Dual Enzymatic Detection by Bulk Electrogenerated

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Dual enzymatic detection by bulk electrogenerated chemiluminescence Anne de Poulpiquet , Beatriz Diez Buitrago, Milena Dumont Milutinovic, Milica Sentic, Stephane Arbault, Laurent Bouffier, Alexander Kuhn, and Neso Sojic Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01434 • Publication Date (Web): 23 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016

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Analytical Chemistry

Dual enzymatic detection by bulk electrogenerated chemiluminescence

Anne de Poulpiquet, Beatriz Diez-Buitrago, Milena Dumont Milutinovic, Milica Sentic, Stéphane Arbault, Laurent Bouffier, Alexander Kuhn* and Neso Sojic*a [a]

University of Bordeaux, ISM, UMR 5255 CNRS, ENSCBP, 33607 Pessac, France. E-mails: [email protected] ; [email protected]

Abstract The combination of enzymes, as recognition elements for specific analytes, and of electrogenerated chemiluminescence (ECL) as a readout method has proven to be a valuable strategy for sensitive and specific analytical detection. However, ECL is intrinsically a 2D process which could potentially limit the analysis of inhomogeneous samples. Here we show how a bulk ECL signal, generated by thousands of carbon microbeads remotely addressed via bipolar electrochemistry, are implemented as a powerful tool for the concomitant ECL sensing and imaging of two enzymatic substrates. We selected two enzymes (glucose dehydrogenase and choline oxidase) that react with their respective model substrates and produce in situ chemical species (NADH and H2O2) acting as coreactants for the ECL emission of different luminophores ([Ru(bpy)3]2+ at λ = 620 nm and luminol at λ = 425 nm, respectively). Both enzymes are spatially separated in the same capillary. We demonstrate thus the simultaneous quantitative determination of both glucose and choline over a wide concentration range. The originality of this remote approach is to provide a global chemical view through one single ECL image of inhomogeneous samples such as a biochemical concentration gradient in a capillary configuration. Finally, we report the first proof-of-concept of dual biosensing based on this bulk ECL method for the simultaneous imaging of both enzymatic analytes at distinct wavelengths.

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Introduction ECL is the light-emission process triggered by an exergonic electron-transfer reaction between electrogenerated chemical species.1 It combines intimately electrochemistry and photochemistry and has several key characteristics. First, as the ECL signal depends on the applied potential, the light-emitting reaction can be controlled both in time and space. This is particularly interesting when biomolecules are used,2 because it allows performing the measurements after the biorecognition reactions have taken place. Second, the ECL emission intensity is proportional to the concentration of the involved reactants, affording an accurate measurement in a wide dynamic concentration range. Moreover, ECL is very sensitive because the absence of any excitation light compared to photoluminescence results in a low background.3-7 These advantages have been exploited to develop ECL assays with remarkably high sensitivity and extremely wide dynamic range. Currently, more than 150 ECL immunoassays are commercialized on the diagnostic market, such as for cardiac and infectious diseases, maternal care, thyroid and tumour markers, etc. and they account for hundreds of millions of dollars in sales per year.8 The ECL mechanism involved in such bioassays is based on the so-called co-reactant pathway, in which the light is emitted by a luminophore, whose excited state is populated through its reaction with a co-reactant.4 Interestingly, several ECL-active co-reactants are either a coproduct or a cofactor of an enzymatic reaction and this allows the development of enzymatic ECL assays.2,9 Typical examples of such tandem luminophore/co-reactant systems are [Ru(bpy)3]2+/β-nicotinamide adenine dinucleotide (NADH) or luminol/H2O2.2,4,10-12 The light emitted is proportional to the concentration of the co-reactant generated during the enzymatic reaction, the latter being correlated to the initial concentration of the analyte of interest, which is recognized as the enzyme substrate. For example, NADH coenzyme is generated by NAD+-dependent enzymes, like dehydrogenases.10 Some enzymes of this class are (1) glucose dehydrogenase allowing glucose sensing in case of diabetes, (2) alcohol dehydrogenase, used for evaluating blood alcohol concentration, or (3) lactate dehydrogenase, used for the determination of lactate in blood. The reduced form, NADH, initiates ECL reaction in the presence of [Ru(bpy)3]2+ in contrast to its oxidized form, NAD+. The aliphatic tertiary amine group of NADH undergoes ECL emission whereas aromatic amines, such as the pyridine ring of NAD+, do not generate ECL with [Ru(bpy)3]2+ (λmax ~ 620 nm). The substrate concentration is thus measured by recording the ECL intensity, which is directly related to the variations of the NADH concentration. On the other hand, hydrogen peroxide is generated by several oxidases like glucose oxidase, cholesterol oxidase, or ethanol oxidase, that couple the oxidation of their substrate to the 2-electron reduction of dioxygen. In aqueous alkaline solution, the oxidation of luminol in the presence of H2O2 generates 3-aminophthalate in the excited state which emits a characteristic blue light (λmax = 425 nm).13-16 Thus


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the ECL intensity measured at this wavelength is directly proportional to the concentration of the substrate of the oxidase enzyme. ECL is however intrinsically a two-dimensional process, as the light-emitting reaction can only occur in the vicinity of the electrode surface where the reactive intermediates are electrogenerated. This drawback could limit the analysis of inhomogeneous or complex samples. To circumvent this limitation, our group recently proposed a new method that allows performing bulk ECL with a suspension of conductive carbon microbeads or carbon nanotubes individually addressed by bipolar electrochemistry (BPE).17,18 In this case, ECL is generated in a wireless manner at the surface of conductive objects dispersed in solution and acting as an ensemble of single ECL emitting reporters. BPE is based on the generation of asymmetric electrochemical reactions at opposite poles of a conductive object (bipolar electrode) exposed to an electric field, but without any direct electrical contact to an external circuit.19,20 Practically, a uniform electric field is generated in a solution by applying a voltage between two feeder electrodes. This leads to an interfacial potential difference established between the electrolyte solution and the bipolar electrode. Electrochemical reactions can then be driven by the cathodic and anodic overpotentials resulting at the opposite poles. The promotion of ECL on bipolar electrodes was reported initially on large conducting objects.21-29 This remains true at the micrometric scale23,30-33 and we recently demonstrated that a sufficient electric field promotes ECL of both [Ru(bpy)3]2+ and luminol dyes at individual micro- and nano-objects homogeneously dispersed in the volume of a solution, thus leading to bulk ECL.17,34 Here, we demonstrate how this method can be further extended to the design of enzymatic biosensing using ECL as a readout method. Bulk ECL is combined for the first time with enzymatic coreactant production. Each microbead is addressed electrochemically by BPE and generates ECL light only in presence of the enzymatic substrate. Our approach allows switching from heterogeneous diffusion-limited reactions to quasi-homogeneous reactions with much higher kinetic rates and improved analytical performances. Indeed, the enzymatically-generated co-reactants have to diffuse only over a short distance to reach the surface of bipolar electrodes (i.e. carbon beads) dispersed in solution in comparison to the classic configuration. Therefore, it allows the sensitive remote detection and imaging of two different analytes, namely choline and glucose, in inhomogeneous sample.

Experimental section Materials. All chemicals were of analytical grade and were used as received. Enzymes were stored at –20 °C and used without further purification. Solutions were prepared using Milli-Q water (resistivity



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18.2 MΩ.cm). Glucose dehydrogenase (GDH), choline oxidase (ChOx), tris(2,2’-bipyridyl)dichloro ruthenium(II) hexahydrate, 2,3-aminophthalhydrazide, β-nicotinamide adenine dinucleotide, hydrogen peroxide, glucose, choline, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate and agarose were purchased from Sigma Aldrich. Glassy carbon beads (22 ± 4 µm in diameter) were purchased from Alfa Aesar. It is a powder of non-graphitizing carbon containing a random combination of basal plane and edge plane. The electrochemical and ECL properties of commercial glassy carbon electrodes and of home-made electrodes composed of the same material as the carbon beads are identical. The carbon particles have not been treated with any chemicals so the surface charge density (i.e. carboxylic groups) is low. It is confirmed by the experiments performed in PBS solution without gel where we have never observed any migration of the carbon particles when applying the potential at the feeder electrodes. Measurement procedures. The enzymatic reactions were conducted before the enzymatic systems were mixed with other constituents for the bulk ECL emission. For the [Ru(bpy)3]2+/GDH system, unless otherwise specified, 58 U.mL–1 GDH was mixed with 10 mM NAD+ and varying concentrations of glucose in a 30 mM phosphate buffer pH 7.4 and left 30 minutes at 37°C. For the luminol/ChOx system, 3.3 U.mL–1 ChOx was mixed with varying concentrations of choline in a 30 mM phosphate buffer pH 7.4 and stirred at ω = 6000 rpm during 30 minutes at 35°C. After the enzymatic reaction, the enzymatic system was mixed with the luminophore, agarose and the glassy carbon beads, and the mixture was left 10 min to gel in a glass capillary (outer diameter 6 mm, length 2.5 cm). Agarose gel was used to avoid the sedimentation and aggregation of the particles and to keep them well-separated during the detection step. It is an important point because aggregates will act as single emitters and generate ECL at lower potential due to its bigger size in comparison to a particle. Unless otherwise specified, the final composition of the mixture in the glass capillary was: 1.4 % agarose gel in 3 mM phosphate buffer pH 7.5; 100 µM [Ru(bpy)3]2+ (respectively 25 µM luminol and 20 mM NaOH); approximately 300,000 carbon beads per mL and the products of the enzymatic reaction diluted by a factor of 10. The glass capillary was then placed between two compartments filled with agarose gel containing the graphite feeder electrodes (Scheme 1). All lights were turned off before the voltage was applied between both feeder electrodes. The ECL intensity values are averaged results recorded in at least 4 different experiments. The applied electric field was 0.75 kV.cm–1 and 1 kV.cm–1 for the experiments with luminol and [Ru(bpy)3]2+, respectively. These values were calculated based on the fundamental equations of BPE19 as reported in previous works.10,17,18,34,35 The polarization voltage difference between the two sides of a bead has to be at least equal to the difference in formal potentials of the two redox reactions that are involved at the


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anodically and cathodically polarized sides of the object. For the luminol system, the oxidation of luminol occurs around +0.5 V vs Ag/AgCl on the anodically polarized side, whereas the reduction of oxygen takes place for potentials around -1 V vs Ag/AgCl.18,34 This means that a potential difference of 1.5 V is needed between the two sides of a bead so that both reactions can occur simultaneously. Taking a diameter of 20 µm for one bead, this leads to an electric field of 0.75 kV.cm–1 which should be applied throughout the cell in order to have even the smallest beads emitting light. For the [Ru(bpy)3]2+ system, ECL emission occurs around +1 V vs Ag/AgCl and similar calculations leads to an electric field of 1 kV.cm–1.35 In the presence of ions in the gel (i.e. buffer, ruthenium complex, coreactant), the gel behaves as an ionic conductor with a conductivity which is directly related to the ionic strength. Thus it constitutes an ohmic resistance and there is a potential drop along the gel inside the capillary, which can be assumed in a first order approximation to be linear where the carbon beads are located. There is also a non-linear potential drop at the feeder electrodes, but as the capillary has a much smaller section than the rest of the cell, it will constitute the part of the setup with the highest potential drop. Instrumentation. A DC generator (Heinzinger 10000-200 pos) was used to apply the electric field. The white light and ECL images were recorded with a commercial digital camera (Canon 60 D, Raw mode imaging in full frame resolution) equipped with a lens for macrophotography (Canon 100 mm – f:2.8). ECL images were obtained during the first few seconds of the experiment in order to obtain the best reproducibility. For longer times, the current, which flows through the bipolar cell, induces ohmic heating which can lead to a melting of the gel and therefore sedimentation of the suspended carbon beads. If long term experiments need to be conducted, it would be necessary to replace the present gel based on agarose (melting point 30-40 °C) by gels with a higher melting point. The intensity of emitted light (calibration curves) was measured with a Hamamatsu photomultiplier tube R5070A connected to a µ-Autolab potentiostat controlled by the software GPES. ECL spectra were acquired with a Princeton Instruments Model Acton SpectraPro 2300i system equipped with a CCD camera.

Results and discussion The general principle of the setup is presented in Scheme 1. A suspension of carbon microbeads is used to fill a glass capillary positioned between two graphite feeder electrodes. A uniform electrical field is generated by applying a voltage between the feeder electrodes. A gradient of potential difference is thus created between the surface of the conductive microparticles and the surrounding solution, resulting in a polarization voltage between the opposite poles of every individual carbon microbead.19 When this polarization voltage becomes large enough, asymmetric redox reactions start to occur at the extremities of these bipolar electrodes.35 Each microparticle is thus remotely



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addressed by BPE and may act as a single ECL emitter in the presence of the appropriate tandem system composed of a luminophore and its co-reactant.17,35 In this case, all the beads may generate ECL and their collective behavior enables to collect an overall strong light emission in the whole volume of the solution. The concentration of glassy carbon beads is a major analytical parameter and it has been optimized previously.18 In the present work, we used a concentration of approximately 300,000 carbon beads per mL suspended in the capillary which corresponds to the optimal concentration to achieve strong ECL signals.

Scheme 1. Principle of bulk ECL generated by enzymatic systems in a suspension of carbon microbeads. (A) Scheme of the setup. The capillary is filled with a suspension of carbon microbeads, a luminophore and the corresponding enzymatic system. The microbeads are polarized by the electric field generated between the feeder electrodes, allowing anodic and 2+

cathodic reactions to take place at the opposite poles. (B) Reaction scheme of the [Ru(bpy)3] /glucose dehydrogenase +

(GDH) system. In the presence of glucose, GDH reduces NAD to NADH which acts as a co-reactant leading to ECL emission of [Ru(bpy)3]2+. (C) Reaction scheme of the luminol/choline oxidase (ChOx) system. In the presence of choline, ChOx reduces O2 to H2O2 which generates the 3-aminophthalate excited state and thus blue ECL emission.


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For the detection of two different analytes, namely glucose and choline, we first studied both enzymatic systems independently. The first one, that we will refer to as [Ru(bpy)3]2+/glucose dehydrogenase (GDH) system in the following, is based on NAD+-dependent GDH which catalyzes the oxidation of glucose with the cofactor NAD+ as an electron acceptor (Scheme 1B).36 The enzymatic reaction generates NADH, the reduced form of NAD+, and the actual NADH concentration is proportional to the initial concentration of glucose substrate. As NADH contains a tertiary amine, it can act as a co-reactant for generation of ECL by the [Ru(bpy)3]2+.9,37,38 Upon application of a suitable electric field between the feeder electrodes, NADH and [Ru(bpy)3]2+ are both oxidized at the anodic pole of each bipolar microbead. The resulting cation radical NADH•+ then deprotonates to form the strongly reducing NAD• radical which reacts with [Ru(bpy)3]3+ to generate the excited state [Ru(bpy)3]2+*. Upon de-excitation, the latter returns to the ground state by emission of a photon at a given wavelength (λ = 620 nm), while NAD+ is regenerated.2,39 The global electroneutrality across the conductive object imposes an opposite redox reaction to take place at the cathodic pole of the microbead, in this case, the reduction of dioxygen dissolved in the buffer solution. Indeed, at the level of an individual bead, all the electrons which are liberated by the oxidation reaction at the anodically polarized side are consumed by the reduction reaction at the cathodically polarized side. It means that there is still a net electron flow through every bead, from the anodically polarized side to the cathodically polarized side, which needs to be supported by a global current flowing through the cell and also through the feeder electrodes. To study the cathodic reactions, we recorded the voltammetric responses (data not shown) of both solutions used for the bulk ECL ([Ru(bpy)3]2+ and luminol) with a home-made electrode composed of the same materials than the carbon beads. The cathodic waves disappear after bubbling nitrogen in both solutions, indicating that dioxygen reduction is the reaction occurring at the cathodic pole of the bipolar beads (Scheme 1 B-C). The detection of glucose was studied because it is a model analyte due to its relevance in diabetes, but it is noteworthy that this mechanism could be triggered by any NAD+-dependent dehydrogenase, and thus used for a wide variety of biorelevant analytes. The second enzymatic system is based on the oxidation of choline to betaine by choline oxidase (ChOx) with O2 as an electron acceptor (Scheme 1C). This system will be referred to as luminol/ChOx. During the enzymatic reaction, O2 is reduced to H2O2 in amounts proportional to the quantity of choline oxidized. H2O2 co-product then acts as a co-reactant for ECL emission of luminol. In basic or neutral conditions, luminol can be electrochemically oxidized to form an intramolecular diazoquinone which is further chemically oxidized by an oxidized form of H2O2 (HOO• or the superoxide radical O2•–) to the excited state of 3-aminophthalate*.16,40,41 Upon its de-excitation, light



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with a typical wavelength λ = 425 nm is emitted. The reaction occurring at the cathodic pole of the microbead is again the reduction of dissolved O2 to H2O. Considering the enzymatic reaction and both redox reactions occurring at the opposite poles of the carbon beads, one can see that the concentration of O2 remains constant on the anodic side of the bead since the enzymatic reaction which consumes O2 in a stoichiometric way is counter-balanced by the anodic bipolar reaction which generates O2. Therefore the overall concentration of oxygen is not changing on this side of the bead (it is only regenerated). On the cathodically polarized side of the bead, the concentration of O2 is decreasing due to the reduction. Thus in both enzymatic systems (1B and 1C) the global oxygen concentration will decrease as a function of time exactly like what would be observed during a normal enzymatic reaction. Therefore, there is no risk of bubble formation which may affect the stability of the ECL signal. Experimentally we also have not observed the formation of bubbles.

Figure 1. Bulk ECL produced by an enzymatic system in a suspension of carbon microbeads. Images of the [Ru(bpy)3]2+/GDH system (A) under white light before applying the electric field and (B) of the bulk ECL in the dark after applying an electric –1

field of 1 kV.cm . (C) Image in the dark of the ECL emitted by the luminol/ChOx system when applying an electric field of –1

0.75 kV.cm .

Representative optical images of the light emitting processes are displayed in Figure 1. Figure 1A shows a white-light image of the capillary filled with the suspension of carbon microbeads in an agarose gel, which contains also a luminophore and the corresponding enzymatic system after the


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enzymatic reaction has taken place. It is possible to visually distinguish discrete black dots that correspond to the microbeads. Images are then recorded in the dark during the application of an electric field generating a sufficient polarization voltage difference ∆V between both poles of the microbead. (Eq. 1)

∆V = E.d

with E being the intensity of the electric field and d the diameter of the bead. ∆V must be high enough to allow the oxidation reactions leading to ECL emission to take place at the anodic pole, and also the reduction of O2 at the cathodic pole. Figure 1B shows the red light emitted by the [Ru(bpy)3]2+/GDH system when E = 1 kV.cm–1 is applied. A bright ECL emission can be observed with the naked eye or recorded with a commercial camera, similarly to what was previously described for bulk ECL emission by [Ru(bpy)3]2+with diisobutylaminoethanol as a co-reactant.18 Figure 1C shows the bulk ECL emission recorded with the luminol/ChOx system upon application of E = 0.75 kV.cm–1. However, even if ECL is generated in 3D, both pictures recorded with the camera present solely a 2D distribution of the ECL signals where light intensity measured at a given point represents the cumulative contribution of the ECL-emitting beads depending on their position and distance to the objective as well as on the optical characteristics of the photodetector. Information on the third spatial dimension might be obtained by using confocal microscopy or tomography experiments. Compared to the first system, the voltage applied to the feeder electrodes for the luminol/ChOx is lower because the ECL emission of luminol occurs at an overpotential η = 0.5 V vs. Ag/AgCl whereas 1.0 V vs. Ag/AgCl is necessary for the [Ru(bpy)3]2+ emission. Although some slightly brighter zones can be observed in the optical image, it is clear that the light emission process is well-distributed in the bulk. This validates for the first time that both enzymatic systems are able to generate an efficient bulk light emitting process by using the enzymatic co-product or co-factor as ECL co-reactants.



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Figure 2. Calibration curves of the bulk ECL produced in a suspension of carbon microbeads placed in the capillary. Dependence of ECL intensity on the concentration of (A) NADH and (B) hydrogen peroxide with [Ru(bpy)3]2+ and luminol, respectively. Variation of the ECL intensity with the concentration of (C) glucose and (D) choline for the [Ru(bpy)3]2+/GDH and luminol/ChOx systems, respectively.

In a first set of quantitative experiments, we tested both co-reactants (i.e. NADH and H2O2) in this bulk ECL configuration. Using a photomultiplier tube (PMT), the intensity of ECL emission was measured for various initial concentrations while all other parameters were kept constant. Figure 2 displays the calibration curves obtained for both systems. In both cases, the linear relation between bulk ECL intensity and the concentration of co-reactant was established before the enzymatic sensing was developed further. A good linear relationship is observed for the evolution of [Ru(bpy)3]2+ ECL intensity with NADH concentration (Figure 2A) over the whole concentration range explored (0-200 µM). Similarly, the evolution of luminol ECL intensity with H2O2 concentration (Figure 2B) is linear in the same concentration range. It is noteworthy that such a linear response which is typical in conventional 2D ECL is also observed in our bulk format. In the next step, bulk ECL experiments with the enzymes in presence of their respective substrates were carried out. As expected, the ECL intensity for both enzymatic systems was found to be linearly correlated to the initial concentration of their respective substrates. For the [Ru(bpy)3]2+/GDH


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system, the linearity can be observed for glucose concentrations up to 5 mM (Figure 2C). The limit of detection (LOD), defined as the concentration corresponding to a signal to noise ratio equal to 3, is ≈ 25 µM. For the luminol/ChOx system, linearity can be observed for choline concentrations ranging from 0 to 500 µM, with a LOD of 50 µM (Figure 2D). The small bias observed on Figure 2B and 2D is also present on Figure 2A and 2C. It is just related to the background, mainly of the photodetector. Although the sensitivity of the presented detection scheme is not very low in this first proof-ofprinciple work, it can be noted that it is sensitive enough for the detection of glucose for example, because the measurement of glucose concentration in blood samples typically demands sensitivities in the millimolar range. Furthermore, as indicated in the experimental section, the products of the enzymatic reaction have been diluted by a factor of 10 before the measurement and thus for analytes with very low concentrations this dilution step can be avoided, which would improve the detection limit by an order of magnitude. To reach lower detection limits several parameters may also be optimized such as the collection of the ECL light emitted in a long capillary using a collimating lens, the sensitivity of the photodetector, the nature and the size of the bipolar emitters (beads, nanotubes) and the concentration of the ECL reagents.

Figure 3. Bulk ECL produced by a bi-enzymatic system in a suspension of carbon microbeads. (A) Image of the system under white light before the application of the electric field. The grey part on the left of the capillary corresponds to the zone filled 2+ with the luminol/ChOx system, whereas the orange part on the right contains the [Ru(bpy)3] /GDH system. (B) Image in the –1 dark of the ECL emitted by the bi-enzymatic system when applying an electric field of 1 kV.cm .

To image the spatial distribution of both analytes (i.e. choline and glucose), we recorded the bulk ECL picture of the capillary containing both enzymatic systems with a consumer digital camera. One half of the capillary was filled with the [Ru(bpy)3]2+/GDH system and the other half with the luminol/ChOx



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system as shown in Figure 3A. To optimize the ECL process, an electric field of E = 1 kV.cm–1 was applied. This value allows generating simultaneously ECL emission of both luminophores. This dual setup results in a red ECL emission at the [Ru(bpy)3]2+/GDH part of the capillary and a blue ECL emission at the luminol/ChOx part of the capillary (Figure 3B). Each color corresponds to a different luminophore and therefore to a different enzymatic substrate. However, one can observe a black region between the two ECL zones, where luminescence of [Ru(bpy)3]2+ should be electrogenerated. This non-emitting region could be attributed to the diffusion of different species such as hydrogen peroxide or hydroxide anions from the luminol zone towards the [Ru(bpy)3]2+ one. The diffusion of hydrogen peroxide may inhibit the ECL emission of the ruthenium complex since the addition of H2O2 to the [Ru(bpy)3]2+/NADH system also leads to the extinction of ECL (data not shown). The increase of the local pH value in this zone by diffusion of hydroxide anions may also contribute to a decrease of the ECL efficiency. Indeed, ECL of [Ru(bpy)3]2+/trialkylamine is very sensitive to the pH value of the medium with an optimal pH around 7-8.42,43 Outside of this zone, the optical image demonstrates the simultaneous detection of two different analytes in the same bulk ECL setup, and paves the way for the simultaneous enzymatic imaging of multiple analytes.

Figure 4. Analysis of the light emission by the bi-color ECL systems (capillary filled with luminol/ChOx on the left and [Ru(bpy)3]2+/GDH on the right). Images in the dark of the ECL emitted by the bi-color system, differentiated by the applied –1 –1 electric field: (A) E = 0.75 kV.cm and (B) E = 1 kV.cm . (C) Full ECL spectra recorded with the same bi-color enzymatic


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system. Each ECL light has been collected by an optical fiber bundle connected to the spectrograph. The arrows indicate the region where the optical fiber was positioned in front of each zone of the capillary to collect both ECL signals.

As the PMT only measures the global number of photons emitted, a fully quantitative measurement allowing a deconvolution of both ECL contributions was not possible with a PMT. Therefore, we show in the following that both analytes could be eventually distinguished either by applying different voltages (Figure 4A) and using a PMT, or by recording the ECL spectrum (Figure 4C). Since the oxidation reactions that trigger the ECL of luminol occur at a lower overpotential than those inducing [Ru(bpy)3]2+ ECL, it was possible to address first selectively the luminol system by choosing a lower voltage for which the light emission is mostly due to the luminol (Figure 4 A). The blue curve centered at 425 nm corresponds to the light emission from 3-aminophthalate* and no ECL corresponding to [Ru(bpy)3]2+ emission is observed at this voltage. By applying a higher voltage in the same capillary, ECL was emitted simultaneously by both luminophores in the presence of their respective enzymatically-produced co-reactants (Figure 4 B). The red curve centered at 620 nm is mainly the light emission during [Ru(bpy)3]2+* relaxation. However, in this case, one can also notice that residual blue ECL light is collected by the optical fiber bundle used to record the spectra even if it is positioned in front of the [Ru(bpy)3]2+ zone. Alternatively, as the light is emitted at two distinct wavelengths, recording the full emission spectrum easily gives access to the respective contribution of both systems in the global light emission (Figure 4B). In this contribution, we demonstrated the detection of two analytes using the emission of two ECL luminophores. However, to detect simultaneously more than two analytes, various other ECL luminophores with distinguished emission wavelength are required. Over the last decade, numerous researchers have reported various ECL luminophores exhibiting a wide range of emission wavelengths that can be tuned through changes in the structure, in the central metal atoms and in the nature of the ligands.44-47 These complexes or their mixture may lead to different color emission when applying specific potentials, thus creating new possibilities for multiplexed ECL detection systems.48 Furthermore, silica-doped nanoparticles and quantum dots may also be exploited as ECL nano-emitters and allow tuning the emission wavelengths.49-51 Therefore, a large variety of ECL emitters covering wavelength emission ranging from UV to near IR can be used with our original bulk ECL imaging approach.



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Figure 5. A) ECL imaging of a concentration gradient of choline established in the capillary. The entire capillary is filled with the carbon bead dispersion and luminol but, on the right side, choline and ChOx were also added. The applied electric field was 0.75 kV.cm-1. B) ECL intensity profile calculated from the image above.

Since the ECL intensity depends on the local concentration of the enzymatic substrate, the presented approach is also suitable to image variations of substrate concentration in space. Indeed, the ECL intensity reflects the local concentrations of H2O2 or NADH which are directly related to the enzymatic activity of ChOx or GDH, and thus to the concentration of choline and glucose, respectively. In other words, ECL will only be emitted at locations where the enzymatic substrate is present. To demonstrate this ability to resolve spatially the distribution of inhomogeneous samples, the left part of the capillary is first filled with the gel containing the carbon beads and luminol. Then another gel solution containing the carbon beads, the enzyme and its substrate is introduced in the right part. After waiting for 20 min, a lateral concentration gradient is established in the capillary by diffusion of the substrate, of the enzyme and mainly, of the chemical signature of the enzymatic reaction (i.e. H2O2 or NADH). Figure 5A presents the ECL image recorded when an electric field of 0.75 kV.cm-1 was imposed. One can observe clearly a black part on the left where no ECL is generated due to the absence of H2O2 produced by the enzymatic reaction. It shows also that the background signal is very low in this bulk ECL configuration. By contrast, strong ECL emission is visible in the right


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zone where choline is present. Eventually, in the middle of the capillary, ECL intensity increases steadily from 0 to a constant value by passing from one region to the other. The ECL intensity profile represented on Figure 5B was calculated by averaging the intensity of the pixels perpendicular to the capillary axis in a region of interest defined in the middle of the capillary. As expected, a linear profile is observed in this part of the capillary. Similar ECL profiles were extracted from the images recorded using the same procedure but with glucose, GDH and [Ru(bpy)3]2+ (data not shown). Therefore, the ECL images enable a direct mapping of the spatial distribution of these substrates in the capillary. This series of experiments demonstrates the ability of the approach to resolve different enzymatic analytes in space and thus to analyze locally inhomogeneous samples.

Conclusion Here, we demonstrated for the first time that combining ECL readout with the wireless features of BPE at the level of dispersions of conducting beads gives the possibility to measure simultaneously and selectively relevant analytes. A quantitative analysis for two different analytes was achieved by exploiting the intrinsic enzymatic selectivity, namely glucose by using the [Ru(bpy)3]2+/GDH system, and choline with the luminol/ChOx system. For both tandem systems, a linear relationship between the concentration of analyte and the ECL intensity was observed over a broad concentration range. It is noteworthy that the method is highly versatile since numerous analytes can be metabolized by NAD+-dependent dehydrogenases or by oxidases. It could also be extended to the detection of more complex analytes through a cascade of enzymatic reactions, where the later involves either a NAD+dependent or a H2O2-producing enzyme. Moreover, the detection of both analytes present at the same location should be possible by immobilizing different ECL luminophores on the surface of the beads. Beads with different types of luminophores could then be mixed and when applying a ramp of electric field the first population of beads (those requiring the lowest polarization potential) would emit first and subsequently beads with another luminophore would emit at higher electric fields. In addition, using transparent and conductive beads (e.g. ITO beads) should allow improving the detection limit by avoiding the screening effect of the carbon beads. The presented approach allows the analysis of inhomogeneous samples with a straightforward visual readout. Finally, such an approach could be used in the future for healthcare diagnostics, notably the detection of diseaserelated proteins like cancer markers by enzyme-linked immunosorbent assay (ELISA) that couples the high affinity and specificity of antibodies towards antigens to all the advantages of the bulk ECL technique described above.



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Dual Enzymatic Detection by Bulk Electrogenerated

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