Imaging Redox Activity at Bipolar Electrodes by Indirect Fluorescence

Letter pubs.acs.org/ac

Imaging Redox Activity at Bipolar Electrodes by Indirect Fluorescence Modulation Laurent Bouffier,*,†,‡ Thomas Doneux,§ Bertrand Goudeau,†,‡ and Alexander Kuhn†,‡ †

Univ. Bordeaux, ISM, UMR 5255, F-33400 Talence, France CNRS, ISM, UMR 5255, F-33400 Talence, France § Chimie Analytique et Chimie des Interfaces, Faculté des Sciences, Université Libre de Bruxelles, Boulevard du Triomphe, 2, CP 255, B-1050 Bruxelles, Belgium ‡

S Supporting Information *

ABSTRACT: Bipolar electrochemistry (BPE) is nowadays well-known but relatively underexploited and still considered as unconventional. It has been used, among others, in the frame of materials science and most importantly has also found very promising applications in analytical chemistry. Here, we extend this emerging field of analytical applications to the development of a new sensing concept based on indirect BPE. This approach is based on the generation of local pH gradients which will allow detecting indirectly redox-active molecules due to a modulation of the fluorescence intensity in the vicinity of a bipolar electrode.

D

Although bipolar electrochemistry (BPE) has been known for decades, it was mainly identified and investigated by the community of chemists working in the fields of electrolysis, corrosion, and batteries.1−3 More recently, this technique has attracted an increasing interest in additional areas. The fabrication of electric contacts between copper particles by means of BPE was reported in the late 90s,4,5 opening the door to numerous investigations in the field of materials science,6,7 based on the selective deposition of electroactive precursors at one extremity of a conducting object. This could be achieved, for example, by direct reduction of metal ions to form the corresponding metal deposit,8−11 reduction of diazonium salts to generate an organic layer,12 or also by oxidation of monomers to electrogenerate polymer films.13,14 BPE has also highly interesting applications in analytical sciences as demonstrated by the seminal work of Crooks at al.15 The possibility to use a bipolar electrode as an analytical platform has been exemplified with the detection of DNA hybridization and the development of electrochemiluminescence (ECL)-based sensing concepts.16−20 The approach was also adapted to the rapid screening of electrocatalysts21,22 and for the generation of surface gradients.13,23−26 ECL quenching at a bipolar electrode has also been reported for the sensitive detection of ferrocenemethanol and molecular oxygen.27 Very recently, we showed that the deposition of nonelectroactive precursors could also be achieved by means of indirect BPE based on a pH-driven local precipitation.28 This strategy has been adapted in the present work to perform analytical tasks by the generation of pH gradients in the vicinity of a bipolar electrode. We propose to detect the presence of

eveloping new approaches allowing delocalized detection of biorelevant molecules is an ongoing challenge in analytical sciences, and electrochemistry offers many opportunities in this context. Conventional electrochemistry is carried out by controlling the potential of the working electrode. Alternatively, it is also possible to control the potential of the solution because electron-transfer reactions are driven by the potential difference between the electrode and the solution. When an external potential (Etot) is applied between two feeder electrodes, a linear potential gradient is established in the electrolyte solution (see Figure 1). Therefore, if a conducting

Figure 1. Principle of bipolar electrochemistry.

material is immersed in the electrolyte, the difference of potential between its two ends (ΔE) is equal to the fraction of Etot which drops over the length of this object. When ΔE becomes large enough, Faradaic reactions will occur simultaneously at both ends of the bipolar electrode, which will act simultaneously as an anode and as a cathode. © 2014 American Chemical Society

Received: February 14, 2014 Accepted: March 21, 2014 Published: March 21, 2014 3708

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Letter

available and also the intensity of fluorescence emission was reported to be sensitive to the pH with a typical enhancement recorded in basic media.31,32 The redox reactions that are coupled across the bipolar electrode when using a 1 mM 1,4naphthoquinone solution are the reduction of the quinone to the corresponding hydroquinone and the oxidation of water, respectively (see Figure 2A). The typical potential difference required between both ends of the bipolar electrode is around ΔE = 1.3 + 0.3 = 1.6 V based on the formal potentials of both redox reactions (Figures S1 and S2 in the Supporting Information). Considering the distance between the feeder electrodes and the length of the bipolar electrode, a difference of at least 16 V will thus be necessary between the feeder electrodes to drive these reactions. Practically, we observed that about 20 V are necessary and this slight experimental deviation from theory could be explained by nonideal conditions. Figure 3A shows fluorescence imaging of the cathodic pole of the

electroactive molecule by this means. The target molecule can be either oxidized or reduced at one extremity of a bipolar electrode, whereas the reduction of protons or oxidation of water molecules will take place at the other side. The resulting basic or acidic gradients forming at the electrode extremities29,30 can then be detected by pH-sensitive fluorescence reporters. Good detection limits can be expected due to the intrinsic sensitivity of fluorescence spectroscopy. As a proof-of-principle, we are reporting the use of such an approach to detect a biorelevant quinone or catechol in the presence of fluorescein, as illustrated in parts A and B of Figure 2 , respectively. It is well established that the dibasic form of

Figure 3. Fluorescence microscopy allowing the visual detection of 1,4-naphthoquinone (1 mM) by applying 10 V (A) and 35 V (B) across a bipolar electrode in a 10 μM fluorescein solution.

Figure 2. Indirect visual detection of 1,4-naphthoquinone (A) and dopamine (B) by localized pH triggered fluorescence enhancement.

bipolar electrode after applying 10 V (part A) and 35 V (part B) for 10 s, respectively. From these images it is clear that below the thermodynamic threshold value, no reaction is observed, whereas a sufficient electric field allows the effective redox coupling. The consumption of protons is very efficient and the pH value is therefore significantly increased locally. The depletion of H+ in the vicinity of the bipolar electrode could be unambiguously visualized at higher polarization as exemplified by Figure 3B (35 V). The influence of the applied electric field was studied in detail, and the relationship between the strength of the polarization and the size of the zone where the fluorescence is enhanced is reported in Figure S5 in the Supporting Information in the range 15−40 V. This modulation of fluorescence intensity on a bipolar electrode is reported here as a proof of principle experiment, but it could be interesting in the future to fully investigate the theoretical framework comparable to what has already been reported for ECL.36 It is noteworthy that the presented data were recorded after applying the electric field for 10 s in order to visualize the fluorescence enhancement in a large area, but it is also possible to perform the measurement at any time (see the Supporting Information, Figure S4). Actually, protons are diffusing from the edge of the bipolar electrode and the experiment could be considered as an indirect visualization of the diffusion layer. A careful examination of Figure 3B reveals that the fluorescence intensity is slightly lower in the direct vicinity of the bipolar electrode when compared to the maximum intensity. This is because the snapshot has been taken after applying the potential for a relatively long time (10 s) in order to enhance the area exhibiting the fluorescence modulation. This is indeed not the case for a very short time delay as the pH gradient starts

fluorescein, F2− (ϕ = 0.93),31 has a higher fluorescence quantum yield than the amphoteric form HF− (ϕ = 0.37),31 the pKa of the latter one being around 6.4. Its fluorescence is thus higher under more basic conditions, and this property is used as an indicator of the occurrence of the electrochemical reactions at the bipolar electrode. In both cases (quinone and catechol), an oxidation is taking place at the anodic pole of the bipolar electrode, while protons are consumed at the cathodic extremity. The consumption of H+ increases locally the pH, which leads to an enhancement of the fluorescence compared to the background intensity.32,33 The strategy could be potentially adapted to other fluorophores in order to tune the pKa value and modulate the efficiency of the pH switch. Finally, the advantage of the approach relies on the fact that it is possible to detect the presence of the biomolecule by simply recording with one’s own eyes changes in the fluorescence. The feasibility of applying indirect BPE for analytical purposes was investigated by using 1,4-naphthoquinone as a model redox-active target. This choice was made because a large number of quinones are biorelevant for acting notably as redox mediators.34,35 The experimental setup consisted in a very simple electrochemical cell made from a conventional glass beaker and two gold feeder electrodes separated by a distance of 4.5 cm (see experimental details). The bipolar electrode was a 4.5 mm long cylindrical graphite rod (diameter = 0.5 mm), positioned on a transparent glass slide between both feeder electrodes. Fluorescein was chosen as a common dye because it is readily 3709

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experimental conditions. In fact, if the same electric field is applied between the feeder electrodes, the fluorescence enhancement is no longer observed (Figure 4C). This is due to the fact that ascorbic acid has a pKa of ∼5. Therefore, the addition of such species shifts the pH value and also increases the ionic strength of the solution, thus favoring the ionic bypass current. Increasing the applied voltage up to 30 V allows recovering an efficient redox coupling (Figure 4D). Therefore, it is possible to distinguish the electrochemical behavior of dopamine with and without ascorbic acid as a competitor by comparing the images recorded at 20 V (part C vs part B of Figure 4). Finally, it is noteworthy that the ideal experimental conditions used to record these data correspond to unbuffered conditions. The reason is because the presence of a large amount of supporting electrolyte does increase the ionic strength of the solution. This results in lowering the fluorescence modulation because of a competition between the amounts of current passing across the bipolar electrode and through the solution (bypass current). To conclude, we report in the present letter the detection of redox-active biomolecules, which can be either oxidized or reduced at one extremity of a bipolar electrode. The analytical event can be easily visualized by taking advantage of the generated pH gradients in combination with a pH-sensitive fluorescent dye such as fluorescein. This approach was illustrated by detecting two different biorelevant targets, namely, 1,4-naphthoquinone and dopamine. Analytical direct BPE has already been reported, especially for the detection of biopolymers such as DNA.16 Here, we use indirect BPE as a complementary sensing concept based on the localized modulation of fluorescence intensity for detecting specific redox activity. This broadens the application potential offered by BPE in the field of analytical chemistry.

to be established right at the extremity of the bipolar electrode (Figure S4 in the Supporting Information). These results clearly demonstrate the possibility of using indirect BPE to detect efficiently a redox-active molecule. Thus we decided to also apply this concept to the detection of dopamine, a well-known neurotransmitter. Dopamine is indeed an essential analytical target which is very often detected by using electrochemical approaches as the ortho-catechol moiety is electroactive with a typical oxidation process occurring at ∼+0.4 V versus Ag/AgCl (Figure S3 in the Supporting Information). A range of techniques such as cyclic voltammetry, differential pulse voltammetry, and chronoamperometry have already been reported.37 The electrochemical detection of dopamine can become difficult in the presence of ascorbic acid, which is also electroactive in the same potential range. Detection of dopamine by indirect BPE is schematized in Figure 2B and can be performed following exactly the same protocol as for 1,4-naphthoquinone. In that case, the two halfreactions that need to be considered are dopamine oxidation to form the corresponding ortho-quinone and proton reduction. The corresponding fluorescence images recorded from a 1 mM dopamine solution in the presence of 10 μM of fluorescein are gathered in Figure 4A,B. Image A was recorded after applying

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EXPERIMENTAL SECTION Chemicals and Materials. Fluorescein (C20H12O5, laser grade 99%) was purchased from Acros Organics. Dopamine hydrochloride (C8H11NO2·HCl) and ascorbic acid (C6H8O6, reagent grade) were purchased from Sigma-Aldrich. All aqueous solutions were prepared from a Milli-Q station (Millipore, resistivity 18.2 MΩ·cm at 25 °C). Microscope slides (76 mm × 26 mm × 1.1 mm) were purchased from Radiospares. Graphite pencil leads (diameter = 0.5 mm, grade HB, BIC criterium trademark) were purchased from Bruneau. Gold feeder electrodes were provided by ACM (1 mm-thick glass slide with a thin NiCr 80/20 coating and a 300 nm-thick Au layer). The electric field was applied with a Keithley high power supply (Electrometer 6517B). Fluorescence was recorded using a microscope Leica DM6000 B LT equipped with a monochromatic digital camera (DFC365FX), an objective lens ×5 magnification (HCX PL Fluotar) together with a filter L5 (excitation BP 480/40 nm, dichroic LP 505 nm, and emission BP 527/30 nm) or I3 (excitation BP 450−490 nm, dichroic LP 510 nm, and emission LP 515 nm).

Figure 4. Fluorescence microscopy for the visual detection of dopamine (1 mM) by applying 10 V (A) and 20 V (B) in a 10 μM fluorescein solution. Same experiment after addition of 1 mM ascorbic acid recorded after applying 20 V (C) or 30 V (D).

an external voltage of 10 V for 10 s between both gold feeder electrodes (i.e., 1 V polarization of the bipolar electrode). It is noteworthy that without dopamine, absolutely no enhancement of the fluorescence due to proton consumption was evident at 10 V. In contrast, the increase of fluorescence intensity could be monitored when 1 mM of dopamine was added to the fluorescein solution (Figure 4A). Indeed, without dopamine, the two reactions which need to take place on the bipolar electrode are water oxidation and proton reduction, respectively. Coupling these two reactions requires at least a ΔE of 2.7 V (Figure S1 in the Supporting Information), meaning that the used experimental conditions are indeed insufficient to observe any significant pH gradient. Figure 4A shows that the addition of dopamine favors the electrochemical coupling as it is much easier to oxidize the catechol than H2O molecules. At 20 V, the zone where the pH is modulated is further enhanced (Figure 4B). Interestingly, the detection could also be achieved in the presence of 1 mM of ascorbic acid by adjusting the

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ASSOCIATED CONTENT

* Supporting Information S

Cyclic voltammetry experiments, additional fluorescence microscopy data, and analysis of the variation of the fluorescence modulation as a function of the applied electric field. This material is available free of charge via the Internet at http://pubs.acs.org. 3710

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Letter

(27) Wang, T.; Fan, S.; Erdmann, R.; Shannon, C. Langmuir 2013, 29, 16040. (28) Loget, G.; Roche, J.; Gianessi, E.; Bouffier, L.; Kuhn, A. J. Am. Chem. Soc. 2012, 134, 20033. (29) Aora, A.; Eijkel, J. C. T.; Morf, W. E.; Manz, A. Anal. Chem. 2001, 73, 3282. (30) Bouffier, L.; Kuhn, A. Nanoscale 2013, 5, 1305. (31) Sjöback, R.; Nygren, J.; Kubista, M. Spectrochim. Acta A 1995, 51, L7. (32) Bowyer, W. J.; Xie, J.; Engstrom, R. C. Anal. Chem. 1996, 68, 2005. (33) Rudd, N. C.; Cannan, S.; Bitziou, E.; Ciani, I.; Whitworth, A. L.; Unwin, P. Anal. Chem. 2005, 77, 6205. (34) Rau, J.; Knackmuss, H.-J.; Stolz, A. Environ. Sci. Technol. 2002, 36, 1497. (35) Van der Zee, F. P.; Cervantes, F. J. Biotechnol. Adv. 2009, 27, 256. (36) Mavré, F.; Chow, K.-F.; Sheridan, E.; Chang, B.-Y.; Crooks, J. A.; Crooks, R. M. Anal. Chem. 2009, 81, 6219. (37) Kim, Y.-R.; Bong, S.; Kang, Y.-J.; Yang, Y.; Mahajan, R. K.; Kim, J. S.; Kim, H. Biosens. Bioelectron. 2010, 25, 2366.

AUTHOR INFORMATION

Corresponding Author

*E-mail: laurent.bouffi[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by the Centre National de la Recherche Scientifique (CNRS). The support from WallonieBruxelles International and the Fonds de la Recherche Scientifique, the Ministry of Foreign Affairs, and the Ministry of Higher Education and Research is gratefully acknowledged in the frame of the Hubert Curien Partnerships (Program Tournesol No. 29120QE).

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