Development of a Modular Ratiometric Fluorescent Probe for the Detection of Extracellular Superoxide

Article abstract of DOI:10.1002/chem.201700563

Extracellular detection of endogeneous analytes (e.g., superoxide) can provide important insights into mechanisms of homeostasis and diseases, such as tumorigenesis. A ratiometric probe with a fluorescent reference and an analyte-specific switch-on dye was developed. Detection of ROS in the extracellular milieu was ensured by connecting the two fluorophores with a modular peptide-nucleic-acid-based linker. The ROS-sensing ability was assessed and validated in cell-free assays and in cell culture.

Full text of DOI:10.1002/chem.201700563

A Journal of
Accepted Article
Title: Development of a Modular Ratiometric Fluorescent Probe for the
Detection of Extracellular Superoxide
Authors: Diana Andina, Davide Brambilla, Noah Munzinger, Jasmine
Frei, Cristina Zivko, Jean-Christophe Leroux, and Paola
Luciani
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201700563
Link to VoR: http://dx.doi.org/10.1002/chem.201700563
Supported by

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COMMUNICATION
Development of a Modular Ratiometric Fluorescent Probe for the
Detection of Extracellular Superoxide
[
a]
[a]
[a]
[a]
[a]
Diana Andina, Davide Brambilla, Noah Munzinger, Jasmine Frei, Cristina Zivko, Jean-
[
a]
[a,b]
Christophe Leroux, * and Paola Luciani*
Abstract: Extracellular detection of endogeneous analytes (e.g.
superoxide) can provide important insights into mechanisms of
homeostasis and diseases, such as tumorigenesis. A ratiometric
probe with a fluorescent reference and an analyte-specific switch-on
dye was developed. Detection of ROS in the extracellular milieu was
ensured by connecting the two fluorophores with a modular peptide
nucleic acid-based linker. The ROS-sensing ability was assessed and
validated in cell free assays and in cell culture.
c.[28] Additionally, these probes detect intracellular superoxide and,
●
-
to the best of our knowledge, extracellular detection of O
2
has
not been attempted with ratiometric fluorescent probes. As the
most specific and used technique is still the colorimetric
cytochrome c reduction,[ a fluorescent tool to study extracellular
superoxide specifically can be of great value. Therefore, we
present a new modular, bio-polymeric platform in a ratiometric
probe set-up to sense extracellular superoxide.
28]
In this work, a switch-on probe was made ratiometric by linking a
ROS-sensitive dye to a stably emitting reference fluorophore. The
spacer connecting the imaging units was designed to spatially
separate the two dyes, increase solubility and reduce cellular
uptake. Linking the dyes also eliminates the risk of a possible
uneven tissue distribution of the two molecules, which would
result in an impaired sensing ability of the system. The spacer unit
was covalently linked to the two fluorophores in an initial approach
Oxidative stress is the consequence of an imbalance in the redox
state of the body often originating from a dysregulated immune
response.[1,2] Reactive oxygen species (ROS), such as
●
-
superoxide (O
2 2 2
) or hydrogen peroxide (H O ), have long been
identified as harmful molecules and were regarded as
a
necessary evil. By identifying various signaling functions of ROS,
the interest in studying these transient molecules increased.[3,4]
Distinct roles were ascribed to the different reactive species, that
can even vary depending on their location.[5,6] Extracellular ROS
were shown to have a distinct function in inflammation,[6] wound
healing,[ and to be an important regulator in ROS-mediated
apoptosis in tumor cells.[8] Thus, discriminating between extra-
and intracellular ROS is pivotal to better understand their complex
and diverse functions.
(
Fig. 1a), but was then further improved by introducing a peptide-
based DNA mimic (peptide nucleic acid, PNA) that allowed
handling of the two building blocks separately and increased the
modularity and versatility of the sensor design. The final probe
was assembled by annealing two fluorescently-labelled
complementary strands (Fig. 1b).
First, two model imaging units, a reporter dye and a ROS sensor,
were selected. For the latter, hydrocyanine5 (HCy5) was chosen.
Hydrocyanines are a class of non-fluorescent sensors that were
7]
The methods to detect ROS range from the quantification of
specific biomolecule and lipid oxidation by mass spectrometry to
the utilization of spin trapping, electrochemical, colorimetric,
luminescent and fluorescent approaches.[9–13] Due to the high
spatio-temporal resolution, fluorescent probes represent a unique
tool to detect changes in analyte concentration in real-time using
inexpensive equipment.[14,15] Stimuli-responsive switch-on
sensors are used to enhance the signal-to-noise ratio but also to
ensure specific detection of different ROS.[13,16,17] Their general
drawback is an inherent concentration dependence, which can be
overcome by ratiometric probe designs, in which a change in the
ratio of emission intensities is monitored.[18–20]
●
-
shown to switch-on upon reaction with O
2
and hydroxyl radicals
●
(
OH ) specifically but to be insensitive towards H
2
O
2
(Figs. S1-
[29,30]
2).
Any cyanine dye can be converted into its corresponding
hydrocyanine by a single step reduction with sodium borohydride
NaBH ). This feature favors the introduction of the ROS sensing
(
4
moiety in the last step of the synthesis, which implies that the
reference dye should not be affected by the reducing conditions.
In a preliminary screening several possible reporter fluorophores
4
were exposed to NaBH and their stability was assessed.
Chromis500 (Chromis, Fig. S1b) proved to be the most resistant
candidate towards reducing conditions (Fig. S3) and was selected
as reference dye.
Although selective superoxide detection has been improved
recently,[
13,21–23]
the sensing unit used in ratiometric probe designs
For the initial approach, that covalently linked Cy5 with Chromis,
a spacer containing ten glutamic acid residues (poly(-glutamic
acid), PGA) was synthesized by Fmoc-solid phase peptide
synthesis (SPPS). The model dyes were coupled to each
extremity of the PGA. Cy5, underwent reaction with the N-
terminus directly on the resin, whereas one lysine residue was
introduced at the C-terminus, to later condensate activated
Chromis with the primary amine. The ratiometric probe was
purified and analyzed by HPLC, MS and fluorescence
spectroscopy (SI section 4).
is mostly hydroethidin (HE).[24–27] The reliability of this fluorophore
indeed is plagued by several side reactions, e.g. with cytochrome
[
[
a]
b]
D. Andina, Dr. D. Brambilla, N. Munzinger, J. Frei, C. Zivko,
Prof. Dr. J.-C. Leroux, Prof. Dr. P. Luciani
Swiss Federal Institute of Technology (ETHZ), Department of
Chemistry and Applied Biosciences
Vladimir-Prelog-Weg 1-5/10, 8093 Zurich (Switzerland)
E-Mail: jleroux@ethz.ch
Prof. Dr. P. Luciani
Biologisch-Pharmazeutische Fakultät, Institut für Pharmazie,
Friedrich-Schiller-Universität Jena
Excitation of Chromis at 470 nm resulted in two fluorescence
emission peaks, one at 510 nm for Chromis and the other at 670
nm for Cy5 (Fig. 2a). The peak for Cy5 at 670 nm was attributed
to fluorescence resonance energy transfer (FRET).[31] A FRET
effect was undesired for this system as it only occurs when the
07743 Jena (Germany)
E-Mail: paola.luciani@uni-jena.de
Supporting information for this article is given via a link at the end of
the document.
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COMMUNICATION
Figure 1. General design of the ratiometric sensors a) schematic representation of a ROS-sensor that is covalently linked to the reporter dye, inset: chemical
structures of 1 and 2; b) schematic representation of the modular approach using PNA to combine the building blocks giving 3 and 4, inset: chemical structures of
the building blocks.
ROS sensor is switched-on, considerably complicating the final
signal read-out. Taking into account that FRET efficiency is
inversely proportional to the sixth power of the distance from
donor to acceptor, the inter-fluorophore distance was increased
subsequently. A library of probes with one to six discrete
poly(ethylene glycol10) (PEG10) units was prepared to compare
the effect of spacer length (Fig. 1a inset). The PEG10 units were
reduction of FRET was observed with increasing PEG10 units (Fig.
molecule was investigated by adding different equivalents of
NaBH to a solution of 1. The fluorescence intensity of Chromis
4
almost doubled (which could be related to some disaggregation
of the dye), then plateaued and stabilized at higher intensity also
during all further experiments (Figs. 2c and S5). On the contrary,
the Cy5 emission was rapidly suppressed, which confirmed the
successful conversion into the ROS-sensor 2 without interfering
with the spectral properties of the reporter dye.
[
31]
●
-
2a, Table S1). Incorporating six PEG10 units resulted in a
The ROS-sensing properties of 2 were validated generating O
2
negligible FRET effect and, thus, Chromis-PGA-(PEG10
was used in all further experiments.
)
6
-Cy5 1
in a cell-free assay. Two different concentrations (50 and 100 M)
of superoxide anions were produced and variations in the
fluorescence intensity at 10 M were monitored over 2 h (Fig. S6).
After 1 h a 1.9- and 2.3-fold increase of the fluorescence intensity
ratio (ICy5/IChromis) was observed (Fig. 3a). The limit of detection
(LOD) was calculated to be 3.6 M and the ratio at a
physiologically relevant concentration (50 M) showed a low
coefficient of variation (CV, 5%).[ The conjugation of HCy5 with
the polymer reduced its sensitivity towards the detection of
hydroxyl radicals compared to free HCy5 (Fig. S7) in this cell free
assay.[ These findings show that the optimized setup of the
covalently linked ratiometric ROS-sensing probe Chromis-PGA-
The design of 1 with its macromolecular size, negative charges
and hydrophilicity should restrict its internalization by cells. This
working hypothesis was validated by uptake studies with
differentiated Caco-2 cells, a well-established model of the
intestinal epithelial barrier.[32,33] After 1 h incubation of the free
dyes and 1, the cell monolayer was trypsinized and analyzed by
flow cytometry (Fig. S4). A considerably reduced uptake of 1 (Fig.
34]
2b) in comparison with the uncoupled dyes Chromis and Cy5 was
29]
observed, confirming that the probe remains largely extracellular.
In a last step the ROS-sensing ability was introduced by reducing
Cy5 to HCy5. The effect of reducing agent on the ratiometric
6
Figure 2. Characterization of 1 a) effect of spacer lengths on FRET efficiency, ex = 470 nm; b) Chromis-PGA-(PEG10) -Cy5 (1), Cy5 and Chromis uptake by
differentiated Caco-2 cells at 3 M after 1 h at 37 °C (Mean + SD, n=9) *** p≤0.001; c) effect of NaBH on Chromis and Cy5 (10 M, Mean ±SD, n=9).
4
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COMMUNICATION
compared with the free dyes Cy5
and Chromis (Figs. 4a and S11),
confirming that the probe design
still prevents internalization by
cells, although the overall
hydrophilicity of the sensor was
altered by introducing the
relatively hydrophobic PNA
moiety.
To
verify
the
successful
annealing of the two strands and
to show that it is not affected by
the reduction of the cyanine dye,
a native PAGE was used to
analyze the reduced 4 and the
non-reduced sensor 3 (Fig. 4b).
The yellow band, being an
overlay of green (Chromis) and
•
-
2
sensing with a) 2 (n=9) and b) 4 (n=6) in cell-free assays. c) ROS sensing of 4 in cell
Figure 3. ROS-sensing: O
culture (n=9), both 2 and 4 are used at 10 M (Mean + SD) *** p≤0.001, ** p≤0.01, * p≤0.05.
(
PEG10
)
6
-HCy5 stays extracellular and specific superoxide
red (Cy5), confirmed annealing of the modified strands. The lack
of other bands or a band corresponding to Cy5-(PEG ) -PNA
detection is possible (Fig. S8).
1
0 3
With the aim of improving the design, a modular unit that allows
to manipulate each dye separately (e.g. reduction of Cy5) and
combine them as last step of synthesis was introduced (Fig. 1b).
The versatile moiety was implemented in the spacer and
(red band) showed that most of the probe was successfully
hybridized in a 1:1 ratio and no undesired structures are built (e.g.
triplex). Chromis-PNA does not have negative charges and, thus,
did not show any electrophoretic mobility (Fig. S9). The same
position of the green (4) and yellow band (3) indicated that both
imaging probes have similar molecular weights and negative
charges, which proved that the reduction of Cy5 to HCy5 did not
affect the hybridization of the ratiometric PNA probe.
The sensitivity towards superoxide was tested at 50 and 100 M
and revealed a 1.3-fold and 2.2-fold increase, respectively, after
1 h (cprobe = 10 M, Figs. 3b and S12). The CV was low for both
concentrations (4% and 3%) and the LOD was 15.3 M. The
sensitivity towards hydroxyl radicals was assessed but found to
be lower than for superoxide radicals (Fig. S13). As superoxide
radical detection was possible, oxidative stress sensing in vitro
consisted of PNA,
a DNA/RNA analogue in which the
nucleobases are connected to an uncharged polyamide
backbone. Due to the lack of electrostatic repulsion, PNAs form
stronger Watson-Crick base pairing than DNA or RNA, and the
polyamide backbone stabilizes the oligomers in biological media
and to enzyme degradation.[35,36] Due to their robustness PNAs
were applied in many different areas such as FISH probes,[37]
aptamer sensors[38] and bio-barcode systems.[39,40] Exploiting the
complementarity of the nucleobases as a combinatorial unit, the
final ratiometric probe can be assembled by annealing the two
PNA strands, both of them derivatized at the 5´ end either with the
reporter dye or the sensing unit (Fig. 1b).
was
investigated
using
2,2'-azobis(2-amidinopropane)
A lysine residue was added to the sequence of one PNA strand
and activated Chromis underwent reaction with the primary amine
of the lysine in a condensation reaction resulting in PNA-Chromis
dihydrochloride (AAPH) and H O -stimulated Caco-2 cells. AAPH
2
2
showed a 1.4-fold increase after 1 h with a CV of 6%. A steady
increase was observed reaching the same level as H O -
2
2
(
Fig. 1b inset). The other PNA strand was directly modified at the
2 2
stimulated cells after 2 h (Fig. S14). For H O -stimulated cells a
N-terminus on the resin, as PNA synthesis conditions are
compatible with Fmoc-SPPS. A similar synthetic strategy, as
described for the PGA, was used for a modified peptidic unit (Ala-
doubling of the ratio was observed immediately (Fig. 3c) with a
CV of 10%. Overall, the sensing capacity of 4 was as good as the
covalently linked ratiometric probe 2 in the cell-free assays, which
corroborates that the design of modular platform increases the
flexibility of the sensor with equal ROS detection ability.
Additionally, 4 was successfully used to detect extracellular
oxidative stress in cell culture.
x
Glu10-(PEG10) -Cy5). To study the impact of the spacer length on
the FRET efficiency, the number of PEG10 units was varied from
zero to four. The five protected peptidic units were reacted with
the PNA linked to a resin using common coupling conditions at
room temperature for (PEG10
)
0-3 and microwave assisted
Despite the interesting results obtained with our modular
prototype for sensing extracellular superoxide radicals,
derivatization of HCy5 seems to decrease the switch-on ability of
the HCy5 in cell-free assays.[29,30] Indeed, as also reported by two
other groups that modified hydrocyanines for imaging purposes
(derivatizing HCy5 with PEG[41] or coupling them to a nano-
carrier[42]), the detected response towards superoxide and
hydroxyl radicals was lower compared to the original report.[41,42]
All these findings suggest that coupling the (hydro)cyanines to a
macromolecule may influence the fluorescence properties and/or
sensing ability and may account for the reduced sensing capacity
and a high LOD (3.5 and 15.3 M) compared to other examples
conditions at 70 °C for (PEG10 . Annealing of the strands resulted
)
4
in the ratiometric sensor in the unreduced form (Fig. 1b, Fig. S9).
The energy transfer was measured by excitation of Chromis at
470 nm and recording a fluorescence emission scan. The
observed differences between the various spacer length were
small (Fig. S10, Table S2). The presence of PNA led to an overall
reduced FRET effect compared to Chromis-PGA-(PEG10
0 6
) - -Cy5.
As PNA-(PEG10 -Cy5 showed the lowest FRET, it was used in all
)
3
further experiments. The uptake in differentiated Caco-2 cells was
assessed by incubating the free dyes and 3 for 1 h, followed by
flow cytometer analysis. The uptake of 3 was significantly reduced
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