Evaluation of Ebselen-azadioxatriangulenium as redox-sensitive fluorescent intracellular probe and as indicator within a planar redox optode

Article abstract of DOI:10.1016/j.dyepig.2019.107866

Redox homeostasis is essential for cellular survival and is therefore tightly regulated. Visualizing changes in the local redox state is essential for detecting oxidative stress, especially with the help of red, long emission lifetime probes. We synthesized a new reversible, redox-sensitive fluorescent probe, ebselen-azadioxatriangulenium (Ebselen-ADOTA), and characterized its potential use for intra- and extracellular redox sensing. The probe consists of a photostable triangulenium-based fluorophore (emission maximum at 564 nm) modulated by a redox-active ebselen motif. The reduced form only shows around 1/10 of the emission intensity of the oxidized form. The Ebselen-ADOTA probe exhibited reversible oxidation and reduction in solution under extracellular conditions. In addition, Ebselen-ADOTA can visualize an increase in oxidative stress within anaerobic bacteria when exposing them to air, the probe did not readily respond to changes in the redox environment inside mammalian cells, probably due to unfavorable localization, aggregation or binding to cysteine containing proteins. When incorporated inside a polymer matrix (hydrogel D4 mixed with poly(acrylic acid)), the probe showed a rapid and fully reversible response to redox changes, thus enabling the preparation of a redox-sensitive optode.

Full text of DOI:10.1016/j.dyepig.2019.107866

Journal Pre-proof
Evaluation of Ebselen-azadioxatriangulenium as redox-sensitive fluorescent
intracellular probe and as indicator within a planar redox optode
Klaus Koren, Nina Katharina Gravesen Salinas, Marco Santella, Maria Moßhammer,
Marie-Caroline Müller, Ruslan I. Dmitriev, Sergey M. Borisov, Michael Kühl, Bo W.
Laursen
PII:
S0143-7208(19)31384-1
DOI:
https://doi.org/10.1016/j.dyepig.2019.107866
DYPI 107866
Reference:
To appear in: Dyes and Pigments
Received Date: 14 June 2019
Revised Date: 5 September 2019
Accepted Date: 6 September 2019
Please cite this article as: Koren K, Gravesen Salinas NK, Santella M, Moßhammer M, Müller M-
C, Dmitriev RI, Borisov SM, Kühl M, Laursen BW, Evaluation of Ebselen-azadioxatriangulenium as
redox-sensitive fluorescent intracellular probe and as indicator within a planar redox optode, Dyes and
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107866.
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Evaluation of Ebselen-Azadioxatriangulenium as
Redox-Sensitive Fluorescent Intracellular Probe and
as Indicator within a Planar Redox Optode
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Klaus Koren , Nina Katharina Gravesen Salinas , Marco Santella , Maria Moßhammer , Marie-
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Caroline Müller , Ruslan I. Dmitriev , Sergey M. Borisov , Michael Kühl and Bo W. Laursen
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a Aarhus University Center for Water Technology, Department of Bioscience – Microbiology, Aarhus
University, 8000 Aarhus, Denmark
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b Nano-Science Center & Department of Chemistry, University of Copenhagen, Copenhagen,
Denmark
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c School of Biochemistry and Cell Biology, University College Cork, College Road,
Cork, T12 K8AF, Ireland
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d Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology,
Stremayrgasse 9, 8010 Graz, Austria
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e Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Helsingør,
Denmark
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f Climate Change Cluster, University of Technology Sydney, Ultimo, New South Wales 2007, Australia
g International Max Planck Research School of Marine Microbiology, Bremen, Germany
* Corresponding authors
Klaus Koren klaus.koren@bios.au.dk
Bo W. Laursen: bwl@chem.ku.dk
Keywords: indicator, redox chemistry, fluorescence, optical sensor, triangulenium
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Abstract:
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Redox homeostasis is essential for cellular survival and is therefore tightly regulated. Visualizing
changes in the local redox state is essential for detecting oxidative stress, especially with the help of
red, long emission lifetime probes. We synthesized a new reversible, redox-sensitive fluorescent
probe, ebselen-azadioxatriangulenium (Ebselen-ADOTA), and characterized its potential use for
intra- and extracellular redox sensing. The probe consists of a photostable triangulenium-based
fluorophore (emission maximum at 564 nm) modulated by a redox-active ebselen motif. The
reduced form only shows around 1/10 of the emission intensity of the oxidized form. The Ebselen-
ADOTA probe exhibited reversible oxidation and reduction in solution under extracellular conditions.
In addition, Ebselen-ADOTA can visualize an increase in oxidative stress within anaerobic bacteria
when exposing them to air, the probe did not readily respond to changes in the redox environment
inside mammalian cells, probably due to unfavorable localization, aggregation or binding to cysteine
containing proteins. When incorporated inside a polymer matrix (hydrogel D4 mixed with
poly(acrylic acid)), the probe showed a rapid and fully reversible response to redox changes, thus
enabling the preparation of a redox-sensitive optode.
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1. Introduction
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Maintaining redox homeostasis is essential for cellular function and survival [1]. While elevated
levels of reactive oxygen (ROS) or nitrogen (RNS) species are considered harmful [2], those species
are also involved in essential signaling pathways within the cell [1]. Therefore, it is important to
understand the redox environment within a cell, which is maintained by several enzymes (e.g.
catalase or superoxide dismutase) as well as antioxidants (e.g. glutathione) [3]. Malfunction of this
cellular machinery and therefore changes in the redox environment have dramatic consequences for
the cell and consequently the organism [2]. Additionally, the extracellular environment can influence
redox homeostasis and act as a source of ROS. Therefore, redox sensing is desired both within cells
and on a larger scale in solutions and polymers surrounding cell aggregates, tissues or biofilms.
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The intra- and extracellular redox environment of cells [4] can be evaluated by measuring the redox
state of certain intrinsic, redox-active substances (e.g. NADH and NADPH [5]) or by measuring the
presence of various reactive species [6,7]. Over the years, many specific molecular probes [8–11] as
well as genetically encoded probes (like HyPer and roGFP [12,13]) were developed for particular
reactive species, such as hydrogen peroxide. In general, most of the developed and commonly used
probes rely on a non-reversible change in optical property (typically an increase in probe
fluorescence), when the probe reacts with ROS [14]. Unfortunately, many of the used probes are not
selective to a specific reactive species and react with several types [15–17]. Frequently, they display
irreversible reaction mechanism, which makes difficult to quantify dynamic and real-time changes in
cellular ROS production [18,19].
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An alternative strategy for redox sensing is to develop reversible probes that measure the redox
balance rather than a specific reactive species [20]. This approach has the advantage that not only
increasing concentrations can be measured, but also dynamic changes in the redox state. So far, only
a limited number of reversible redox probes have been reported [21,22] and these probes have not
been used and tested as extensively as the common reaction-based probes. Reversible redox probes
often contain a dye molecule linked to a redox-sensitive motif such as a near infrared-emitting Cy7
analogue [23] or a naphthalimide [24] coupled to ebselen. Ebselen, or 2-phenyl-1,2-
benzisoselenazol-3(2H)-one, is known to be a redox-active compound with glutathione peroxidase-
like properties [25]. Ebselen can be oxidized by various ROS [26] and reduced by a variety of thiol
compounds [27]. It was shown that Cy-O-Eb can be used to monitor changes in the redox state of
cells. Additionally the recent naphthalimide based probe showed that the formation of seleninic acid
is essential for the increase in fluorescence after oxidation [24].
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In this study we investigated the redox sensitivity of the ebselen group and its potential application
as a reversible fluorescent redox probe for intra- and/or extracellular sensing, by fusing an ebselen
group to a highly photostable azadioxatriangulenium (ADOTA) dye [28–30] (see scheme 1). Besides
high photostability, ADOTA dyes also show an unusually long fluorescence decay time (up to around
20 ns) [31] making them interesting candidates for fluorescence lifetime-based imaging, which e.g.
enables efficient suppression of background fluorescence [32] and multiplexing with other probes
both in the intensity and time domains (FLIM microscopy). The prepared indicator was tested in
solutions as well as in bacterial and mammalian cells, and was also immobilized in a polymer for
construction of a redox optode for extracellular measurements.
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2. Materials and Methods
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If not stated otherwise, chemicals were purchased from Sigma-Aldrich and used as received.
Solvents were of HPLC grade and used as received. Benzene was dried over sodium. 2,2´-
dicarboxydiphenyl diselenide (1) [26], N-(4-Aminophenyl)-azadioxatriangulenium tetrafluoroborate
(Amino-ADOTA, (3) [30]), and the reference compounds N-phenyl-azadioxatriangulenium
tetrafluoroborate (Phenyl-ADOTA) and N-propyl-azadioxatriangulenium tetrafluoroborate (Pr-
ADOTA) were prepared according to literature [29]. Thin layer chromatography (TLC) was carried out
on commercially available plates (silica 60) pre-coated with fluorescence indicator. Chromatographic
purifications were performed on silica gel (SiO ) with a pore-size of 60 Å and a particle size of 40-63
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μm.
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2.1 Spectroscopy
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Mass spectrometry (MS) was performed using matrix-assisted laser-desorption ionization time-of-
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flight (MALDI-TOF) spectrometry using dithranol as a matrix. H and C NMR spectra were acquired
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on a 500 MHz instrument equipped with a (non-inverse) cryoprobe. All chemical shift values in H
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and C NMR spectra are referenced to the residual solvent peak (CD CN δH = 1.94 ppm, δC = 1.32
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ppm). Solvents used for optical spectroscopic measurements were of highest purity grade and used
as received. The UV-VIS absorption spectra were recorded on a Perkin-Elmer Lambda 1050
UV/VIS/NIR double beam spectrometer using the pure solvent as baseline. Fluorescence spectra
were recorded at a Cary Eclipse Fluorescence Spectrophotometer (Agilent) or a FluoTime 300
spectrometer (PicoQuant). All spectra were recorded in 1 cm path length cuvettes using slit widths
of 1 nm and 1 nm steps. Fluorescence lifetimes were determined at a FluoTime 300 spectrometer
using a pulsed laser (510 nm) both from PicoQuant (Germany) and analyzed using the provided
software (FluoFit).
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2.2 Synthesis
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Scheme 1: Synthesis of Ebselen-ADOTA (4)
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2.2.1 Synthesis of 2-(Chloroseleno)benzoyl Chloride:
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2,2´-Dicarboxydiphenyl diselenide (1) (150 mg, 0.38 mmol) was suspended in 6 mL of dry benzene.
100 µL of dimethylformamide (DMF) and 300 µL of SOCl (4.1 mmol) were added to the suspension
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and the resulting mixture was refluxed until the solid compound (1) has reacted quantitatively and a
light yellow solution formed (after approx. 2-3 hours). After removing excess SOCl and solvents
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under vacuum, the prepared 2-(chloroseleno)benzoyl chloride (2) was used for the next step without
further purification.
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2.2.2 Synthesis
of
N-(2-phenyl-1,2-benzisoselenazol-3(2H)-one)-azadioxatriangulenium
(Ebselen-ADOTA):
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Amino-ADOTA (3) (153 mg, 0.29 mmol) was dissolved in 15 mL of acetonitrile. Subsequently 100 µL
of triethylamine (TEA) were added. The entire crude product of (2) obtained in the previous step was
dissolved in 4 mL of acetonitrile and added dropwise to the stirring solution of Amino-ADOTA. The
reaction was carried out at room temperature until no trace of (3) could be observed by TLC (eluent
10% methanol in dichloromethane (DCM)). After completion of the reaction, the mixture was
quenched by pouring the reaction mixture into approx. 800 mL of acidified (0.05M HCl), aqueous
0.2M KPF . The precipitated product was collected by filtration and dried. The crude material was
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purified by column chromatography on silica gel (eluent 10% methanol in DCM). 101 mg of Ebselen-
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ADOTA (5) were obtained (49%). Purity was confirmed via H NMR (500 MHz, Acetonitrile-d3) δ 8.21
– 8.13 (m, 2H), 7.96 – 7.90 (m, 1H), 7.70 – 7.67 (m, 0H), 7.66 – 7.62 (m, 1H), 7.56 – 7.53 (m, 1H), 7.49
– 7.44 (m, 1H), 6.97 (d, J = 8.8 Hz, 1H). and HRMS (m/z calculated: 557.0399 mz/ found: 557.0399)
(spectra in Supporting information figure 1 and 2).
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2.3 Characterization and Testing of Ebselen-ADOTA
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A 1 mM stock solution of Ebselen-ADOTA was prepared in dimethyl sulfoxide (DMSO) or acetonitrile
and used in all experiments. If not stated otherwise, experiments were performed at 37°C in
buffered solutions (PBS or HEPES Buffer) at pH 7.4. Hydrogen peroxide (H O ), glutathione (GSH) or
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ascorbic acid (AA) were used as oxidizing or reducing agents respectively, and were prepared fresh
every day as stock solutions within the buffer. For MS measurements, 10 µM Ebselen-ADOTA was
incubated in Eppendorf tubes at 37°C for 2h within either pure buffer (HEPES at pH 7.4), buffer with
30 µM GSH, or buffer with 200 µM H O .
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Cyclic voltammetry measurements were performed in dry DCM with 2 mM of Ebselen-ADOTA (or
Phenyl-ADOTA as a reference) and 0.05 M tetrabutylammonium perchlorate as the supporting
electrolyte. A glassy carbon electrode was used as the working electrode, and Pt and Ag wires served
as the counter and pseudo reference electrode, respectively. The reference potential was calibrated
versus the ferrocene/ferrocenium (Fc/Fc+) couple. The measurement cell and the solution were
purged with argon prior to the measurements. All measurements were done inside an argon-filled
glovebox (MBraun) using a Biologic SP-300 electrochemical workstation.
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Sensor foils, i.e., planar redox-optodes, were prepared as followed: 1 mg of Ebselen-ADOTA
dissolved in 100 µL of acetonitrile was mixed with 50 mg of poly(acrylic acid) (PAA, MW≈ 100.000,
obtained from Sigma-Aldrich), before addition of 100 mg of the hydrogel D4 (obtained from
AdvanSource Biomaterials) and 2 g of THF. The mixture was stirred until fully dissolved and was then
knife-coated on a dust free PET foil (thickness of 125 µm; Goodfellow) resulting in a ~10 µm thick
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sensor foil after solvent evaporation. The redox-optode was mounted in a custom-built flow-cell and
monitored using a fiber-optic fluorimeter (FireSting-Mini customized for green excitation and
detection of emission >550 nm, using an OG 550 longpass filter (SCHOTT); PyroScience, Aachen,
Germany) equipped with a 1 mm PMMA optical fiber.
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2.4 Cell Culture Studies and Microscopy
2.4.1 Mammalian cell culture studies
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Sterile cell culture grade plasticware was obtained from Sarstedt (Wexford, Ireland). ‘µ-slide
Angiogenesis’ multi-well chambers were purchased from Ibidi GmbH (Martinsried, Germany). Wild-
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type human colorectal carcinoma HCT116 cells (ATCC) and SCO2 knock-out cell line [33] were
handled as described previously [9,34]. Briefly, cells grown in McCoy’s 5A medium, supplemented
with 10% FBS, l-glutamine, penicillin-streptomycin and 10 mM HEPES-Na, pH 7.2, were seeded onto
collagen / poly-D-lysine pre-coated 35 mm dishes. They were allowed to reach 50-75% confluence
and stained with Ebselen-ADOTA (3 µM, 4 h). After staining, cells were washed with growth medium
and used for live fluorescence lifetime imaging microscopy (FLIM). FLIM was performed on an
upright laser scanning Axio Examiner Z1 microscope [34] equipped with 5x/0.25 Zeiss Fluar and
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63x/1.0 W-Plan Apochromat dipping objectives (Carl Zeiss), with integrated T (T=37 C) and Z-axis
control, a pulsed diode BDL-SMNI 488 nm laser, a DCS-120 confocal TCSPC scanner, a photon
counting detector, and operatedby dedicated SPCM and SPCImage software (Becker & Hickl GmbH).
Ebselen-ADOTA was excited with a 488 nm laser (30-50% power set by Galvano Scanner Control)
with emission collected at 565-605 nm. For solution-based measurement, 1 µL of 3 mM dye in DMSO
was placed in the well of ‘µ-slide Angiogenesis’ multi-well chamber (Ibidi), measured using a 5x/0.25
air objective. Subsequently, 10 µL of freshly made glutathione (10 mM, H O) and H O (200 mM,
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H O) were added and measured. Cells were measured using a 63x/1.0 W-Plan Apochromat dipping
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objective in 2 mL of total media volume.
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Fluorescence lifetimes were calculated assuming mono-exponential decay (longer component, T1 =
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essentially as described previously [35].
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2.4.2 Bacterial cell culture studies
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Desulfomicrobium baculatum (Dbac) stock culture was purchased from the Leibniz institute DSMZ.
Dbac was grown in a Tris-buffered chemically defined medium (MOLS4) that consists of 30 mM
Na SO , 60mM Na-lactate, 8mM MgCl , 20mM NH Cl, 2mM K HPO -NaH PO , 30 mM Tris-HCl as well
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as solutions of trace elements, Thauer’s vitamins and rezasurin as an oxygen indicator. The pH was
adjusted to 7.2 with HCl and the bacteria were grown in a 20 mL serum bottle. The bottles were
crimp sealed with butyl rubber stoppers, and oxygen in the headspace was removed by flushing with
pure nitrogen gas. After gassing, individual crimp-sealed medium bottles were sterilized in an
autoclave. All further steps were performed in anoxic conditions. After inoculation, the bacteria
were grown at 30°C for several days. Bacterial staining was performed in a glovebag flushed with N2
gas. 1 ml Dbac culture was stained with 1µl ADOTA-Ebselen (1 mM in DMSO) and incubated for 15
min at room temperature (RT), protected from direct light. After the staining, the cells were
centrifuged, the supernatant was discarded and 900 µL deoxygenated nutrient broth (NB) medium
was added. The cells were resuspended and DAPI staining was performed by the addition of 100 µL
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DAPI (100 µg mL ). The cells were incubated 5 min at RT and afterwards centrifuged and
resuspended in 1 ml deoxygenated NB. For microscopy, approximately 1 ml of stained Dbac
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suspension was taken up in a syringe, which was then transferred out of the anaerobic bag and
quickly applied in small drops on a microscope cover slip. Immediately afterwards microscopy was
performed with a Zeiss Axiovert 200M microscope using a standard Cy5 filter set.
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3. Results and Discussion
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Assessing the redox state within a cell or in the extracellular environment is essential to unravel
dynamics of oxidative stress [20,21]. Useful probes have to be able to compete in terms of reactivity
with the proteins and antioxidants within the cell or environment that ensure redox homeostasis
[3,36,37]. In this study, we synthesized a new redox sensitive probe based on an ebselen moiety and
an ADOTA dye. The synthesis shown in scheme 1 yielded the desired product in high purity (see
Supporting figure 1 and 2) and good overall yield (~50%). The fluorescent properties of this probe
were studied under reducing and oxidizing conditions. Ebselen-ADOTA showed a typical
azadioxatriangulenium absorption spectrum [31] (figure 1A) with its most red-shifted absorption
band at 530 nm. The emission peaks at 564 nm and its intensity was strongly affected by oxidation
or reduction (figure 1B)
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Figure 1: A) Normalized absorption and emission spectra of Ebselen-ADOTA in acetonitrile (10 µM).
B) Emission spectra of Ebselen-ADOTA (10 µM) in HEPES Buffer (pH=7.4) in its oxidized form (excess
of H O ) or reduced form (excess of GSH), excitation at 510 nm.
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Upon reduction, the emission intensity dropped to around 1/10 of its maximum. A typical feature of
ADOTA based dyes is the relatively long fluorescence decay time; in this case, the oxidized form
showed τ = 15 ns (supporting figure S4) in buffered solution, potentially enabling background
fluorescence suppression by time-gating [32,38]. After reduction, the fluorescence decay retained a
contribution from a long (14 ns) component but was clearly dominated by a fast 0.5 ns component
(see supporting figure S5). The fast component was assigned to the quenched fraction and the long
component to an unreacted (oxidized) population. The change in fractional amplitudes and lifetimes
upon reduction was in good agreement with the total intensity drop. Absorption, fluorescence and
fluorescence lifetime measurements of ADOTA without the ebselen moiety were unaffected by the
used reducing and oxidizing conditions (see supporting figure S6), showing that the observed effects
were solely related to the state of the ebselen moiety. The kinetics of reduction and oxidation of
Ebselen-ADOTA were studied in a buffered solution. As seen in figure 2, the fluorescence read-out of
the probe changed reversibly in response to addition of oxidants (H O ) and reductants (GSH). We
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observed that continuous illumination of the probe in its reduced form caused a gradual increase in
fluorescence intensity (figure 2, first 20 min), which was not observed in the absence of continuous
irradiation (see supporting figure S7). We tentatively assign this to a photo-oxidation of the ebselene
moiety. Addition of H O led to a 10-fold increase in fluorescence intensity within a few minutes,
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presumably due to nearly full oxidation of the ebselen moiety. Once fully oxidized, the oxidized form
could be immediately reduced by adding GSH. Once all the GSH was consumed, the signal increased
again due to photo-oxidation (see 30-45 min in figure 2). Further additions of GSH brought the
intensity back to the starting value (reduced form). When fully reduced, an excess of H O was added
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and it took >30 min for the dye to reach its maximum intensity again, in contrast to the previously
observed fast conversions. When DMSO was used as solvent for the stock solution, the oxidation
and reduction reaction occurred at a slower rate compared to when acetonitrile was used.
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Figure 2: Fluorescence time trace of 10 µM Ebselen-ADOTA in HEPES Buffer (pH=7.4) at 37°C upon
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addition of GSH or H O (concentrations as in the graph). Excitation at 510 nm; emission collected at
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565 nm.
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The experimental kinetics indicated that the first reaction with GSH was fastest and that a relatively
stable product formed upon full reduction with GSH. To investigate this in more detail MALDI MS
data from Ebselen-ADOTA exposed to i) pure buffer, ii) 200 µM H O in buffer, or iii) 30 µM GSH in
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buffer were acquired (supporting figure S8). For both the H O -containing and the pure buffer
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sample oxidized forms of the probe were found (molecular mass increased by 16 and/or 32). In
contrast, the sample containing an excess of GSH did not show any oxidized forms. Dimers were
seen mainly in the GSH-containing sample (and traces in the pure sample). This led to the conclusion
that oxidation of the selenium atom [39] within the ebselen motif resulted in inhibition of
quenching, and turn-on of the fluorescence. This is in full agreement with a recent study focusing on
a naphthalimide-based ebselen probe, where seleninic acid was found to be the major reaction
product upon oxidation that leads to a decrease in photo-induced electron-transfer (PET) quenching
[24]. Our measurements also showed that dimer formation can occur in the reduced state [25],
explaining the slow re-oxidation of the probe once fully reduced or dimerized. Broadening of peak of
the reduced form within the absorption spectrum (supporting figure S3) also indicated aggregation
of the reduced form within aqueous environments, but this can be mitigated by increasing the
fraction of organic co-solvent (as done e.g. for the fluorescence lifetime measurements).
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Cyclic voltammetry (supporting figure S9) confirmed that ebselen can undergo a variety of redox
reactions with various ROS and thiols as well as other reducing agents [25,39,40]. In contrast to the
reference Phenyl–ADOTA, the novel redox probe showed a variety of electrochemically irreversible
oxidations and reductions. In a natural environment (e.g. within cells), the oxidation products of
Ebselen-ADOTA can thus be reduced e.g. by GSH and recycled [23,24].
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3.1 Evaluation of Ebselen-ADOTA in Cell Culture
3.1.1 Intracellular Redox Sensing in Mammalian Cell Cultures
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Mammalian cell culture experiments were performed in order to evaluate Ebselen-ADOTA as
potential intracellular redox probe within a complex intracellular environment. Using a previously
described setup [35], we evaluated the performance of Ebselen-ADOTA as prospective redox probe
in FLIM. First, we measured fluorescence lifetimes in solutions with reduced and oxidized forms of
Ebselen-ADOTA (figure 3A-B), respectively. We observed apparent polarity- as well as redox-
dependent changes, as reflected in the observed differences in fluorescence decay kinetics ranging
from ~4.3 to ~ 11 ns. Next, we tested Ebselen-ADOTA with a cancer HCT116 cell line [9,33,34], which
has strong oxidative phosphorylation and displays robust changes to metabolic stimulation. We
found that Ebselen-ADOTA was able to efficiently and quickly cross cell membranes and stained
various intracellular compartments (figure 3C). To discern the effects of redox environment and test
the performance of Ebselen-ADOTA as an intracellular redox probe, we used both the wild-type (WT)
-
/-
and a SCO2 knock-out cell line deficient in oxidative phosphorylation and thus experiencing strong
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/-
oxidative stress and elevated NAD(P)H levels [9,33,34]. WT and SCO2 cells were compared for
fluorescence lifetime changes in Ebselen-ADOTA, both at rest and under stimulated conditions, as
well as upon treatment with glutathione and peroxide. The comparison of lifetime distributions
upon these conditions showed no clear responses to changes in the intracellular redox status and
rather similar intracellular values, in contrast to expected decrease (GSH) and increase (H O ) (figure
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2
3D-E). This could be due to strong solvent-dependent changes in the reaction kinetics and
fluorescence lifetime of Ebselen-ADOTA, also seen in solution-based experiments (figure 3B). This
suggests that after internalization, Ebselen-ADOTA could become bound to lipophilic compartments
such as the mitochondrial membrane or protein domains, which blocks the ability to sense
cytoplasmic redox molecules. This behavior of cell localization and lifetime reduction somewhat
resembles that of the recently reported red-shifted triangulenium derivative CDATA [38] and pH-
sensing DAOTA [41] In addition, aggregation of the probe within the cellular environment cannot be
excluded as a reason for the lack of redox-related response within mammalian cells. Therefore, we
looked if the Ebselen-ADOTA fluorescence lifetime depends on the staining concentration and found
that over the range of 3-10 uM the dye had very similar interactions with intracellular environment
(Fig. S11).
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Figure 3. Evaluation of Ebselen-ADOTA as a probe for extra- and intracellular FLIM-based redox
measurements. A - B: solution-based measurement of fluorescence lifetime in DMSO, and after
addition of excess of GSH (10 mM) and H O (200 mM). A: examples of fluorescence decay curves.
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IRF – instrument response function (laser pulse). Note that the sample was diluted with GSH (10
fold) and H O (20 fold). B: normalized distribution histograms for fluorescence lifetimes at different
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redox states generated in DMSO solution. C-E: evaluation of Ebselen-ADOTA with HCT116 cells. C:
-
/-
typical fluorescence image of stained cells (3 µM, 4 h). D: FLIM images of cells (SCO2 and wild-type)
at rest and after sequential addition of GSH and H O . E: Normalized distribution histogram of
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intracellular fluorescence lifetimes observed during experiment D. Scale bar is in µm.
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3.1.2 Redox Sensing in Anaerobic Bacterial Cultures
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Next, we evaluated Ebselen-ADOTA for redox sensing within bacterial cells. In order to produce
excessive ROS-related stress, the culture of anaerobic gram-negative sulfate reducing bacteria
Desulfomicrobium baculatum (Dbac) was exposed to ambient air, while being monitored on a
fluorescence microscope. As seen in figure 4, a Dbac suspension previously stained with Ebselen-
ADOTA showed a fast increase in fluorescence upon exposure to air. Already after 10 min of air
exposure, the fluorescence signal increased and reached a maximum after around 37 min. This
indicates that the O diffusing into the bacterial suspension changes the redox environment within
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the bacterial cells dramatically; as expected. Thus, Ebselen-ADOTA has the potential to monitor the
redox state within bacterial cells.
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Figure 4: Fluorescence images of Desulfomicrobium baculatum stained with Ebselen-ADOTA (A and
C) and DAPI (B and D) after exposure to ambient air for 0 min (A and B) and 37 min (C and D). E:
Average fluorescence intensity (with standard deviation) of the Ebselen-ADOTA signal within the
bacterial suspension over time.
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3.1.3 Ebselen-ADOTA for intracellular redox sensing and the interplay between extra and intracellular
ROS
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We found that while Ebselen-ADOTA successfully reported on oxidative stress within bacteria, it was
not suitable for experiments with mammalian cells. As several pathways tightly regulate redox
balance within cells, manipulating the intracellular redox state is not trivial. The bacterial experiment
was chosen as a positive control as anaerobic bacteria are prone to be highly sensitive to oxidative
stress. In general, the observed behavior of Ebselen-ADOTA was similar to many other reported
redox probes [42]. We found a slight pH sensitivity of the probe (supporting figure S10) but within
the measured pH range, the fluorescence ratio of the reduced to the oxidized form remained at ~10.
This is similar or even better when compared to many reported redox probes such as commonly
used Hyper fluorescent proteins [43], which emphasizes the importance of parallel monitoring of pH,
with the redox probe.
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Imaging the extra-cellular redox state (e.g. in the growth medium) is an interesting and relatively
unexplored area. As the cell-membrane hinders the diffusion of H O into the cell it is known that
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the applied extracellular H O concentration is around 100 times (depending on the cell and
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environment) higher than the internal [7]. This means that imaging the extracellular H O can give
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some information on the actual intracellular H O . Furthermore, the extracellular redox environment
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2+
3+
affects other redox active substances. Those can be metal ions (e.g. Fe /Fe , with activating pH-
dependent Fenton reaction) or other small molecules[44]. Currently, the extracellular redox
environment can only be assessed with redox-microsensors [45] and two-dimensional redox imaging
with an redox optode has hitherto not been realized.
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3.2 Ebselen-ADOTA Based Redox Optode
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While planar sensor ‘optode’ films exist for a variety of chemical and physical parameters [46],
designated redox optodes are not available. As a proof of concept we prepared the first redox
optode, consisting of the positively charged Ebselen-ADOTA entrapped in a polymer mixture of the
negatively charged poly(acrylic acid) (PAA) and the hydrogel D4. This approach was chosen to reduce
leaching of the probe from the matrix due to electrostatic interactions between the probe and PAA
as shown for other charged indicators [47]. Figure 5A shows a redox sensor film under UV
illumination with a university logo painted on it with a reducing solution (ascorbic acid). We found
this process fully reversible as illustrated by the supporting video (Supporting Video 1).
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We also tested such redox optodes within a flow through system, where the sensor fluorescence
was altered in a controlled way by flushing either reducing or oxidizing solutions through the flow
cell. The optode showed a fast and reversible response to redox changes (figure 5B). Despite
attempts to minimize dye leakage by electrostatic interaction, a constant signal decrease and dye
leakage into the saline buffer solution was still observed. With some further improvements (e.g.
covalent coupling to the matrix), Ebselen-ADOTA can be used as a fast responding reversible optical
redox sensor. Alternatively, the ADOTA fluorophore could be replaced by a diazaoxatriangulenium
(DAOTA) containing a further reactive site or a hydrophobic anchor within the fluorophore for
covalent attachment to the polymer [48] and physical entrapment [41], respectively. Such an
improved sensor material could be put on the tip of an optical fiber resulting in an optical
microsensor to be used for sensing the redox environment in tissues or biofilms [49].
B
A
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Figure 5: A: Planar redox optode containing the Ebselen-ADOTA dye under UV illumination. A sketch
of the University of Copenhagen logo was painted on the planar optode with a reducing ascorbic
acid solution. The oxidized part of the foil remained fluorescent, while the application of reducing
agent promoted fluorescence quenching in selected areas. A video visualizing the reversibility of the
process can be found as supporting information (Supporting Video 1). B: Fluorescence (detected at
>550 nm) of a redox optode mounted in a flow-through system and exposed to various amounts of
oxidant (H O ) and reductant (AA).
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4. Conclusion
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We synthesized and characterized a new reversible fluorescent redox probe based on redox active
ebselen and fluorescent azadioxatriangulenium units. The probe showed promising photophysical
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properties and response to oxidizing (e.g. H O ) and reducing agents (e.g. GSH or AA) in solution. Our
2 2
study of the reaction mechanism confirms that oxidation of the selenium leads to a reduction in PET
efficiency and consequently to increase of fluorescence [24]. The prepared redox probe efficiently
stained both mammalian and bacterial cells. While our experiments showed no clear response to
elevated ROS levels within mammalian cells, stressing anaerobe bacteria by exposing them to O2
showed a clear signal increase indicative of oxidative stress. Furthermore, a first proof-of-concept
redox optode was prepared by mixing the positively charged redox probe with a negatively charged
polymer. With such a planar redox optode, reversible changes in the redox environment can be
visualized in 2D. However, the current optode preparation suffers from dye leaching and needs
further optimization. In summary, we have shown that triangulenium-based dyes can be converted
to redox probes by linking them to a redox sensitive motif. High photostability, long decay times and
high yield synthesis are some of the arguments to further investigate triangulenium based indicators
for sensor development [41,50–52] as well as imaging purposes.
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Acknowledgements:
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We thank Sofie Jakobsen (University of Copenhagen) for technical support; Dr. Eleonore Siham
Mourad and Christian Leypold are thanked for support with CV measurements. André Pellerin
(Aarhus University) is thanked for providing the studied bacteria. This study was supported by a
Sapere-Aude Advanced grant from the Independent Research Fund Denmark (DFF-1323-00065B;
MK), the Danish Council of Independent Research (DFF – 6111-00483), grants by the Independent
Research Fund Denmark | Technical and Production Sciences (DFF-8022-00301B and DFF-4184-
00515B ; KK, MK), a grant by the Poul Due Jensen Foundation (KK) and a grant from the Science
Foundation Ireland 13/SIRG/2144 (RID).
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Highlights
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Synthesis and photophysical characterization of the redox probe Ebselen-ADOTA
Detailed characterization of the reaction kinetics and products
Intracellular redox sensing within bacterial and mammalian cells
Preparation of the first redox optode

Title:
Evaluation of Ebselen-Azadioxatriangulenium as Redox-Sensitive Fluorescent Intracellular Probe and as
Indicator within a Planar Redox Optode
Conflict of interest:
We have no conflicts from any authors of the article.
Corresponding Author:
Klaus Koren