Pauline Lefrançois

Fanny Girard-Sahun

Vasilica Badets

UPPA, IPREM Franck Clément

Stéphane Arbault
email: stephane.arbault@u-bordeaux.fr

+ V Badets

] P Lefrançois

Electroactivity of Superoxide Anion in Aqueous Phosphate Buffers Analyzed with Platinized Microelectrodes

Keywords: superoxide, ultramicroelectrode, hydrogen peroxide, reactive oxygen species, cold atmospheric plasma

The reactivity of platinized ultramicroelectrodes (Pt-black UMEs) towards superoxide anion O 2 *À , an unstable Reactive Oxygen Species (ROS), and its relatives, H 2 O 2 and O 2 , was studied. Voltammetric studies in PBS demonstrate that Pt-black UMEs provide: i) a wellresolved reversible redox signature for O 2 *À detected in both alkaline and physiological buffers (pH 12 and 7.4); ii) irreversible oxidation and reduction waves for H 2 O 2 at pH 7.4. The oxygen reduction reaction (ORR) at Pt-black surfaces solely yields H 2 O 2 (2 electrons/2 H + ) at physiological pH. Consequently, Pt-black UMEs allow to sense different ROS including superoxide anion for future biomedical or physico-chemical investigations.

Introduction

The Reactive Oxygen and Nitrogen Species (ROS, RNS, merged as RONS) are known to play crucial roles in multiple biological processes, including innate immunity, redox signaling, vasodilation, neuro-modulation, carcinogenesis, etc. [1][2][3][4][5]. ROS are produced by aerobic cells owing to diverse enzymatic systems (NADPH oxidase, Xanthine oxidase, etc.) and by side reactions such as during the reduction the conversion of dioxygen (O 2 ) to water by the mitochondrial respiratory chain (semiquinone intermediate reduction). Bioactive ROS mostly comprise superoxide anion (O 2 *À ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO disproportionation occurs at rate constants of 5.10 À 5 M À 1 •s À 1 and 10 À 9 M À 1 •s À 1 at pH 7.4, either spontaneously or when catalyzed by superoxide dismutases (SOD), respectively. The disproportionation follows the reaction [7]:

2O �À 2 þ 2H þ ! O 2 þ H 2 O 2 (1)
H 2 O 2 is stable over hours in aqueous solution at pH 7.4 and is degraded in presence of metal ion traces (iron or copper ions) [8,9], and catabolic enzymes, namely catalase and peroxidases. The ROS can be involved further in reactions with nitrogen derivatives including nitric oxide (NO * ) to form highly reactive, nitrosative and nitrative species, such as the peroxynitrite anion (ONOO À ), nitrogen dioxide (NO 2 ) and the nitrosonium cation (NO + ) [10,11].

The RONS can also be produced by other physicochemical processes involved in atmospheric pollution or developed for sterilization, chemical reactions, etc. [12][13][14][15] This is the case of Cold Atmospheric Plasmas (CAPs), which are complex ionized gases composed of electrons, neutral atoms or molecules, ions and excited atoms or molecules. CAPs can be produced experimentally at atmospheric pressure and room temperature and are used for a large range of biomedical applications: skin regeneration, skin tumor treatment, teeth whitening, etc. [15][16][17]. The majority of the observed biological effects induced by CAPs are due to RONS.

For these reasons, there is still a large interest for the detection and quantification of the biologically active RONS. However, this remains a challenge, mainly because their short lifetime does not allow enough accumulation. Therefore, their subsequent detection by analytical techniques is very limited [18]. This is partic-ularly true when RONS are produced in aqueous buffers under CAPs exposure. We have recently reported the electrochemical detection of kinetically stable species, including H 2 O 2 , NO 2 À and NO 3 À species, accumulating in PBS during its constant exposure to a CAP [19]. The detection in plasma-treated solutions of H 2 O 2 at concentrations reaching hundreds of μM [20,21], as well as the detection of peroxynitrite ONOO À (tens of μM) by UVvisible absorption spectroscopy [19,20] have strongly suggested the prior existence of superoxide in these solutions. Unfortunately, the hypothesis was not confirmed since the direct detection of O 2 *À in aerobic and physiological conditions remains very challenging. The objective of the present work is to propose an electroanalytical approach to go beyond this limitation.

A wide variety of electrochemical sensors for the detection of RONS have been reported, particularly based on redox enzymes (peroxidase, catalase, etc.) [22][23][24][25]. The specific detection of O 2 *À was shown by using cytochrome c-modified gold electrodes [26]. Ultramicroelectrodes (UMEs) which carbon or platinum surface could be modified by platinum nanostructured rough deposits, quoted as platinum black (Pt-black coating) were later developed [27,28]. Their electrocatalytic properties provide UMEs with the ability to selectively detect several ROS and RNS [29][30][31]. In addition, platinized UMEs are particularly useful for sensitive measurements in micrometric volumes including at single cells [32] and in microsystems (channels and chambers) [33][34][35][36]. Pt-black UMEs display a fast response-time and offer linear current variations over large concentration domains of e. g. H 2 O 2 (from tens of nM to mM) [37]. In addition, we recently observed that Pt-black UMEs allow to detect superoxide anion in alkaline conditions (PBS at pH 12) [37].

The aim of this paper is to further investigate the electroanalysis of O 2 *À as function of the pH in phosphate buffers (pH 12 to 7.4). The reactivity of Pt black UMEs towards the by-products of superoxide disproportionation, H 2 O 2 and O 2 , was studied at pH 7.4 for each species and in solutions containing both species. We demonstrate that superoxide and hydrogen peroxide can be detected selectively at different potentials in aerobic PBS. Finally, we show the direct monitoring of superoxide, when produced and accumulating in alkaline PBS exposed to a CAP. Thus, this study will serve as a reference work to decipher on the complex production of RONS by CAPs and biological sources as well.

Experimental

Chemicals

PBS solution at 10 mM, pH 7.4, was prepared by dissolving the content of a ready-made salt mix (ref. P5368, Sigma Aldrich) in 1 L of pure milliQ water (Millipore, Integral 3, 0.22 μm MilliPak filter). PBS solution at 10 mM and pH 12 was prepared by additions of 1 M sodium hydroxide in PBS pH 7.4 (10 mM). Solutions of H 2 O 2 were prepared by dilutions of a 100 mM stock solution in PBS, freshly prepared from a 9.8 M commercial solution (30 % hydrogen peroxide, Sigma). A stock solution of superoxide was prepared by solubilizing potassium dioxide (ref. 278904 Sigma Aldrich) in ice-cold 30 mM NaOH. The obtained concentration was calculated following a protocol reported elsewhere [37,38]. Briefly, a ~1 M superoxide stock solution was prepared, which corresponds to 5 M KO 2 (Caution! Potassium superoxide reacts violently with water). The 1 : 5 ratio was proposed by Marklund et al. and originates from the high reactivity of O 2 *À [17]. Superoxide dismutase (ref. S5639, Sigma Aldrich) stock solution was prepared at 30 kU.mL À 1 in PBS (10 mM, pH 7.4), aliquoted and stored at À 20 °C. Aliquots were thawed prior to use.

Fabrication and Surface Modification of UMEs

UMEs were prepared by heat-sealing Pt micro-wires (25 μm diameter, 99 % purity, Goodfellow) in glass capillaries (1 mm diameter, WPI) (Figure 1). Each UME was polished with coarse-and fine-grade polishing papers before use or further surface modification. Platinum-black electrodeposition (Pt-black) on UME surface was obtained in potentiostatic mode, at a potential of À 0.06 V vs. Ag/AgCl/NaCl 3 M, by reducing a hydrogen hexachloroplatinate solution (115 mM in 10 mM PBS, pH 7.4 with 0.76 mM of lead acetate). The electrodeposition was stopped when the total reduction charge reached ~0,1 μC μm À 2 (37 μC total charge), which corresponds to a previously defined optimum for the detection of hydrogen peroxide by Pt-black UMEs [39] (Figure 1C).

Electrochemical Measurements

Cyclic Voltammetry

All the characterizations of ROS electrochemical responses were achieved by cyclic voltammetry (CV). Experiments were performed using a gas tight electrochemical cell comprising typically 20 mL of solution. The three electrodes (WE: Pt-black UME, REF: Ag/AgCl wire, CE: Pt wire) were inserted in the septum stopper of the electrochemical cell. When needed, a slight gas flow was injected using a needle for 7-10 min to saturate the solution with either nitrogen or oxygen. The effective solution saturation in N 2 or O 2 was estimated by following the signal evolution in CV. When no modification of the solubilized gas was performed, we will quote the experimental condition as 'aerated' or will not specify it. Measurements were obtained in steady-state conditions at a 20 mV s À 1 scan rate, within different potential ranges, specified for each experiment. All the potentials are computed with respect to an Ag/AgCl (saturated KCl solution)-type reference electrode. All electrochemical measurements were carried out using a bipotentiostat (BioLogic, VSP-300, EC-Lab software) equipped with low current modules and filters set at 5 Hz. The analyses were performed in a Faraday cage to minimize the electric noise.

Collector-generator Experiment

For collector-generator experiments, we prepared platinized carbon fiber-based UMEs, which outer diameter was smaller and easier to manipulate. To prepare a carbon UME, an individual carbon fiber (10 μm diameter, Cytec Engineered Materials, ref. Thornel P-55S) was inserted in a glass capillary (1 mm diameter, WPI) and sealed by heat pulling (Narishige, model PC10). The protruding part of carbon fiber was insulated by electrochemical deposition of poly-oxyphenylene (see [40] for details). The tip of the insulated carbon fiber was then polished at a 45 * angle on a microgrinder (WPI, model 48000, 1 μm roughness) to expose a clean, elliptical carbon surface. The polished carbon surface was then platinized following the same procedure as for Pt UMEs described in the previous section.

In these experiments, two different electrochemical measurements were performed at each UME, by using cyclic voltammetry on one side (from + 0.16 to À 0.34 V) and chrono-amperometry at a fixed potential (+ 0.35 V) on the other one, or chrono-amperometry at a different potential on each UME. A three-electrode electrochemical configuration was used for each UME, composed of the platinized carbon UME as working electrode, an AgCl-coated-Ag wire as pseudo-reference electrode (REF; 1 mm diameter) and a Pt wire as counter electrode (CE; 1 mm diameter). Each UME was coupled to an individual CE whereas a common REF was used for the two UMEs.

An inverted microscope (Leica STP 6000, 20x objective) was used to observe the position of one UME to the other. The experiment was carried out on a clean microscope slide on which a rectangular PDMS frame was placed. All the electrodes were positioned in 1 mL of PBS solution (10 mM, pH 7.4, Sigma). The position of the two UMEs was set with two micromanipulators (ThorLabs, PCS-5200) facing each other at a few micrometers distance.

Analysis in Solution Exposed to a Cold Atmospheric Plasma

The plasma setup used in these experiments has been described previously [20,21]. Briefly, a surrounding gas device allows to control the gaseous environment of the gas phase (plasma). We used helium He as the working gas (1.67 slm rate) and the environment gas was composed of 100 % N 2 (0.03 slm rate; Linde, 99.9995 %), for a total gas flow of 1.7 slm. The production of ionization waves was achieved using a pulsed high voltage power supply with rise and fall times of pulses in the order of 100 ns. The electrical parameters are as following: positive pulses of 7.5 kV amplitude, 1 μs duration, 10 kHz repetition frequency and 1 % duty cycle. 6 mL of PBS at pH 11 was exposed to the plasma in a homemade quartz cuvette (external diameter 44 mm, inner diameter 41 mm, depth 40 mm). A distance of 20 mm was fixed between the end tube of the plasma device and the solution. Moreover, the cuvette was placed over a metallic support connected to the ground in order to ensure a constant potential to the sample. The working WE Pt-black UME, as well as the REF and CE electrodes, were inserted within two insulated-earthed containers in order to protect them from the surrounding high electric field transported by the plasma. Each container was made of a quartz glass capillary (few centimeters long, internal diameter 7-9 mm) covered by a conductive sheath connected to the ground of the building, and covered by an insulating layer of retractable polymer sheath for power cables. Containers (first with WE and REF; second with CE) were placed in the PBS solution and continuous CV was performed at the WE before and during the solution exposure by the CAP to monitor the generation of ROS including superoxide.

Results and Discussion

Redox Behavior of H 2 O 2

First, the electrochemical reactivity of Pt-black UMEs was analyzed in H 2 O 2 solutions. We previously reported the characterization of the oxidation waves of hydrogen peroxide on these surfaces for large concentrations (up to 100 mM) and positive potential ranges [37]. Herein, we focused on lower concentrations (< 1 mM) in order to get closer to the range of concentrations measured in PBS exposed to CAPs [20,21]. Cyclic voltammetry analyses were performed between À 0.2 V and + 0.5 V, allowing to observe both the reduction and oxidation processes for H 2 O 2 . Results are depicted on Figure 2. We observe in these conditions two well separated, irreversible oxidation and reduction waves, which plateaus start around + 0.35 V vs. Ag/AgCl and À 0.15 V vs. Ag/AgCl, respectively. We hypothesize that the oxidation reaction of H 2 O 2 follows the equation (Equation 2):

H 2 O 2 ! O 2 þ 2H þ þ 2e À (2)
(redox couple O 2 /H 2 O 2 )
, while the reduction of H 2 O 2 follows (Equation 3):

H 2 O 2 þ 2H þ þ 2e À ! 2H 2 O (3) (redox couple H 2 O 2 /H 2 O
). Indeed, the amplitudes of both waves are directly dependent on the concentration of H 2 O 2 . The plateau current of each wave was plotted as a function of H 2 O 2 concentration (see Figure S1). The response follows a linear evolution both in oxidation and reduction, providing good calibration curves for Pt-black UMEs in each potential domain. However, the slope is slightly lower in reduction, possibly because of two processes: the interference from phosphate anions, which adsorb on platinum and compete with other species; the formation of platinum oxides and their involvement in the oxidation process [41]. Nevertheless, both waves display similar current ranges for the same concentrations, showing that 2 electrons are involved in each electrochemical reaction. This is very unlikely, thermodynamically and kinetically, at physiological pH that 1e-reduction or oxidation processes occur. The proton concentration is sufficiently high to drive the reactions. Kinetically, hydroxyl radicals would not be stable enough to be observed, as well as for superoxide radical anions, which is demonstrated in the ensuing collector-generator experiments. In addition, H 2 O 2 is a redox amphoteric species, thus the same diffusion coefficient applies for the reduction and oxidation reactions.

Redox Behavior of O 2

The second set of experiments was designed to study the electroactivity of dioxygen on Pt-black UMEs in the potential range of hydrogen peroxide reactions. CV responses were recorded in PBS solutions containing different amounts of dissolved oxygen: either aerated, N 2or O 2 -saturated. Results are presented on Figure 3. A reduction wave, which starts at 0 V and is kinetically slow, appears when the PBS solution is saturated with O 2 , whereas N 2 -saturated PBS response shows solely the PBS background current (capacitive signal). The aerated PBS response shows an intermediate response, corresponding to the 220 μM concentration of solubilized dioxygen in equilibrium in aerated solutions. The Oxygen Reduction Reaction (ORR) catalyzed by platinum-based electrodes is a well-studied reaction with a major focus on the products, being either H 2 O 2 or H 2 O in aqueous solutions [42][43][44][45]. However, little is known about the ORR product on Pt-black surfaces, which have rarely been used for that purpose.

In this study, H 2 O 2 and O 2 are key species (mother or daughter species of superoxide anion). Therefore, it was necessary to know what the product of ORR [46,47] in our experimental conditions is (PBS pH 7.4, Black-Pt UMEs). To do so, we designed a collector-generator experiment that aimed to detect in situ the product of ORR [48] (Figure 4). In this experiment, a reduction reaction and an oxidation reaction can be performed simultaneously but independently at the surface of two electrodes (Figure 4A). When the two electrodes are close enough, their respective diffusion layers overlap. It allows the electro-generation of a species on one electrode (generator) that is detected on the other electrode (collector). Cyclic voltammetry measurements were made at 20 mV s À 1 , between + 0.16 V and À 0.34 V vs. Ag/AgCl, in order to reduce O 2 on UME 1 (Figure 4A, left: generator), while current intensity variations were monitored by chrono-amperometry on UME 2 (Figure 4A, right: collector) at the oxidation potential of H 2 O 2 (+ 0.35 V vs. Ag/AgCl). The reduction wave detected on UME 1 is similar to the one observed in Figure 3 (Figure 4B). This process generates a species that is detected on UME 2, as shown by the simultaneous current variations (Figure 4C). The only candidate is H 2 O 2 in comparison with our previous studies [28,49,50].

Additionally, we confirmed that the species produced at UME 1 is effectively diffusing in solution before being detected at UME 2 by varying the distance between UME 1 and UME 2 (see Figure S2). In this experiment, both UME were set at a fixed potential: a continuous generation on UME 1 (chronoamperometry at À 0.34 V vs. Ag/AgCl) occurred simultaneously with a continuous collection on UME 2 (chronoamperometry at + 0.35 V vs. Ag/AgCl). When the distance between the two electrodes was about 1 μm, the current on UME 2 was maximum (0 .03 nA) whereas it decreased drastically when the distance was superior to 10 μm (~0.015 nA, i. e. close to the background current). Indeed, the diameter of the electrode being of 25 μm, the size of the diffusion layer is about 10-15 μm (in the Nernst approximation). Therefore, the species produced by the reduction of O 2 on UME 1 is no longer detected on UME 2. Overall, the collector-generator experiments demonstrated that H 2 O 2 is the major product of the O 2 reduction reaction on Ptblack electrodes at physiological pH (PBS, pH 7.4).

Redox Behavior of H 2 O 2 and O 2 Mixture

From results depicted on Figures 2 and3, we observe that both O 2 and H 2 O 2 are reduced on Pt-black electrodes at similar potentials, below 0 V vs. Ag/AgCl. As these two species should be produced together during superoxide disproportionation (Equation 1), we studied how they could possibly be discriminated. To answer this question, cyclic voltammetry experiments were realized in N 2 -and O 2 -saturated H 2 O 2 solutions (1 mM) between À 0.2 V and + 0.5 V (Figure 5). When the H 2 O 2 solution was saturated with dioxygen, the reduction current increased compared to the N 2saturated one. It is clear from these curves that the reduction wave of oxygen superimposes with the one of hydrogen peroxide, though a slight shift may be distinguished; the plateau potential for O 2 is observed ~30 mV further than the one of H 2 O 2 . Nevertheless, the total faradaic current at À 0.2 V is indeed the sum of at least the two reduction currents of hydrogen peroxide and dioxygen. Consequently, the two species cannot be discriminated based solely on electro-reduction responses although figure 5 shows also that in the potential range of + 0.1 V and + 0.5 V, the oxidation current is specific to H 2 O 2 . Finally, the electroactivities of H 2 O 2 and O 2 on Ptblack UMEs are intimately related but hard to discriminate upon their reduction waves unless the solution would be analyzed before and after degassing (nitrogen or argon).

Redox Behavior of Superoxide at pH 12 and its Fate at pH 7.4

The next step consisted in studying the response of Ptblack UMEs towards superoxide anion itself. As described in the experimental section, O 2 *À was prepared by solubilizing KO 2 salt in alkaline phosphate buffer (pH 12) in order to stabilize it and therefore facilitate its electrochemical study. Cyclic voltammetry experiments were thus performed in 5 mM superoxide basic solutions (10 mM PBS, pH 12) (Figure 6).

The superoxide anion voltammogram shows a welldefined electrochemical reversible signal on Pt-black surface, which is centered at E * = À 0.085 V vs Ag/AgCl and follows the redox equation (Equation 4, O 2 /O 2 *À redox couple):

O �À 2 $ O 2 þ e À (4)
The dependence between the detected electrochemical wave and the superoxide species was further demonstrated by studying the evolution of this response in CV as function of the pH. We first recorded successive CVs in a 5 mM superoxide solution in phosphate buffer at pH 12 for nearly 30 min (40 cycles at 20 mV s À 1 ). A progressive decrease of the wave amplitude was observed (see Figure S3A) even in such basic conditions. O 2 *À disproportionates (Equation 1) into dioxygen and hydroperoxide anion (HO 2 À , the basic form of H 2 O 2 ). We performed in parallel a similar experiment in a phosphate buffer solution at pH 12 containing only the hydroperoxide anion and observed that the loss of response was more important for the O 2 *À solution (~35 %) than for HO 2 À at pH 12 (~3 %; Figure S3B), as HO 2 À does not follow the disproportionation reaction of O 2 *À . This observation was reinforced by switching rapidly the pH of a 5 mM O 2 *À solution from 12 to ~7.4, as shown on Figure 6. The evolution was monitored by CV in a potential window where all the different species can be detected, i. e. À 0.2 V to + 0.5 V. The pH of the superoxide solution was shifted from pH 12 (blue trace) to physiological values (~7.4-7.6) by additions of concentrated HCl (yellow trace). It led to an increase of the reduction current, due to the appearance of O 2 and H 2 O 2 formed by the O 2 *À decomposition (see Equation 1), while the oxidative current decreased and displayed the oxidation wave of H 2 O 2 observed previously (Figure 2). In order to confirm that no more superoxide remained in the solution, SOD (55 U.mL À 1 final) was added. A very slight increase of reduction and oxidative currents were noticed (purple trace, to be compared with the yellow one), revealing that the major part of superoxide had disproportionated just after the pH change. The shapes of the yellow and purple curves are similar to the one corresponding to an O 2 -saturated H 2 O 2 solution, as described in Figure 5 (red trace). Thus, at this end stage of the experiment, H 2 O 2 and O 2 should be the only species present in solution.

To verify this later assumption, catalase was added to the solution in order to consume H 2 O 2 (orange trace). Compared with the purple trace, the oxidative current due to H 2 O 2 oxidation is vanished and the reduction current is decreased leading to a reduction wave attributable to dioxygen (orange trace). Indeed, H 2 O 2 was consumed by the catalase producing a stoichiometric amount of O 2 that is reduced at low potentials (see Figures 3 and5). Overall, this set of experiments unambiguously demonstrates that the reversible wave detected initially at pH 12 is due to superoxide anion.

Moreover, since the electrochemical reaction (equation 4) corresponding to this wave does not involve any proton, superoxide might be detected at the same potentials, with the same Pt-black surfaces, regardless of pH, assuming that the species is sufficiently chemically stable to be detected by CV. When considering the results reported above about the electrochemical responses of superoxide and its relatives, dioxygen and hydrogen peroxide, we may figure out how to discriminate one from the others as a function of the potential window, in particular at pH 7.4. We observed that at potentials below À 0.1 V, all three species are reduced on Pt-Black UMEs and cannot actually be discriminated from their CV response. Nevertheless, the addition of scavengers and enzymes (SOD, catalase) would allow to make a decision since the only species remaining after enzymatic conversions is O 2 . However, as shown on Figures 2,5 and 6, at neutral potential, i. e. between 0 and + 0.1 V, hydrogen peroxide and oxygen are not detected, only a background current is observed (with slightly negative current values due to the reduction of platinum oxides). Therefore, we can assume that an oxidation current between 0 V and + 0.1 V recorded at pH 7.4 would only correspond to superoxide (if produced in sufficient amount). At higher potentials, above + 0.1 V, up to + 0.5 V herein, O 2 *À and H 2 O 2 can be both detected meaning that the oxidation current due to H 2 O 2 would add to the plateau current of the superoxide oxidation wave already detected at + 0.1 V. Consequently, as function of the CV shape and current amplitudes in the range of positive potentials, we might be able to decipher on the contributions of O 2

*À and H 2 O 2 .
This set of analyses opens avenues for the detection of superoxide in aqueous solutions treated by CAPs, for two reasons: 1/ superoxide has already been detected indirectly in plasma-treated solutions [51,52]) and is present transitorily at high concentrations; 2/ CAPs are considered as a source of superoxide (and other RONS) which dissolve at the interface between the gas phase and the liquid solution and does not change significantly the pH of the PBS [19], contrary to the release of superoxide in PBS using the KO 2 source.

In this context, we recently reported [21] the results of in situ measurements, with shielded Pt-black UMEs, of species accumulating in PBS exposed to a CAP. We observed in PBS pH 7.4 the progressive appearance of chemically stable species, namely H 2 O 2 and NO 2 À , as well as the rise of an oxidation wave and current near 0 V vs Ag/AgCl. This species was detected after long exposure durations (typically 30 min.) of the PBS corresponding to its accumulation and increase of its net flux versus reactions involved in its disappearance. The same experiment was performed in PBS at pH 11, a condition which increases superoxide stability and should favor its steadystate detection. In situ monitoring of O 2 *À along its generation and accumulation was performed with a shielded Pt-black UME during the solution exposure to a He CAP (see material and methods for details about the setup). As shown in Figure 7, a reversible wave appears after 20 min. and rises in amplitude over the exposure duration. The wave, e. g. detected at 26 min., is very similar in characteristics to the one detected in Figure 6 with the same WE in a solution supplied with the superoxide species. This demonstrates the in situ detection of O 2 *À , which concentration at this time point can be estimated to reach about 400 μM. Moreover, the chemical evolution of superoxide solutions, as discussed above, was also observed in the exposed PBS since a second wave that can very likely be attributed to H 2 O 2 was observed after 30 min. The kinetic evolutions of exposed PBS solutions at pH 11 were slower, as expected, than what was observed recently at pH 7.4 [21]. Finally, based on the set of studies reported herein, we draw the conclusion that the species detected in CAP-treated PBS might be superoxide anion. No other RONS (including H 2 O 2 , NO * , ONOO À , HNO and NO 2 À ) has shown the same electrochemical and chemical features, particularly when detected with Pt-black modified electrodes.

Conclusions

In this study, we analyzed the redox behavior of O 2 *À in a physiological phosphate-buffered saline (PBS) solution, the most commonly used in bioanalyses. As superoxide is not stable and rapidly disproportionates into H 2 O 2 and O 2 , it was necessary to decipher on the reactivity of Ptblack UMEs towards these species when they are mixed in solution. We showed that H 2 O 2 has a clear redox signature in both oxidation and reduction. Nevertheless, since O 2 is naturally dissolved in solution when working in open environment, the detection by reduction of H 2 O 2 appeared to be more complex because its wave overlaps with the one of O 2 reduction. Moreover, we showed that the ORR at Pt-Black (pH 7.4) is a 2 electrons and 2 H + process leading to H 2 O 2 . Thus, oxygen cannot be specifically measured by Pt-black UMEs if hydrogen peroxide is present. However, we have identified that H 2 O 2 can be selectively detected with Pt-black UMEs in the potential range of + 0.1 V to + 0.5 V owing to its first oxidation wave.

The redox response of superoxide on Pt-black UMEs was studied first in alkaline conditions, which favors its stabilization, and then in physiological conditions by changing the pH of the solution. Despite the presence of H 2 O 2 and O 2 in solutions at both pH 7.4 and 12, we have identified that 0 V (no overpotential vs Ag/AgCl) is an adequate potential to specifically detect superoxide by Ptblack UMEs based on its oxidation response, regardless of pH. This result opens a range of possibilities concerning the electroanalysis of O 2 *À in physiological conditions, for instance when produced by cells in NAPDH oxidasedependent activities, as well as for biomedical applications of Cold Atmospheric Plasmas (CAPs) produced in air.

Fig. 1 .

 1 Fig. 1. Multiscale view of a platinized platinum ultramicroelectrode (Pt wire Ø 25 μm, total external diameter 1 mm) used as WE in the experiments. (A) Side view of the whole electrode. (B) Top view of the electrode. The Pt wire (encircled) is sealed in a glass capillary. Scale bar = 100 μm. (C) Platinum-black nanostructure observed by scanning electron microscopy. Scale bar = 5 μm.

3. 1

 1 Redox Behavior of Hydrogen Peroxide and Oxygen in PBS pH 7.4

Fig. 2 .Fig. 3 .

 23 Fig. 2. Cyclic voltammetry responses of H 2 O 2 solutions using Ptblack UMEs. H 2 O 2 solutions from 0.2 to 1 mM concentration are prepared in aerated physiological PBS at pH 7.4. Scan rate: 20 mV s À 1 . Fig. 3. Cyclic voltammetry responses on a Pt-black UME of aerated (black trace), N 2 (blue trace) and O 2 (red trace) saturated PBS solutions (10 mM, pH 7.4). Scan rate: 50 mV s À 1 .

Fig. 4 .

 4 Fig. 4. A collector-generator experiment with two UMEs was performed to decipher on the ORR mechanism at Pt-black surfaces. A. Schematic representation describing the experiment principle. Inset: Image of the two electrodes used for the generator-collector experiment observed in transmitted light (bright field, inverted microscope). The distance between the two UMEs was set using two micromanipulators. B. Cyclic voltammogram of UME 1 at potentials varying between + 0.16 V to À 0.34 V (generator) at 50 mV s À 1 . C. The induced current is recorded on UME 2 (black trace). The potential variations applied to the generator are displayed (dashed grey trace) to correlate with the collector signal.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Cyclic voltammetry responses on Pt-black UMEs of N 2 -(blue trace) and O 2 -(red trace) saturated H 2 O 2 solutions (1 mM) prepared in PBS (10 mM, pH 7.4). Scan rate: 20 mV s À 1 .Fig.6. Evolution of the cyclic voltammetry response (on Pt-black UMEs) in a superoxide basic solution (blue trace, t = 0), when pH is changed to physiological values (yellow trace), followed by SOD (55 U mL À 1 final, purple trace, t = 13 min) and catalase (> 100 U mL À 1 final, orange trace, t = 17 min) additions. Scan rate: 20 mV s À 1 .

Fig. 7 .

 7 Fig. 7. Evolution of the cyclic voltammetry response in a PBS solution at pH 11 (gray trace, t = 0), along its exposure to a cold atmospheric plasma (He working gas; 100 % N 2 environment). Measurements were performed directly, in situ (6 mL cell volume), with a shielded-grounded electrochemical cell (Pt-black UME, REF and CE placed in shielded tubes). Scan rate: 20 mV s À 1 .

www.electroanalysis.wiley-vch.de © 2020 Wiley-VCH GmbH Electroanalysis 2020, 32, 1 -10 6These are not the final page numbers! ��

Acknowledgements

This work was financially supported by the Centre National de la Recherche Scientifique (CNRS), the French Ministry of Research (MESR) and the Agence Nationale de la Recherche (ANR, PLASMAREGEN project, no. ANR-14-CE16-0007-01).

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

FULL PAPER

P. Lefrançois, F. Girard


Jingjing Zhang

Stéphane Arbault

Neso Sojic
email: sojic@enscbp.fr

Dechen Jiang
email: dechenjiang@nju.edu.cn

Electrochemiluminescence Imaging for Bioanalysis

Keywords: Electrochemiluminescence imaging, spatial/temporal resolutions, multiplex bioassay, single cells/particles, co-reactant pathway, bipolar electrochemistry

whether they are published or not. The documents may come   L'archive ouverte pluridisciplinaire

In the past ten years, thanks to the improvement of single photon counter devices, microscopy objectives with greater numerical aperture (N.A.) and optimized light transduction pathways, the detection efficiency of weak luminescence was greatly improved. Moreover, the appearance of new materials with excellent electrochemical properties accelerates the reaction rate of luminophore emitting higher luminescence. Coupled with novel electrode designs and detection strategies, the spatial and temporal resolutions in ECL imaging reach nanometer level and millisecond level, respectively. As a result, high throughput and/or local study in bioanalysis using ECL imaging is realized. In this review, the mechanisms, the electrode designs and the bioanalytical applications, including at single cells and particles, are summarized and discussed.

Mechanisms and Apparatus of ECL imaging

2.1 Co-reactant pathway in ECL imaging ECL processes can be divided into two main dominant mechanisms: the annihilation pathway and the co-reactant pathway. The earliest ECL reactions were reported by the annihilation mode where an oxidized and a reduced species are electrochemically produced at an electrode surface by applying alternating pulsed potentials [START_REF] Hercules | Chemiluminescence resulting from electrochemically generated species[END_REF][START_REF] Visco | Electroluminescence in solutions of aromatic hydrocarbons[END_REF][START_REF] Santhanam | Chemiluminescence of electrogenerated 9,10diphenylanthracene anion radical[END_REF] . Then, these two radicals react together in the diffusion layer according to an annihilation reaction to generate the excited state of the luminophore. Finally, it relaxes to the ground state and emits a photon. However, a major limitation for bioanalytical applications is that this pathway is limited to aprotic solvents. An alternative mode of ECL generation with more complex mechanisms is based on the use of co-reactants. In the early 1980s, the discovery of ECL in aqueous phase involving [Ru(bpy) 3 ] 2+ as a luminophore and a sacrificial co-reactant opened a new area [START_REF] Rubinstein | Electrogenerated chemiluminescence. 37. Aqueous ECL systems based on tris(2,2'-bipyridine)ruthenium(2+) and oxalate or organic acids[END_REF][START_REF] Noddsinger | Generation of chemiluminescence upon reaction of aliphatic amines with tris(2,2'-bipyridine)ruthenium(III)[END_REF][START_REF] Leland | Electrogenerated chemiluminescence: an oxidative-reduction type ECL reaction sequence using tripropyl amine[END_REF] . The large majority of ECL imaging in bioanalysis is based on a mechanism involving the reaction of a co-reactant species with the luminophore by applying a single anodic potential. One of the most widely used ECL imaging systems is currently the organometallic [Ru(bpy) 3 ] 2+ complex with amine-based co-reactants, such as tripropylamine (TPrA) or 2-(dibutylamino) ethanol. Since [Ru(bpy) 3 ] 2+ species could be linked to proteins, this ECL imaging system is mainly applied for immunoassays. Another classic ECL imaging system is luminol (5-amino-2, 3-dihydro-1,4-phthalazinedione) which reacts with hydrogen peroxide to produce light. The luminescence is positively dependent on the amount of hydrogen peroxide, and thus, this imaging system attracts the interest for bioanalytical applications in the context of detection of reactive oxygen species and oxidative stress. In addition, it allows monitoring the enzymatic activity of oxidases which generate hydrogen peroxide in presence of their substrate.

ECL mechanism depends on numerous experimental parameters: electrode material, solvent, pH, presence of surfactant, respective concentrations of the co-reactant and of the ruthenium complex, hydrophobicity of the electrode surface, etc. The different competitive mechanistic pathways of [Ru(bpy) 3 ] 2+ /TPrA couple for ECL imaging can be classified into two main groups depending on how [Ru(bpy) 3 ] 2+ is oxidized. The first "direct" route, as demonstrated in Figure 1A, requires the direct oxidation of the ruthenium complex at the electrode surface to generate ECL. However, these mechanistic routes cannot account for the ECL imaging reported at low oxidation potentials and also for the excellent sensitivity of the bead-based immunoassays. A second "revisited" route involving the diffusing TPrA radicals, as shown in Figure 1B, explains how ruthenium complexes located at micrometric distances from the electrode surface produce ECL. [START_REF] Deiss | Multiplexed sandwich immunoassays using electrochemiluminescence imaging resolved at the single bead level[END_REF] In this mechanism, only the coreactant TPrA is oxidized at the electrode and the resulting radicals, TPrA •+ and TPrA • , diffuse and react with the luminophore to generate an excited state and photon emission (~ 615 nm). [START_REF] Miao | Electrogenerated chemiluminescence 69: the Tris(2,2'bipyridine)ruthenium(II),(Ru(bpy) 3 2+ )/Tri-n-propylamine (TPrA) system revisited-A new route involving TPrA •+ cation radicals[END_REF][START_REF] Sentic | Mapping electrogenerated chemiluminescence reactivity in space: mechanistic insight into model systems used in immunoassays[END_REF] ECL of this system is obtained at physiological pH and the luminophore is regenerated during the process.

Different mechanisms about luminol/hydrogen peroxide system have been proposed depending on the applied potentials and the experimental conditions. [START_REF] Sakura | Electrochemiluminescence of hydrogen peroxide-luminol at a carbon electrode[END_REF][START_REF] Lin | Potential-resolved electrochemiluminescence of luminol in alkaline solutions at glassy carbon and platinum electrodes[END_REF][START_REF] Cui | Multi-channel electrochemiluminescence of luminol in neutral and alkaline aqueous solutions on a gold nanoparticles self-assembled electrode[END_REF] Classically, luminol deprotonates in basic solution to form an anion that is first oxidized to a diazaquinone form and the ensuing 3aminophthalate in the excited state at the electrode surface. This latter decay to the ground state and emits typical light at 425 nm. Dedicated to the imaging in neutral solution for bioanalysis, a luminol analog, 8-amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4 (2H,3H) dione sodium salt (L012), is frequently used, because this anion can be electrochemically oxidized and emit much stronger luminescence than luminol, as displayed in Figure 1C. With a co-reactant such as hydrogen peroxide, oxygen radicals are electrochemically produced at the electrode and accelerate the generation of the excited 3-aminophthalate for enhanced ECL. In this system, luminol undergoes an irreversible oxidation, which means that it can produce photon just once in the process and not electrocatalytically as the [Ru(bpy) 3 ] 2+ luminophore. On the other hand, luminol ECL requires a lower anodic potential (i.e. ~ 0.4 V) than [Ru(bpy) 3 ] 2+ , which is favorable for the imaging of living cells.

Apparatus for ECL imaging

Since no excitation light is involved, the setup of ECL imaging is relative simple and can be adjusted based on the applications. Figure 1D shows the schematic illustration of a basic ECL imaging setup, which is assembled with a simple bright field microscopy, an electrochemistry module (including a cell and a potential generator) and a charge coupled device (CCD) recording the image.

The typical electrochemical module is based on a classical two or three-electrode system applied with a direct current (DC) potential. [START_REF] Yamazaki-Nishida | Direct current electrogenerated chemiluminescence observed with [Ru(bpz) 3 2+ ] in fully aqueous solution[END_REF] Many well-developed potential protocols to boost the electrochemical property of the electrode surface or generate more radicals in the electrochemical process have been adopted for enhanced luminescence during the imaging. We proposed, a periodic switching between positive and negative potential applied at indium tin oxide (ITO) slides was hypothesized to minimize the metallic surface passivation and provide stronger luminescence from luminol/hydrogen peroxide system. [START_REF] Zhou | Electrochemiluminescence imaging for parallel single-cell analysis of active membrane cholesterol[END_REF] Also, a bi-potentiostatic mode was exerted on two pieces of ITO slides that were assembled in a thin-layer cell, in which one piece was in charge of yielding enough oxygen radicals under low potential and another boosted the oxidative intermediate of luminol under high potential. [START_REF] Ming | A new strategy for exciting the electrochemiluminescence of luminol by double-static potential[END_REF] As a result, a stronger and constant ECL intensity was obtained through energy transfer between those active species. Furthermore, an alternating current (AC) potential was introduced at the electrode surface to produce the reversible switched bias polarity between anode and cathode. The generation of oxidized and reduced species at the same electrode surface resulted in the efficient generation of annihilation ECL without long-distance diffusion of the relevant species providing a high-intensity emission. [START_REF] Eßmann | Alternating current-bipolar electrochemistry[END_REF] An objective with a high numerical aperture (N.A.) to increase the collecting efficiency and a high sensitive CCD camera to enable single photon detection are two other critical components for high spatial resolution ECL imaging. Due to the weak luminescence generated, the spatial resolution can be compromised by the magnification of the object and the smallest size generating a measurable luminescence. It is noted that, if the luminophore is not attached to the sample, the ECL intermediates could be present in microseconds during this process. This diffusion of these species induces the delocalization between the bright-field and ECL images affecting the spatial resolution of ECL image. [START_REF] Zhou | Electrochemiluminescence imaging for parallel single-cell analysis of active membrane cholesterol[END_REF] Therefore, the current spatial resolution of ECL imaging is restricted to a few micrometer, and the achievement of nanometer resolution imaging is still challenging.

For the visual detection of bio-molecules that does not need the spatial distinction, a simple CCD or even a mobile phone camera is sufficient to image the luminescence. By analyzing the pixel intensity in the digital ECL image, di-n-butylaminoethanol (DBAE) as low as 250 μM was detected using the mobile camera. [START_REF] Hao | Portable thermo-powered highthroughput visual electrochemiluminescence sensor[END_REF][START_REF] Delaney | Electrogenerated chemiluminescence detection in paper-based microfluidic sensors[END_REF] Most importantly, as compared with high sensitive CCD with two colors only, the digital camera has the inherent color selectivity that could be applied to exploit the distinct excitation and emission properties of concomitant ECL. This feature permits multiplexed ECL detection for the development of low-cost and portable clinical diagnostic devices. [START_REF] Doeven | Red-green-blue electrogenerated chemiluminescence utilizing a digital camera as detector[END_REF] 3. ECL imaging at wired and wireless electrodes for bioanalysis.

Wired electrodes

The ECL signal depends directly on the concentration of the luminophore as well as of the coreactant. Therefore, both can be potentially detected and quantified as their concentrations directly 2A) [START_REF] Zhou | A novel biosensor array with a wheel-like pattern for glucose, lactate and choline based on electrochemiluminescence imaging[END_REF] . These enzymes could be co-included in a photopolymer of sepharose beads charged with the different enzymes and of luminol supporting beads. [START_REF] Marquette | Electrochemiluminescent biosensors array for the concomitant detection of choline, glucose, glutamate, lactate, lysine and urate[END_REF] The particular performances of each biosensing spots (choline, glucose, glutamate, lactate, lysine and uric acid) were measured with equivalent analytical performances in comparison to mono-enzymatic detection without a significant cross reactivity.

Our group fabricated nanoneedle electrodes that could generate visible ECL at the nanometer-sized tip through enhanced mass transfer of luminophore and/or co-reactants at the tip. [START_REF] Sentic | Microscopic imaging and tuning of electrogenerated chemiluminescence with boron-doped diamond nanoelectrode arrays[END_REF][START_REF] Zhang | Localized electrochemiluminescence from nanoneedle electrodes for very-high-density electrochemical sensing[END_REF] Glucose oxidase was embedded in the Nafion coated electrode surface and the generated ECL intensity of all the nanoneedles was mapped. The concentration of glucose ranging from 0.5 to 5 mM was quantified that demonstrated a very high-density (over 4 × 10 3 spots/mm 2 ) electrochemical analysis (Figure 2B). [START_REF] Zhang | Localized electrochemiluminescence from nanoneedle electrodes for very-high-density electrochemical sensing[END_REF] The sensitive detection of biomarker proteins is of fundamental importance for early diagnostics and therapy monitoring. Currently, a large number of immunoassays are commercialized for cardiac and infectious diseases, thyroid and tumor markers, etc. ECL imaging has been exploited by Rusling et al. in an array format to detect two cancer biomarker proteins (IL-6 and PSA) in patient sera. [START_REF] Sardesai | Carbon nanotube microwell array for sensitive electrochemiluminescent detection of cancer biomarker proteins[END_REF] Their approach involved microwells incorporating antibody-coated SWCNT forests surrounded by hydrophobic polymer walls. Such nanostructured electrode surfaces provided high functionalized surface areas for the attachment of large populations of capture antibodies in sandwich immunoassays.

In addition, silica nanoparticles containing [Ru(bpy) 3 ] 2+ were employed to enhance the ECL signals.

The detection limits for IL-6 and PSA were equivalent to or better than commercial bead-based protein measurement systems. The spatial distribution of ECL-active sites of the array in ECL imaging present a significant advantage over voltammetric arrays in that individual electrical connections to the individual wells or spots are not necessary. This approach has been extended for the detection of 4 proteins with microfluidic channels. [START_REF] Kadimisetty | Automated multiplexed ECL immunoarrays for cancer biomarker proteins[END_REF] Genotoxicity screening was also reported using ECL imaging of array spots containing DNA, various isoforms of cytochrome P450, and ECL-active metallopolymer [Ru(bpy) 2 PVP 10 ] 2+ (Figure 2C). [START_REF] Hvastkovs | Electrochemiluminescent arrays for cytochrome P450-activated genotoxicity screening. DNA damage from benzo[a]pyrene metabolites[END_REF] The thin-film spots were exposed to hydrogen peroxide to activate the enzymes, which generate metabolites. Several human isoforms of cytochrome P450 were simultaneously tested for their activity toward the procarcinogen benzo[a]pyrene. Up to 50 distinct enzyme/DNA spots in an array were simultaneously imaged allowing to estimate relative rates of DNA damage associated with the enzymes. Relative genotoxicity from bioactivated metabolites is thus assigned to specific enzymes.

Various amplification strategies are designed and implemented in ECL immunosensor arrays.

These strategies are essential in ECL imaging to increase the signal-to-noise ratio (contrast) in the images. For example, the ECL intensity was enhanced by associating polymer dots and a dual DNA amplification strategy. [START_REF] Wang | Electrochemiluminescent imaging for multi-immunoassay sensitized by dual DNA amplification of polymer dot signal[END_REF] It was achieved on the immunoarrays by rolling circle amplification of oligonucleotide strands and then enzymatically cyclic release of polymer dots from self-quenched probes. The polymer dots showed 11 times higher ECL efficiency than previously reported systems,

and the deposition of gold on ITO enhanced the ECL signal by 100-fold. In addition, the high quenching efficiency of black hole quencher provided low background for "off-on" imaging.

Su et al. reported a very original application of ECL imaging by visualizing latent fingerprints in

both negative and positive modes. [START_REF] Xu | Imaging latent fingerprints by electrochemiluminescence[END_REF] In the negative-imaging mode, the ITO electrode bearing a sebaceous (sebum-rich) fingerprint make the underlying surface ECL inert or less active. Thus, the ECL emission was generated mainly from the bare surface in presence of TPrA. This mode does not involve any pre-/post-treatment, which is usually laborious and requires in most of current detection methods. In the positive mode, the fingerprint ridge deposit was tagged with a ruthenium derivative.

These immobilized ECL-generating luminophores react with freely diffusing co-reactants to generate locally ECL. This strategy was further extended to the immuno-imaging of secretions in human perspiration present in latent fingerprints using horseradish peroxidase and luminol as labels and luminophores, respectively. [START_REF] Xu | Electrochemiluminescence imaging of latent fingermarks through the immunodetection of secretions in human perspiration[END_REF] Figure 2D shows an ECL image of an human immunoglobulin G (hIgG) groomed fingermark. This ECL imaging approach enables both identification of an individual and recognition of the secretions in the human perspiration.

Wireless electrodes

Bipolar electrochemistry (BPE) is an electrochemical approach based on the polarization of a conducting object placed in an electric field, which generates the potential difference between the two extremities. With sufficient potential difference, opposite redox reactions are triggered at these two extremities. [START_REF] Fosdick | Bipolar electrochemistry[END_REF][START_REF] Loget | Shaping and exploring the micro-and nanoworld using bipolar electrochemistry[END_REF] Since Fleischmann et al. demonstrated the water splitting using bipolar microelectrodes, multiple applications in the fields of sensors, electrocatalyst, fluorescence, material enrichment and separation have been developed. [START_REF] Loget | Bipolar electrochemistry: from materials science to motion and beyond[END_REF] The attractive feature of bipolar electrodes is the absence of the connection of the electrode with the external power, which facilitates electrochemistry to be carried out on the microelectrodes.

The first combination of ECL with BPE in a microfluidic system by Manz et al. in 2001. [START_REF] Arora | A wireless electrochemiluminescence detector applied to direct and indirect detection for electrophoresis on a microfabricated glass device[END_REF] In the next year, Crooks et al. improved the detection principle and used the anodic reaction solely as an ECL reporter of events occurring at the cathode. [START_REF] Zhan | Electrochemical sensing in microfluidic systems using electrogenerated chemiluminescence as a photonic reporter of redox reactions[END_REF] Accordingly, the electrochemical reduction of the analyte, benzyl viologen (BV 2+ ), could be indicated by the luminescence intensity from the oxidation of Ru(bpy) 3 2+ in presence of TPrA. The successful coupling of ECL and BPE provides a convenient and sensitive means for direct photonic readout of electrochemical reactions.

Taking the advantage of ECL imaging, a current flowing in multiple bipolar electrodes is observed as light emission simultaneously. In the first work, an electrochemical DNA microarray was fabricated within a microfluidic channel as an "open" BPE system. [START_REF] Chow | Wireless electrochemical DNA microarray sensor[END_REF] The interfacial potential of multiple, well-defined bipolar electrodes were controlled by two wires regardless of the electrode number (Figure 3A). By measuring the luminescence intensity in the image, the hybridization of DNA oligonucleotides was sensed at the array. [START_REF] Khoshfetrat | Wireless electrochemiluminescence bipolar electrode array for visualized genotyping of single nucleotide polymorphism[END_REF] The current electrode density in the array for ECL imaging was reported to be ∼2000 sensing elements/cm 2 , which was comparable to the typical density for a fluorescence-based microarray. [START_REF] Chow | A large-scale, wireless electrochemical bipolar electrode microarray[END_REF] The physical separation of the ECL reporting cocktail and the solution containing the target is important to prevent chemical interferences between the two extremities. To address this issue, a twochannel microelectrochemical sensor that communicates between separate sensing and reporting microchannels via one or more BPEs was designed as the "closed" BPE system. When the sensing pole was activated by a target, ECL was emitted at the reporting pole. [START_REF] Chang | Two-channel microelectrochemical bipolar electrode sensor array[END_REF] ECL signal obtained at the anode of bipolar electrode in the closed BPE was observed to be much stronger than that in the open BPE, confirming the enhanced potential difference between solution/BPE interface in the closed BPE.

To expand the application range of the [Ru(bpy) 3 ] 2+ -TPrA anodic ECL reaction, the three-channel and BPE configuration was designed to include all the oxidants, reductants, or chemicals so that all of them can be detected in a single device. [START_REF] Zhang | Full-featured electrochemiluminescence sensing platform based on the multichannel closed bipolar system[END_REF] Biorecognition elements (aptamer or antibody) and a probe (e.g. thionine) can be immobilized at the cathodes by the recognition of different target analytes. In presence of more analytes, the electrochemical conversion of thionine increases, enhancing the ECL signals due to the charge neutrality of bipolar electrode. As a result, multiplex detection of cancer biomarkers was realized on the cathodic poles of BPEs that was reported in the ECL images. [START_REF] Wu | Visual electrochemiluminescence detection of cancer biomarkers on a closed bipolar electrode array chip[END_REF] The gap between the two ITO bands could be regarded as an electrical switch, the conductivity of which were regulated by the protein (e.g. prostate cancer antigen, PSA) guided silver particles. In this strategy, an electronic circuit between the adjacent BPEs was achieved by the deposition of silver particles in presence of PSA. Accordingly, a sensitive visual measurement of PSA was realized by the imaging of ECL at BPE (Figure 3C). [START_REF] Wu | Electrochemiluminescence on bipolar electrodes for visual bioanalysis[END_REF] In the previous examples, the multiplexing capabilities of ECL imaging rely mainly on the fabrication of arrays of sensing spots. But the performances may be improved by tuning the ECL emission colors. Indeed, multiple, spectrally distinct ECL luminophores add a new dimension for multi-analyte imaging and increase the complexity of the analytical platforms. 62,63, Bidirectional color change from blue-green to red was observed by selective excitation of iridium and ruthenium complexes by scanning the electrode potential. [START_REF] Wang | Bidirectional electrochemiluminescence color switch: an application in detecting multimarkers of prostate cancer[END_REF][START_REF] Li | Spatially-resolved multicolor bipolar electrochemiluminescence[END_REF] Imaging the color switching from blue-green to red enabled the measurement of multiple prostate cancer biomarkers (PSA, circulating microRNA-141, and sarcosine) (Figure 3D). [START_REF] Wang | Bipolar electrode based multi-color electrochemiluminescence biosensor[END_REF] Such array platforms using enzymes or proteins show the interest of performing highly multiplexed assays using ECL imaging.

Both wireless features and asymmetric redox reactivity of BPE is exploited, which offers the possibility to design analytical ECL swimmers. The application of an electric field across the solution promoted simultaneous reduction of O 2 at the cathodic pole of a conductive bead and oxidation of ECL reagents at its anodic pole. Thus it induced both motion and ECL-light emission from the bead in a capillary. [START_REF] Sentic | Light-emitting electrochemical "Swimmers[END_REF][START_REF] Bouffier | Lighting up redox propulsion with luminol electrogenerated chemiluminescence[END_REF] In this case, ECL imaging provides a direct monitoring of the swimmer motion, which could be very useful for localizing micromotors. An analytical task was also associated to such ECL swimmers to explore and report the local glucose concentration by combining it with an enzymatic reaction. [START_REF] Sentic | Electrochemiluminescent swimmers for dynamic enzymatic sensing[END_REF] Glucose dehydrogenase oxidized glucose to gluconolactone with the concomitant conversion of NAD + to NADH, which acted as a co-reactant in the ECL process. The simultaneous oxidation of the luminophore and of the enzymatically-produced NADH led to ECL emission with a glucose-dependent light intensity. A concentration gradient of glucose was explored by the ECL swimmer and, as it was moving towards a region of higher glucose concentration, its ECL intensity increased. Thus ECL allowed to visualize and measure remotely the local concentration of the enzymatic substrates. [START_REF] Sentic | 3D electrogenerated chemiluminescence: from surface-confined reactions to bulk emission[END_REF] This concept was later extended to 3D ECL by generating light at the level of millions of micro-emitters dispersed in solution. [START_REF] Sentic | 3D electrogenerated chemiluminescence: from surface-confined reactions to bulk emission[END_REF][START_REF] Poulpiquet | Dual enzymatic detection by bulk electrogenerated chemiluminescence[END_REF] Each single object was addressed remotely by BPE and they generate collectively the luminescence in the bulk. Therefore, the entire volume of the solution produces light and ECL imaging was used as a readout method to visualize the concentration gradients in a single ECL image.

ECL imaging of single objects.

Single cells.

High throughput and spatial/temporal analysis at single living cells provide valuable information about the cell features that are associated with cellular heterogeneity. Although fluorescence imaging is popular to investigate the distribution and content of bio-molecules, the design and synthesis of specific fluorescence probe for small molecules are time-consuming, and the specificities of these probes often subject to caution. Moreover, high background signal in fluorescence image of cells is defavorable to visualize low-content membrane proteins. While, ECL imaging involves commercially available oxidases to react with the corresponding small molecules generating hydrogen peroxide.

Hydrogen peroxide produced enhances ECL from aqueous luminol and the luminescence intensity is proved to be correlated with the amount of target molecules. [START_REF] Ma | Luminol electrochemiluminescence for the analysis of active cholesterol at the plasma membrane in single mammalian cells[END_REF][START_REF] Tian | Fast serial analysis of active cholesterol at the plasma membrane in single cells[END_REF] The detection strategy is illustrated in Figure 4A. More importantly, ECL imaging could efficiently minimize the background signals for the sensitive measurement of small molecules and membrane proteins at cells. [START_REF] Richter | Electrochemiluminescence (ECL)[END_REF][START_REF] Hesari | Review-electrogenerated chemiluminescence: light years ahead[END_REF] To perform single cell ECL imaging, cell-sized microwells were fabricated at ITO slides to retain individual living cells. Visible ECL from luminol and hydrogen peroxide were imaged at bare ITO regions in the microwells. The modification of g-C 3 N 4 nanosheets at ITO regions catalyzed the decomposition of hydrogen peroxide resulting in more •OH radical and enhanced ECL. [START_REF] Xu | C 3 N 4 nanosheet modified microwell array with enhanced electrochemiluminescence for total analysis of cholesterol at single cells[END_REF] Accordingly, the lowest visualized concentration reported by our group was 500 nM that guaranteed the detection of low concentrated cholesterol at single cells in parallel. During single cell imaging, the consumption of membrane molecules by oxidases was completed in a few minutes. The continuous generation of hydrogen peroxide from cellular surface and the escape (free diffusion) of hydrogen peroxide from the microwells to the bulk solution are proved to offer a near steady-state concentration at bare ITO regions in a certain time period. [START_REF] Tian | Fast serial analysis of active cholesterol at the plasma membrane in single cells[END_REF] By recording the luminescence in this period (Figure 4B), membrane cholesterol at 64 cells was quantified and a heterogeneity as high as 35.7% for cholesterol content was observed exhibiting the significance of single cell analysis.

The expansion of single cell ECL imaging to intracellular analysis is challenging, but important, for the biological study because the cellular activity is strongly associated with the fluctuation of intracellular molecule amount. Triton X-100 was co-added with oxidase to break plasma membrane of individual cells in the microwells. The intracellular molecules, such as glucose and cholesterol ester, were released and then reacted with the oxidases to generate hydrogen peroxide in the microwell. [START_REF] Xu | Analysis of intracellular glucose at single cells using electrochemiluminescence imaging[END_REF] The luminescence from 64 individual cells on one ITO slide was visualized, which enlights the feasibility of intracellular ECL imaging at single cells. Further evidence exhibited that the removal of membrane cholesterol by cholesterol oxidase maintained the cellular membrane intact. [START_REF] Zuo | Determining the composition of active cholesterol in the plasma membrane of single cells by using electrochemiluminescence[END_REF] Therefore, the same individual cells could be exposed to different oxidases in multiple steps providing the images of membrane and intracellular cholesterol. [START_REF] Xu | C 3 N 4 nanosheet modified microwell array with enhanced electrochemiluminescence for total analysis of cholesterol at single cells[END_REF] This co-detection of multiple molecules should offer more information for the study of cholesterol-related pathways at single cells.

Although single cell imaging by ECL could provide high-through analysis, applying this technique for clinic tests requires simple fabrication of the chips. Currently, multiple-step fabrication of photoresist based microwells involving the coating and partial removal of photoresist from ITO slide is well-developed, but still complicated. A simple protocol using a silica stamp was developed to prepare PDMS microwells that take place of the previous photo-resist microwells. [START_REF] Xia | Gold-coated polydimethylsiloxane microwells for high-throughput electrochemiluminescence analysis of intracellular glucose at single cells[END_REF] After a simple spraying of an Au layer on the PDMS chip, a clean conductive surface was produced to induce higher luminescence intensity for single cell glucose analysis. This two-step fabrication does not involve the exposure and washing process that simplifies the chip production.

Another technical goal of single cell ECL imaging is to obtain high spatial (nanometer level) and temporal (millisecond level) measurement at one cell so that the subtle information associated with the cellular activity could be monitored without any labeling. The tentative work was conducted to image the efflux of hydrogen peroxide from single living cells. [START_REF] Zhou | Electrochemiluminescence imaging for parallel single-cell analysis of active membrane cholesterol[END_REF] When the cells were cultured in the physiological buffer with luminol, the existence of membrane ruffling created small space between the lower surface of the cell and ITO slide retaining some amount of luminol. Under the electrochemical control, the released hydrogen peroxide reacted with luminol in these spaces emitting the luminescence so that the efflux of hydrogen peroxide beneath the cell was imaged (Figure 4C). The addition of cholesterol oxidase into the buffer generated hydrogen peroxide from the reaction with membrane cholesterol, and thus, offered ECL images of cholesterol distribution at the cellular surface.

However, the limited amount of luminol beneath the cells and the steric hindrance of luminol from the solution to these space restrict the luminescence at the cells affecting the quality of ECL image. The introduction of permeable chitosan film at ITO slides increased the space between the cells and the electrode surface. Consequently, more luminol was present below the cells, which improved the sensitivity of single cell imaging. [START_REF] Liu | Direct Electrochemiluminescence imaging of a single cell on a chitosan film modified electrode[END_REF] Despite that the efflux of hydrogen peroxide and the distribution of membrane cholesterol are imaged, the diffusion of these ECL intermediates is unavoidable affecting the spatial characterization.

Our result showed ~ 2 µm blurry luminescence edge surrounding the cells that revealed the existence of lateral diffusion. [START_REF] Zhou | Electrochemiluminescence imaging for parallel single-cell analysis of active membrane cholesterol[END_REF] Therefore, the diffusion of these species limits the spatial resolution of ECL imaging with a few micrometers. An alternative strategy to achieve the local imaging of cellular information might be the usage of micro/nano-tips in ECL imaging. Our group fabricated a comprehensive Au-luminol-microelectrode that was inserted into the cell. The bright luminescence observed at the tip visualized local concentration of hydrogen peroxide inside the cells (Figure 4D). [START_REF] He | Electrochemical visualization of intracellular hydrogen peroxide at single cells[END_REF] Besides small molecules in the cells, membrane proteins at single cells were imaged using ECL as a surface-confined microscopy. [START_REF] Valenti | Single cell electrochemiluminescence imaging: from the proof-of-concept to disposable device-based analysis[END_REF] By contrast with classic fluorescence microscopy, ECL emission was only detected from the [Ru(bpy) 3 ] 2+ labels immobilized on the cell membrane located in the immediate vicinity of the electrode surface (Figure 4E). This result is intrinsically associated to the unique ECL mechanism of TPrA due to the limited lifetimes of the electrogenerated TPrA radicals (Figure 1B). The potential diagnostic applications of this approach were reported by using carbon nanotubes (CNT)-based inkjet-printed disposable electrodes for the direct ECL imaging of a labeled plasma receptor over-expressed on tumor cells. The ECL luminophore was linked to an antibody targeting an important membrane receptor, the epidermal growth factor receptor (EGFR), which was a plasma membrane receptor over-expressed in several cancer cells. This important work allows the development of new surface-confined microscopy and of ultrasensitive single cell imaging assays. To improve the ECL imaging sensitivity of tumor markers expressed at the cell level, BPE and functional nanoprobes of heterogeneous [Ru(bpy) 3 ] 2+ @SiO 2 /Au nanoparticles were combined for visual immunoassay of prostate specific antigen (PSA) at single cells. [START_REF] Cao | Immuno-electrochemiluminescent imaging of single cell based on functional nanoprobes of heterogeneous Ru(bpy) 3 2+ @SiO 2 /Au nanoparticles[END_REF] Such silica NPs enabled the immobilization of a large amount of labels, and the Au NPs decorated on the surface of [Ru(bpy) 3 ] 2+ @SiO 2 NPs increased the amount of loaded antibody so that more luminescence was obtained.

Single particles

Micro/nano-particles with high electron-transfer rate and electrocatalytic features are being incorporated as the new electrode materials for basic electron-transfer study, electrochemical sensing and electrocatalysis. The examination at the single particle level should contribute to elucidate the correlation between the particle structure and function. [START_REF] Anderson | Single-nanoparticle electrochemistry through immobilization and collision[END_REF][START_REF] Mirkin | Electrochemistry at one nanoparticle[END_REF] Due to the challenge of separating the signal at an individual particle from the surrounding particles and/or environment, single particle electrochemistry is combined with optical techniques providing a solution to image electrochemical signal at multiple particles in parallel. Since ECL has near-zero background readout and the corresponding high-sensitivity, single particles ECL imaging becomes a highly promising tool in this field.

The pioneering work on single-nanoparticle ECL analysis was conducted by the group of Bard.

They immobilized sub-25 nm single polymer nanoparticles at ITO slides and observed ECL from single particles in the presence of a co-reactant. The ECL photons as high as 1500 photons during 100 s from individual nanoparticles exhibits the feasibility of this technique for the bio-sensing. [START_REF] Chang | Electrogenerated chemiluminescence of single conjugated polymer nanoparticles[END_REF][START_REF] Fan | Electrogenerated chemiluminescence of partially oxidized highly oriented pyrolytic graphite surfaces and of graphene oxide nanoparticles[END_REF] The early work of single particle ECL imaging for biosensing was realized at ~ 3.1 µm polystyrene microbeads modified with antigens. [START_REF] Deiss | Multiplexed sandwich immunoassays using electrochemiluminescence imaging resolved at the single bead level[END_REF] Upon the recognition by [Ru(bpy) 3 ] 2+ tagged antibodies, the visual luminescence emitted from the entire bead demonstrated the possibility of performing multiplexed immunoassays using ECL imaging (Figure 5A). The possibility of performing highly multiplexed assays by ECL was demonstrated because all the individual sensing beads in the array were simultaneously imaged and individually resolved by ECL. Such a three electrode configuration for ECL imaging was continuously optimized using 8 µm polystyrene beads coated with a Ru(bpy) 3 2+ complex. [START_REF] Dolci | Development of a new device for ultrasensitive electrochemiluminescence microscopy imaging[END_REF] The concentration of Ru(bpy) 3 2+ complex as low as 1 × 10 -19 mol/ µm 2 at the microbeads could be imaged.

Furthermore, single particle ECL imaging was also achieved to visualize electrocatalytic reaction activity at single quantum dots (QDs) loaded silica microparticle involved in water oxidation. [START_REF] Chen | In situ visualization of electrocatalytic reaction activity at quantum dots for water oxidation[END_REF] Under anodic voltage, the active redox centers at CdSeTe QDs induced the generation of hydroperoxide surface intermediates as co-reactants to remarkably enhance ECL emission from luminol for imaging (Figure 5B). A linear relation between ECL intensity and turn over frequency suggests that ECL intensity can be used as a visual signal to exhibit the electrocatalytic activity of single particles. A quasi-zero background luminescence is obtained in the full potential window, which is advantageous versus the other optical imaging with the fluctuation of background signal under high voltage.

To realize ECL imaging of single nanoparticles (NPs), the metal materials have received attention due to their good ability for reaction catalysis and electron transfer, and good biocompatibility. Au nanoparticles could catalyze the oxidation of TPA to enhance ECL from Ru(bpy) 3 2+ . As a result, the luminescence from single Au NPs was firstly imaged (Figure 5C). [START_REF] Pan | Observation of local redox events at individual Au nanoparticles using electrogenerated chemiluminescence microscopy[END_REF] The luminescence intensity at individual nanoparticles was positively dependent on the particle size from 30 to 300 nm, and was affected by the local chemical and charge transfer environment of the NPs. The achievement should permit a massively parallel direct readout of local redox activities on single nanoparticles. However, the obtained ECL from single NPs was vanished within several seconds because of the surface oxidation of Au NPs. To overcome the surface oxidation of Au NPs in aqueous environments, a polymer blend was coated at single Au nanowires resulting in readily measurable ECL. [START_REF] Wilson | Imaging electrogenerated chemiluminescence at single gold nanowire electrodes[END_REF] ECL image quality and reproducibility over multiple redox cycles could be improved with thicker polymer layer.

Recently, Au-Pt Janus NPs, meaning particles with different surfaces or asymmetric reactivity, were prepared exhibiting enhanced ECL intensity and stability. [START_REF] Zhu | Electrogenerated chemiluminescence imaging of electrocatalysis at a single Au-Pt janus nanoparticle[END_REF] Different heterogeneous electrontransfer rate constants at Au and Pt results in a concentration difference arose at the asymmetric bimetallic interface. The fluid slip around the Janus particle enhanced local redox reactions and protected the particle surface from passivation. Therefore, in comparison to the monometal NPs, the Janus particles weakened the surface poisoning and exhibited much better long-time stability that improved the quality of ECL imaging significantly.

Single-particle collision is another exciting direction to examine the electrochemical features of individual NPs, which takes place on a macroscopic electrode at exceedingly high frequencies.

Classically, single particle events can be temporally resolved using a microelectrode as the recording electrode. The combination of ECL imaging and single-particle collision should help to investigate the dynamic redox behavior of multiple NPs at the electrode/solution interface for more detail information. To achieve this aim, ECL imaging with sufficient spatial and temporal resolutions is critical to track the fast diffusional motion of the NPs at the interface. The currently fastest temporal resolution of ECL imaging was reported to be 200 ms from the imaging of ~ 62 nm Ru(bpy) 3 2+ doping silica nanoparticles. [START_REF] Ma | Dynamically imaging collision electrochemistry of single electrochemiluminescence nano-emitters[END_REF] Several discrete ECL spots were observed successively in different ROIs, which provided the direct evidence for the ECL visualization of collision electrochemistry (Figure 5D).

Two major categories "spike" and "staircase", of the ECL signals from the nanoparticles suggest the elastic and sticking collision, respectively.

Remarks

The electrochemical mapping of the distribution of bio-molecules at heterogeneous and microscopic interfaces is significant for high spatio-temporal bioanalysis. SECM is a classic approach to obtain spatially resolved electrochemical signals. However, due to the use of scanning electrodes, high temporal resolution is not realized. Plasmonic-based electrochemical microscopy allows widefield imaging of the interfacial distribution of an electrochemical signal from a planar electrode with simultaneous submicron spatial resolution and sub-millisecond temporal resolution, but the sensitivity is being improved. ECL imaging is a fast-developing technology that owes the advantages of high detection sensitivity and spatial/temporal resolutions in bioanalysis. Various electrode materials and architectures, and imaging strategies have been realized to achieve lower detection limits, better resolutions and image quality.

Despite the successes of ECL imaging, many challenging, such as single molecule detection, super-resolution imaging and the application in clinic test, are present in the field of ECL imaging.

The modern biological study has revealed that many biological events at single molecule level occur at nanometer sized region in nanoseconds. To fulfill the requirement, a series of revolutionary developments in fluorescence microscopy have circumvented the limit of light diffraction and provided an image of single molecules with subcellular spatial resolution below 200 nm. However, the current detection limit and the spatial/temporal resolutions in ECL imaging are far less than the biological requirements that need the breakthrough in the detection principle, setup and strategy.

The performances of the ECL imaging depends on several factors related directly to the ECL process such as the sensing strategies, the efficiency of the ECL generation, the ECL mechanisms of the luminophores and the co-reactants as well as the optical detection. Therefore, the endeavor to In addition, the current co-reactant ECL is commercialized for more than 100 immunoassays, which are vital for early diagnosis and treatments of the corresponding pathologies. The continuous coupling of ECL imaging strategies and these immunoassays offers exciting opportunities and promising applications in ultrasensitive ECL-based imaging assays for clinic test.

Disclosure statement.

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

Figure 1 .

 1 Figure 1. Main ECL mechanistic pathways for [Ru(bpy) 3 ] 2+ /TPrA and luminol/H 2 O 2 systems. (A)[Ru(bpy) 3 ] 2+ and TPrA are both oxidized at the electrode surface in the "oxidative-reduction" mechanism. (B) ECL emission generated only by TPrA oxidation at the electrode surface and involving the homogeneous reaction of the ruthenium luminophore with the TPrA radicals. Reprinted with permission from ref 8. (C) Luminol anion and hydrogen peroxide are both electrochemically oxidized at the electrode surface emitting the luminescence. (D) Schematic setup used for ECL imaging.

Figure 2 .

 2 Figure 2. (A) Illustration of the biosensor array with choline oxidase (blue spots), glucose oxidase (green spots) and lactate oxidase (red spots) diagonally immobilized on the working electrodes, and ECL images of choline, glucose and lactate. Reprinted with permission from ref 40. (B) Luminescence image of glucose oxidase-modified nanoneedle electrodes for the response of glucose. Inset: SEM images of nanoneedle electrodes. Reprinted with permission from ref 43. (C) ECL image of the array with 49 individual RuPVP/DNA/enzyme spots containing cyt P450 1B1. Reprinted with permission from ref 46. (D) Representative ECL images of the natural fingermarks treated by the multiple-HRP route for the detection of dermcidin. Reprinted with permission from ref 49.

Figure 3 .

 3 Figure 3. (A) Top-view schematic illustration and ECL imaging of the bipolar electrodes at 22.0 V. Reprinted with permission from ref 55. (B) Micrograph of an interchannel BPE spanning parallel sensing and reporting microchannels (a) and ECL imaging obtained with 0.10 mM (b) and 5.0 mM (c). Reprinted with permission from ref 58. (C) Schematic illustration of the electrical switch fabricated by sandwich immune complex in the channels. Reprinted with permission from ref 61. (D) ECL images on BPE array with increased concentrations of PSA, miRNA-141and sarcosine. Reprinted with permission from ref 66.

Figure 4 .

 4 Figure 4.(A) Illustration of ECL imaging for parallel measurement at single cells using luminol/hydrogen peroxide system; (B) ECL imaging of intracellular glucose at single cells retained in the microwells. Reprinted with permission from ref 75; (C) ECL imaging of the efflux of hydrogen peroxide from single cells. Reprinted with permission from ref 34. (D) ECL imaging of intracellular hydrogen peroxide using luminol modified microelectrodes . Reprinted with permission from ref 80. (E) PL (a), ECL (b) and the overlaying (c) images of CHO cells grown on a GC electrode. Reprinted with permission from ref 81.

Figure 5 .

 5 Figure 5. (A) ECL imaging of multiplexed sandwich immunoassays to a sample solution containing IL-8, TIMP-1 and VEGF. Reprinted with permission from ref 27; (B) ECL images of single QDs loaded particle in L-012 solution with different applied potentials. Reprinted with permission from ref 88; (C) A full ECL image (50 × 50 μm 2 ) collected from Au NPs with an average particle size of 29 nm deposited onto an ITO electrode. Reprinted with permission from ref 89; (D) ECL snapshots of three RuDSNs colliding with the electrode simultaneously. Reprinted with permission from ref 92.

  prepare novel electrodes with higher electron transfer rate and luminophores with high luminescence efficiency should be targeted to obtain significant enhancement in ECL signals. And, the optical detection relies on the instrumentation and the optical components (objectives, 2D photodetectors, etc.) and ECL imaging should benefit also from the fast progresses in the microscopy field. Moreover, imaging the ECL reactivity on single objects should provide insights into the ECL mechanistic routes

	allowing to select new co-reactants and luminophores with improved sensitivity and to develop new
	imaging strategies. As already mentioned, two co-reactant ECL systems ([Ru(bpy) 3 ] 2+ /TPA and
	luminol/H 2 O 2 ) are mainly used for imaging purposes. However, the developments of original
	strategies by combining ECL with wireless addressing methods and by using multicolor ECL emission
	and nanoluminophores such as QDs or doped nanoparticles open new avenues to develop original
	ECL-based microscopies.

Acknowledgements.

We apologize in advance to all the investigators whose research could not be cited due to space limitations. We are grateful for financial support from the National Natural Science Foundation of China (grants 21327902 and 21575060) and from the Agence Nationale de la Recherche (NEOCASTIP ANR-15-CE09-0015-03).