Dye-doped silica nanoparticle probes for fluorescence lifetime imaging of reductive environments in living cells

Article abstract of DOI:10.1039/c6ra21427d

Fluorescence detection sensitivity can be drastically improved by the application of nanoparticles (NPs) because of their superior brightness compared to organic dyes. Here, using dye-doped silica NPs (SiNPs), we developed FRET-based nanoparticle probes for the detection of reductive environments in living cells. To this end, we designed three FRET acceptors based on black hole quenchers (BHQs). Their polarity was tuned by introducing hydroxyl, PEG and sulfate groups. To conjugate them to NPs, we used an original pre-functionalization approach, where the quencher was coupled by a "click" reaction to Pluronic F127 and further used for the preparation of silica NPs. This approach enabled easy preparation of silica NPs functionalized with varying amounts of quenchers by simple mixing of functionalized and parent Pluronic F127 in different mol%. The increase in the quencher concentration at the SiNPs surface produced a rapid drop in the fluorescence intensity with 80% quenching and a 2-fold drop in the emission lifetime for 16 mol% of the quenchers. Then, to obtain turn-ON sensing of reductive environments, the quenchers were coupled to the NPs through a disulfide linker using the same pre-functionalization strategy. The obtained nano-probes showed a >10-fold increase in their fluorescence in the presence of reductive agents, such as tris(2-carboxyethyl)phosphine (TCEP) and glutathione. Remarkably, BHQ quencher bearing sulfate group showed the highest turn-ON response, probably due to its superior capacity to escape from the NP surface after disulfide bond cleavage. The obtained best nanoprobe was successfully applied for detection of reductive environments inside living cells using fluorescence lifetime imaging (FLIM). This work provides insights for FRET acceptor design and its controlled grafting, which enables preparation of the first redox-sensitive silica nanoparticle probe for lifetime imaging.

Full text of DOI:10.1039/c6ra21427d

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Dye-doped silica nanoparticle probes for fluorescence lifetime imaging of reductive environment in
living cells
Luca Petrizza,a,b Mayeul Collot,a,* Ludovic Richert,a Yves Mely,a Luca Prodi,b Andrey S. Klymchenkoa,*
aLaboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de
Pharmacie, 74, Route du Rhin, 67401 ILLKIRCH Cedex, France.
bDipartimento di Chimica “Giacomo Ciamician”, Università degli Studi di Bologna, via Selmi 2, 40126
Bologna, Italy.
*Corresponding authors: andrey.klymchenko@unistra.fr; mayeul.collot@unistra.fr
Abstract
The fluorescence detection sensitivity can be drastically improved by application of nanoparticles (NPs)
because of their superior brightness compared to organic dyes. Here, using dye-doped silica NPs (SiNPs) we
developed FRET-based nanoparticle probes for detection of reductive environment in living cells. To this
end, we designed three FRET acceptors based on black hole quencher (BHQ). Their polarity was tuned by
introducing hydroxyl, PEG and sulfonate groups. To conjugate them to NPs, we used an original pre-
functionalization approach, where the quencher was coupled by “click” reaction to Pluronic F127 and further
used for preparation of silica NPs. This approach enabled easy preparation of silica NPs functionalized with
varied amount of quenchers by simple mixing of functionalized and parent Pluronic F127 in different mol%.
The increase in the quencher concentration at SiNPs surface produced rapid drop in the fluorescence
intensity with 80% quenching and 2-fold drop in the emission lifetime for 16 mol% of the quenchers. Then,
to obtain turn-ON sensing of reductive environment, the quenchers were coupled to the NPs through a
disulfide linker using the same pre-functionalization strategy. The obtained nano-probes showed >10-fold
increase in their fluorescence in the presence of reductive agents, such as tris(2-carboxyethyl)phosphine
(TCEP) and glutathione. Remarkably, BHQ quencher bearing sulfonate group showed the highest turn-ON
response, probably due to the best capacity to escape from NPs surface after disulfide bond cleavage. The
obtained best nanoprobe was successfully applied for detection of reductive environment inside living cells
using fluorescence lifetime imaging (FLIM). This work provides insights for FRET acceptor design and its
controlled grafting, which enabled preparation of the first redox-sensitive silica nanoparticle probe for
lifetime imaging.

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Introduction
Fluorescent probes are essential tools in current biomolecular research.1-3 They enable detection of bioactive
molecules and monitoring biomolecular processes with highest specificity and sensitivity. However, the ever
problem of fluorescent molecular probes is their brightness, due to limited values of their extinction
coefficient and quantum yields.4 Therefore, fluorescent nanoparticles gained recent attention because they
can be 10-100 fold brighter than individual dyes.5-7 So far, a variety of fluorescent NPs has been developed:
15, 16
quantum dots (QDs),8, 9 dye-doped silica NPs (SiNPs),10-13 nanodiamonds,14 conjugated polymer NPs,
dye-doped polymer NPs,7, 17-19 etc. Within this list, dye-loaded silica NPs present particular interest due to the
high brightness and photostability they can reach, combined with the very low toxicity of many of their
formulations. Moreover, their high versatility offered by the possible synthetic strategies and by the
introduction of effective collective photophysical processes18,
makes them a powerful toolbox for
20
addressing many different imaging and sensing applications in the field of nanomedicine.
The most common mechanism in the design of fluorescent probes is Förster Resonance Energy Transfer
FRET.21 It is particularly suitable for molecular probes because dyes are very small entities, which enables
preparation of FRET pairs with sufficiently small inter-fluorophore distances that ensures efficient FRET.22-
24
In contrast, NPs are less efficient FRET donors or acceptors because frequently their size is much larger
than the Förster radius. Thus, earlier works on quantum dots showed that efficient FRET requires ≥10 FRET
acceptors at the periphery of the organic shell,25, 26 though there are examples for high FRET with fewer
acceptors, but located just next to the inorganic core of NPs.27 Dye-doped silica NPs are particularly
promising FRET donors for two reasons. At first, recent protocols that use organic surfactant Pluronic as
template enable preparation of ultrasmall NPs down to 10 nm size.11 Second, at high dye loading energy
hopping within the encapsulated dyes can further improve the efficiency of the energy transfer.20, 28 As we
have demonstrated, this process, due to the energy transfer among chromophores of the same nature (the so-
called “homo-FRET”) plays in fact a prominent role in distributing the excitation energy among several
donor dyes, including the closest ones to the final acceptors.20 In this way, the silica NPs can be employed as
“excitation energy reservoirs”, leading to nanoarchitectures that can be advantageously designed, for
example, as nanosensors for ions,29, 30 pH,31 small bioactive molecules,32, 33 nucleic acids,34 etc as well as
photoswitchable systems,35 large Stokes shift emitters,36, 37 and fluorescent drug delivery systems.38-40
Herein we describe the development of FRET-based nanosensors using rhodamine-doped silica
nanoparticles (SiNPs) linked to amphiphilic BHQ-2 type quenchers through a reducible disulfide (S-S)
group. Disulfide bond has been proven to be a robust cleavable bond in the reductive environment of the
cells.41-43 We varied the polarity of the quenchers by adding different polar heads and studied their
interaction with fluorescent SiNPs as well as their ability to quench their fluorescence by direct adsorption
and by covalent linkage. These nanosensors displayed a fluorescence increase (turn-ON effect) upon
reduction of the disulfide bond by tris(2-carboxyethyl)phosphine (TCEP) or gluthatione that separate the
BHQ quencher from the particles. Moreover, this quenching system showed significant effect on the
fluorescence lifetime of the SiNPs, which allowed us to use this system in Fluorescence Lifetime Imaging
Microscopy (FLIM) experiments in live cells.

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Results and discussion
Design of the nanoprobes. We designed a system where a fluorescent SiNP bears covalently linked
quenchers through a disulfide bond. Upon reduction of this bond, the quenchers separate from the particle
and provoke a fluorescence increase as the particle recovers its initial fluorescence. As a particle we used
previously reported rhodamine B-doped SiNPs, prepared using Pluronic F127 as a scaffold.36 These NPs
feature ~10 nm core diameter and ~5 nm size PEG shell. In order to achieve an efficient turn-ON of these
SiNPs, the quencher must fulfil several properties. First, the overlap between the particle’s emission
spectrum and the absorbance spectrum of the quencher must be sufficient to insure an appropriate quenching
effect. Therefore, Black Hole Quencher-2 (BHQ-2) was chosen as FRET acceptor. Then, the quencher
should present an intermediate polarity, so that it could locate inside the PEG shell and thus close to the
fluorescent core of the SiNPs to ensure efficient FRET. On another hand, once the disulfide bond get
cleaved, the quencher moiety must be sufficiently hydrophilic so that it can escape from the particle in order
to recover the fluorescence of SiNPs providing an optimal turn-ON effect. However, BHQ quenchers are
non-charged apolar molecules and therefore have a lipophilic nature that could be detrimental for their
escape from SiNPs. We consequently designed and synthesised three different functionalizable and
amphiphilic quenchers: BHQ-OH, BHQ-SO3 and BHQ-PEG that bear, in addition to a “clickable” alkyne
group, different polar heads, i.e., hydroxyl, sulphate and methoxy-PEG8 groups, respectively (Figure 1).
At the first step of the synthesis of BHQ-OH, N-(2-Hydroxyethyl)aniline was reacted with propargyl
bromide to introduce a clickable group. The obtained aniline was reacted with Fast Black K giving rise to
BHQ-OH (Scheme 1). BHQ-OH was efficiently converted to BHQ-SO3 by sulfation using the SO3.pyridine
complex with a yield of 87%. BHQ-OH was clicked to a methoxy PEG8 moiety to afford compound 3. The
latter was then converted into an active carbonate 4, which was coupled to propargylamine to obtain BHQ-
PEG (Scheme 1).
Figure 1. Structure of the 3 amphiphilic functionalizable BHQs. Polar heads are highlighted in red, the
functionalizable site is highlighted in blue.

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Scheme 1. Synthesis of the 3 “clickable” BHQs : i. propargyl bromide, NaHCO3, DMSO ; ii. Fast Black K
salt, dioxane/water; iii. SO3.NEt3, DMF; iv. SOCl2, pyridine, CHCl3, NaN3, DMF (2 steps); v. 2,
CuSO4.5H2O, AscNa, dioxane/water; vi. 4-Nitrophenyl chloroformate, pyridine, DCM; vii. Propargylamine,
DIEA, DMF.
Model FRET experiments. These amphiphilic quenchers were first used to study their ability to interact
non-specifically with the surface of SiNPs. Increasing amounts of BHQs were added to an aqueous solution
of rhodamine B doped SiNPs and the fluorescence spectra were recorded. It was found that the quenching
efficiency was dependent on the structure of the quencher (Fig. 2). At a ratio of two BHQ molecules per
SiNP (10 rhodamine B dyes per SiNP, calculated as previously reported36), BHQ-OH, BHQ-PEG and BHQ-
SO3 quenched 30, 40 and 55% of the SiNP fluorescence, respectively. At 10 quenchers per NP, nearly 80%
of the SiNP fluorescence was quenched for all three quenchers. Thus, all three quenchers displayed capacity
to bind SiNPs non-specifically, though this behaviour depended slightly on the nature of the polar group.
Figure 2. Effect of the adsorption of the 3 BHQs on the SiNPs’ fluorescence intensity. (excitation
wavelength= 530 nm) Concentration of SiNP = 200 nM.
FRET to covalently grafted quenchers. In a second step, the quenching ability of these amphiphilic BHQs,
being covalently attached to the surface of SiNPs, was investigated. In one approach, rhodamine B doped
SiNPs bearing an azide group at the terminal end of the Pluronic F127 (Pluronic) were prepared.44 The
functionalization of the SiNPs with the clickable quenchers by CuAAC “click” chemistry was not efficient,
as we could not see significant differences between the samples with and without Cu-catalyst. This could be
explained by the poor efficiency of the “click” reaction at low concentrations in the presence of oxygen.
Higher efficiency would require the use of a special chelated Cu(I) catalyst.45 In a second approach,
Pluronic-N3 was directly functionalised with the quenchers by CuAAC click chemistry prior to the
formulation of SiNPs. The reaction proceeded smoothly and provided pure bi-functionalised Pluronic F127

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(Scheme 2) according to NMR. Then, the modified Pluronic polymer was used to prepare SiNPs using the
same protocol. Previously, it was estimated that a single SiNP was constituted of ~50 Pluronic polymers,46, 47
which corresponded to ~100 functionalizable sites per particle. Therefore, SiNPs bearing 0, 2, 4, 8 and 16
BHQs at their surface were readily obtained by mixing Pluronic and Pluronic-BHQ in appropriate ratios
prior to formulation, i.e. 0, 2, 4, 8 and 16% of Pluronic-BHQ in total Pluronic, respectively. Dynamic light
scattering (DLS) measurements showed that the Pluronic-BHQ molecules did not affect the size (24 nm) and
the low polydispersity value (typically <0.1) of the obtained SiNPs. This important result shows that
Pluronic modified with quencher can be readily used as a building block to prepare functionalized SiNPs
with preserved size characteristics.
Table 1. Hydrodynamic diameter and polydispersity of the different SiNP as measured by DLS.
NPs
BHQ
(%)
0
Diameter
(nm)
22
Polydispersity
(PDI)
0.02
SiNPs
SiNPs-N3
0
22
23
25
36
24
24
25
24
29
26
0.1
0.01
0.1
SiNPs-BHQ-OH
16
16
32
16
16
32
16
16
32
SiNPs-SS-BHQ-OH
SiNPs-SS-BHQ-OH
SiNPs-BHQ-PEG
SiNPs-SS-BHQ-PEG
SiNPs-SS-BHQ-PEG
SiNPs-BHQ-SO3
SiNPs-SS-BHQ-SO3
SiNPs-SS-BHQ-SO3
0.28
0.01
0.03
0.06
0.01
0.25
0.3
Scheme 2. Two different approaches for the preparation of functionalised SiNPs by CuAAC click chemistry.
i. MsCl, NEt3, DCM ; ii. NaN3, CH3CN.
The increase in the fraction of Pluronic-BHQ during particle synthesis resulted in the gradual increase in the
absorbance of the obtained SiNPs as well as broadening of the absorption band with appearance of a
shoulder above 600 nm (Fig. 3A). These spectral changes correspond to the increased contribution of the

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absorption of the BHQ quenchers, which confirms their successful grafting to SiNPs. Fluorescence
measurements of the obtained SiNPs-BHQs showed that an increase in the number of BHQ molecules per
particle leads to a gradual decrease of the fluorescence intensity (Fig. 3B,C). Despite their different polar
groups, the quenching efficiency was similar for all three quenchers, so that 16 BHQ molecules per particle
quenched more than 80% of SiNPs fluorescence in all cases (Fig. 3C). The average fluorescence lifetime
also showed a gradual decrease upon an increase in the number of BHQ quenchers (Fig. 3D), though this
effect was slightly stronger for BHQ-OH. Overall, the lifetime decreased from ~3.7 ns in the parent SiNPs
up to 1.9-2.1 ns in SiNPs bearing 16 quenchers per particle. It should be noted that the decrease in the
lifetime is smaller than the decrease in the fluorescence intensity, namely, <2-fold and ~5-fold, respectively.
This discrepancy indicates that FRET is probably not the only mechanism of quenching of SiNPs by BHQ.
We expect that other mechanisms (electron transfer and excitonic interactions) could produce dark species
that decrease the fluorescence intensity without detectable change in the lifetime measurements. Moreover,
we cannot exclude a non-homogeneous distribution of dyes and/or quenchers in SiNPs, which could also
affect differently fluorescence intensity and the lifetime. Clearly, these experiments show that NPs could be
efficiently quenched by a relatively small number of BHQ quenchers and this process can be followed by
both intensity and lifetime measurements.
Figure 3. Spectroscopic properties of SiNPs with grafted BHQ quenchers. Absorption spectra (A) and
emission spectra (B) of SiNP-BHQ-SO3 (200 nM) with various amounts of BHQ per particle. Excitation
wavelength was 530 nm. (C-D) Quenching effect of the covalently grafted BHQ quenchers on the
fluorescence intensity (C) and the average lifetime (D) of SiNPs.
Preparation of nanoprobes and their validation in solution. Encouraged by the developed synthetic
methodology and the quenching efficiency of the new amphiphilic BHQs, we prepared a fluorogenic
nanoprobe. The new BHQs quenchers were attached to the terminal PEG chains of the Pluronic F127

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through a linker containing a disulfide bond. For this purpose, bromopropanol was converted into alcohol
azide 5, which was activated by carbonyldiimidazole 6. The latter was coupled to cystamine to give an
amino-azide linker 7. Pluronic F127 was converted to a di-active carbamate 8 that was subsequently reacted
with 7 to afford the Pluronic azide derivative 9. The latter was clicked to the 3 different amphiphilic BHQs
(Scheme 3). The reducible Pluronic-quencher conjugates were then mixed with Pluronic F127 in ratios 4/21
and 8/17 prior to the formulation to obtain SiNPs with respectively 16 and 32 BHQs at their surface. These
ratios were chosen to insure an efficient quenching and therefore an enhanced turn-ON effect upon
reduction.
Scheme 3. Synthesis of the reducible SiNPs nanosensors. i. NaN3, DMF ; ii. Carbonyldiimidazole, EtOAc.
Iii. Cystamine, DCM ; iv. 4-Nitrophenyl chloroformate, pyridine, DCM; v. DIEA, CH3CN; vi. BHQ,
CuSO4.5H2O, AscNa, dioxane/water. vii. Pluronic F127, Rhodamine B-Si(OEt)3, AcOH, TEOS, TMSCl.
As expected, the fluorescence intensity decreased many-fold for all three quenchers and the effect was
particularly strong with 32% of quenchers per particle (Fig. S1). To evaluate the ability of these nano-
constructs to sense a reductive environment, they were incubated with 20 mM TCEP (pH was adjusted to 7.4
by sodium hydroxide) or 40 mM glutathione and their fluorescence intensity was monitored over one hour.
For all quenchers used at 32%, TCEP produced a strong increase in the fluorescence intensity on the time
scale of 1h (Fig. 4A). The fluorescence increase was lower for SiNPs with lower % of the quencher (16%)
and no increase was observed for SiNPs without grafted quencher (Fig. 4B, S2 and S3). Addition of
glutathione also produced fluorescence increase when the quencher was grafted to SiNPs, though the
fluorescence intensity increased slower, probably because gluthatione is a weaker reducing agent than TCEP
(Fig. S4). Thus, for SiNPs with 32% BHQ-SO3 in presence of 40 mM glutathione, the fluorescence intensity
reached a plateau only after 10 h with a 12-fold enhancement (Fig. S5), which is close to the value obtained
in the presence of TCEP in 1 hour (Fig. 4A). The fluorescence intensity of the nanosensors (with S-S bond)
in the absence of reducing agent was nearly invariant for at least 20 min, which demonstrated their stability
in the phosphate buffer medium (Fig. S6). An invariant fluorescence over time was also observed for BHQ-
free SiNPs in the presence of glutathione, thus, excluding a direct effect of the reducing agent on SiNPs (Fig.
S7). Finally, in our control SiNPs-BHQ-OH without reducible linker, no fluorescence increase was observed
in the presence of glutathione (Fig. S8). Taken together, these results suggest that the reducing agents cleave
the S-S bonds, which leads to the liberation of the quenchers and thus to the increase in the SiNPs
fluorescence.
The three nanosensors (at 32% of grafting) displayed significantly different turn-ON effect efficiencies upon
reduction as their fluorescence enhancement varied from 3- to 13-fold after one hour in the presence of
TCEP (Fig. 4A) and 1.5 to 3.5-fold after one hour with glutathione (Fig. S4). The following ranking of BHQ
efficiency in terms of turn-ON effect for the nanosensors could be drawn up: BHQ-SO3 > BHQ-OH > BHQ-
PEG. This tendency can be explained by the fact that, in these systems, an efficient turn-ON effect is ensured

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by three synergetic factors: (1) the accessibility of the S-S bond; (2) the capacity of BHQ to quench NPs and
(3) the capacity of the BHQ to escape from SiNPs after S-S bond reduction. For all three quenchers we
expect that the access to S-S bond to the reductive agent should be similar. Moreover, all three quenchers at
high percentage of grafting showed similar quenching efficiency. Therefore, the observed differences are
probably related to the differences in desorption of the quenchers from the SiNPs after S-S bond reduction.
The presence of a negative charge brought by the sulfate group of BHQ-SO3 is likely favorable to the
dissociation of the BHQ from the fluorescent SiNP, that have a negative zeta potential, leading to an efficient
de-quenching of this latter. By contrast, BHQ-PEG and BHQ-OH are non-charged which can explain their
lower efficiency compared to BHQ-SO3 due to less efficient desorption from SiNPs after S-S bond
reduction. More unexpected was the significant difference in efficiencies between BHQ-PEG and BHQ-OH.
Indeed BHQ-OH was shown to be >2 times more efficient than its PEGylated version despite the fact that
the latter bears two triazole moieties as well as a PEG8 which enhance its polarity.
Figure 4. Effect of TCEP with time on the fluorescence of the fluorogenic SiNPs. A) Fluorescence increase
as a function of time of BHQ-SO3 SiNPs (20 nM) in the presence of 20 mM TCEP in phosphate buffer 20
mM pH 7.4. B) Fluorescence increase as a function of time of SiNPs bearing 32% BHQ-OH (red), BHQ-
SO3 (black) and BHQ-PEG (green) (20 nM) in the presence of 20 mM TCEP in phosphate buffer 20 mM pH
7.4. Excitation and emission wavelengths were 530 nm and 580 nm, respectively.
Validation in living cells. The efficient turn-ON effect observed in the SiNP nanoprobes bearing 32% BHQ-
SO3 led us to investigate their ability to sense the reductive environment in living cells. HeLa cells were
incubated for 2 hours in the presence of SiNPs with no quencher and SiNPs with 32% of BHQ-SO3. In both
cases the cells showed intracellular dotted fluorescence, which can be ambiguously assigned to internalized
SiNPs. The internalization of these nanoprobes probably took place by endocytosis, as suggested by the
earlier data using analogous SiNPs.48 As SiNPs with 32% BHQ-SO3 are very poorly fluorescent in the intact
form, the observation of fluorescence inside the cells comparable to that of control NPs without grafted
quencher indicates that the internalized nanoprobes probably turned ON their fluorescence due to the
reductive environment present in cytosol and endosomes of living cells.41, 49 However, as intensity response
cannot provide definite answer regarding the particle turn-ON, we performed Fluorescence Lifetime Imaging
Microscopy (FLIM), which can provide direct information about changes in FRET.50, 51

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Figure 5. Confocal laser scanning microscopy images of SiNP-0%BHQ (A) and SiNP-32%-SS-BHQ-SO3
(B) incubated at 50 nM for 2h in the presence of HeLa cells. SiNPs were excited at 561 nm and were
detected in the 567-700 nm range (red color). The plasma membrane was stained by WGA-AlexaFluor®488
(green). Scale bar is 10 µm.
In our FLIM studies, we first compared the images of suspensions of control SiNPs and SiNPs bearing 32%
of BHQ-SO3 quenchers (Fig. 6). They showed clearly different pseudo-colors (blue and yellow-green), with
average lifetimes around 2.9 and 1.8 ns. Thus, in line with our time-resolved studies (see above), the BHQ-
bearing SiNPs showed significantly shorter lifetime due to FRET quenching process. Then, we incubated
these two types of SiNPs with HeLa cells for 2h. The obtained FLIM images showed strong intracellular
fluorescence in form of dots, indicating good internalization of both types of SiNPs (Fig. 6). However, in the
case of SiNPs bearing BHQ-SO3, the pseudo-color showed clear heterogeneity (Fig. 6D), ranging between
long lifetime of control SiNPs (blue) and the short lifetime of SiNPs with 32% of BHQ-SO3 (yellow-green).
This result suggests that after internalization, some part of the nanoprobes underwent reductive cleavage at
the S-S bond, which produced de-quenched SiNPs with lifetime close to control SiNPs. Thus, FLIM images
validated the response of our nanoprobes to the reductive stimulus in the living cells. Moreover, we showed
that FLIM imaging applied to FRET nanoprobes is complementary to intensity measurements, because
FLIM provides information about FRET efficiency, thus describing the probe response to the reductive
environment.
Figure 6. Intensity (A,B) and FLIM (C,D) images of SiNP-0%BHQ (A,C) and SiNP-32%-SS-BHQ-SO3
(B,D) incubated at 50 nM for 2h in the presence of HeLa cells. Insets of C and D at the top left corner show
the FLIM image of corresponding SiNPs without (SiNP-0%BHQ) and with BHQ quencher (SiNP-32%-SS-
BHQ-SO3) in solution. Scale bar is 10 µm.

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Materials and methods
Milli-Q water (Millipore) was used in all experiments. All starting materials for synthesis were purchased
from Alfa Aesar, Sigma Aldrich or TCI Europe and used as received unless stated otherwise. Synthesis of
the BHQ and the Pluronic F127 derivatives are described in the ESI. NMR spectra were recorded on a
Bruker Avance III 400 MHz spectrometer. Mass spectra were obtained using an Agilent Q-TOF 6520 mass
spectrometer. DLS measurements were performed on a Zetasizer Nano series DTS 1060 (Malvern
Instruments S.A.). Absorption and emission spectra were recorded on a Cary 400 Scan ultraviolet–visible
spectrophotometer (Varian) and a FluoroMax-3 spectrofluorometer (Horiba Jobin Yvon) equipped with a
thermostated cell compartment, respectively. For standard recording of fluorescence spectra, the excitation
wavelength was set to 530 nm and emission was recorded from 540 to 700 nm. The solutions of TCEP and
glutathione were prepared in 20 mM phosphate buffer solutions (pH 7.4) and the pH was readjusted to 7.4.
Cell culture. HeLa cells (ATCC CCL-2) were grown in DMEM (Gibco–Invitrogen), supplemented with
10% fetal bovine serum (Lonza) and 1% antibiotic solution (penicillin–streptomycin, Gibco–Invitrogen) at
37°C in humidified atmosphere containing 5% CO2. Cells were seeded onto a chambered coverglass (IBiDi)
at a density of 5×104 cells per well 24 h before the microscopy measurements. For imaging, the culture
medium was removed and the attached cells were washed with Opti-MEM (Gibco–Invitrogen). Next, a
freshly prepared solution of NPs (50 nM) in Opti-MEM was added to the cells, which were incubated for 2
or 24 h. The medium was then removed and washed 2 times with HBSS and replaced by HBSS for imaging.
Cell plasma membrane staining with wheat-germ agglutinin-Alexa488 (50 nM) was done at room
temperature 10 min before the measurements.
Fluorescence Lifetime measurements in solution. Time-resolved fluorescence measurements were
performed with the time-correlated, single-photon counting technique using the excitation pulses at 480 nm
provided by a pulse-picked frequency doubled Ti-sapphire laser (Tsunami, Spectra Physics) pumped by a
Millenia X laser (Spectra Physics). The emission was collected through a polarizer set at the magic angle and
an 8 nm band-pass monochromator (Jobin-Yvon H10) at 582 nm. The single-photon events were detected
with a micro-channel plate photomultiplier (Hamamatsu) coupled to a pulse pre-amplifier HFAC (Becker-
Hickl GmbH) and recorded on a time correlated single photon counting board SPC-130 (Becker-Hickl
GmbH). The instrumental response function (IRF) was recorded using a polished aluminum reflector, and its
full-width at half-maximum was ~40 ps. Experimentally measured fluorescence decays were deconvoluted
with the instrumental response function and fitted to retrieve the most probable lifetime distribution using the
maximum entropy method (Pulse 5 software).52, 53 In all cases, the
optimal fit.
χ2 values were close to 1, indicating an
Fluorescence Lifetime Imaging Microscopy. Fluorescence microscopy experiments were performed on a
home-built two-photon laser scanning setup based on an Olympus IX70 inverted microscope with an
Olympus 60× 1.2NA water immersion objective.54 Two-photon excitation was provided by a Ti-sapphire
(Tsunami, Spectra Physics) or an Insight DeepSee (Spectra Physics) laser. Imaging was carried out using
two fast galvo-mirrors operating in the descanned fluorescence collection mode. Photons were detected with
an avalanche photodiode (APD SPCM-AQR-14-FC, PerkinElmer) which was connected to a time-correlated
single photon counting (TCSPC) module (SPC830, Becker and Hickl) operating in reversed start−stop mode.
The typical acquisition time was 60 s with an excitation power around 5 mW (830 nm) at the laser output.
Subsequent data analysis using the software SPC Image (Becker and Hickl) allowed us to extract the
fluorescence lifetimes from the decays, and their visualization in a FLIM image, using an arbitrary color
scale.
Conclusions
Preparation of FRET-based sensors using nanoparticles remains a challenge because particle size is generally
larger than the Förster distance. The problem can be solved by using very small NPs and a large number of
quencher moieties. Here, we designed a nanosensor where fluorescent dye-doped silica NPs (SiNP) bear
covalently linked quenchers through a disulfide bond. We prepared ~20 nm silica NPs doped with rhodamine
B dyes and grafted with BHQ-2 type quenchers. It was found that optimal quenching (>10-fold) requires 16
to 32 quenchers at the SiNP surface. Moreover, the amphiphilic nature of the quencher was found to play a
key role in the design of these nanosensors. Time-resolved fluorescence measurements suggested in addition
to the expected FRET mechanism, the presence of dark species due to static quenching. In the presence of

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TCEP or glutathione, SiNPs strongly increase their fluorescence due to the reduction of the disulfide bonds
and separation of the quenchers from the particle. Laser scanning confocal imaging showed that particles can
be internalized in cells. Fluorescence lifetime measurements (FLIM) revealed populations of internalized
NPs with short and long lifetimes corresponding to weakly and strongly reduced SiNPs, respectively. Our
data confirmed that the SiNPs nanoprobes lose their quenchers in response to the intracellular reductive
environment. This work provides insights into design of FRET-based nanoparticle probes and demonstrates
suitability of FLIM for characterization of these nanoprobes in living cells.
Acknowledgements
This work was supported by the European Research Council ERC Consolidator grant BrightSens 648528,
Université de Strasbourg (IdEX 2015, W15RAT68), Global Grant Spinner 2013, Emilia-Romagna region
and by Marco Polo grant, University of Bologna.
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Dye-doped silica nanoparticle probes with rationally designed FRET acceptors enable
fluorescence lifetime imaging of reductive environment in living cells.