Elsevier

Electrochimica Acta

Volume 305, 10 May 2019, Pages 370-377
Electrochimica Acta

Combined SECM-fluorescence microscopy using a water-soluble electrofluorochromic dye as the redox mediator

https://doi.org/10.1016/j.electacta.2019.03.069Get rights and content

Abstract

A combined experiment of fluorescence and electrochemical microscopies was performed to highlight the possibility of using a unique water-soluble molecule as the redox mediator and fluorescence reporter. Resorufin was selected as it owns a highly luminescent redox state which can be easily converted into a non-emissive one by reduction at moderately negative potentials. In the feedback mode of Scanning Electrochemical Microscopy (SECM), it is shown that the fluorescence modulation amplitude is sensitive to the nature of the substrate as well as to the tip-substrate distance and can thus be utilized to record optical approach curves. Changing the polarization of the ITO substrate enables to move from positive to negative feedback and the fluorescence modulation amplitude is also sensitive to this change. Substrate generation-tip collection mode was also investigated showing that fluorescence intensity can be used to detect at the tip the species produced at the substrate with a higher accuracy than the electrochemical current.

Introduction

The combination of optical techniques with electrochemistry has known a dramatic development in the two past decades especially at the microscopic level [1]. Optics in general allows imaging and investigating phenomena at the single event level, where its intrinsically high signal to noise ratio compared to electronics becomes a great advantage. Besides, the optical signal can be modulated at high frequency allowing detection of short-lived species. Several optical techniques have been recently coupled to electrochemistry among which holography [2], surface plasmon resonance (SPR) [3] and of course fluorescence, which displays both bright imaging and high sensitivity features. Fluorescence microscopy either in epifluorescence, confocal [4], or TIRF [5] configuration is a well-known imaging technique widely used to investigate chemical or biochemical events. Its coupling with electrochemistry is now well established and documented [6,7]. It was demonstrated to be an efficient way for reporting redox reactions when one species is emissive in one redox state either when directly involved [8,9] or when coupled to it through bipolar electrodes [10]. On the other hand, electrochemical microscopy known as SECM [11,12] has also become popular for various applications like surface analysis, etching, that is surface writing and readout processes. It has become more recently a tool of choice for nanoelectrochemistry due to the improvement in the design of nanoelectrodes [13]. Combining optical techniques with SECM has been a topic of interest as well, for instance using Surface Plasmon Resonance (SPR) [14], ECL [15] or fluorescence to image processes that were triggered by an electrochemical signal [16,17] or even simply using a tip incorporating an optical fiber for dual electrochemical and optical scanning [18,19]. More recently, SECM and fluorescence microscopy were successfully coupled to investigate biochemical events like detection of ROS in human cells [20] or ion permeation through nuclear pore complex [21], highlighting the power of such hyphenated techniques to investigate complex phenomena. It is worth mentioning that in most cases the electrochemical and fluorescence responses are considered independently as they come from different probes. However there are examples where using a single moiety as the fluorescent and redox reporter are especially useful [22]. On our side we recently published the use of such a fluorescent and redox active (that is electrofluorochromic, EF) species in a combined in situ SECM and fluorescence microscopy experiment [23]. However, this example based on a tetrazine derivative was restricted to organic solvents due to its solubility and possible reactivity in water. In the present paper, we wish to extend the concept to aqueous solutions, which is especially suited when dealing with biological issues. We selected resorufin as the EF dye since, beyond its use as a probe in several biological events [[24], [25], [26], [27]], it was also recently reported in fluorescence microscopy coupled to electrochemistry experiments [28]. Resorufin (RF) is highly fluorescent with an emission maximum at 592 nm in its anionic form and becomes non-fluorescent in its reduced protonated-anionic dihydroresorufin (DH) form (see Scheme 1). Thus the electrode potential can be used to reversibly switch RF fluorescence in a similar way as already reported for tetrazines [29], but with the advantage of being useable in water. Moreover, the properties of the ITO-electrolyte interface may dramatically change from organic solvent to water [30]. This is also a reason why investigating the electrochemically monitored fluorescence switch at the ITO surface in aqueous solution is definitely of interest in comparison with previous reports in acetonitrile. In the present paper, we will demonstrate that RF can behave both as a redox mediator and fluorescent reporter in a combined SECM-fluorescence microscopy experiment, using either feedback or substrate generation-tip collection mode.

Section snippets

Chemicals

Resorufin (Sigma Aldrich, dye content 95%) was investigated at the concentration of 1.1 mM in a 0.05 M carbonate buffer solution (25 mM NaHCO3, 25 mM Na2CO3, pH 10) prepared with ultrapure water (Milli-Q system, Millipore). Before each experiment, argon bubbling was used to remove dissolved oxygen from this solution. The argon flow was then maintained at low rate above the solution during all the acquisitions. Resorufin, sodium bicarbonate (VWR chemicals) and sodium carbonate (VWR chemicals)

Results and discussion

The electrochemical modulation of the RF fluorescence intensity was recorded and analyzed in the various SECM modes: negative feedback, positive feedback and generation-collection. In parallel fluorescence images are recorded within the ROI around the SECM tip (Fig. 1A) and the various images can be seen respectively in Fig. 1B for insulating substrate and Fig. 1C for conductive substrate.

Conclusion

Combination of SECM with fluorescence microscopy can be performed using a unique water-soluble species as fluorophore and redox mediator. In all the SECM modes investigated (feedback and generation-collection), the modulation amplitude, i.e. the normalized difference between the on and off intensities is very sensitive to the tip-substrate distance on one hand and to the reaction likely to occur on the substrate on the other hand. This proof of concept opens new possibilities for exploring

Acknowledgments

CHARMMMAT Labex and Region Ile-de-France (C'Nano network) are acknowledged for financial support of the setup.

The University of Michigan, promotor of the program ‘Optics in the city of lights’ is acknowledged for funding the internship of I.M. Gonzalez Ojeda in PPSM laboratory.

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