, East-Grinstead, UK) fitted with a microfocused and monochromatic Al Kα X-ray source (1486.6 eV, spot size: 400 μm, 12kV, 6mA as emission intensity). The pass energy was set to 150 and 40 eV for the survey and the high-resolution spectra, respectively. The spectra were calibrated against the C-C/C-H C1s component set at 285 eV. The chemical composition was determined with version 5.9902 Avantage software by using the manufacturer sensitivity factors. The K-Alpha + system is equipped with an Argon MAGCIS (Mono-Atomic and Gaz Cluster Ion Source, Ar + and Ar n + ) dual ion source. To performed profile analysis, we have used accelerated Ar 2000 + gas cluster ion beam at an energy of 4000 eV, for specified ionic bombardment durations and with an ion beam size leading to a crater close to 4 mm 2 . The ion gun was placed at an incidence angle of 30° with respect to the surface of the samples, with no rotation. During ion beam bombardment, the X-ray source was blanked.

Water Contact Angle (WCA)

Water contact angles of modified gold wafers used a custom-built angle goniometer and a water droplet volume of ~ 3 μL. The measurements were collected over a 10-second time span at a 0.66 second intervals. Determination of WCA was carried out automatically, using software (SFECam2008, KSV Instruments Ltd., Helsinki, Finland). Multiple measurements (minimum of 3)

were taken from each sample, the mean angle was obtained from the data set and used as the final value for the measurement.

Determination of interfacial shear strength (IFSS)

To determine the mechanical properties of the composite with modified fiber, interfacial shear strength was employed. The methods used are consistent with our and other's previously published work (refer to SI).[25]

Statistical Analysis

A Student's T-test was undertaken to determine statistically significant changes in data. This assumed equal variance and a P-value of less than 0.05 was considered statistically significant. To denote statistical significance throughout the manuscript we have used asterisks (*).

Results and Discussion

Electrografting of AQ on gold and carbon fibers.

Grafting of gold surfaces with the diazonium salt of 2-aminoanthraquinone (AQ-N 2 )

Initially, preliminary experiments were carried out on flat gold wafers, as this is a common practice for surface modifications and allows for easy comparison to the literature. While a gold surface may be considered a poor representation of the carbon fiber surface, it is worth noting that our previous work in the covalent sizing of carbon fibers used a similar adaptation of electrochemical procedure from gold to carbon with great success. [16] In particular, the use of a flat, gold surface allows the collection of better IRRAS spectra, and grants a greater ability to measure film thickness than carbon fiber surfaces.

The electrochemical grafting of the diazonium salts of 1-and 2-aminoanthraquinone (as the corresponding tetrafluoroborate salt) has been investigated, in MeCN. [12,[START_REF] Ceccato | Using a Mediating Effect in the Electroreduction of Aryldiazonium Salts To Prepare Conducting Organic Films of High Thickness[END_REF][START_REF] Mooste | Electrochemical properties of gold and glassy carbon electrodes electrografted with an anthraquinone diazonium compound using the rotating disc electrode method[END_REF][START_REF] Mooste | Oxygen reduction on thick anthraquinone films electrografted to glassy carbon[END_REF][START_REF] Chernyy | Elucidation of the Mechanism of Redox Grafting of Diazotated Anthraquinone[END_REF][START_REF] Assresahegn | Advances on the use of diazonium chemistry for functionalization of materials used in energy storage systems[END_REF] The reduction of the diazonium salt on a gold electrode is observed at 0.18 V/SCE that disappears on the second scan;

it is followed by the first reversible wave of anthraquinone at E° = -0.93 V/SCE corresponding to the formation of the radical anion (Figure 2, AQ •-). This wave is followed by a second at more negative potential E° = -1.59 V/SCE corresponding to the formation of the dianion (Figure 2, AQ 2-). [12] Figure 2 The reduction of anthraquinone in aprotic and protic medium.

In this medium, with anthraquinonediazonium and 4-nitrobenzenediazonium, a very interesting phenomenon was reported by Daasjberg et al. [12,[START_REF] Ceccato | Using a Mediating Effect in the Electroreduction of Aryldiazonium Salts To Prepare Conducting Organic Films of High Thickness[END_REF][START_REF] Chernyy | Elucidation of the Mechanism of Redox Grafting of Diazotated Anthraquinone[END_REF] upon repetitively scanning the diazonium reduction wave and the first wave of anthraquinone. The latter increases upon repetitive scanning, indicating the repeated grafting of anthraquinone groups, generating a covalently bound film composed of many AQ units. This corresponds to thicker films up to 50 nm. This reduction of the anthraquinone moiety to its radical anion permits the transfer of electrons to the surface of the film, and therefore, the reduction of more diazonium salts, resulting in the growth of the film. This is a very interesting method for obtaining thick films with a redox behaviour.

In our work, the diazonium salt of 2-aminoanthraquinone (AQ-N 2 ) was grafted to a gold electrode in to 0.1 M TBAPF 6 in MeCN solution (scanning between 0.1 and -1.5

V/(Ag/AgCl)), obtaining sample Au-AQ 20. After grafting of the gold electrode, rinsing and transfer to a fresh electrolytic solution (0.1 M TBAPF 6 in MeCN) the reversible voltammogam of Au-AQ, is observed at E° = -0.89 V/(Ag/AgCl) (Figure 3) in good agreement with the redox potential of AQ itself in MeCN: E° = -0.93V/SCE. [START_REF] Lehmann | Anomalous behavior in the two-step reduction of quinones in acetonitrile[END_REF] Once films were grafted to the gold surface, we measured the thickness of the film obtained by repeated reduction of AQ-N 2 , in MeCN (scanning between 0.1 and -1.5 V/(Ag/AgCl)). The film thickness was measured to be 14±1.03 nm, and it is worth noting that in our previous experience in the translation of surface modification from gold plates to carbon fibers, film thicknesses measured on gold are magnified on carbon fibers. This was described in our previous work, where the thickness of an electrochemical initiation of a covalent polymer was measured at ~150 nm on a metallic surface, but was increased to at ~250-350 nm on carbon fibers. [16,[START_REF] Deniau | Surface Electroinitiated Emulsion Polymerization: Grafted Organic Coatings from Aqueous Solutions[END_REF] This is attributed to the gold surface being effectively 2-dimensional when compared with the typical curved and striated surface of carbon fibers, though faster electron transport may also be responsible.

Grafting of Carbon Fibers with AQ-N 2

The films of anthraquinone were achieved using 5 and 20 cycles (scan rate = 1.0 Vs -1 , from 0.1 to -1.5

V/ (Ag/AgCl), behind the peak of anthraquinone), in 0.1 M TBAPF 6 /MeCN (CF-AQ 5 ). These modified carbon fibers were carefully rinsed and transferred to an anhydrous solution of 0.1 M TBAPF 6 /MeCN. The reversible voltammogram of CF-AQ 5 (Figure 4) presents a partly reversible voltammogram, compared to that of the AQ on a gold surface. This disparity in reversibility is likely due to the inherent presence of -COOH groups on the surface of the carbon fiber, installed during manufacture, that protonate the radical anion. Despite only partial reversibility, the presence of these peaks affirms for the successful grafting of anthraquinone moieties to the fiber surface. In search of thicker films, the number of scans was increased to 20 sweeps (CF-AQ 20 ). The FT-IR spectrum (Figure SI3) of CF-AQ 20 shows strong peaks appearing at 1726 cm -1 , significantly higher than 2aminoanthraquinone itself, which presents carbonyl bands at 1689 cm -1 . It is possible that this peak is a combination of the carbonyl of the grafted AQ species and the carbonyl of the native carboxylic acids present on the fibers, as has been reported previously. [18] Notably, the absence of signal in the 3000-3500 cm -1 region indicates that there are no OH groups and that the film is entirely formed of anthraquinone groups, as hydroquinone presents a strong band at 3262 cm -1 due to the presence of phenolic moieties. Taken together, these data support the successful grafting of AQ to the fiber surface under two different conditions (CF-AQ 5 and CF-AQ 20 ). Unfortunately, the innate curvature and striated surface of the fibers (functionalized or otherwise) prevents the determination of film thickness using such techniques as ellipsometry for comparison with the cyclic voltammograms. With these in hand, our focus turned to assessing the physical characteristics of the fibers to ensure that their fundamental and desirable properties had not decreased as a result of surface modification. The pristine fibers (unsized and not surface treated) were found to have a tensile strength 3.71±0.07 GPa, and a Young's modulus of 262.6±0.73 GPa. Functionalization to give the CF-AQ 5 fibers returned a tensile strength of 3.70±0.11 GPa and a Young's modulus of 265.4±5.56 GPa (Figure 5). Thus, showing no degradation of the fiber properties as a result of this surface treatment, only the introduction of redox properties. Interestingly, the tensile strength of the CF-AQ 20 fibers was statistically indistinguishable (3.75±0.06

GPa) to control fibers, though surface modification had occurred at the expense of stiffness, as this had been reduced to 251.0±3.37 GPa. Despite this slight reduction (~5%) in fiber stiffness, these fibers are still suitable as polymer reinforcement and thus suggests that as the functionalization process had minimal impact on fiber properties. Finally, the determination of interfacial shear strength of the functionalized fibers was undertaken using the single filament fragmentation test (SFFT) in an epoxy resin, as per our previous publications. [START_REF] Arnold | Enhancing interfacial shear strength via surface grafting of carbon fibers using the Kolbe decarboxylation reaction[END_REF][START_REF] Stojceveski | Effect of Tow Size and Interface Interaction on Interfacial Shear Strength Determined by Iosipescu (V-Notch) Testing in Epoxy Resin[END_REF][START_REF] Eyckens | Fiber with Butterfly Wings: Creating Colored Carbon Fibers with Increased Strength, Adhesion, and Reversible Malleability[END_REF] The pristine fiber samples showed a very poor IFSS (18.1±0.7 MPa), as would be expected, as this sample had not undergone any surface treatment or sizing.

When evaluating IFSS for the CF-AQ 5 and CF-AQ 20 functionalized fibers, significant increases in IFSS of 64% (29.6±1.8 MPa) and 151% (45.4±6.8 MPa), respectively, were observed (Figure 6). The increased IFSS for CF-AQ 20 was attributed to the thicker film of small molecules grafted to the fiber surface enhancing the degree with which these grafted species penetrated into the interphase. Previously, we have shown that a large degree of the IFSS increases observed are the result of the energy expended by the lateral drag of the surface tethered molecules through the composite interphase. [START_REF] Randall | Designing carbon fiber composite interfaces using a 'graft-to' approach: Surface grafting density versus interphase penetration[END_REF][START_REF] Eyckens | Using molecular entanglement as a strategy to enhance carbon fiber-epoxy composite interfaces[END_REF] This effect is quite sensitive to thickness and degree of penetration, consistent with the thicker films resulting in greater enhancement of this effect. In this instance, this would be complemented by the surface grafted molecules possessing both fully conjugated aromatic structure and the presence of the carbonyl (ketone) units would potentially interact with the epoxy by hydrogen bonding. With this encouraging data in hand, our attention turned to the sequential modification of the carbon fiber surface to generate multi-layered surfaces which could potentially imbue the carbon fibers with unique functionality while not compromising on adhesion. While these anthraquinone units do not, in this instance, compromise on adhesion, their physical penetration into the epoxy interphase may compromise their electrochemical activity. Thus, our interest was to grow an additional layer of polymer on top of the anthraquinone layer to introduce a spacing unit between the resin and the fiber surface. Recently, following the work of Deniau, [START_REF] Tessier | Surface Electroinitiated Emulsion Polymerization (SEEP): A Mechanistic Approach[END_REF] we described the surface modification of carbon fibers that had been subjected to an in situ polymerization process in aqueous acidic solution. In that instance, the growth of poly(acrylic acid) had been effected on the surface of the carbon fibers, leading to IFSS being improved by significantly (i.e. > 200%). [16] Therefore, we hypothesized that these AQ modified fibers could be utilized in initiating this same polymerization, then this would result in a layered interface (Figure 7), significantly increasing IFSS. Additionally, this concept would serve as a platform upon which solid or polymeric electrolytes could be covalently linked to carbon surfaces for enhanced performance in structural batteries and capacitors.

Electro grafting of a bilayer AQ /acrylic acid on gold and carbon fibers

Figure 7 Using the AQ layer as a means to initiate the reduction of a second aryldiazonium salt in the presence of acrylic acid to generate a layered interface. This process occurs by using the redox activity of AQ to reduce the incoming 4-nitrophenyldiazonium salt (Step A), which can both attach to the already installed AQ layer (Step B), or diffuse into the solution to initiate the polymerization of acrylic acid (Step C and D), which then diffuses to the surface of the fiber to covalently bind to the modified surface (Step E). Note that during this process a portion of the nitrophenyl groups are concomitantly reduced to the corresponding NH 2 group (in the presence of COOH groups of acrylic acid). The exact point of this occurring is unknown and thus the above schematic is illustrative only.

Diazonium salts are stable in an acidic medium, but the reduction of anthraquinone takes place through a reversible 2e -+ 2H + as presented in Figure 2. In aqueous solution, on an edge plane pyrolytic graphite (EPPG) electrode, the E° of AQ-SO 3 H shifts with pH. [START_REF] Gamage | Ultraviolet-visible and electron paramagnetic resonance spectroelectrochemical studies of the reduction products of some anthraquinone sulphonates in aqueous solutions[END_REF][START_REF] Batchelor-Mcauley | Voltammetric Characterization of DNA Intercalators across the Full pH Range: Anthraquinone-2,6-disulfonate and Anthraquinone-2sulfonate[END_REF][START_REF] Batchelor-Mcauley | Voltammetric Responses of Surface-Bound and Solution-Phase Anthraquinone Moieties in the Presence of Unbuffered Aqueous Media[END_REF] When 2aminoanthraquinonesulfonate is grafted to an EPPG electrode, the reversible system shifts from approximately -0.2 V/SCE at pH 0 to -0.9 V/(Ag/AgCl) at pH 14. [START_REF] Batchelor-Mcauley | Voltammetric Characterization of DNA Intercalators across the Full pH Range: Anthraquinone-2,6-disulfonate and Anthraquinone-2sulfonate[END_REF][START_REF] Batchelor-Mcauley | Voltammetric Responses of Surface-Bound and Solution-Phase Anthraquinone Moieties in the Presence of Unbuffered Aqueous Media[END_REF] For Glassy Carbon (GC) modified electrodes with 1-or 2-aminoanthraquinone groups, a similar behaviour is observed and a reversible wave is observed at E°~ -0.15 V/SCE in acidic medium . [START_REF] Weissmann | Electrochemical study of anthraquinone groups, grafted by the diazonium chemistry, in different aqueous media-relevance for the development of aqueous hybrid electrochemical capacitor[END_REF] In 0.05 M H 2 SO 4 , the reduction wave of the diazonium salt (AQ-N 2 ) has been observed at 0.24 V/(Ag/AgCl).

An electrode was modified in an aqueous acidic (0.01 M H 2 SO 4 ) solution of AQ-N 2 (2 mM). After grafting the electrode was rinsed and transferred to 0.1 M TBAPF 6 in MeCN solution to check the redox activity of the aqueous grafted AQ, the reversible voltammogam of Au-AQ, is observed at E° = -0.82 V/(Ag/AgCl) consistent with earlier observations within this work and as reported for AQ in MeCN (E° = -0.93 V/SCE; Figure SI2). [START_REF] Lehmann | Anomalous behavior in the two-step reduction of quinones in acetonitrile[END_REF] This shows that when grafted in 0.01 M H 2 SO 4 the AQ moiety retains its redox behaviour, therefore facilitating the first step (Figure 7, Step A) of the proposed polymerization mechanism; the reduced form AQH 2 being able to reduce the diazonium salt

AQ-N 2 .[30]
Therefore, grafting with 4-nitrophenyldiazonium tetrafluoroborate (2 mM) and acrylic acid (0.7 M) (-1.5 V vs. Ag/AgCl) was conducted, as per our previously reported procedures. [START_REF] Randall | Expanding the scope of surface grafted polymers using electroinitiated polymerization[END_REF] A cyclic voltammogram shows the reversible system of AQ followed by the reduction of the nitrophenyl group and the reduction of H + to H • at more negative potentials (Figure SI4).

The reaction was also performed on 1×1 cm gold surface to obtain surface data such as FT-IR, film thickness, and water contact angle, which as mentioned previously are challenging on carbon fiber surfaces. An Au-AQ 20 plate was modified under the above conditions. Note that at this final potential (-1.5 V/(Ag/AgCl)) in acidic aqueous medium both AQ and 4-nitrophenyl groups are reduced to AQH 2 and 4-aminophenyl groups (Figure 7). Analysis of this double layered electrode Au-AQ 20 -AA revealed that the top film (polyacrylic acid) was 8 ± 3.4 nm, and the bottom film 14 ±1.0 nm, giving a total film thickness of approximately 22 nm. Analysis by FTIR (Figure 8) showed the presence of carbonyl peaks at 1677 cm -1 that includes both the C=O signal of acrylic acid (1705 cm -1 ) and anthraquinone (1680 cm -1 ).

Figure 8 FT-IR analysis of the double layered electrode (Au-AQ 20 -AA), the strong presence of a two carbonyl peaks carbonyl peak at 1677 cm -1 corresponding to both acrylic acid and AQ. * The 1323 cm -1 and 1293 cm -1 band correspond to C-N group and the -NO 2 signals are not observed.

The band at 1593 cm -1 is assigned to the vibration of the aromatic band of anthraquinone, as the anthraquinone itself appears at 1592 cm -1 .

[44] The -NO 2 signals are not observed due to their reduction, but COOH (from acrylic acid), OH (from AQH 2 ) and NH 2 (from the reduction of nitrophenyl groups) bands are observed in the 3000-4000 cm -1 range. To corroborate that the surface modification process had been successful, atomic force microscopy (AFM) was conducted on this multi-layered surface. The formation of a film is clearly observed in the AFM images of the modified gold surface (Figure 9). The main characteristic of this image is the uniform aspect of the film and the absence of obvious pinholes at this scale (1 µm scale with a grainy surface and an RMS roughness of 2.2 nm), though of course this does not preclude their presence on a smaller scale (vide infra). Wavenumber (cm -1 ) Abs Using water contact angle (WCA) as an alternative means to determine the nature of the outermost layer was useful as the AQ and poly(acrylic acid) layers should possess vastly different surface polarities. Indeed, the Au-AQ 20 functionalized electrode gave a WCA of 67.1±4.8°, while after the growth of the second acrylic acid polymer layer this was significantly decreased to 51.0±5.3°, as would be expected, due to the increased polarity of poly(acrylic acid).

Finally, to ensure that the installation of a second polymer layer on top of the AQ priming layer, does not hinder its redox behavior, the cyclic voltammetry was examined. The Au-AQ 5 -AA electrodes were then transferred to an MeCN with 0.1 M TBAPF 6 solution and analyzed using cyclic voltammetry.

This revealed (Figure 10) that the underlying AQ moieties still present a redox behavior E° = -0.95 V (vs. Ag/AgCl), despite the presence of a bonded poly(acrylic acid) phase that could provide protons, note however that the electron transfer is slower Ep = 0.25 mV by comparison to Figure 3 Ep = 0.18 mV. A slight shift to more negative potentials was observed relative to our previous examination (Figure 3). This is not unexpected given that the chemical environment of the electrode surface has been significantly changed and the structure of the AQ is likely to have been altered during the grafting of the 4-nitrophenyldiazonium reduction (Figure 7). In the N1s spectrum, the only discernible peak is located at 400 eV and not the expected 406 eV, typical of highly oxidized NO 2 groups. The absence of this peak is due to the in situ reduction of the NO 2 group during grafting and under the XPS beam. Indeed, under identical XPS conditions the reduction of NO 2 to NH 2 is possible, in this instance it seems that the abundance of acidic protons from the poly(acrylic acid) layer favoured this reduction to all observed nitrogen in the film. With this baseline spectrum in hand, the depth-profile of the dual layered film was examined.

The surface of the gold electrodes was etched for intervals of 30 seconds with Ar n + argon clusters and an XPS spectrum was recorded at each interval. Assuming that gold electrode possesses two layers of polymer, it would be expected that the C1s (289 eV, COOH) peak would be detected in a high concentration initially. Though, as erosion of the top layer occurs, the relative amount of C=O contribution from the carboxylic acid should decrease relative to the other constituents, such as the C=O contribution from the AQ and the Au4f surface.

Analysis of the XPS profile data shows that is indeed what is observed (Figure 11), the signal attributable to acrylic acid decreases while the C=O signal of AQ remains constant (i.e. relative ratio increases). Also as would be expected, the Au4f signal also increases with increasing etch time. This data, in addition to the WCA and FT-IR data described above, is consistent with the installation of a dual layer polymer on the surface of the gold electrode.

With confidence in the ability to install a secondary layer on top of the initial AQ film, we focused on applying this methodology to carbon fibers. In doing this we noted that (after installation of the second layer) the fibers became rigid, similar to commercially available fibers which possess a heavy thermoplastic sizing, an effect we have observed previously, when covalently grafting the fibers with only poly(acrylic acid). relative to the pristine fibers (3.71±0.07 GPa), a 15% increase, and retained a similar Young's modulus (256.05±2.43 GPa) (Figure 12). This significant increase in tensile strength, we propose, is due to the growth of the polymer from the surface of the carbon fibers. Unlike a sizing or coating applied to the fiber surface, the approach used in this work uses the innate conductivity of the carbon fibers to initiate the polymerization process. As such, polymerization can initiate from within defects within the fiber structure, this serves to reinforce these defects, therefore providing less stress concentrations for fiber failure to initiate when under tension. Interestingly, when evaluating the IFSS of these fibers (Figure 13) , the CF-AQ 5 -AA sample showed an additional 90% improvement relative to the parent AQ 5 fibers, a cumulative improvement of 153%, relative to pristine fibers (45.7±9.4 MPa vs. 18.1±0.7 MPa, respectively). Surprisingly, the CF-AQ 20 -AA, possessed a similar IFSS compared to just the AQ 20 functionalized sample, prior to polyacrylic acid growth. While this is counter-intuitive, we attributed this result to the thicker AQ films slowing down the electron transfer to the nitrophenyl diazonium salt, thus limiting the extent to which poly(acrylic acid) is growth from the surface. This outcome is advantageous for the processing of carbon fiber via this methodology in a continuous line, as the initial installation of the AQ conductive layer using only 5 sweeps can be carried out very rapidly. A similar grafting procedure was attempted on pristine carbon fibers, taking the potential back to -2.1 V (vs. Ag/AgCl) for both 5 and 20 cycles. This potential is much more negative than the onset of reduction of H + in acidic aqueous solution for the AQ 5 and AQ 20 samples and was investigated for the potential to improve electron transfer through the grafted AQ films. These samples were then subjected to the same polymerization conditions (again using -2.1 V (vs. Ag/AgCl)) to graft the polyacrylic acid to both, unfortunately no noteworthy results were observed compared to this work at lower reductive potentials. While disappointing, this outcome defines a working potential that would be suitable for these materials as applied to structural energy storage applications.

Conclusions

In conclusion, we have surface modified carbon fibers, in acidic aqueous solution, with anthraquinone films, able to undergo multiple redox processes. Carbon fibers with reversible oxidation-reduction batteries. [START_REF] Weissmann | Electrochemical study of anthraquinone groups, grafted by the diazonium chemistry, in different aqueous media-relevance for the development of aqueous hybrid electrochemical capacitor[END_REF] We show that the grafting of these AQ units using varying the number of potential sweeps can lead to significant gains in IFSS. Further we demonstrate the ability to use this conductive film as an anchor for the covalent attachment of a secondary polymer, in this case polyacrylic acid, to the carbon fibers. This was initially shown on a gold electrode, and characterized using FT-IR, water contact angle, and depth-profiling XPS. Adaptation of this process to the AQ functionalized fibers conferred an additional 90% improvement in IFSS to fibers bearing just the surface grafted AQ.

Attempts at using a more aggressive surface modification protocol, in the form of more extreme reductive potentials, highlighted a deleterious effect on IFSS, attributed to the degradation of the surface bound moieties.

Figure 3

 3 Figure 3 Reversible redox peak of the grafted anthraquinone moiety on a gold wafer (Au-AQ 20 ).

Figure 4

 4 Figure 4 Cyclic voltammetry of CF-AQ 5 transferred to 0.1 M TBAPF 6 in MeCN solution (vs. Ag/AgCl), scan rate = 1.0 Vs -1 .

  vs. Ag/AgCl))

Figure 5

 5 Figure 5 Tensile strength and Young's modulus of pristine, CF-AQ 5 and CF-AQ 20 surface modified carbon fibers. Error bars show standard deviation, error bars for pristine sample (Young's Modulus) is too small to be seen, * denotes statistical significance (P<0.05).

Figure 6

 6 Figure 6 The interfacial shear strength of CF-AQ 5 and CF-AQ 20 surface modified carbon fibers. Error bars show standard error, * denotes statistical significance (P<0.05).

Figure 9 A

 9 Figure 9 A) AFM sampled area; B) Height variations along AFM tip course; C) Distribution of events versus topography; D) Water droplet on Au-AQ 20 surface; E) Schematic of the surfaces being examined by water contact angle, and the corresponding angles; F) Water droplet on Au-AQ 20 -AA.

- 1 .Figure 10

 110 Figure 10Cyclic voltammogram showing the retention of redox activity for the AQ groups tethered to the fiber surface after a poly(acrylic acid) layer has been grafted on top. Cyclic voltammogram of Au-AQ 20 -AA in 0.1 M TBAPF 6 in MeCN (vs. Ag/AgCl), Scan rate = 1.0 V s -1 , reversible wave is observed at E°~ -0.95 V (vs. Ag/AgCl).

[ 16 ]Figure 11 D

 1611 Figure 11 Top: generic representation of the gold electrode surface after installation of the dual polymer layer before ablation (t = 0 seconds) and after ablation (t = 180 seconds); Bottom: Intensities of the C1s contributions at a) 289.2 eV (-COOH) and b) 287.0 eV (C=O, c), N1s and d) Au4f over etching time, indicating the passage through the poly(acrylic acid) layer towards the AQ and aryl nitro species present from initial diazonium salt reduction.

Figure 12

 12 Figure 12 Tensile strength and Young's modulus for Pristine and CF-AQ 5 -AA and CF-AQ 20 -AA. Error bars show standard deviation, * denotes statistical significance (P<0.05).

Figure 13

 13 Figure 13 IFSS for CF-AQ 5 -AA and CF-AQ 20 -AA grown on the surface. Error bars show standard error, * denotes statistical significance (P<0.05).

  value to areas such as environmental remediation, energy storage and structural

Acknowledgements

The authors gratefully acknowledge Deakin University, this research was conducted with support from the Australian Government via the Australian Research Council World Class Future Fiber Industry Transformation Research Hub (IH140100018), ARC Training Centre for Lightweight Automotive Structures (IC160100032) and Discovery projects (DP180100094)). This work was also partially supported by the Office of Naval Research (N62909-18-1-2024) and was performed in part at the Deakin and Swinburne University node of the Australian National Fabrication Facility. A company established under the National Collaborative Research Infrastructure Strategy to provide nano-and microfabrication facilities for Australian researchers. ANR (Agence Nationale de la Recherche) and CGI (Commissariat à l'Investissement d'Avenir) are gratefully acknowledged for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices), ANR-10-LABX-096 and ANR-18-IDEX-00.


Gabriel Dumy
email: gabriel.dumy@espci.fr

Mauricio Hoyos
email: mauricio.hoyos@espci.fr

Jean-Luc Aider
email: jean-luc.aider@espci.fr

Observation of selective optical manipulation of particles in acoustic levitation

Acoustic Radiation Force is commonly used to create stable large-scale aggregates of particles in levitation (so-called "acoustic levitation") in a micro cavity. We show in the following work that this well-known and well-controlled aggregation process can be reversed without contact or external flow if the aggregated particles are enlightened with the proper optical wavelength. This coupled optics and acoustics effect has been observed with various kinds of particles and different optic wavelengths, showing high reproducibility. The phenomenon is studied using fluorescent micro-metric polystyrene particles without flow, and the effects of acoustic energy and illumination power have been quantitatively assessed. It is then exploited to separate a mix of particles with identical mechanical properties, based on their different optic absorption. If the phenomenon is not well understood, we propose and discuss some possible mechanisms that could be responsible for the rapid ejection of the objects in levitation from the illuminated area. Since it is a tag free phenomenon which does not need high energies to happen and since it works with biological objects such as algae, red blood cells and bacteria, it may open the way to a broad range of applications.

I. INTRODUCTION

Acoustic manipulation of micro-objects (particles, cells, bacteria, micro-bubbles, etc.) can be realized using ultrasonic standing waves in a fluidic or microfluidic resonator [START_REF] Coakley | Analytical scale ultrasonic standing wave manipulation of cells and microparticles[END_REF][START_REF] Nilsson | Review of cell and particle trapping in microfluidic systems[END_REF][START_REF] Dron | Varying the agglomeration position of particles in a microchannel using acoustic radiation force beyond the resonance condition[END_REF][START_REF] Hulström | Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip[END_REF] . Acoustic focusing of suspensions (or "acoustophoresis") is a complex phenomenon which has been the subject of many theoretical studies during the last decade. It occurs in a micro-channel or a micro-cavity when an acoustic wave of frequency f ac , emitted into the cavity through a vibrating wall and reflected by the opposite wall, matches the resonance condition λ ac = 2h, h being the height of the device (Fig. 1). In this case, particles such as plastic beads or living cells are drawn toward the acoustic pressure node created at mid-height of the cavity by the so-called Acoustic Radiation Force (ARF).

The ARF is the primary effect of the standing ultrasonic wave on the particles in suspension, and it rises from a second-order non-linearity. The ARF is different for each particle because it depends on their physical characteristics and size. The second effect of the ultrasonic standing wave is called acoustic streaming [5][START_REF] Zarembo | Acoustic streaming[END_REF][START_REF] Nomura | Streaming induced by ultrasonic vibration in water vessel[END_REF] . The acoustic wave transfers momentum to the fluid, leading to global motions of the particles through the Stokes force. The third effect is a Bjerknes force. It is basically a short distance force between particles [START_REF] Apfel | Acoustic radiation pressure -principles and application to separation science[END_REF] , attractive in most of the cases for the kind of particles used here, that contributes to the stabilization of the aggregate.

There are different theoretical models describing the ARF, based on more or less simplifying hypotheses [START_REF] König | Hydrodynamisch akustisch untersuchungen vol. 1[END_REF][START_REF] Rayleigh | On the pressure of vibrations[END_REF][START_REF] Bjerknes | Fields of Force[END_REF][START_REF] King | On the acoustic radiation pressure on spheres[END_REF][START_REF] Yosioka | Acoustic radiation pressure on a compressible sphere[END_REF][START_REF] Gor'kov | On the forces acting on a small particle in an acoustic field in an ideal fluid[END_REF][START_REF] Doinikov | Theory of acoustic radiation pressure for actual physics[END_REF] . King [START_REF] King | On the acoustic radiation pressure on spheres[END_REF] was the first to propose computations on the forces on particles in a sound field based on several simplifying hypotheses. In particular, he considered the particles as rigid spheres. In the following, we will use the Yosioka model [START_REF] Yosioka | Acoustic radiation pressure on a compressible sphere[END_REF] which takes into account the compressibility of the spherical particles in the modeling. In this case, the primary Acoustic Radiation Force (ARF) F ac responsible of particle acoustic levitation can be defined as:

- → F ac = π 4 E ac k d 3 p F Y sin (2 k ac z) - → e z (1)
where -denotes time averaging, d p is the particle diameter, E ac is the time-averaged acoustic energy density inside the channel, k = 2π λac = 2πfac c is the wave number of the acoustic plane wave of frequency f ac , F Y is the contrast factor, a positive (for polystyrene particles in water) numerical constant, and z is the axial (or vertical) position of the particle, z = 0 being at the bottom of the channel and z = h being at the top of the channel (Fig. 1).

The z axis also corresponds to the wave propagation direction.

The acoustic contrast factor of a given particle of density ρ p in a medium of density ρ f is defined as:

F Y = 1 + 2 3 1 - ρ f ρp 2 + ρ f ρp - ρ f c 2 f 3 ρ p c 2 p ( 2
)
where c p is the speed of sound in the particles material and c f is the speed of sound in the fluid. In this investigation we are dealing with an ultrasonic standing wave with a frequency c f = 1500 m.s -1 ), F Y equals 0.2244, and if particle diameter is 1 µm, the primary acoustic radiation force equals 2.8 × 10 -13 N.

f ac ∼ 2
Once the particles have reached the nodal plane, the axial component of the force becomes null and the transverse component F T is no longer negligible. Indeed it has been shown that this component is about one hundred times weaker than the axial component and is then the ARF for a radially symmetric acoustic wave in the nodal plane:

F T = d 3 p 3(ρ p -ρ f ) ρ f + 2ρ p ∇ E ac (3)
This transverse component depends directly on the radial gradient of the acoustic energy ∇ E ac and is responsible for the aggregation of particles in the levitation plane toward the local maximum of acoustic energy, which are the final equilibrium positions that we observe.

After a short description of the experimental setup, we will present the first observations of the ejection of particles in acoustic levitation when illuminated at specific optical wavelengths. The ejection velocity of the particles from the illuminated area is then studied quantitatively, especially by varying the amplitude of the acoustic force field and the power of the light wave. The phenomenon is then applied to a binary mixture of particles, illustrating the great potential of this phenomenon as a new highly selective separation technique. Finally the different possible mechanisms that could explain our observations and measurements are discussed, before coming to the conclusion.

II. MATERIALS AND METHODS

A. Acoustic resonator

In the following, all the experiments have been carried out in the same acoustic resonator whose dimensions are summarized on Fig. 2 a). We use a homemade aluminum circular cavity of diameter D = 20 mm and height h = 400 µm. The cavity is closed by a round silica wafer (transmitter thickness h trans = 0.30 mm). The cover plate is easily removable and allows fast changes of the medium. The 2 MHz piezoeletric transducer (PZT) (square ceramic, 1 mm thick, 10 mm wide) is glued directly on the silica wafer using a water soluble glue. The PZT is powered by a wave generator (Tabor Electronics 5200) connected to an amplifier (Tabor Electronics 3222) allowing the variation of the applied voltage between a few mV to more than 10 V peak to peak. A correlation is made between the acoustic energy carried by the generated standing ultrasonic field and the voltage applied to the piezoelectric transducer, by measuring the axial focusing velocity v z (z) of a spherical bead, which is directly proportional to the acoustic energy. Using polystyrene spheres of known compressibility and density, we can find the acoustic energy density [START_REF] Dron | Acoustic energy measurement for a standing wave in a microchannel[END_REF] . We will thus speak of acoustic energy when referring to the amplitude of the acoustic field in the remaining of the article. The frequency f ac of the sinusoidal signal is tuned to maximize the acoustic radiation force which corresponds to the resonance condition λ ac = 2h. We find this resonance by monitoring the reflected electric signal through an oscilloscope (LeCroy WaveAce 214), and minimizing the amplitude of said signal. In our case, the optimal frequency is f ac The images resulting from experiments were post-treated with the free software ImageJ (https://imagej.nih.gov/ij/), to adjust contrast, crop areas of interest and to produce the spatio-temporal diagrams illustrated on Fig. 6. The intensity delivered by the illumination lamp has been measured with a ThorLabs PM16-130 Probe (Spectral range 400 to 1100 nm, 1 cm × 1 cm silicon sensor area, optical power range 5 pW -500 mW) connected to the same computer controlling the other instruments.

III. CREATION OF LARGE-SCALE AGGREGATES UNDER ACOUSTIC RADI-ATION FORCE

As explained in the previous sections, the ARF is a simple and fast process to create large aggregates of particles in acoustic levitation. Once the ultrasonic wave is generated with the proper frequency into the cavity, the ARF forces the suspended particles to move Once the aggregate is formed, it can be kept in acoustic levitation as long as needed and will remain stable, with the same spatial organization [START_REF] Bazou | Controlled cell aggregation in a pulsed acoustic field[END_REF] . The aggregation process is efficient on particles as well as cells or active matter (bacteria). It can be used as a tool for creating an acoustic trap, especially for self-propelled organisms like bacteria that can be held in levitation in acoustic confinement [START_REF] Gutiérrez-Ramos | Induced clustering of escherichia coli by acoustic fields[END_REF] .

In previous works [START_REF] Bazou | Controlled cell aggregation in a pulsed acoustic field[END_REF] we demonstrated that it is also possible to generate and sustain a 2D aggregate of particles using burst mode acoustic, or pulsed acoustic, i.e. applying a low-frequency square wave modulation with a given duty-cycle. We also demonstrated that it helps suppressing the acoustic streaming for particles smaller than 1 µm. It is also of great possible thermal heating of the piezoelectric element when running long-time experiments, therefore reducing the potential thermal detuning of the resonator. It will be useful in the following to evaluate the possible influence of acoustic streaming on the phenomenon.

IV. BREAKUP OF AGGREGATES

As mentioned previously, once an aggregate of passive particles is formed using ARF, it remains stable and can be kept in levitation as long as needed. Without the use of an external flow or force, there is no way to break it up, apart from lowering enough or turning off the acoustics. In that case the aggregate either disintegrates in small aggregates and particles which all sediment, or the entire aggregate sediments, with particles still being stuck to each other by short distance hydrodynamic interactions.

There can be other behaviors with active matter, for instance when aggregating bacteria (active) or Janus particles (pseudo active). Indeed, when aggregated bacteria kept in levitation are released from the confinement imposed by the acoustic field, the aggregate inner active pressure triggers an explosion-like breakup, radially ejecting bacteria confined in the acoustic field [START_REF] Gutiérrez-Ramos | Induced clustering of escherichia coli by acoustic fields[END_REF] . In the case of Janus particles, as demonstrated by Takatori et al. [START_REF] Takatori | Acoustic trapping of active matter[END_REF] , the relaxation of an active crystal has been observed and described when the acoustic confinement is released.

What we shall report in the following is a breakup process of aggregates of particles kept in acoustic levitation using specific illuminations. We use fluorescent polystyrene particles of diameter d p = 1.62 µm which can be excited with green light and fluoresce in red light (λ abs = 545 nm and λ em = 620 nm). Large aggregates of particles are created in acoustic levitation in the cylindrical resonator described on Fig. 2 using f ac = 1.86 MHz resonant frequency. As mentioned previously, the aggregates can be kept stable as long as needed when observed with a white light and at standard microscope illumination power, below 5 mW.mm -2 (The regular aggregation process can be seen in Fig. 3 a) ).

Interestingly, when using a monochromatic (λ = 545 nm) illumination, and gradually increasing the light intensity, the aggregation process is stopped. For even higher intensities, the aggregate starts to eject particles from its periphery. This ejection process goes on until disruption of the entire aggregate. If the light intensity is still increased, then this ejection process takes the form of an explosion, as can be seen on Fig. 3 b) and associated movie (see SI Movie 1, 4). One astonishing effect is that the particles escape the aggregate while remaining in the focal plane of the camera, i.e. in levitation. This result suggests that the primary radiation force (responsible for the acoustic focusing) is not affected, but that transversal and Bjerknes forces seems to be completely counterbalanced or screened by another force. MM.1. Observation of the standard process of formation of an aggregate in acoustic levitation. It is then disrupted under monochromatic illumination. The aggregate is reformed quickly when the white light illumination is restored. This is a file of type "mp4" (4.5 Mb)

The disaggregation only occurs in acoustic levitation (no effect observed on beads that are on the bottom or top of the cavity) and when the particles are illuminated with the proper wavelength corresponding to the fluorescent particle absorption wavelength. In this case, for instance, the aggregate remains unchanged if illuminated with a blue light (λ = 488 nm).

Another interesting point is that the effect is reversible: once the light intensity is lowered under a given threshold, then the standard acoustic aggregation process starts again with the same dynamics (as demonstrated at the end of SI Movie 1).

This phenomenon is highly reproducible and not limited to a given type of fluorescent other. To further investigate the dependence of the effect on the amount of light injected in the aggregate, we did vary the light intensity for a given aggregate of 1.62 µm beads, at a fixed amplitude of the acoustic field (f ac = 1.85 MHz). In the following the amplitude of the acoustic force is considered proportional to the acoustic energy inside the resonator (cf.

Eq. 1). In this case, this energy is constant ( E ac = 10 J.m -3 ).

To quantify the disaggregation dynamics, we use spatio-temporal diagrams as explained in Fig. 6. A line crossing the stable aggregate is chosen before illumination. The aggregate being a bright spot over a dark background, the line crossing it shows a clear frontier between the aggregate and surrounding fluid. The time-evolution of these lines is then plotted as a function of time, leading to a spatio-temporal diagram (left part of Fig. 6). The disruption of the aggregate is then visualized as a cone in the spatio-temporal diagram. The slope of the cone frontier in the spatio-temporal diagram leads directly to the expulsion velocity v ej .

It is then possible to measure the ejection velocity for each illuminations. The evolution of v ej as a function of the illumination power P light is plotted on Fig. 7 a). A linear evolution is found suggesting a direct proportionality of the ejection velocity with the injected illumination power: v ej (P light ) ∝ P light .

C. Influence of the amplitude of the ARF

Similarly, the influence of the acoustic force amplitude has been investigated. We also found that the phenomenon was no longer observed when the acoustic field is turned off.

This time, we did explore a range of ARF amplitudes ranging form 8 to 200 J.m -3 at f ac = 1.85 MHz for a given illumination power (50 mW.mm -2 ), all other parameters being kept constant.

Using the same spatio-temporal analysis, we plot on Fig. 7 b) on logarithmic scale the evolution of v ej as a function of the acoustic energy in J.m -3 for a given illumination power P light . In this case, a linear evolution is found suggesting a scaling of the ejection velocity as a 3/4 power law of the acoustic energy: v ej ( E ac ) ∝ E ac 3/4 . Combining both results, and considering that both parameters are independent, one can define the following scaling law for the ejection velocity: v ej (P light , E ac ) ∝ P light • E ac 3/4 .

D. Influence of acoustic streaming

Acoustic streaming is a common phenomenon when using standing acoustic waves in resonant cavities. It could be considered as a possible explanation for the breakups of the aggregates, so we did investigate this effect.

As briefly described in section III, it is possible to control the onset of acoustic streaming in our resonators using a burst signal. The duty cycle DC of the ultrasonic wave can be of the excitation :

DC = T ON T OF F + T ON (4)
When we vary this parameter, we are modifying the temporal average of the acoustic energy that plays a role in the ARF. Fig. 8 represents the ejection speed of 1.62 µm polystyrene particles, when submitted to a 1.908 MHz ultrasonic standing field, with an acoustic energy of 160 J.m -3 , and enlightened with a light power of 50 mW.mm -2 . The acoustic field is then modulated by a square signal of period T burst = 1 ms, and we then vary the duty cycle of the resulting field. As can be seen on this figure, at low duty cycles, we observe a linear evolution of the ejection speed with an increase of the duty cycle.

Defining a threshold for the acoustic streaming [START_REF] Castro | Study of the onset of the acoustic streaming in parallel plate resonators with pulse ultrasound[END_REF] (below which no acoustic streaming is affecting the aggregates in levitation), we can find a critical duty cycle for these burst parameters, that is represented by a gray bar on Fig. 8. Below this threshold, we do not observe any streaming in the cavity [START_REF] Bazou | Controlled cell aggregation in a pulsed acoustic field[END_REF] . Interestingly enough, when we cross the acoustic streaming threshold, we do not observe any change in the evolution of the ejection velocity.

The fact that we do observe breakups of aggregates, with the same coupling between acoustic energy and light power below its threshold means that the origin of this phenomenon is not acoustic streaming.

VI. SEPARATION OF A BINARY MIXTURE

To demonstrate the ability of the opto-acoustic effect to separate a binary mixture, we used a mixed solution of two colloidal particles: polystyrene particles of mean diameters d p1 = 1.62 µm and d p2 = 0.883 µm, with absorption wavelengths λ 1 = 532 nm and λ 2 = 450 nm with an equal volume fraction of 0.025 %. First we created a large aggregate in acoustic levitation, following the same process as in the previous experiments (1.903 MHz and 200 J.m -3 ) and using the same resonator. After a relatively short focusing time, the aggregate becomes stable in acoustic levitation. The aggregate contains an homogeneous mixture of both particles.

The upper row of Fig. 9 shows the aggregates in acoustic levitation but using different filters. Fig. 9 a) corresponds to an observation without filters. It shows a large aggregate in stable acoustic levitation. When using a green (Fig. 9 b) or red filter (Fig. 9 c) one can see that both types of particles are rather homogeneously distributed over the entire aggregate.

To our knowledge, it is impossible to separate such a mixture of particles once they are so well mixed using acoustics alone.

The aggregate has then been illuminated with a blue light (λ light = 488 nm) at a constant power of 30 mW.mm -2 during 10 seconds, then the illumination has been turned off. The resulting aggregate has then been observed once the particles have aggregated again under the influence of ARF. The result is shown on the lower part of Fig. 9. Fig. 9 d) shows the aggregate after a few seconds without any optical excitation. One can see that the aggregate has changed from its original shape. Fig. 9 e) and Fig. 9 f) shows the pictures of the aggregate when observed respectively with a green and red filter. We can clearly see that the aggregate no longer contains an homogeneous mixture of both particles. The red fluorescent beads are now on one side of the aggregate while the green are now on the other side of the aggregate. This is a clear demonstration that the two types of particles have indeed been separated by the photoacoustofluidic effect. The separation was very fast (a few seconds) and efficient. The particles absorbing the illumination wavelength (the smaller ones in this case) have indeed been extracted from the aggregate before being attracted again toward the aggregate of larger particles, which did not breakup, because of the ARF. power to reach the same ejection speed. One possible explanation for this behavior is that for these high acoustic energies, the acoustic streaming is no longer negligible, and may interact with the expulsion process, leading to some errors in the estimation of the initial ejection velocity (leading to a no longer isotropic ejection). speed contour is the thermal agitation speed at experiment temperature (20 degrees Celsius) and has been identified as the threshold between stable aggregate and aggregate breakup zones.

phenomenon. Particles need to absorb light at the specific optic excitation wavelength used to be ejected from the aggregates, and to be trapped at the same time in an acoustic field carrying enough acoustic energy. This means that the optic energy absorbed by the particles needs to play a role in the explanation of the phenomenon.

B. Influence of heating on particles properties

The most plausible explanation is that optical illumination at specific wavelength leads to absorption and conversion of incident light (E λ ) into thermal heating. One basic explanation could be that while absorbing light, particles heat up, thus modifying their mechanical properties, potentially affecting the sign of the acoustic contrast factor in Eq. 2. However, after calculation, compressibility and density of polystyrene and water do not change that much between 20 and 100 degrees Celsius. Moreover, if the acoustic contrast factor were to change sign, then the principal component of the ARF (responsible for the vertical focusing of the particles) would also be affected, and we do not observe a movement out of the focal plane.

C. Heating of the fluid itself

One could argue that it is the fluid itself that heats up when exposed to a strong illumination, under the confinement of the acoustic field. Having the fluid directly react to the coupled excitation could then create a large scale flow dragging away the particles. To try this, we have performed experiments using coloured beads. Two types of particles having the exact same mechanical properties (same radii = 10 µm, made of polystyrene), in the same acoustic field ( E ac = 106 J.m -3 , f ac = 1.90 MHz), have been illuminated with the same optical wave (λ = 550 nm or 580 nm, P = 50 mW.mm -2 ). The only difference between the two populations was their optical absorption, one of the particle type being reddyed while the other was transparent (plain polystyrene). Everything else being equal, the acoustic force is the same on the two species. When illuminated with the same wavelength and power, the dyed particles are almost instantly dispersed, while the transparent ones are not affected by the light (see SI Movie 3, 12). That confirms that the particles need only absorbing the illumination wavelength and that they are the source of the phenomenon, and not the fluid itself. with the same optical wave (580 nm, P=50 mW.mm -2 ). As the aggregate made of dyed particles is instantly disersed, nothing happens to the particles that do not absorb the optical wave.

MM.3. Side to side comparison of an aggregate of plain polystyrene particles and another one being red dyed, under the same acoustic confinement and optical illumination. This is a file of type "mp4" (6.3 Mb)

D. Soret effect

Another effect that could affect the stability of the aggregate in acoustic levitation is related to the Soret effect, or Ludwig-Soret effect 25 , or thermodiffusion, namely, particle migration under temperature gradients. The Soret coefficient is the ratio between the thermal diffusion coefficient and the molecular coefficient of colloidal objects suspended in a liquid.

The Soret coefficient is well understood in gases when molecules of different size differentially migrate under a thermal gradient. Nevertheless, Soret effect is visible and measurable in suspensions of polymers in organic solvents, but the theoretical models are still poorly developed. Concerning Soret effect of non colloidal particles (10 µm) in water, to our knowledge only one study has been undertaken 25 . The thermophoretic mobility of 10 µm polystyrene particles has been estimated in water, in a thermal gradient corresponding to 0.25°C/ µm, yielding to a migration velocity of 5 µm/s. In our case, the red fluorescent particles being 1.62 µm in diameter, 10 µm is a good approximation of our thermal layer thickness. Having even a few additional degrees in the aggregate will yield thermal gradients on this order of magnitude. At the periphery of the aggregate, particles could be pushed away by a Soret-like effect. When illuminated, the temperature in the aggregate is higher than that in the surrounding liquid. With a Soret effect of non-colloidal species, particles migrate from higher to lower temperatures, in qualitative agreement with our observations. Once particles are far apart from each other, with distances longer than the Bjerknes acoustic forces range [START_REF] Garcia-Sabaté | Experimental study on inter-particle acoustic forces[END_REF][START_REF] Castro | Determination of the secondary Bjerknes force in acoustic resonators on ground and in microgravity conditions[END_REF] , the aggregate starts to disrupt. Unfortunately, thorough investigations would be needed to precisely quantify the thermal gradient generated in the boundary layer surrounding the particles in levitation and illuminated with the proper wavelength.

E. A thermal boundary fluid layer

Nevertheless, another thermal effect may originates from the heating of a layer of fluid directly surrounding the particles, leading to a local change in the fluid properties this time.

It would create around the particle a soft shell-like boundary layer, reminding the results obtained by Yasuda and Kamakura 28 . While in their work magnitude of forces are discussed, it is probable that the local increase of the temperature on the upper part of the particles leads to a local increase of the temperature of the fluid layer close to the particle. This would lead to a local change of the fluid density around the particles. A "convection-like" instability could be triggered with a decrease of the fluid density which would then lead to an upward motion to the fluid layer. We did not observe an axial motion of the particles.

It would be possible that the axial motion is stopped by the much stronger axial radiation force (justifying the acoustic coupling), leading to a lateral escape motion of the fluid layer, dragging the particles while staying in the levitation plane.

F. Acoustic instability of the thermal boundary layer

It has been shown recently that fluid layers with even a relatively small contrast factor can undergo ARF effects [START_REF] Karlsen | non-Brownian particles in pure liquids and estimation of their thermophoretic mobility[END_REF] . This is a side effect that can be a strong limitation to the separation of species if they are transported in different layers of fluid of different densities.

If the temperature of a particle T part is different from the one of the surrounding fluid then a thermal boundary layer δ therm is created around the particle, or around an aggregate of particles (See Fig. 11). It is then possible that there exist a fluid layer with a density ρ f (T 1 ) lower than the one of the surrounding fluid ρ f (T 0 ). Gradients of fluid density ∇ρ f will appear, leading to the following expression for the volume force F ac-f lu exerted on the fluid (we denote ρf the dimensionless perturbation in the density, following the analytical development stated above 24 ) : F ac-f lu = -cos(2ky)E ac ∇ ρf (5) One can see that if there is density gradient, then the fluid layer will undergo an acoustic force aligned with density gradients, leading to a possible motion of the fluid layer. It may be that this fluid layer drags particle alongs, while these ones are still subject to the axial acoustic force (much larger than the lateral one), thus moving sideways. The resulting ejection speed would then be a competition between the transverse component of the acoustic force and the viscous drag imposed by this fluid layer. The scaling in this case would be interesting, since as described in Eq. 3 the lateral acoustic force scales with d 3 p , and the drag at this R e would scale with d p only.

G. Heating process and size

If the motion origin is indeed related to an instability, either thermal or acoustic, of the surrounding fluid layer, one could expect that the effect will be weaker for larger particles.

Indeed, the thickness of the thermal boundary layer will be the same, if all parameters are kept constant apart from the radius of the particle. The ratio δ ac /r p will decrease and the entrainment of the particles will become weaker.

We carried out preliminary experiments with a given type of fluorescent beads, made from the same material (polystyrene, green fluorescent), trapped in the same acoustic field ( E ac = 106 J.m -3 for a frequency of f ac = 1.90 MHz) and illuminated with the same

We observed a reduction of the ejection velocity when increasing particle diameter, as can be seen on Fig. 13. The aggregate made of 1 µm particles undergoes the expansion effect, as described before. But this effect is less noticeable on the 3 µm particles aggregate after the same exposition time, and the 10 µm aggregate seems not to have been affected at all after the same manipulation. This result can be considered as a first hint of a possible mechanism associated to the heated fluid layer surrounding the particle. However, it could also stem from a competition between a fluid drag and the transverse component of the ARF as denoted in VII F.

  FIG. 1. (color online) Side view of an acoustic resonator showing the principle of acoustic focusing

1. 85

 85 MHz, but day-to-day fluctuations can occur, caused by small room temperature change, or slight errors in the position of the quartz cover plate. These fluctuations are on the order of 0.5%f ac 10 kHz.

FIG. 2 .

 2 FIG. 2. (color online) a) Sketch of the acoustic resonator, showing the piezoelectric transducer

  toward the nodal plane, at mid-height of the cavity. Once the particles have reached the focusing plane, the transverse component of the ARF forces the particles to move toward the location of the maximum of acoustic energy and then create an aggregate. When the particles are close enough from each other then the Bjerknes force keep the particles close together, making the aggregate more compact and stable. The creation of a large aggregate of particles is illustrated on Fig.3 a) with 1.62 µm polystyrene beads. The focusing time T f oc can be very short (a few tenths of second), depending on the amplitude of the ARF.

FIG. 3 .

 3 FIG. 3. (color online) Time steps between frames is 200 ms. a) The three first pictures show the

FIG. 4 .

 4 FIG. 4. Movie (MM1)showing the formation of an aggregate made of 1.62 µm beads under the

  FIG. 5. Movie (MM2) showing the disruption of an aggregate made of 1.75 µm beads under the

FIG. 7 .

 7 FIG. 7. Evolution of the ejection velocity as a function of the illumination power (constant E ac =

FIG. 8 .

 8 FIG. 8. Influence of the duty cycle on the ejection speed. The grey bar indicates the critical duty

FIG. 9 .

 9 FIG. 9. (color online) Separation of a binary mixture of 1.62 µm red fluorescent particles, and

1 FIG. 10

 110 FIG. 10. (color online) Determination of the phase diagram for an assembly of 1.62 µm red

FIG. 11 .

 11 FIG. 11. (color online) Illustration of how the particle (or an aggregate, as depicted on the right

FIG. 12 .

 12 FIG. 12. Movie (MM3) Side to side comparison of the evolution of (left) an aggregate of plain

FIG. 13 .

 13 FIG. 13. Evolution of the aggregate behavior in acoustic levitation under illumination for different

ACKNOWLEDGMENTS

The authors are grateful to Ecole Doctorale Frontières du Vivant (FdV) Programme Bettencourt for supporting G. Dumy's PhD thesis. The authors would also like to thank L. Bellebon for his important contribution on the manipulation and analysis of the acoustic energy of the resonators, and M. Mounaix for the lend of the optical power probe used for light characterization.

VII. DISCUSSION

A. Coupled dependency on acoustic and illumination

The previous experiments show a clear correlation between acoustics energy and illumination power for the breakdown of aggregates. To evaluate this coupling, we studied the evolution of the ejection speed from the aggregates when varying the illumination power and amplitude of the ARF for a given suspension of particles (d p = 1.62 µm). The result is shown on Fig. 10 as a phase diagram: we represent the initial ejection speed of particles from the aggregate as a function of illumination power and acoustic energy applied to the aggregate, i.e. a contour plot of v ej (P light , E ac ), the color bar being proportional to v ej . The 1 µm.s -1 speed contour line is highlighted in red, and corresponds to the Brownian motion speed at experiment temperature (20 degrees Celsius) for these particles and has been identified as the threshold between stable aggregate and aggregate breakup zones. As one can observe, the aggregate is locked in a stable state, provided that the illumination power is below 10 mW.mm -2 , or the acoustic energy of the ultrasound field is below 25 J.m -3 .

Once this threshold is crossed (solid red line on the phase diagram, corresponding to the Brownian motion at the experiment temperature, for these particles), then we have a more or less linear evolution of the ejection speed as light power or acoustic energy is increased.

Interestingly, there seem to be a threshold on acoustic energy density at 160 J.m -3 , above which the light dependence of the ejection speed changes. It seems to be an optimum on illumination power dependence, as it is the point where the highest ejection speeds are reached using the lowest illumination power. After this threshold, we need to put more illumination We mentioned and discussed many possible explanations for the light induced ejection of particles in acoustic levitation. A plausible scenario seems to be the heating of the surrounding fluid layer leading to instability mechanisms. To further clarify this hypothesis, more experiments are needed, especially temperature controlled ones.

VIII. CONCLUSION

Choosing the proper geometric and acoustic parameters, it is possible to control the aggregation process of micro particles using our in-house ultrasonic resonators. Once the aggregates are formed, it is possible to keep them in acoustic levitation as long as desired.

The only way to break the aggregates is to turn off the ultrasounds. Nevertheless we show that it is possible to destroy an aggregate of fluorescent particles just by illuminating it at the proper absorption wavelength. This phenomenon depends on both the acoustic force amplitude and the illumination power : the expulsion of the particles can only be observed in acoustic levitation, suggesting a strong coupling between acoustics and photonics effects.

Above a given critical illumination power, the aggregate literally explodes. The expulsion of the particles from the illuminated area can be very fast with an expulsion velocity which depends on the injected power (acoustic amplitude and illumination power). Early observations also show that the phenomenon is affecting living cells such as red blood cells, opening the path to many applications of these findings. This phenomenon, which may be named optoacoustophoresis due to its dependence on both excitations, opens the path to many new manipulations and sorting processes of suspensions, combining both acoustic and optical properties of particles or cells. This new branch of acoustofluidics could be called optoa-