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2D Electrochemiluminescence: Light Emission Confined at the Oil/Water Interface in Emulsions Stabilized by Luminophore-Grafted Microgels Remy Bois, Sabina Scarabino, Valerie Ravaine, and Neso Sojic Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01585 • Publication Date (Web): 02 Jul 2017 Downloaded from http://pubs.acs.org on July 4, 2017

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2D Electrochemiluminescence: Light Emission Confined at the Oil/Water Interface in Emulsions Stabilized by Luminophore-Grafted Microgels Rémy Bois, Sabina Scarabino, Valérie Ravaine*, Neso Sojic* Univ. Bordeaux, Bordeaux INP, ISM, UMR CNRS 5255, 33607 Pessac, France Corresponding Authors *Valérie Ravaine: [email protected] ORCID iD: 0000-0002-1192-7974 *Neso Sojic: [email protected]. ORCID iD: 0000-0001-5144-1015

Abstract We describe a method to confine electrochemiluminescence (ECL) at the oil-water interface of emulsion droplets which are stabilized by luminophore-grafted microgels. These hydrogel nanoparticles incorporating covalently-bound Ru(bpy)

as the luminophore are irreversibly adsorbed at the interface of micrometric oil droplets dispersed in a continuous aqueous phase. We study the electrochemical and ECL properties of this multiscale system, composed of a collection of droplets in close contact in the presence of two types of model coreactants. ECL emission is observed upon oxidation of the coreactant and of the luminophore. ECL imaging confirms that light is emitted at the surface of oil droplets. Interestingly, light emission is observed more than 100-µm far from the electrode. It is possibly due to the interconnection between redox-active microgels making an entangled 2D-network at the dodecane/water interface and/or to some optical effects related to the light propagation and refraction at the different interfaces in this multiphasic system. Confining ECL in such an inhomogeneous medium should find promising applications in the study of compartmentalized systems, interfacial phenomena, sensors and analysis of single oil droplets.

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Introduction Electrochemiluminescence (ECL) is the process of light emission by the excited state of a luminophore triggered by an initial electron-transfer reaction at the electrode surface.1 The excited state is produced by a highly exergonic electron-transfer reaction between electrogenerated intermediates. It relaxes to the ground state by emitting light which is the analytical signal. ECL is a remarkable analytical method which has led to various applications, especially in bioassays.2, 3 To improve the sensitivity and the versatility of ECL, numerous studies have been focused on the development of new ECL luminophores and original sensing designs.4, 5, 6, 7, 8 In view of life science where media are often heterogeneous, it

is

essential

to

provide

better

understanding

of

ECL

processes

in

multiphasic/compartmentalized systems. For example, liposomes encapsulating tris(2,2′bipyridyl)ruthenium(II) complex have been studied9 and proposed recently for ECL immunosensing.10,

11

Moreover, Dick et al. reported the ECL of common hydrophobic

luminophores such as pyrene or rubrene using an oil-in-water emulsion system.12 The oil droplets were used to dissolve the organic-based luminophores and thus to generate their ECL emissions while the coreactant was present in the aqueous continuous phase. This approach has been extended to the ECL detection of single droplet collisions at the surface of an ultramicroelectrode.13 To the best of our knowledge, these 2 reports are the only ones on ECL in emulsions. On the other hand, chemiluminescence reactions have been widely studied in microheterogeneous media such as emulsions.14 Contrarily to these 2 recent works where the ECL luminophores were homogeneously dissolved in the oil droplets,12,

13

we propose an

alternative strategy allowing to confine the ECL emission in a 2D format. In this paper, we demonstrate that ECL is produced strictly at the interface of droplets which are connected together and stabilized by flattened luminophore-grafted microgels (Figure 1). We selected prototypical thermo-responsive microgels based on poly(N-isopropylacrylamide) (pNIPAM) 15 to covalently attach the model ECL luminophore Ru(bpy)

. The electrochemical and ECL 15, 16 behaviors of Ru(bpy)

anchored at the oil/water interface in -functionalized microgels

such oil-in-water emulsions are presented.


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Figure 1. a) Schematic representation of the Pickering emulsion, where the oil droplets are connected together and stabilized by flattened pNIPAM-Ru microgels. The aqueous phase contains the supporting electrolyte and the coreactant, whereas the Ru(bpy)

luminophore is covalently bound to the microgels. b) TEM view of the dried microgels. c) Optical microscopy image of the emulsion droplets. d) Cryo-SEM image of the polymeric matrix obtained after sublimation of an emulsion droplet which was initially covered by the pNIPAM-Ru microgels.

Experimental Materials. All the reagents were purchased from Sigma-Aldrich unless otherwise noted. Nisopropylacrylamide (NIPAM) was recrystallized from hexane (ICS) and dried under vacuum prior to use. The cross-linker N,N′- methylenebis(acrylamide) (BIS), and the initiator 2,2′azobis(2-amidinopropane) dihydrochloride (V-50) were used as received. Ruthenium(II) (43 ACS Paragon Plus Environment

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vinyl-4′-methyl-2,2′-bipyridine)bis(2,2′-bipyridine)bis-(hexafluorophosphate)

(Ru(bpy)

monomer) was synthesized according to the procedure described by Spiro et al.17 The coreactants, tri-propylamine (TPrA) and sodium oxalate, were purchased from SigmaAldrich. Deionized water, obtained with a Milli-Q system, was used for all synthesis reactions, purification and solution preparation.

Microgel synthesis and characterization. The microgels were obtained by a method similar to an aqueous free-radical precipitation polymerization classically employed for the synthesis of thermoresponsive pNIPAM microgels. The incorporation of the ruthenium complex derivative was performed by the copolymerization

of

Ru(bpy)

monomer,

Ruthenium(II)

(4-vinyl-4′-methyl-2,2′-

bipyridine)bis(2,2′-bipyridine)bis(hexafluorophosphate), with NIPAM and BIS as a crosslinker agent. Polymerization was performed in a 100 mL three-neck round-bottom flask equipped with a magnetic stir bar, reflux condenser, thermometer, and argon inlet. The total monomer concentration was held constant at 70 mM. The cross-linker content was 1.5 mol % of the total monomer. The monomers except the Ru(bpy) derivative were dissolved in 47 mL of [NaCl] = 30 mM. Ru(bpy) monomer (0.6 mol %) was dissolved in 1 mL of acetone and added to the previous solution. The whole solution was heated to 70 °C and thoroughly purged with nitrogen for at least 30 min prior to initiation. Free radical polymerization was then initiated with V50 (1.5 mM) dissolved in 2 mL of water and degassed during 10 min. The successful initiation was indicated by the occurrence of slight turbidity. The solution was allowed to react for a period of 6 h under nitrogen. Following synthesis, the pNIPAM-Ru microgels were purified by dialysis (dialysis membrane: MCWO 10 000, Orange Scientific) against water (three changes per day for 1 week at room temperature). At the end of the process, a stock microgel solution containing 0.6 %wt. in polymer was stored for further use. Particle sizes and polydispersity index were determined by dynamic light scattering (DLS) with a Zetasizer Nano S90 Malvern Instruments equipped with a HeNe laser at 90°. Hydrodynamic diameters were calculated from diffusion coefficient using the Stokes-Einstein equation. All correlogram analyses were performed with the software supplied by the manufacturer. The polydispersity index is given by the cumulant analysis method.

Emulsion preparation. Typical emulsion batches were composed of 7 mL of an aqueous phase containing pNIPAM-Ru microgels at a concentration of 0.05%wt and 3 mL of dodecane. This mixture 4 ACS Paragon Plus Environment

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was then stirred with an Ultra- Turrax T25 mixer equipped with an S25 kV-25F shaft at constant speed (9500 rpm) for 30 s. The temperature was kept constant and equal to 25 °C. The created droplets rapidly creamed due the density mismatch but did not coalesce after reaching contact. For further electrochemical and ECL experiments, the subnatant, containing most of the aqueous phase, was replaced by the supporting electrolyte (in general PBS 20 mM unless otherwise noted). This operation was done three times, with gentle shaking each time. Electrochemical, PL and ECL experiments. An Autolab PGSTAT30 potentiostat, a Hamamatsu photomultiplier tube (PMT) equipped with KEITHLEY 6485 picoammeter and high voltage power supply Hamamatsu Photonics model C9525 were used for electrochemical and ECL experiments. All electrochemical and ECL experiments were performed using a Ag wire as a pseudo-reference electrode and a platinum wire as counter electrode. The carbon working electrode was inserted a few mm lower than the top surface, in the creamed layer containing concentrated droplets. DPV signals were recorded with the following scan parameters: 5 mV for the step potential, 10 mV for the pulse amplitude, 0.05 sec. for the pulse width and 0.5 sec. for the pulse period. Photoluminescence (PL) analysis was performed on a spectrofluorometer CARY 100 Scan. ECL spectra were recorded using a Princeton Instruments Acton SpectraPro 2300i after the CCD camera cooled to –115°C with liquid N2. The electrochemical cell is built with a glass slide on the bottom in order to record the ECL signal. The optical fiber connected to the device is placed close to this glass slide in front of the working electrode. The instrument used for PL and ECL imaging was a modified epifluorescence microscope (BX-30, Olympus) equipped with a 20x objective. PL and ECL emission was recorded by an Electron Multiplying Charge Coupled Device (EM-CCD) Camera (Hamamatsu, 9100-13). PL and ECL images were recorded with an exposure time of 1 and 10 sec., respectively.

Results The Pickering emulsion stabilized by microgel particles is a multiscale-organized system at submicrometric and micrometric scales (Figure 1a). Indeed, hydrogel nanoparticles containing the ECL luminophore are situated at the interface of micrometric oil droplets with a continuous aqueous phase. The hydrodynamic diameter of these hydrogel pNIPAM-Ru particles was measured in PBS by Dynamic Light Scattering revealing a value of 300 nm (Figure S1), as also illustrated by TEM (Figure 1b). It was previously demonstrated that 5 ACS Paragon Plus Environment

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pNIPAM microgels are very efficient Pickering emulsion stabilizers, typically providing oilin-water emulsions which are stable at rest for months.18, 19 Microgels are soft particles and can thus adopt different conformations at the interface. They can be either flattened at the interface or compressed, depending on their concentration and on their kinetics of adsorption.20 Here, we used pNIPAM-Ru microgels as the sole stabilizers. Emulsions were obtained by mixing vigorously together 7 mL of aqueous phase containing 0.05% wt. of microgels and 3 mL of dodecane, using a high shear rate for 30 s. After preparation, the emulsions rapidly formed a creamed layer at the top of the vial because of the differential density between oil and water and of the large droplet size. In this creamed layer, the droplets were in contact but remained stable at rest. The observation in optical microscopy (Figure 1c) showed that their diameter was about 40 µm in average with a relatively low polydispersity. Indeed, these emulsions were prepared in the so-called limited coalescence regime.21 In these conditions, the system is emulsified with a low amount of particles and the newly created droplets are insufficiently protected by the particles. When the agitation is stopped, the droplets coalesce, thus reducing the total amount of oil/water interface. Since the particles are irreversibly adsorbed, the coalescence process stops as soon as the oil/water interface is sufficiently covered, and the resulting emulsions exhibit remarkably narrow size distributions of the droplets (P < 30%).22 The final average droplet diameter is thus inversely proportional to the amount of particles, with the assumption that all particles are adsorbed. In our case, the aqueous phase was very transparent, indicating the absence of excess microgels in the continuous phase and thus confirming that all the microgels were adsorbed at the interface of the droplets. The organization of the pNIPAM-Ru microgels at the interface of oil droplets was characterized by cryo-SEM (Figure 1d). The polymeric film was revealed after partial sublimation of the oil and water phase. In agreement with previous observations, the microgels formed a monolayer made of hexagonally packed microgels (Figure 1d). The lattice parameter was about 400 nm. This value is higher than the diameter of the microgels in solution (300 nm), because the microgels are soft and deformable and were flattened at the oil/water interface. This conformational change results from the balance between chain spreading - driven by surface activity - and microgel internal elasticity – promoted by crosslinking junctions. Polymeric filaments between adjacent microgels were also visible (Figure 1d). They indicate the presence of entanglements between peripheral dangling chains. The interconnected flattened microgels make an elastic 2D interfacial network around oil droplets, which protect them against coalescence. The thickness of this film was not measured precisely here. However, in previous studies,23 it was found to be equal to 400 nm for 6 ACS Paragon Plus Environment

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microgels twice bigger than the ones used here. It can be supposed that the thickness is in the order of a few hundred of nanometers. In addition, the emulsions were flocculated, because some microgels can adsorb simultaneously to adjacent droplets making bridges.24 In a first set of electrochemical experiments, the aqueous phase of the emulsion was replaced by the supporting electrolyte containing eventually other free redox species. After at least three washings with the electrolyte solution, the creamed layer containing the oil droplets was delicately transferred to a vial and an electrode was inserted in the medium (a few millimeter from the top surface). In such conditions, it could be considered that the oil volume fraction was comprised between 50 and 60% vol. To study the electrochemical properties of the emulsions, we added a well-known outer-sphere redox couple, /

Ru(NH )

, in the continuous aqueous phase of the emulsions stabilized by pNIPAM-Ru

microgels. This compound was selected as a redox probe since it is a non-adsorbing species with a very high electron-transfer rate.25 Moreover, since Ru(NH )

is positively charged, this species is soluble only in the aqueous phase whereas the Ru(bpy)

complex grafted to the pNIPAM matrix of the microgels is confined at the dodecane/water interface. In addition, the redox potentials of both species are well separated and do not overlap. Therefore, Ru(NH )

can be considered as an internal reference for measuring the potential of the studied system.

1.4 1.2 1

Current / µA

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0.8 0.6 0.4 0.2 0 -0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Potential / V vs. Ag

Fig. 2. DPV signal of a dodecane-in-water emulsion stabilized by 0.05% wt pNIPAM-Ru microgels. The water phase was a 20 mM PBS solution (pH 7.4) containing 0.1 mM

Ru(NH )

species was added as an internal reference

. The redox-active Ru(NH ) compound. The working electrode was glassy carbon (GC). 7 ACS Paragon Plus Environment

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Figure 2 shows a differential pulse voltammogram (DPV) of a dodecane-in-water emulsion stabilized by pNIPAM-Ru microgels and containing Ru(NH )

in the aqueous phase. Well-defined oxidation peaks are observed at - 0.35 V vs. Ag and at 0.95 V vs. Ag. A control DPV experiment was carried out in dodecane-in-water emulsion stabilized by pNIPAM microgels which did not contain the Ru(bpy)

complex and no redox process was visible in the explored potential range. In the present case, the potential of the first peak at

0.35 V vs. Ag is in agreement with a one-electron oxidation of Ru(NH )

to Ru(NH ) in

water. The second anodic peak at 0.95 V vs. Ag corresponds to the oxidation of the Ru(bpy)

complex covalently bound in the microgels. Since we used a Ag pseudo-reference

electrode, the redox potentials of both electroactive species (i.e. Ru(NH )

and Ru(bpy) )

are shifted by approximately 200 mV in comparison to a Ag/AgCl/KCl saturated reference electrode. One can also notice that the peak intensity of the oxidation of the freely-diffusing Ru(NH )

is 2.3 higher than that of the Ru(bpy)

centers grafted to the microgels.

However, it would be too hypothetical to extract some quantitative information on the fraction of the electroactive Ru(bpy)

sites from the respective values of the peak intensity. Indeed, as already mentioned, the pNIPAM-Ru microgels are anchored at the oil/water interface whereas Ru(NH )

diffuses in the continuous phase so it corresponds to two completely different mass transport and electrochemical situations. Whatever, DPV shows clearly that the Ru(bpy)

redox centers in the microgels stabilizing the emulsions or a fraction of them are electrochemically accessible. The Faradaic process observed at the level of the redox active microgels raised the questions about the localization of the electron-transfer process and the charge neutrality in the studied system. In the electrochemical reactions of redox active microdroplets and emulsions,26, 27, 28, 29, 30, 31, 32 the charge neutrality is maintained in the oil phase during the electron-transfer reaction by an accompanying ion transfer across the oil/water interface.26 Herein, we employed dodecane as the oil phase which is an organic solvent with very low dielectric constant (ε = 2). The ions of the phosphate buffer remain thus solely in the aqueous phase and we did not add any electrolyte soluble in dodecane. Hence, in light of these observations and the DPV results, it is concluded that the oxidation of grafted Ru(bpy)

can occur only in the aqueous side of the microgels confined at the interface. In other words, the dodecane droplets can be considered as an electro-inactive phase in the DPV experiments. It allows simplifying the different electrochemical processes in the studied


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quadruple phase boundary between the oil droplet, the pNIPAM-Ru microgels, the continuous aqueous phase, and the electrode surface (i.e. oil|microgel|aqueous|electrode). 40

0.2

a)

b)

35 0.15

ECL intensity / a.u.

30

Current / µA

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25 20 15 10

0.1

0.05

5 0

0 0

0.5

1

1.5

0


0.5

1

1.5


Fig. 3. Voltammetric (a) and ECL (b) signals of dodecane-in-water emulsion stabilized by 0.05% wt pNIPAM-Ru microgels. The water phase was a 20 mM PBS solution (pH 7.4) containing initially either 10 mM oxalate (green curve) or 10 mM TPrA (red curve) as coreactants. The working electrode was GC. Scan rate: 0.05 V.s-1.

In the current study, the ECL behavior of the pNIPAM-Ru emulsions was examined using two model coreactant species: oxalate and tri-n-propylamine (TPrA). We selected them because they follow different mechanistic ECL routes and are distributed predominantly either in the oil or in the aqueous phase. Indeed, the values of their partition coefficients are very different. On one hand, oxalate (i.e. C O ) is a negatively charged coreactant and it is dissolved only in the water phase. On the other hand, TPrA is a hydrophobic compound which is better soluble in organic phases. Dick et al. recently reported the distribution of TPrA between toluene and water using electrospray ionization-mass spectrometry.12 Using the same method, we measured the TPrA concentrations in the organic and aqueous phase. It was found to be 5.5 mM and 11.5 mM in the water and in dodecane, respectively. In agreement with the report of Dick et al.,12 a low concentration of TPrA remains in the continuous aqueous phase. Figure 3a shows the cyclic voltammograms of the emulsions stabilized by pNIPAMRu microgels in the presence of 10 mM oxalate (green curve). The irreversible anodic wave starting at 0.8 V vs. Ag is attributed to the oxidation of oxalate which is present in the continuous phase. The ECL emission starts to increase from 0.9 V vs. Ag concomitantly with the oxidation of the Ru(bpy)

grafted in the microgels, reaching a maximum at 1.07 V vs.


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Ag before decaying progressively (Figure 3b). Oxalate follows an “oxidative-reductive” route where both the luminophore Ru(bpy)

in the microgels and the coreactant C O are both

oxidized at the electrode surface. Then, upon bond cleavage, C O● forms a strong reducing species (CO● ) that diffuses in the aqueous phase, reduces the Ru(bpy)

attached to the microgels, and generates the excited state Ru(bpy) ∗ . It relaxes to the ground state and ECL light is emitted. The ECL mechanism involving oxalate requires explicitly the oxidation of the Ru(bpy)

centers and this explains why ECL starts to be generated at the potentials where Ru(bpy)

is oxidized at the electrode surface. To further assess our approach, in a second set of experiments, TPrA was added as an ECL coreactant to the dodecane-in-water emulsions stabilized by pNIPAM-Ru microgels. In this case, both voltammetric and ECL behaviors differ from the ones with oxalate (Figure 3). Faradaic current starts to increase progressively at 0.65 V vs. Ag (red curve). One can notice a small shoulder around 1.05 V vs. Ag where Ru(bpy)

centers are oxidized and the current continues to increase progressively at higher potentials. On GC electrode material, oxidation of TPrA occurs at potentials less anodic than oxalate and Ru(bpy)

. However, only a very small current corresponding to TPrA oxidation is measured in the potential range 0.7-0.9 V vs. Ag and it is much smaller than the welldefined oxalate oxidation wave, even if equal concentration of TPrA and oxalate were dissolved initially in the aqueous phase. This is unexpected because their concentration differs only by a factor 2 and their diffusion coefficients are relatively comparable in water.33, 34, 35 However, this apparent contradiction could be explained by the relative charge of the hydrogel interfacial layer compared to the charge of the coreactant. In a previous work,36 we have shown that the positive charge of the hydrogel film had a strong impact on the transport of charges through the pNIPAM-Ru films: cationic coreactants diffuse less in the hydrogel film than anionic ones. Unlike TPrA, the electrostatic interactions between C O and the positively-charged microgels increase the local concentration of the coreactant in the pNIPAM-Ru matrix where electrocatalytic oxidation reaction occurs. Therefore, the contribution of electrostatics could explain why the current corresponding to TPrA oxidation is much smaller than that of oxalate coreactant. The ECL behavior using TPrA as a coreactant is also very different from that using oxalate (Figure 3b). ECL starts to increase at the same potential for both oxalate and TPrA, i.e. at 0.9 V vs. Ag where Ru(bpy)

centers are oxidized. This indicates that both ECL mechanistic pathways require the direct oxidation of the Ru(bpy)

centers. It is remarkable that, even if the TPrA is mainly in the oil phase, the resulting ECL intensity is stronger in the 10 ACS Paragon Plus Environment

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emulsions with TPrA in comparison to oxalate (Figure 3b). This can be explained by the higher ECL efficiency of this coreactant.35, 37 The ECL emission observed with TPrA consists of two waves around 1.04 V vs. Ag and 1.28 V vs. Ag. The first one occurs at the Ru(bpy)

oxidation potential and the corresponding ECL process can be described by the following mechanism: TPrA - e TPrA●+

(1)

pNIPAM—Ru(bpy)

- e pNIPAM—Ru(bpy)

(2)

●+

pNIPAM—Ru(bpy)

+ TPrA pNIPAM—Ru(bpy) + TPrA

(3)

TPrA●+ TPrA● + H+

(4)

● ∗ pNIPAM—Ru(bpy)

+ P1 + TPrA pNIPAM—Ru(bpy)

(5)

pNIPAM—Ru(bpy)

pNIPAM—Ru(bpy) ∗ + hνECL

(6)

where P1 is the product of TPrA● oxidation and pNIPAM—Ru(bpy)

represents the ruthenium complex attached to the pNIPAM matrix in the microgels as depicted on Figure 1a.

TPrA is oxidized either directly at the electrode surface (Eq. 1) or in a homogeneous reaction by electrogenerated Ru(bpy)

(Eqs. 2-3). After deprotonation (Eq. 4), a strongly reducing species TPrA● is generated and reacts with the oxidized luminophore to lead to the excited state (Eq. 5) and finely to the ECL emission (Eq. 6). Control ECL experiments were carried out in dodecane-in-water emulsion stabilized by pNIPAM microgels (i.e. without grafted Ru(bpy) ). As expected, in the absence of the luminophore in the emulsion, the ECL signal was completely negligible with oxalate or TPrA (data not shown). The second ECL wave arises at more positive potentials and is concomitant with the increase of the faradaic current (Figure 3). It may be related to the oxidation of the TPrA34 or of the Ru(bpy) centers located in the dodecane. Since these events may happen in the oil phase, the electrontransfer reaction has to be coupled with an ion-transfer to counter-balance the charge: a cation from the oil phase to the aqueous phase or an anion in the opposite direction. This can explain why this second process occurs at more positive potentials. To investigate further the luminescence properties, we recorded the ECL spectrum of the emulsions (Figure 4). Similar ECL and photoluminescent (PL) spectra were obtained with an emission at ~610 nm which is typical for the metal-to-ligand charge transfer (MLCT) transition of the grafted Ru(bpy) complex. It shows that the same excited state is reached by the electrochemical stimulation in the emulsions and by photo-excitation of the microgels 11 ACS Paragon Plus Environment

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in PBS. In fact, a slight red-shift of 10 nm is observed for the PL of the microgels in PBS solution in comparison to the ECL of the emulsion. But it is probably related to the different spectrometers used in ECL and PL experiments. Indeed, in the case of a more hydrophobic environment, a blue shift is expected.38, 39 It shows that the ECL emission process occurs in the aqueous phase and that only the Ru(bpy) emitters located in the aqueous side of the interface contribute to the ECL signal.

Luminescence intensity / a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

0.8

0.6

0.4

0.2

0 450

500

550

600

650

700

750

800

Wavelength / nm

Fig. 4. Comparison of the normalized ECL spectrum (black curve) of dodecane-in-water emulsion stabilized by 0.05% wt pNIPAM-Ru microgels and of PL spectrum (blue curve) of pNIPAM-Ru microgels dissolved in PBS solution. For the ECL experiments, the water phase was a 20 mM PBS solution (pH 7.4) containing initially 10 mM TPrA. For the PL spectrum, λexc= 452 nm.

We used ECL imaging40, 41, 42 to study the spatial distribution of the light emission at the level of the emulsion. Figure 5a shows first a PL image of the droplets forming the dodecane-in-water emulsion recorded with an epifluorescence microscope in a side-view configuration. The dodecane droplets were stabilized by the pNIPAM-Ru microgels and the water phase contained initially 10 mM TPrA. The coreactant TPrA was tested because it is a model system which leads to more intense ECL signal than oxalate. The GC electrode was gently placed in the emulsion and is located at the top of the image (hatched zone). In PL mode, one can clearly see the dodecane droplets. Some brighter spots were also observed. They are supposed to correspond to interfacial zones connecting two adjacent droplets. Indeed, as already mentioned, the droplets were bridged together by microgels adsorbing simultaneously to two adjacent droplets. In those area constituted by a monolayer of microgels separating two oil compartments, it was previously demonstrated that the packing of microgels was different from that at free oil-water interface.24 The local concentration of 12 ACS Paragon Plus Environment

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microgels was higher, which could explain the brighter luminescence. Then, the excitation light was turned off and ECL imaging experiments were performed.

Fig. 5. PL (a) and ECL (b) images of the same ROI of the dodecane-in-water emulsion stabilized by 0.05% wt pNIPAM-Ru microgels. The water phase was a 20 mM PBS solution (pH 7.4) containing initially 10 mM TPrA. ECL emission is generated by applying a potential of 1.2 V vs. Ag at the GC working electrode. The hatched zone materializes the position of the electrode. The ECL image was coded according to the color scale (right). White color represents high luminescence intensities. Scale bar: 50 µm.

Figure 5b displays the false color ECL image of the same region of interest (ROI) recorded a few seconds after the PL. ECL image was recorded with an exposure time of 10 seconds. We imposed a constant potential of 1.2 V vs. Ag which is high enough to oxidize both the TPrA coreactant and the Ru(bpy) luminophore. The dodecane droplets are visible and well-defined in the ECL mode. Each droplet can be resolved individually. A total of ~50 droplets were observable in Figure 5. The spatial distribution of ECL emission follows a pattern very similar to the PL image. Indeed, there is a good correspondence between the droplets visible in PL and ECL modes (Figure S2). The slight difference between the position of the droplets in Figure 5a and 5b is due to the fact that an emulsion is a dynamic system in which the droplets can move during the few seconds required to switch from PL to ECL imaging mode. Some brighter ECL spots are also visible at the level of the droplets surface and it might correspond to the contact zone of bridged droplets, as already mentioned. Remarkably, light emission is observed more than 100-µm far from the electrode surface 13 ACS Paragon Plus Environment

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along the z-axis (Figure 5). This result is unexpected because the ECL-emitting region extends spatially over a few micrometers next to the electrode due to the limited life-times of the generated TPrA radicals when the ruthenium complex is immobilized.41, 43 However, in the present system, the redox-active microgels are interconnected at the dodecane/water interface, as revealed by the cryo-SEM image (Figure 1d). Consequently, charge diffusion may occur in such redox polymeric films involving an electron-hopping process from site to site during the 10-second exposure time of the EM-CCD camera (i.e. Ru(bpy) during the application of the constant potential 1.2 V vs. Ag required to generate ECL and to collect the corresponding ECL image).36, 44, 45, 46 This process may explain the extension of the ECL-emitting region in the solution. However, we cannot exclude that the ECL image was distorted by some optical effects related to the light propagation and refraction at the electrode surface and at the different interfaces in this multiphasic system. On the other hand, if one would consider light refraction as the dominant phenomenon, the background of the ECL image should be much more important. In microscopy, depth of field is very short with a 20x objective and usually measured in units of microns. Therefore, contribution of the light emitted out of the focal plane should be negligible. Instead, in the focal plane, we can see that the outer surface of the droplets is very bright, light does not simply come from the contour. ECL imaging experiments at the single droplet level may clarify the observed phenomenon. Whatever, this result shows that ECL emission can be observed in the solution far from the electrode surface. It represents a very significant advantage with respect to classical ECL configurations, where the emission is intrinsically confined to the close vicinity of electrodes. We can see also that the ECL-emitting zones follow the curvature of the droplet and that ECL is localized at the droplet surface. Indeed, ECL is generated at the dodecane/water interface because the luminophore is confined strictly in this region. In other words, ECL is emitted only in the very thin pellicle of the microgel monolayer located around each oil droplet. The thickness of the ECL-emitting layer corresponds to the thickness of the flattened microgels so approximately 200 nm. Therefore, the ECL process is confined at the droplet interface and it results into a 2D ECL light emission.

Conclusions. In this work, we demonstrated that ECL emission can be confined at the interface of oil droplets. Although the role of the water-soluble coreactant has been evidenced, the 14 ACS Paragon Plus Environment

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electrochemical and ECL signatures appear to be more complex than in homogeneous medium, highlighting the possible electrochemical activity of the oil-soluble coreactant. The unusual extent of ECL profile is also original and points out to the possible role of the interconnected 2D network of microgels. From a fundamental point of view, various questions were raised that will deserve further work to better understand the mechanistic aspects. The study at the single droplet level should shed some light on these questions. From an applied point of view, this work opens new opportunities to design sensing in multiphase medium, with the possibility to track interfacial phenomena modifying the electrochemical or the luminescent activity.

Acknowledgment. This work was supported by the Agence Nationale de la Recherche (NEOCASTIP ANR-15CE09-0015-03). The authors thank Michel Martineau from Placamat for cryoSEM imaging, Christelle Absalon from CESAMO for ESI-MS analysis. Supporting Information. The following are available free of charge: materials, microgel characterization, emulsion characterization, PL and ECL imaging experimental section, determination of the TPrA concentrations. Notes. The authors declare no competing financial interests.

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