NanoSOSG: A Nanostructured Fluorescent Probe for the Detection of Intracellular Singlet Oxygen

Article abstract of DOI:10.1002/anie.201609050

A biocompatible fluorescent nanoprobe for singlet oxygen (¹O₂) detection in biological systems was designed, synthesized, and characterized, that circumvents many of the limitations of the molecular probe Singlet Oxygen Sensor Green (SOSG). This widely used commercial singlet oxygen probe was covalently linked to a polyacrylamide nanoparticle core using different architectures to optimize the response to ¹O₂. In contrast to its molecular counterpart, the optimum SOSG-based nanoprobe, which we call NanoSOSG, is readily internalized by E. coli cells and does not interact with bovine serum albumin. Furthermore, the spectral characteristics do not change inside cells, and the probe responds to intracellularly generated ¹O₂ with an increase in fluorescence.

Full text of DOI:10.1002/anie.201609050

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Communications
Chemie
International Edition: DOI: 10.1002/anie.201609050
German Edition: DOI: 10.1002/ange.201609050
Sensors
NanoSOSG: a Nanostructured Fluorescent Probe for the Detection of
Intracellular Singlet Oxygen
Rubꢀn Ruiz-Gonzꢁlez+, Roger Bresolꢂ-Obach+, ꢃscar Gulꢂas, Montserrat Agut,
Huguette Savoie, Ross W. Boyle,* Santi Nonell,* and Francesca Giuntini*
In memory of Giulio Jori
Abstract: A biocompatible fluorescent nanoprobe for singlet
oxygen (1O2) detection in biological systems was designed,
synthesized, and characterized, that circumvents many of the
limitations of the molecular probe Singlet Oxygen Sensor
Greenꢄ (SOSG). This widely used commercial singlet oxygen
probe was covalently linked to a polyacrylamide nanoparticle
core using different architectures to optimize the response to
1O2. In contrast to its molecular counterpart, the optimum
SOSG-based nanoprobe, which we call NanoSOSG, is readily
internalized by E. coli cells and does not interact with bovine
serum albumin. Furthermore, the spectral characteristics do
not change inside cells, and the probe responds to intra-
in a very short lifetime[6] and reducing the probability of it
being captured by the probe.
There is an emerging trend of using nanoparticles (NPs)
for drug delivery,[7] sensing,[8] and imaging.[9] However, only
a few reports exist on the use of 1O2 traps associated with NPs.
Among the emerging nanomaterials for biomedical applica-
tions, polyacrylamide has attracted much interest owing to its
biocompatibility and chemical versatility. Successful exam-
ples of its use have recently been published in several fields.[10]
The synthesis of polyacrylamide NPs is straightforward and it
allows tailoring of both the size and functionalization of the
nanospecies to suit the needs of specific applications. The
porosity of polyacrylamide allows analytes (e.g., ions or small
molecules) to diffuse within the NP and interact with the
sensing moiety, thus making it an ideal support for nano-
sensors.[11] Numerous examples of biosensors based on
polyacrylamide NPs have been reported.[12] In this work, we
present results on the synthesis, photochemical behavior and
performance of polyacrylamide-based 1O2-fluorescent nanop-
robes.
1
cellularly generated O2 with an increase in fluorescence.
S
inglet oxygen (1O2) is the lowest excited electronic state of
molecular oxygen and is endowed with atypical but appealing
physicochemical properties and behavior that puts it at the
forefront of research in many different disciplines.[1] Great
efforts have been made in developing techniques and/or
methods that enable not only detection, but also quantifica-
tion of the generation of 1O2. Chemical probes react with 1O2,
leading to changes in a measurable physical property such as
absorption, fluorescence, chemiluminescence, or spin signal.[2]
Most 1O2 probes are highly hydrophobic and thus lack
sufficient water solubility and cell permeability. Although
water-soluble chemical probes exist,[3] they often fail to
produce reliable results inside cells,[4] or present marked
synthetic complexity.[5] Furthermore, a large fraction of the
1O2 produced inside a cell is rapidly quenched, thus resulting
As an initial approach, we developed a sensor in which 1O2
chemical traps were directly attached to the NP, as depicted in
Scheme 1A. Amino-functionalized NPs were covalently
[*] R. Ruiz-Gonzꢀlez,[+] R. Bresolꢁ-Obach,[+] ꢂ. Gulꢁas, M. Agut, S. Nonell
Institut Quꢁmic de Sarriꢃ, Universitat Ramon Llull
Via Augusta 390, 08019 Barcelona (Spain)
E-mail: santi.nonell@iqs.url.edu
H. Savoie, R. W. Boyle, F. Giuntini
Department of Chemistry, University of Hull
Cottingham Road, Kingston upon Hull, HU6 7RX (UK)
E-mail: R.W.Boyle@hull.ac.uk
F. Giuntini
School of Pharmacy and Biomolecular Sciences
Liverpool John Moores University, Liverpool, L3 3AF (UK)
E-mail: F.Giuntini@ljmu.ac.uk
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201609050.
Scheme 1. Conjugation of ADPA or SOSG to functionalized polyacryl-
amide NPs directly (A) or via a spacer (B), and with positively-charged
trimethylphosphonium groups (C).
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bound to Singlet Oxygen Sensor Greenꢀ (SOSG)[3f] through
its most reactive carboxylic group.[4b] SOSG is an anthracene–
fluorescein dyad in which the fluorescence is quenched by
photoinduced intramolecular electron transfer between the
two moieties. Upon anthracene endoperoxidation in the
to the proteins kinetically competing with SOSG for 1O2
molecules. Moreover, they were able to show that SOSG
could in fact be internalized by HeLa cells in protein-free
culture medium.[4b] However, intracellular SOSG shows addi-
1
tional problems that further detract from its use as a O2
1
presence of O2, electron transfer is blocked, thereby restor-
reporter: its fluorescence spectrum is red-shifted compared to
aqueous solutions, intense fluorescence is still observed prior
ing the intrinsic fluorescence of the fluorescein.[4b] According
to the data supplied by the manufacturer, SOSG is a probe
with a high specificity towards singlet oxygen,[13] and there is
no chemical rationale for expecting alteration of its specificity
in the nanoparticle because we linked it through formation of
an amide bond at a site that is rather remote from the reactive
anthracene moiety.[14] Aqueous solutions containing
2 mgmLÀ1 of these nanoprobes and 1 mm of new methylene
blue (NMB) as 1O2 source were irradiated and the probe
fluorescence changes observed over time. The response of the
nanoprobe was poor when compared to the free probe; only
a modest 10% fluorescence increase was observed (Fig-
ure S1C,D in the Supporting Information). Even in deute-
rium oxide (D2O), where the 20-fold longer 1O2 lifetime
facilitates the reaction,[1] the fluorescence enhancement was
below 40%. In order to rule out any effects of light scattering
by the NPs, molecular SOSG solutions were irradiated in the
presence of 1 mm NMB and increasing amounts of NPs
(Figure S2). The results show that for concentrations up to
5 mgmLÀ1, SOSG behaves almost equivalently in the pres-
ence and absence of added free NPs.
1
to O2 exposure, and it is difficult to obtain systematic and
reproducible results.[4b] As shown below, the performance of
the NanoSOSG probe is not affected by such shortcomings.
Interaction with proteins, which strongly affects the
fluorescence of SOSG,[16] causes only marginal fluorescence
quenching in NanoSOSG at the highest BSA concentration
(Figure S3), thus indicating that interaction of the nanoprobe
with proteins is prevented by the NP scaffold. We show in the
Supporting Information that SOSG actually forms 1:1 and 2:1
complexes with BSA, each with distinct spectra and binding
constants (Figure S4). Similar situations are likely to occur in
protein-rich environments such as in cells. This evidence
suggests NanoSOSG to be more suited than molecular SOSG
1
as a fluorescent probe for O2 in biological media.
1
The ability of NanoSOSG to respond to intracellular O2
was then assessed through a series of assays using wild type
(wt) E. coli and a genetically modified E. coli strain that
expresses miniSOG, a flavin-binding fluorescent protein with
1
a strong capacity to sensitize O2 production inside cells.[17]
Figure 1 presents the behavior of the free SOSG probe in
both types of cells.
The poor performance of this early nanoprobe was
ascribed to non-specific interactions occurring between the
probe and the polyacrylamide matrix, leading to decreased
reactivity of the SOSG molecule bound to the polymer
network. In addition, it is worth noting that the initial
fluorescence of the SOSG-NPs was substantially higher than
that of free SOSG, which suggests that the SOSG micro-
environment in the nanoprobe impairs efficient electron-
transfer quenching.
Figure 1A shows that the fluorescence spectrum of SOSG
in the presence of wt E. coli cells matches that in PBS, thus
indicating that it is not bound to proteins. The effects of
irradiation at 420 Æ 20 nm (see Figure S5 for details) are
shown in Figures 1B,C. In wt cells, the fluorescence of SOSG
increases linearly as a result of its well-known self-sensitized
photooxidation.[3d] In miniSOG-expressing cells, the rate of
fluorescence increase shows two distinct regions: up to 15 min
irradiation, the fluorescence increases at a rate similar to that
observed in wt cells, whereas after that, the rate increases by
approximately 2.5-fold compared to the wt cells. The viability
of wt cells is not compromised by irradiation of SOSG,
whereas cells expressing miniSOG are killed very effectively,
with more than 90% being killed after just 15 min (Fig-
ure 1C). Taken together, these results indicate that SOSG and
miniSOG are not in close proximity at the early stages of
irradiation, that is, SOSG is not internalized by E. coli cells.
The fluorescence increase observed in both cells at these early
times is due solely to SOSG self-sensitized 1O2 production in
the aqueous medium and is therefore independent of intra-
To address this problem, spacers of different lengths were
introduced to separate the polyacrylamide scaffold and the
probe. To this end, alkyne-functionalized polyacrylamide
particles were prepared to enable orthogonal copper-cata-
lyzed azide–alkyne cycloaddition (CuAAC) coupling to the
amino-azide-functionalized linkers.[15] Following CuAAC, the
SOSG probe was attached to the free end of the linker
through amide bond formation (Scheme 1B). Linkers of
different lengths were tested (S, M, and L). The inclusion of
linkers improved the performance of the nanoprobes. The
1
best O2 trapping efficiency was observed for the nanoprobe
with the medium-sized linker (M, 7.1 ꢁ), which showed
a fluorescence enhancement of up to 3.2-fold in D2O after
50 min irradiation. This nanoprobe (henceforth called Nano-
SOSG) was selected for further experiments. Its surface was
further functionalized with cationic groups to facilitate
cellular uptake (Scheme 1C).
1
1
cellular O2. In miniSOG-expressing cells, intracellular O2
damages the cell from the inside,[18] and eventually miniSOG
is released to the external medium where it enhances the rate
of SOSG photooxidation. An alternative explanation, such as
photochemical internalization of SOSG,[19] can be ruled out
because no spectral changes are observed after irradiation
(Figure 1A).
SOSG is described as cell-impermeant,[13] which detracts
1
from its usefulness as an O2 probe in cells. Gollmer et al.
concluded that this property is the result of extensive protein
binding in the culture medium. Indeed the presence of bovine
serum albumin (BSA) resulted in red-shifted fluorescence
and lower responsiveness to 1O2, which the authors attributed
The corresponding results for NanoSOSG are also shown
in Figure 1. As expected, NanoSOSG does not interact with
cell proteins either before or after extensive irradiation
(Figure 1D). The rate of fluorescence increase in wt and
2
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molecular probe SOSG. NanoSOSG is successfully internal-
ized by E. coli cells, does not show appreciable toxicity in the
dark, and correctly responds to intracellularly generated 1O2.
This “nano” approach has thus proven useful to extend the
utility of an existing and valuable fluorescent probe to
complex biological systems.
Acknowledgements
Financial support for this research was obtained from the
Spanish Ministerio de Economꢂa
y
Competitividad
(CTQ2010-20870-C03-01 and CTQ2013-48767-C3-1-R).
R.B-O thank the Generalitat de Catalunya (DURSI) and
the European Social Fund for predoctoral fellowships (2015
FI_B 00315). R.R.-G. thanks the Spanish Ministerio de
Educaciꢃn Cultura y Deporte for a “Beca de movilidad
para estudiantes en programas de Doctorado con Menciꢃn
hacia la excelencia” fellowship. OG thanks IQS for his
predoctoral fellowship. FG and RWB thank EPSRC for
funding (EP/H000151/1).
Figure 1. Comparison of SOSG (left panels) and NanoSOSG (right
panels). A) Fluorescence spectra of SOSG in PBS, in E. coli before and
after irradiation, and in the presence of 200 mm BSA. B) Fluorescence
enhancement of SOSG in wt (black) and miniSOG-expressing (red) E.
coli cells as a function of the irradiation time (lexc =420Æ20 nm).
C) Photoinactivation of wt (black) and miniSOG-expressing (red) E.
coli cells pre-incubated with SOSG as a function of the irradiation time
(lexc =420Æ20 nm). D) Fluorescence spectra of NanoSOSG in PBS, in
E. coli before and after irradiation, and in the presence of 200 mm BSA.
E) Fluorescence enhancement of NanoSOSG in wt (black) and mini-
SOG-expressing (red) E. coli cells as a function of irradiation time
(lexc =420Æ20 nm). F) Photoinactivation of wt (black) and miniSOG-
expressing (red) E. coli cells pre-incubated with NanoSOSG as
a function of irradiation time.
Conflict of interest
The authors declare no conflict of interest.
Keywords: fluorescent probes · intracellular sensors ·
nanoparticles · optical sensors · singlet oxygen
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Manuscript received: September 15, 2016
Revised: November 11, 2016
Final Article published: && &&, &&&&
4
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Communications
Sensors
R. Ruiz-Gonzꢁlez, R. Bresolꢂ-Obach,
ꢃ. Gulꢂas, M. Agut, H. Savoie,
R. W. Boyle,* S. Nonell,*
F. Giuntini*
&&&& — &&&&
NanoSOSG: a Nanostructured
Fluorescent Probe for the Detection of
Intracellular Singlet Oxygen
Small particles, big improvement: When
bound to nanoparticles (gray sphere), the
cell-impermeable fluorescent probe Sin-
glet Oxygen Sensor Green (SOSG; green
circles) escapes protein sequestration,
readily undergoes cell uptake, and effi-
ciently reports on intracellular variations
in intracellular singlet oxygen levels.
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