Raman and fluorescence spectroelectrochemical monitoring of resazurin-resorufin fluorogenic system

Dyes and Pigments 172 (2020) 107848

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Raman and fluorescence spectroelectrochemical monitoring of resazurinresorufin fluorogenic system

T

David Ibáñez∗, Daniel Izquierdo-Bote∗∗, Alejandro Pérez-Junquera, María Begona González-García, David Hernández-Santos, Pablo Fanjul-Bolado Metrohm DropSens S.L., Edificio CEEI, Parque Tecnológico de Asturias, 33428, Llanera, Spain

A R T I C LE I N FO

A B S T R A C T

Keywords: Time-resolved spectroelectrochemistry Surface-enhanced Raman scattering (SERS) Fluorescence Resazurin Resorufin

Raman and fluorescence spectroelectrochemistry techniques have been used for the in-situ study of the reaction mechanism of resazurin/resorufin/dihydroresorufin system. Resazurin (RZ) is one of the most highly referenced substances used in studies on cell viability and cytotoxicity for a wide range of biological and environmental systems. Electrochemical behaviour of this tracer is based on the irreversible reduction of RZ, a slightly fluorescent compound, to resorufin (RS), a high fluorescent compound, while a subsequent and reversible reduction of RS generates dihydroresorufin (DHRS), a non-fluorescent compound. Time-resolved Raman spectroelectrochemistry has been used, for the first time, to perform dynamic studies of this system. The continuous optical acquisition provides a large number of spectra during the whole electrochemical reaction and not only at discrete potentials, offering a novel contribution to the literature related to this tracer. The characteristic Raman and fluorescence bands associated with each molecule as well as their evolution with potential allow us to obtain valuable information about the interconversion between RZ, RS and DHRS. In addition, spectroelectrochemistry demonstrates that experimental parameters play an essential role in the fluorescent response of this coloured system. In that way, operando multiresponse techniques shed more light for the complete understanding of RZ, RS and DHRS system, and they yield novel information to explain the electrochemical conversion of these compounds.

1. Introduction Dyes are commonly employed as tracer in the study of biochemical processes for several metabolic and biological studies. For this reason, a huge variety of methods such as colorimetry [1–3], electrochemistry [4–8], spectroelectrochemistry [9–15], fluorescence [16–21], chemiluminescence [22–24], photometry [25,26], etc. have been developed to analyse the behaviour of different systems used in clinical and biological applications. A redox indicator widely used for this purpose is resazurin (RZ), also known as Alamar Blue, a slightly fluorescent dye [27] used as chemical system for measuring the metabolic activity and the proliferation of living cells [28], and testing the cell viability and cytotoxicity in biological materials [29,30]. This tracer shows low toxicity, is a harmless substance and does not kill cells during the assays [31], being suitable for long cell assays. In this way, it allows to reuse the valued cells, saving time and minimizing economic cost. Many of these applications are based on the irreversible reduction of RZ to resorufin (RS), a pink dye and highly fluorescent molecule employed as ∗

fluorogenic tracer. Nevertheless, RS can be reversibly reduced to dihydroresorufin (DHRS), a colourless and non-fluorescent compound (Scheme 1) [32–34]. For this reason, the study of the whole process involving these three compounds is important to provide a better understanding of the evolution of these species during clinical and biological assays. The different fluorescent nature of these three compounds becomes this system a very interesting fluorogenic indicator. For instance, irreversible reduction process from RZ to RS has been employed as fluorimetric test to assess yeast or bacterial contamination in biological fluids and milk [1,35], to study cytotoxicity reliability [31], sperm viability, determination of fertility potential [36,37], cell proliferation on mouse and human lymphocytes [38] and primary neuronal cell culture [39]. Hence, the full understanding of the reaction mechanism is a very interesting issue in order to avoid misinterpretations of the results obtained by spectroscopic techniques. Taking into account the different fluorescent properties of RZ, RS and DHRS, spectroelectrochemistry is one of the most interesting

Corresponding author. Corresponding author. E-mail addresses: [email protected] (D. Ibáñez), [email protected] (D. Izquierdo-Bote).

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https://doi.org/10.1016/j.dyepig.2019.107848 Received 3 June 2019; Received in revised form 28 August 2019; Accepted 28 August 2019 Available online 29 August 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.

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Scheme 1. Structures and reaction mechanism of resazurin (RZ), resorufin (RS), and dihydroresorufin (DHRS) molecules at neutral pH.

techniques for monitoring redox reactions. In-situ fluorescence spectroelectrochemistry provides electrochemical and time-resolved spectroscopic information while interconversion between these species is taking place [33]. Furthermore, the study of this system has also been monitored by Raman spectroelectrochemistry in order to obtain complete information. Fingerprint properties of this technique in combination with the enhancement of the Raman intensity due to Surfaceenhanced Raman scattering (SERS), allows us to differentiate the characteristic Raman spectrum of each molecule during the evolution of the reaction. SERS effect was discovered by Fleishman et al. [40], in 1974, when he observed for the first time a huge enhancement of the Raman signal of pyridine using a roughened silver electrode. The main difference with respect to Raman spectroscopy is the presence of metal nanostructures as fundamental factor to produce the signal enhancement [10,41–43]. Nowadays, it is accepted that SERS effect is explained by the contribution of two mechanisms, electromagnetic and chemical [44–49]. Therefore, not only the sample-light interaction but also the metal nanostructure-light interaction must be considered in SERS effect. In order to detect the generated species during the electrochemical process in the study system, SERS effect plays an essential role, and the presence of AgNPs on the electrode surface is needed. Although RZ has previously been studied by electrochemical techniques applying fixed potentials during long times, to the best of our knowledge, this is the first time that operando Raman spectroelectrochemistry has been used to perform dynamic analysis of this system. Furthermore, the different fluorescent nature of resazurin, resorufin and dihydroresorufin has been studied by fluorescence spectroelectrochemistry, and in-situ and dynamic results demonstrate that experimental parameters, as the oxygen present in solution, are very important in the fluorescent response of this coloured system. Therefore, time-resolved spectroelectrochemistry provides a large volume of data simultaneously recorded with the electrochemical reaction. Optical monitoring, using short integration times, allow us to obtain spectroscopic information of the RZ/RS/DHRS system at all potentials. The main objective of this work is the study of the interconversion mechanism between RZ, RS and DHRS by different spectroelectrochemical techniques. Combination of the spectroelectrochemical results obtained by different spectroscopic techniques contributes to the complete understanding of the reaction mechanism of this tracer under similar experimental conditions of clinical assays. In addition, the dynamic procedure proposed in this work offers a fast and an interesting alternative to overcome the limited information obtained by conventional methods that require long times. In that way, the large number of spectra recorded in our experiments displays suitable time-resolved information to the complete characterization of RZ/RS/DHRS.

grade. Aqueous solutions were prepared using high-quality water (Direct-QTM 5 system, Millipore). All solutions were prepared at neutral pH because most of the biological tests that involve this fluorogenic system are performed at this pH. Neutral pH avoids the oxygen protonation observed in acid media. 2.2. Instrumentation Fluorescence and Raman spectroelectrochemical measurements were carried out with SPELEC and SPELEC RAMAN instruments (Metrohm DropSens), respectively. Both instruments are controlled by DropView SPELEC software. Raman spectroelectrochemistry was performed with SPELEC RAMAN instrument (Metrohm DropSens) which uses a 785 nm laser, a Raman probe (DRP-RAMANPROBE, Metrohm DropSens) and a Raman spectroelectrochemical cell (DRP-RAMANCELL, Metrohm DropSens). Fluorescence spectroelectrochemistry was performed with a fluorescence kit (DRP-FLKIT, Metrohm DropSens), composed of two short optical fibers, two optical filters (one for 230–500 nm and other for 300–750 nm wavelength), two filter holders, a bifurcated reflection probe (DRP-RPROBE, Metrohm DropSens) and a specific cell for screenprinted electrodes (DRP-REFLECELL, Metrohm DropSens) in a nearnormal reflection configuration (Fig. 1). A 395 nm UV LED (DRPLEDVIS395, Metrohm DropSens) was used as excitation light source, and connected to the input filter by a short optical fiber. Adjustment of the input filter connected to the UV LED allows us to remove the light at longer wavelengths than 480 nm, and it ensures that only the LED

2. Material and methods 2.1. Reagents Fig. 1. Fluorescence spectroelectrochemical setup: SPELEC instrument, 395 nm UV LED, two short optical fibers, two optical filters, a bifurcated reflection probe and a spectroelectrochemical cell for SPEs.

Resazurin sodium salt (Sigma-Aldrich), resorufin sodium salt (Sigma-Aldrich) and potassium chloride (KCl, Merck), were used as received without further purification. All chemicals were analytical 2

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potential, one of the most intense Raman bands, at 1613 cm−1, was selected (Fig. 2d). At the beginning of the experiment, from −0.10 V to −0.54 V, this band is not observed because only RS is present in solution. Intensity of this band increases from −0.54 V when the reduction of RS to DHRS starts, and it keeps growing at more negative potential in the forward scan. In the backward scan, Raman intensity increases until the potential reaches a value of −0.53 V when the oxidation of DHRS to RS takes place. Finally, the intensity of Raman band at 1613 cm−1 decreases and it is not differentiated from −0.38 V towards the end of the scan. All bands associated with DHRS show similar behaviour than this band, so it can be considered as representative band to explain the DHRS behaviour. The same experiment was performed using a DRP-110 electrode without AgNPs (data not shown). Although the Raman spectrum of RS is easily differentiated during the first part of the experiment due to its high concentration, DHRS cannot be detected because the low concentration generated is not enough to be observed using unmodified electrodes without SERS features. It demonstrates that metal nanostructures are completely mandatory for SERS effect and it allows differentiating the generated products during electrochemical reactions. Furthermore, the role of the selected metal was analysed using DRP-110 electrode modified with copper nanoneedles (CuNNs). Spectroelectrochemical results obtained with this substrate only show characteristic Raman bands of RS at the beginning at the experiment, but characteristic bands of DHRS after the electrochemical reaction are not observed. Although copper is a metal traditionally used in SERS applications, nowadays there is not a universal SERS substrate and it depends on each system. It is well-known than the enhancement of Raman intensity depends on different factors: metal substrate (type of metal, shape, size, composition, synthesis method, etc), excitation wavelength and sample (concentration, orientation, sample-substrate interaction, etc.). According to our results, CuNNs employed in the RS/ DHRS system do not show SERS properties under our experimental conditions, and on other hand, AgNPs shows a high enhancement factor.

provides the light that arrives to the system. On the other hand, the adjustment of the output filter connected to the spectrometer, enables to remove shorter wavelengths than 480 nm and it ensures that the light collected is not related to the light emitted by the LED and is only associated with the fluorescence of the studied system. Screen-printed Carbon Electrodes (DRP-110, Metrohm Dropsens) consisting of a flat ceramic card with a circular carbon working electrode (4 mm diameter), a carbon auxiliary electrode and a silver pseudo-reference electrode, were employed in all experiments. In order to improve the Raman signal by SERS effect, DRP-110 were modified by drop casting with 25 μL silver nanoparticles (≈1 mg/ml AgNPs aqueous solution, medium size 10.1 nm) and with 25 μL copper nanoneedles (≈0.6 mg/ml CuNNs aqueous solution). SERS properties of AgNPs produce the enhancement of the Raman signal, and then, it circumvents the lack of sensitivity of normal Raman spectroscopy. Fluorescence and Raman measurements were performed using a volume of solution of 80 μL and 60 μL respectively, at room temperature. SPEs were connected to the SPELEC instruments through a specific connector (DRPCAST, Metrohm DropSens). Spectroelectrochemical measurements in thin layer configuration were performed using thin layer flow-cell screen-printed electrodes with a circular carbon working electrode (4 mm diameter), a carbon auxiliary electrode and a silver pseudo-reference electrode (DRPTLFCL110-CIR, Metrohm DropSens). These electrodes have a transparent cover that defines the thickness of the thin channel onto the electrochemical cell. Raman spectroelectrochemistry was performed using DRP-TLFCL110-CIR electrodes modified with AgNPs in order to obtain SERS effect to enhance Raman intensity of RZ, RS and DHRS. DRP-TLFCL110-CIR electrodes were modified with 25 μL AgNPs aqueous solution (≈1 mg/ml AgNPs aqueous solution, medium size 10.1 nm). DRP-TLFCL110-CIR electrodes were used with a spectroelectrochemical reflection flow-cell (DRP-TLFCL-REFLECELL, Metrohm DropSens) for thin layer SPEs and with a specific cable connector (DRPCAC-TLFCL, Metrohm DropSens). 3. Results and discussion

3.1.2. Fluorescence spectroelectrochemistry Fluorescence associated with RS was studied employing the same electrochemical conditions than in the Raman spectroelectrochemical experiment (Section 3.1.1.). In this case, unmodified DRP-110 electrodes were used. AgNPs are not required because they do not enhance the fluorescence signal. Fig. 3a exhibits the same electrochemical behaviour (blue line) than AgNPs modified DRP-110 electrodes (Fig. 2b). Comparison of Figs. 3a and 2b confirms that AgNPs do not affect the electrochemical performance. Red lines in Fig. 3 show derivative fluorescence emission at 590 nm, related to RS, with the potential during first (Fig. 3a) and second cycles (Fig. 3b). Derivative signal was calculated using 800 spectra recorded during the two cycles, hence, it allows us to obtain a complete knowledge of this system. As can be observed in Fig. 3, derivative fluorescence emission is proportional to the faradaic current involved in the redox reaction of RS/DHRS fluorogenic couple. During the forward scan of the first cycle (red line in Fig. 3a), the derivative fluorescence emission at 590 nm exhibits a small maximum at −0.55 V between the two cathodic peaks. This enhancement of fluorescence signal is related to an increase of the fluorescence emission due to oxygen present in solution is reduced. It is well-know that oxygen can behave as a fluorescence quencher for several molecules, including RS. This fact can be observed in the forward scan in Fig. 3a, derivative fluorescence at 590 nm (red line) grows around −0.40 V because dissolved oxygen is reduced, and therefore, fluorescence quenching disappears. Nevertheless, derivative fluorescence emission starts to decrease at −0.55 V because RS is reduced to DHRS, and it remains stable after the second cathodic peak. In the backward scan, the derivative fluorescence increases when RS is reoxidized to DHRS and it reaches the maximum value at −0.55 V. As can be noticed in Fig. 3a, cathodic and anodic scans (blue line) match with

3.1. RS/DHRS system 3.1.1. Raman spectroelectrochemistry Electrochemical behaviour of RS was studied by cyclic voltammetry, scanning the potential from −0.10 V to −1.10 V and back to −0.10 V at 0.02 V s−1 in 5 × 10−3 M RS in 0.1 M KCl solution using AgNPs modified DRP-110 electrodes. Voltammetric profile (Fig. 2a) shows two cathodic peaks in the forward scan, the first one, at −0.50 V, associated with the reduction of dissolved oxygen in solution and the second peak, at −0.65 V, due to the reduction of RS to DHRS. In the backward scan a single anodic peak, at −0.55 V, related to the oxidation of DHRS to RS is noticed. Although Raman spectra were recorded during the whole reaction, in order to simply their understanding, only several spectra are shown in Fig. 2b–d. As can be noticed in spectra plotted during the forward scan (Fig. 2b), intensity and position of Raman bands change with potential. Blue line in Fig. 2b shows Raman bands of RS at −0.50 V prior to the reduction process. At more negative potential than −0.70 V, intensity of Raman bands centred at 466, 579, 725, 1147, 1321, 1475 1506 and 1640 cm−1, related to RS decreases, and on the other hand, new bands at 363, 445, 558, 735, 820, 938, 1102, 1144, 1254, 1282, 1412 and 1613 cm−1, associated with DHRS, are detected. Raman spectra recorded during the backward scan, shown in Fig. 2c, demonstrate the oxidation of DHRS to RS takes place because bands associated with DHRS disappear while the same Raman bands of RS as at the beginning of the forward scan are detected once again. Time-resolved spectroelectrochemistry allows us to perform dynamic measurements of evolving systems and it enables the analysis of the evolution of Raman bands with potential. In order to analyse the spectroelectrochemical behaviour of Raman bands of DHRS with 3

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Fig. 2. (a) Cyclic voltammogram obtained in 5 × 10−3 M RS in 0.1 M KCl solution. Potential was scanned from −0.10 V to −1.10 V and back to −0.10 V at 0.02 V s−1. Raman spectra recorded at several potentials in (b) forward and (c) backward scans. (d) Evolution of Raman intensity at 1613 cm−1 with potential.

variation is only related to the consumption/generation of RS. In order to explain this behaviour with further details, the same experiment was carried out in presence and in absence of oxygen (Fig. 4) in 1 × 10−3 M RS in 0.1 M KCl solution using thin film carbon electrodes (DRP-TLFCL110-CIR). Measurements in absence of oxygen were performed after purging the solution with nitrogen for 10 min, furthermore, thin film electrodes were employed to assure the absence of oxygen in the deoxygenated solution. Experiments in presence of oxygen were carried out by the enrichment of the solution with oxygen by blowing air for 10 min. As can be observed in Fig. 4a (red line),

the derivative response (red line) after oxygen is reduced, and the maximum of derivative fluorescence at 590 nm is achieved just in the oxidation peak of the backward scan. Red line in Fig. 3b shows the derivate fluorescence emission at 590 nm recorded during the second voltammetric cycle (blue line in Fig. 3b). As oxygen initially present in solution is reduced in the first cycle, in the second scan there is not oxygen present in solution and the quenching of the RS fluorescence signal is avoided. Derivative fluorescence signal shows the same behaviour than the voltammetric signal because the oxygen was reduced in the first cycle, therefore, in the second cycle the spectroscopic

Fig. 3. (a) First and (b) second voltammetric cycles (blue lines) obtained in 1 × 10−3 M RS in 0.1 M KCl solution, scanning the potential from −0.10 V to −1.10 V and back to −0.10 V at 0.05 V s−1. Evolution of derivative fluorescence emission at 590 nm with respect to time versus potential (red lines) during (a) first and (b) second cycles. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4

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Fig. 4. Cyclic voltammograms (blue lines) and evolution of fluorescence emission at 590 nm with potential (red line) obtained in 1 × 10−3 M RS in 0.1 M KCl solution in (a) absence and (b) presence of oxygen. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. (a) Cyclic voltammogram obtained in 5 × 10−3 M RZ in 0.1 M KCl solution. Potential was scanned from −0.10 V to −1.10 V and back to −0.10 V at 0.02 V s−1. Raman spectra are represented in three potential ranges: in the forward scan (b) from −0.1 V to −0.58 V and (c) from −0.58 V to −1.10 V and (d) in the backward scan.

in a thin film cell. As can be noticed, the cathodic peak is higher than the anodic one because the oxygen reduction process is overlapped with the RS reduction peak. Furthermore, a high separation of the electrochemical peaks is observed due to the presence of the oxygen. Red line in Fig. 4b demonstrates that fluorescence remains constant in the forward scan until −0.55 V. However, at more negative potential, fluorescence value increases because oxygen is previously reduced and the quenching effect is removed. Nevertheless, the potential is not as negative as is required to reduce RS to DHRS. When the cathodic peak potential is reached, the fluorescence at 590 nm evolves more slightly

evolution of fluorescence emission at 590 nm with potential in absence of oxygen remains constant during the forward scan until the reduction of RS takes place at −0.65 V. This reduction process produces the fluorescence decrease due to the consumption of RS (fluorescent molecule). In the backward scan, spectroscopic emission continues decreasing slightly because the reduction of RS is diffusion controlled. At −0.60 V, the anodic process associated with the reoxidation of DHRS causes that fluorescence increases and achieves the initial intensity at the end of the scan. On the other hand, Fig. 4b displays the evolution of fluorescence emission during cyclic voltammetry in presence of oxygen 5

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Fig. 6. Evolution of Raman band (a) at 1558 cm−1 (associated with RZ and DHRS), (b) at 1321 cm−1 (associated with RS) and (c) at 735 cm−1 (associated with DHRS) with potential.

1633 cm−1 are distinguished (Fig. 5b). As can be noticed in Fig. 5b, Raman bands associated with RZ do not totally disappear because the reduction of RZ from −0.10 V to −0.58 V is not complete and both RZ and RS are present in the sample. RZ still present in solution produces a slight shifting of RS bands, and for that reason, RS bands observed when RS (Fig. 2b) or RZ (Fig. 5b) are used as reagent in the initial solution do not match exactly. Analysis of Raman spectra (Fig. 5c) recorded at different potentials during the second reduction process, from −0.62 V to −1.10 V, demonstrates the generation of DHRS since new Raman bands at 360, 445, 556, 735, 940, 1144, 1254, 1284 and 1622 cm−1 are observed while Raman intensity of RS decreases simultaneously. Finally, spectra recorded in the backward scan (Fig. 5d) demonstrate the reoxidation of DHRS to RS because the intensity of Raman bands associated with DHRS decreases while RS bands are again differentiated. Analysis of the evolution of different Raman bands with potential (Fig. 6) allows us to understand the electrochemical reactions involved in this system. Three bands centred at 1558, 1321 and 735 cm−1 were selected as representative. Fig. 6a shows the behaviour of Raman band at 1558 cm−1 with potential. As can be observed in the forward scan, Raman intensity decreases at −0.25 V because RZ is reduced to RS, but at −0.48 V the intensity increases due to the generation of DHRS starts. In the anodic scan, at −0.54 V, this band decreases due to the oxidation of DHRS is produced. Hence, this band is not only related to RZ but also to DHRS. Evolution of Raman band at 1321 cm−1 (Fig. 6b) associated with RS, shows that Raman intensity increases at −0.26 V in the cathodic scan when the reduction of RZ to RS takes place. However, it decreases at −0.53 V just before the second reduction peak is observed in the cyclic voltammogram. This behaviour suggests the consumption of RS begins before is expected because the cathodic peaks are partially overlapped. Raman intensity decreases at more negative potential in

because at this potential oxygen is removed as well as RS is also reduced to DHRS. In anodic scan, the fluorescence remains constant before oxidation process starts, and at less negative potential than −0.60 V fluorescence increases due to the regeneration of RS. 3.2. RZ/RS/DHRS system 3.2.1. Raman spectroelectrochemistry Electrochemical behaviour of RZ was also studied by cyclic voltammetry, scanning the potential from −0.10 V to −1.10 V and back to −0.10 V at 0.02 V s−1 in 5 × 10−3 M RZ in 0.1 M KCl solution using AgNPs modified DRP-110 electrodes. As previously stablished, AgNPs were employed to increase the SERS effect of the carbon electrode. Cyclic voltammogram, shown in Fig. 5a, displays two reduction peaks; the first one at −0.50 V associated with the generation of RS from RZ but also with the oxygen reduction, and the second peak, at −0.65 V, linked to the reduction of RS to DHRS. In the backward scan, only one peak at −0.55 V related to the oxidation from DHRS to RS is observed. Electrochemical curve agrees with Scheme 1, the reduction of RZ is an irreversible process, while the reduction of RS is reversible. Raman spectra (Fig. 5b–d) simultaneously recorded with the electrochemical curve show the spectral response obtained during the forward (Fig. 5b and c) and backward (Fig. 5d) scans. Initially, only Raman bands at 358, 512, 700, 1167, 1196, 1322, 1403, 1475, 1558 and 1629 cm−1 related to RZ are observed in Fig. 5b (blue line). When the reduction of RZ takes place, the position and intensity of Raman bands change due to the generation of RS on the electrode surface. Considering only the first reduction process from −0.10 V to −0.58 V, not only RZ Raman bands are observed in the spectra, but also RS bands at 335, 387, 469, 577, 670, 728, 745, 817, 860, 1185, 1235, 1400, 1470, 1594 and 6

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generation of RS occurs. Hence, Raman band at 1321 cm−1 is only associated with RS. Finally, Fig. 6c shows that Raman band at 735 cm−1 is not observed initially and only at more negative potential than −0.82 V Raman intensity increases, confirming that this band is only related to DHRS. In the backward scan, the intensity decreases at −0.54 V when the oxidation of DHRS takes place. In order to obtain the characteristic Raman bands of RZ, RS and DHRS, a chronoamperometry was performed to produce the total electrolysis of the solution. Chonoamperometry was carried out applying fixed potentials (−0.45 V and −1.10 V) and using AgNPs modified DRP-TLFCL110-CIR electrodes. Fig. 7 displays obtained Raman spectra of RZ, RS and DHRS. Analysis of Fig. 7 allows us to summarize the position and intensity of Raman bands in Table 1. As can be noticed, SERS is a sensitive technique that differentiates small changes of Raman shift and intensity. In addition, fingerprint features of Raman spectroscopy make possible the accurate spectroelectrochemical characterization of RZ, RS and DHRS. 3.2.2. Fluorescence spectroelectrochemistry Since RZ has been widely employed as a fluoregenic indicator, fluorescence spectroelectrochemistry was selected for studying the reaction mechanism of this tracer. Spectroelectrochemical behaviour of RZ was studied by cyclic voltammetry in 1 × 10−3 M RZ in 0.1 M KCl solution using DRP-110 electrodes (blue line in Fig. 8a). Fluorescence signal was monitored during the electrochemical experiment and evolution of the fluorescence emission at 645 nm, related to RZ, is shown in Fig. 8a (green line). At the beginning of the experiment, from −0.10 V to −0.55 V, no spectral changes are observed, indicating that no reaction takes place. However, at more negative potential, from −0.55 V to −0.65 V, reduction of RZ to RS produces a slight increase of fluorescence intensity. The fluorescence emission increases after the second reduction process due to RS generation still takes place. However, at more negative potential than −0.65 V, the fluorescence intensity increases slower as consequence of the reduction of RS to DHRS. At this potential region, RS behaves as a reaction intermediate between RZ and DHRS and the slope of the fluorescence evolution decreases with respect to the one observed during the first reduction process. Fluorescence increases slowly not only during the cathodic scan, but also in the backward scan until −0.55 V. At this potential, DHRS is reoxidized and a high increase of fluorescence due to the generation RS is noticed. The reaction mechanism was also studied by chronoamperometry (black line in Fig. 8b) applying −0.45 V for 300 s; −1.10 V for 300 s and −0.10 V for 300 s in 1 × 10−3 M RZ in 0.1 M KCl solution using a DRP-TLFCL110-CIR for assuring the bulk electrolysis. Fluorescence emission was concomitantly recorded to the electrochemical experiment and two emission bands centred at 590 and 645 nm were selected as representative. Fig. S1 shows the fluorescence spectra at different times during the chronoamperometry plotted in Fig. 8b. In the first step, −0.45 V was applied for 300 s (green zone in Fig. 8b). At this potential, fluorescence band at 590 nm (red line), related to RS, weakly increases due a small amount of RZ is reduced to RS as well as the oxygen reduction also takes place, however, fluorescence at 645 nm remains constant. After 300 s a potential of −1.10 V was applied (grey zone) and a dramatic increase of fluorescence intensity at 590 nm is observed during the first 20 s. Simultaneously, fluorescence at 645 nm, related to RZ decreases and it suggests that during this time, the most of RZ is reduced to RS. In addition, an isosbestic point observed in Fig. S1b confirms this behaviour. Afterwards, fluorescence begins to disappear due to RS previously formed is reduced to DHRS, a non-fluorescent molecule, and after 300 s there is not fluorescence signal at 590, neither 645 nm. Finally, −0.10 V is applied (blue zone), being this potential positive enough for reoxidizing DHRS to RS, and the fluorescence band centred at 590 nm suffers an abrupt increase and then it remains constant. Fluorescence emission at 645 nm also increases, however this behaviour is due to the high intensity of the band centred at 590 nm

Fig. 7. Raman spectra of RZ (blue line), RS (red line) and DHRS (green line). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Table 1 SERS bands of RZ, RS and DHRS. RZ

RS

DHRS

Raman shift (cm−1)

Intensity

Raman shift (cm−1)

Intensity

Raman shift (cm−1)

Intensity

358 385 461 512 635 700 792 926 1109 1167 1196 1322 1403 1475 1509 1558 1629

m m w s m m w w s s s s s w w m s

315 375 466 579 639 725 813 851 996 1147 1279 1321 1404 1475 1506 1572 1640

w w s s w w w w w s sh s s s s w s

363 445 558 656 735 820 938 1102 1144 1163 1254 1302 1315 1412 1474 1556 1613

s m s w s s s m w w sh s sh m w sh s

s: strong; m: medium; w: weak; sh: shoulder.

the forward scan, it remains constant at the beginning of the backward scan and finally, Raman intensity increases at −0.60 V when the 7

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Fig. 8. (a) Cyclic voltammogram (blue line) and evolution of fluorescence emission at 645 nm with potential (green line) obtained in 1 × 10−3 M RZ in 0.1 M KCl solution. The potential was scanned from −0.10 V to −1.10 V and back to −0.10 V at 0.05 V s−1. (b) Chronoamperogram (black line) and evolution of fluorescence emission at 590 nm (red line, related to RS) and 645 nm (blue line, related to RZ) with potential. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

overlaps 645 nm wavelength, as can be also observed in Fig. S1d.

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4. Conclusions In the present work, RZ/RS/DHRS system has been studied by Raman and fluorescence spectroelectrochemistry. As has been demonstrated, spectroelectrochemistry is a powerful technique that provides information of different nature and combines the advantages of electrochemistry and spectroscopy. In that way, Raman spectroelectrochemistry based on SERS effect allows to define the characteristic bands of RZ, RS and DHRS, as well as, the dynamic monitoring of the interconversion between them. High sensitivity of SERS effect displays a key role in the detection of different compounds in the same sample. For instance, Raman spectra simultaneously recorded to the cyclic voltammetry in RS solution show that the intensity of RS bands decreases concomitantly to the intensity of DHRS bands increases. In addition, if the initial solution only contains RZ, its Raman bands do not totally disappear during the first reduction process because RZ is not completely transformed in RS, and both species are spectroelectrochemically detected. Furthermore, the evolution of representative bands shows that DHRS is generated during the second reduction step. Fluorescence spectroelectrochemistry shows the important effect of oxygen present in the solution. However, a cyclic voltammetry of more than one cycle avoids fluorescence quenching because oxygen is reduced in the first cycle. Furthermore, a high concentration of RS can overlap the fluorescence signal of RZ. Hence, complementary results are obtained by time-resolved Raman and fluorescence spectroelectrochemistry, allowing us to gain knowledge about this fluorogenic system widely used in clinical and biological applications. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107848. References [1] Otsuka G, Nakae T. Resazurin test paper method for determining the sanitary quality of raw milk. J Dairy Sci 1969;52:2041–4. [2] Erb RE, Ehlers MH. Resazurin reducing time as an indicator of bovine semen fertilizing capacity. J Dairy Sci 1950;33:853–64. [3] Mortimer RJ, Varley TS. Synthesis, characterisation and in situ colorimetry of electrochromic Ruthenium purple thin films. Dyes Pigments 2011;89:169–76. [4] Park T-M. Amperometric determination of hydrogen peroxide by utilizing a sol-gelderived biosensor incorporating an osmium redox polymer as mediator determination. Anal Lett 1999;2719:287–98.

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