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Time resolved spectroscopy of infrared emitting

Cite this: Nanoscale, 2017, 9, 2505
Ag2S nanocrystals for subcutaneous thermometry
H. D. A. Santos,a D. Ruiz,b G. Lifante,c C. Jacinto,*a B. H. Juarezb,d and D. Jaque*c

We report a systematic investigation on the temperature dependence of fluorescence decay dynamics of

Received 1st November 2016,
Accepted 9th January 2017
DOI: 10.1039/c6nr08534b
rsc.li/nanoscale
infrared emitting colloidal Ag2S nanocrystals (NCs) with different surface coatings. The drastic lifetime
reduction in the biological temperature range (20–50 °C) makes Ag2S NCs outstanding candidates for
high sensitivity subcutaneous lifetime-based thermal sensing in the second biological window
(1000–1400 nm). Indeed, the lifetime thermal sensitivity of Ag2S NCs has been found to be as large as
3–4% °C−1 at an operating wavelength of 1250 nm. Their application for lifetime-based luminescence
nanothermometry has been demonstrated through simple ex vivo experiments specially designed to
elucidate the magnitude of subcutaneous thermal gradients. Experimental data were found to be in
excellent agreement with numerical simulations.

A. Introduction circulation times in the cardiovascular system.4,7,15–17 Indeed,

The research on new approaches for real three dimensional
anatomical in vivo imaging has boosted interest in new infra-
red emitting nanomaterials. In particular, nanomaterials pre-
senting strong luminescence in the so-called second biological
window (II-BW, extending from 1000 to 1400 nm) have been
demonstrated to be particularly promising. In this spectral
range light is weakly absorbed and scattered by tissues thus
allowing for the achievement of large penetration depths.1–3
This, in turn, allows for the acquisition of highly penetrating
and high resolution in vivo luminescence images of organs
and vessels at the small animal level.4–7 Different nano-
materials have been proposed as efficient luminescent probes
operating in the II-BW including rare earth doped nano-
particles (RE:NPs),8,9 carbon nanotubes (CNTs),5,10,11 PbS
based semiconductor nanocrystals (Quantum Dots, QDs),12
and, even, organic dyes.13,14 Among all of them, Ag2S nano-
crystals (Ag2S NCs) have emerged as being particularly promis-
ing due to their unique combination of outstanding properties
such as high quantum yield, absence of heavy metals, high
physical and chemical stability, emission band centered in the
II-BW, low toxicity and, with suitable surface decoration, long
a
Grupo de Fotônica e Fluidos Complexos, Instituto de Física, Universidade Federal
de Alagoas, 57072-970 Maceió, Alagoas, Brazil. E-mail: cjacinto@fis.ufal.br
b
IMDEA Nanoscience, Faraday 9, Campus Cantoblanco, 28049 Madrid, Spain
c
Fluorescence Imaging Group, Departamento de Fisica de Materiales, Facultad de
Ciencias, Universidad Autónoma de Madrid, Madrid 28049, Spain.
E-mail: daniel.jaque@uam.es
d
Applied Physical-Chemistry Department, Faculty of Science, Universidad Autónoma
de Madrid, Madrid 28049, Spain
Ag2S NCs have been already used for in vivo high resolution
anatomical imaging of small animals, for in vivo tumor target-
ing and, even, for arthrosclerosis imaging.18 Despite the excel-
lent results obtained so far in Ag2S NC based in vivo
bioimaging, the physical mechanisms governing their lumine-
scence have still not been fully understood. When the litera-
ture describing Ag2S NCs is reviewed in detail, it is possible to
find several discrepancies about fundamental questions,
for example, the mechanisms affecting the shape and position
of the emission band or the possible presence of quantum
confinement.15,19–22 In particular, fluorescence decay
dynamics of Ag2S NCs have not yet been explored in detail
because the presence of non-radiative transitions, their poss-
ible correlation with surface states and the existence of
environment induced quenching mechanisms are still to be
investigated. In addition, the temperature dependence of fluo-
rescence decay dynamics in Ag2S NCs would provide infor-
mation about their potential application for lifetime based
luminescence nanothermometry (LT-LNTh).23,24 LT-LNTh has
been validated in different visible emitting semiconductor
nanocrystals,25 but its demonstration in infrared emitting Ag2S
NCs would constitute a new approach for in vivo sub-tissue
contactless thermal sensing, which is in demand for the devel-
opment of minimally invasive thermal therapies as well as for
the design of novel diagnosis tools. Nevertheless, despite
being interesting from both fundamental and applied points
of view, a systematic study of the fluorescence decay dynamics
of Ag2S NCs is still pending.
In this work, the fluorescence decay dynamics of Ag2S NCs
have been systematically investigated. The influence that the
interaction between Ag2S NCs and the environment has on

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decay dynamics is elucidated by comparing the fluorescence
decay curves of colloidal Ag2S NCs in organic and aqueous
media. Quantum confinement effects are explored by compar-
ing the luminescence decay curves obtained at different emis-
sion wavelengths. The temperature dependence of fluo-
rescence decay curves is also reported, and the presence of
temperature induced quenching mechanisms is discussed.
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Finally, the potential use of Ag2S NCs for sub-tissue LT-LNTh
is demonstrated on the basis of representative ex vivo experi-
ments and numerical simulations.
B. Experimental
B.1. Chemicals
Silver(I) diethyldithiocarbamate (AgDDTC, 99%), 1-dodecane-
thiol (DDT, >98%), 1-octadecene (ODE, 90%), poly(ethylene
glycol) methyl ether thiol (PEG-SH, average Mn 6000),
tetrachloroethylene (TCE, >99), dichloromethane (DCE, 99.8%)
and toluene (99.8%) were purchased from Sigma-Aldrich.
Absolute methanol (99.8%) was purchased from Scharlau.
B.2. Synthesis of Ag2S nanocrystals
In this work, the synthesis of Ag2S NCs is based on the
thermal decomposition of silver(I) diethyldithiocarbamate in
the presence of 1-dodecanethiol. The syntheses have been per-
formed with standard Schlenk lines using a temperature con-
troller connected to a PC unit for accurate and reproducible
results. For each synthesis, 0.05 mmol of silver(I) diethyl-
dithiocarbamate, 12.5 mmol of 1-dodecanethiol, and 9 mmol
of 1-octadecene were mixed in a three-neck round bottom flask
and degassed under vacuum at room temperature. The flask is
then backfilled with nitrogen and heated up to 200 °C at a rate
of 17 °C min−1. The solution is stirred for one hour at 200 °C
and then cooled down using a water bath. After the synthesis,
the black colloidal dispersion is washed in several cycles of
precipitation with methanol and re-dispersed in toluene.
B.3. Ligand exchange
In order to obtain water-soluble Ag2S NCs, the hydrophobic
DDT ligands are exchanged with PEG-SH. In a typical pro-
cedure, a toluene dispersion of the NCs is precipitated with
methanol and centrifuged. The supernatant is discarded and a
1 mg ml−1 solution of PEG-SH in dichloromethane is added
onto the NC deposit. The mixture is sonicated at room tempera-
ture for an hour. After that, the NCs are isolated and transferred
to water. The water dispersions present good colloidal stability.
B.4. Luminescence characterization
The luminescence generated by the solution was recorded
using the Fluorimeter NanoLog™ (HORIBA) with double-
grating excitation and two emission monochromators. The
software used in this case was the Fluorescence. The detector
used was a liquid-N2 cooled R5509-73 photomultiplier tube,
operating in the 300–1700 nm range. Luminescence decay
curves were obtained by exciting the colloidal suspensions of
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Ag2S NCs by using a pulsed diode laser (HORIBA, model
DeltaDiode DD-450L) operating at 450 ± 10 nm and providing
pulses of 80 ps duration with a maximum repetition rate of
100 MHz. The fluorescence time decay curve was finally
obtained with time-domain TCSPC using the Datastation soft-
ware. For the measurement of decay curves at different temp-
eratures, the sample was placed on a controlled temperature
stage that was able to tune the sample’s temperature between
20 and 60 °C with 1 °C resolution.
The peak intensity of the 450 nm excitation laser was calcu-
lated to be 100 W cm−2, which is well below the laser intensity
damage threshold (estimated to be 2 × 107 W cm−2).26
For continuous wave excitation a fiber coupled 808 nm laser
(LIMO20-F200-DL808) was used as the excitation source. The
numerical aperture of the fiber was 0.22 and the power density
was set to be below 1 W cm−2.
Surface temperature was acquired with an infrared thermal
camera (FLIR E40).
B.5. Numerical simulations
The simulations of the temperature maps were obtained by
solving the heat equation in two-dimensions for non-homo-
geneous media with internal heat generation, using a numeri-
cal scheme based on finite differences. Dirichlet boundary
conditions were used at the computational edges, except at the
material–air interface, where Newton’s law was applied.
Thermal conductivity (κ), density (ρ) and specific heat capacity
(c) for the substrate (s) and tissue (t) were considered as: κs =
1.05 W (K m)−1,27 ρs = 2200 kg m−3, cs = 700 J (K kg)−1, κt =
0.412 W (K m)−1, ρt = 1120 kg m−3, ct = 4340 J (K kg)−1.28,29 For
numerical simulation purposes, the computational window
was divided with the grid size of Δx = Δy = 0.02 mm, where
propagation time steps of Δt = 0.005 s give good convergence.
C. Results and discussion
C.1. Analysis of room temperature decay curves: decay rates
and quantum confinement
Fig. 1(a) and (b) show the TEM images corresponding to Ag2S
NCs coated with PEG-SH and DDT ligands that make them dis-
persible in nonpolar and polar solvents, respectively. The size
histograms obtained in each case are shown in Fig. 1(c) and
(d), respectively. Average sizes were found to be 4.7 ± 0.7 and
4.3 ± 0.7 nm for the Ag2S NCs coated with PEG-SH and DDT
ligands, respectively. The observed size difference could be
due to the presence of some unreacted precursor or the encap-
sulation of the NCs by the big PEG-SH molecules that could
interact with the DDT existing ligands instead of completely
removing them, thus slightly increasing their size. The
observed size change due to ligand exchange (0.4 nm) is,
nevertheless, within the uncertainty of size determination so it
can be considered as negligible. In both cases TEM images
reveal spherical Ag2S NCs that showed excellent colloidal pro-
perties without signs of precipitation over months (Fig. 1(e)).
Although similar synthetic procedures have been used to
obtain Ag2S NCs, ongoing studies suggest the presence of an
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Fig. 1 TEM images of Ag2S nanocrystals coated with PEG-SH (a) and DDT (b) ligands that make them colloidal in water and toluene, respectively.
The size histograms obtained in each case are shown in (c) and (d) for PEG-SH and DDT coatings, respectively (bars are experimental data and the
solid line is the best fit to a Gaussian distribution). (e) Optical photograph of a colloidal solution of Ag2S nanocrystals in water. (f ) Fluorescence
image of the solution included in (e). Excitation and emission wavelengths were set to 808 and 1200 nm, respectively.

inner core of silver. Under 808 nm excitation, colloidal

solutions showed a bright infrared luminescence centered at
1200 nm (see Fig. 1(f )).
Despite that the colloidal solutions of Ag2S NCs in water
and toluene showed similar emission spectra, their room
temperature fluorescence decay curves are remarkably
different (Fig. 2(a)). Moreover, DDT coated Ag2S NCs dispersed
in toluene showed a single exponential decay (see fits in
Fig. 3). This, according to previous work, may suggest that in
this case, surface related nonradiative de-excitations
(via surface defects or environment assisted multiphonon
relaxations) are not taking place.30 Thus, the observed single
exponential decay obtained for DDT coated Ag2S NCs dis-
persed in toluene would reveal the single contribution of
“bulk” relaxations. Under this assumption we can state that
the room temperature “intrinsic” bulk fluorescence lifetime of
DDT coated Ag2S NCs is close to 180 ns, i.e. τDDTB (RT ) ≈ 180 ns.
The total (radiative plus nonradiative) bulk room temperature
decay probability of DDT coated Ag2S NCs is, therefore, calcu-
−1
lated to be WDDT
B (RT ) = (τDDT
B (RT )) ≈ 5.5 × 106 s−1.
At variance, PEG-SH coated Ag2S NCs dispersed in water
showed an evident nonexponential decay. In this case, the
fluorescence decay curve can be nicely fitted to a double expo-
nential decay curve, an approach widely assumed by authors
in previous studies dealing with fluorescence dynamics of Fig. 2 Room temperature luminescence decay curves of Ag2S nano-
semiconductor nanostructures.31,32 The experimentally crystals in toluene and water. (b) Wavelength dependence of the lumine-
scence lifetime of Ag2S nanocrystals in toluene and water. Symbols are
obtained decay curves of our Ag2S nanocrystals suspended in
experimental data and colored rectangles are just guide for the eye to
water have been fitted to: evidence the wavelength independent value of lifetime. The room temp-
erature emission spectrum of Ag2S nanocrystals is also shown (solid red
em ðtÞ ¼ AB expðt=τ B Þ þ AS expðt=τS Þ
I PEG ð1Þ
PEG PEG
line).

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Fig. 3 Luminescence decay curves at 22 and 48 °C obtained for col-
loidal solutions of DDT and PEG-SH coated Ag2S nanocrystals dispersed
in toluene and water, respectively. Symbols are experimental data and
solid lines are the best fits to single and double exponential functions.
where AB and AS are the amplitude components of slow and
fast decay components, respectively. In expression (1) τPEG
B and
τPEG
S are the fluorescence lifetimes of the slow and fast decay
components, respectively. By fitting the decay curve, at room
temperature, we experimentally obtain τPEG S = 17 ns and τPEG
B =
140 ns as fast and slow decay times, respectively (see fits in
Fig. 3). The fast and slow lifetime values are found to be sig-
nificantly larger than those reported by W. J. Mir et al. (1 and
11 ns) but comparable to the values found by P. Jiang and co-
workers (10 and 75 ns).20,33 According to previous work, the
fast decay component can be attributed to surface nonradiative
de-excitations due to the presence of defect states, surface
inhomogeneities (both created during the ligand exchange
process), and/or to a strong coupling with the environment
phonons (vibrations of water or PEG-SH surface molecules).34
At the same time, the long fluorescence lifetime component
can be correlated with bulk excitonic recombinations. Thus,
we can define, for PEG-SH coated Ag2S NCs dispersed in water,
two characteristic decay times at room temperature, namely
the bulk and surface decay times (τPEG B (RT ) ≅ 140 ns and
τPEG
S (RT ) ≅ 17 ns, respectively). The total bulk radiative prob-
ability of PEG-SH coated Ag2S NCs can be estimated to be
−1
WPEG PEG
B (RT ) = (τB (RT )) = 7 × 106 s−1. The fact that WPEG
B (RT ) ≅
DDT
WB (RT ) evidences that the ligand exchange process did not
affect the bulk recombination dynamics. Therefore, the differ-
ences between fluorescence dynamics of Ag2S NCs in toluene
and water can be attributed to surface-related processes. At
this point, it is important to note that the assignment of the
long lifetime (≈ 140 ns) component to the inverse of the bulk
total de-excitation probability in both PEG and DDT coated
Ag2S NCs is in agreement with previous work. Y. Zhang and
co-workers investigated the fluorescence time decay character-
istics of colloidal Ag2S NCs in chloroform with variable sizes
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ranging from 2.2 up to 7 nm and concluded that, as the size
increased, the fluorescence decay curve was dominated by a
long component with a characteristic decay time of 180 ns
which was also attributed to pure bulk electron–hole
recombinations.19
Fig. 2(b) shows the wavelength dependence of the room
temperature fluorescence lifetime of our Ag2S NCs in toluene
and water (circles and squares, respectively). For comparison,
the room temperature emission spectrum is also shown (red
line). In the case of Ag2S NCs in water the average lifetime is
Ð
IðtÞtdt
defined as ˉτ ¼ Ð , where I(t ) is the emitted intensity at
IðtÞdt
time t after excitation. As can be observed, the fluorescence
lifetime remains, within the experimental uncertainty, wave-
length independent. When dealing with semiconductor nano-
structures, the combination of inhomogeneous broadening
(size dispersion) and quantum confinement (size) leads to
emission wavelength dependent fluorescence decay times.35
Therefore, the experimental evidence of a wavelength indepen-
dent fluorescence lifetime in our Ag2S NCs suggests the
absence of quantum confinement. This was, somehow,
expected since the size of our Ag2S NCs (4–5 nm) is significantly
larger than their Bohr radius. This has been experimentally
estimated by Y. Zhang and co-workers to be 2 nm and theoreti-
cally calculated by S. Lin et al. to be as low as 1 nm.19,21 The
absence of quantum confinement in Ag2S NCs is also in agree-
ment with the work published by V. M. Huxter et al. who
demonstrated that 8 nm Ag2S NCs exhibit optical absorption
spectra similar to the corresponding bulk materials.22
At this point, it should be noted that it is also possible to
find different studies in the literature reporting on strongly
wavelength dependent fluorescence lifetimes for Ag2S NCs. For
instance, W. J. Mir et al. reported on a relevant increment of
the fluorescence lifetime with the emission wavelength, which
is associated with a relevant contribution of emitting defects
at short wavelengths.20 Nevertheless, this behavior was cata-
loged by authors as unusual and, in our opinion, reflects the
relevant role that surface properties, strongly dependent on a
particular synthesis procedure, plays in the fluorescence
dynamics of Ag2S NCs. The determinant role of the surface in
luminescence properties of Ag2S NCs has also been recently
evidenced by R. Gui et al. who demonstrated that slight vari-
ations in the surface decoration could lead to large variations
in the fluorescence quantum yields.36 The strong relationship
between the luminescence properties of Ag2S NCs and capping
agents at their surface was also stated by M. C. Brelle and co-
workers. They claimed the existence of “deep traps” in Ag2S
NCs whose relaxation dynamics after laser excitation were
strongly dependent on capping materials.37
C.2. Temperature dependence of decay curves
Fig. 3 shows the fluorescence decay curves of DDT and PEG-SH
coated Ag2S NCs obtained at two different temperatures
(22 and 48 °C). Fluorescence decay curves were found to be, in
both cases, strongly temperature dependent. Indeed, a relative
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small temperature increment of 30 °C has led to a relevant
(70%) shortening of the decay times. In the case of Ag2S NCs
dispersed in toluene, the temperature induced reduction takes
place while maintaining the single exponential character.
Thus, following the same arguments described in the previous
section, this temperature induced lifetime reduction can be
correlated with the activation of multiphonon assisted recom-
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bination. Fig. 3 also reveals that for DDT coated colloidal Ag2S
NCs dispersed in toluene, temperature increments in the bio-
logical range (20–50 °C) do not lead to the activation of elec-
tronic excitations via neither surface effects nor phononic
coupling with molecules in contact with the surface of NCs.
The temperature dependence of the bulk fluorescence decay
time of DDT coated Ag2S NCs in toluene (τDDTB (T )) is shown in
Fig. 4(a) from which a remarkable temperature induced
reduction is observed from 180 down to 60 ns when the temp-
erature is increased from 20 to 50 °C. At variance with other
semiconductor nanosystems (such as CdTe QDs) the tempera-
Fig. 4 (a) Temperature dependence of “bulk” (τDDTB ) fluorescence life-
times of Ag2S nanocrystals in toluene and temperature dependence of
both “bulk” (τPEG
B ) and “surface” (τS ) fluorescence lifetimes of Ag2S
PEG
nanocrystals in water. (b) Temperature dependence of the relative con-
tribution of “bulk” and “surface” transitions to the net fluorescence
decay curves of Ag2S nanocrystals in water (AB and AS, respectively).
(c) Temperature dependence of the AB/AS ratio as calculated from data
included in (b). Symbols are experimental data and solid lines are guides
for the eye.
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ture induced reduction in the fluorescence lifetime does not
follow a linear trend.25 This, as it will be demonstrated in the
next section, leads to temperature independent thermal sensi-
tivities in the surroundings of room temperature.
When the fluorescence decay curves of PEG-SH coated Ag2S
NCs are analyzed (see Fig. 3) we found that at any temperature
the decay curves could be well described by a double exponen-
tial decay curve. By fitting to double exponential decay profiles
obtained at each temperature, it is possible to access the temp-
erature dependence of both bulk and surface fluorescence
decay times (τPEG B (T ) and τS (T ), respectively). Results are
PEG
shown in Fig. 4(a). Different conclusions can be extracted from
these data. First, the bulk related fluorescence decay time
strongly reduces with temperature, following almost the same
trend as that obtained for DDT coated Ag2S NCs in toluene.
This fact reinforces the assumption that in colloidal Ag2S NCs
the long lifetime component can be associated with bulk elec-
tronic relaxations that, in turn, are strongly favored by the
thermal activation of multiphonon transitions. At the same
time, experimental data shown in Fig. 4(a) reveal a remarkable
temperature induced reduction in the fluorescence lifetime
associated surface recombinations (from 17 down to 6 ns
when temperature is increased from 20 up to 50 °C). This can
be associated with the thermal activation of multiphonon
assisted recombination processes taking place at the surface.
We have also determined the temperature evolution of the con-
tributions of bulk and surface transitions to the net fluo-
rescence of PEG coated Ag2S NCs (AB and AS amplitudes in
expression (1), respectively). Results are shown in Fig. 4(b)
from which it is evident that the relative contributions of both
processes can be considered, in a first order approximation,
temperature independent, with surface transitions being
dominant in the whole temperature range analyzed in this
work. When the temperature dependence of the ratio AB/AS is
calculated in detail, it is observed that the temperature rising
favours surface electronic recombinations, as evidenced in
Fig. 4(c). Experimental data reveal a decrease in the relative
contribution of bulk with respect to surface recombinations
close to 30% when temperature is increased from 20 up to
50 °C. This can be explained in terms of a temperature assisted
energy migration from bulk to surface states, a mechanism
already invoked to explain the size dependence of quantum
efficiency in visible emitting QDs.38
C.3. Lifetime based fluorescence thermometry
As mentioned in the introduction, one of the most interesting
applications of fluorescence nanostructures with a strongly
temperature dependent fluorescence lifetime is their potential
application for remote thermal sensing by lifetime based
luminescence nanothermometry.23,24 The ability of any given
nanostructure for lifetime based luminescence nanothermo-
metry depends on its particular relative thermal sensitivity,
SR, that is defined as:24
1 dτðTÞ
SR ðTÞ ¼ ð2Þ
τðTÞ dT
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where τ(T ) is the fluorescence lifetime of the fluorescence
dτðTÞ
nanostructure at temperature T and is the temperature
dT
derivative of τ(T ). In our case, SR(T ) can be calculated from the
experimental data shown in Fig. 4(a). The thermal sensitivities
obtained for both DDT and PEG-SH coated Ag2S NCs are
shown in Fig. 5(a). It has been found that both DDT and
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PEG-SH coated Ag2S NCs provide very similar values of SR(T )
in the whole temperature range investigated here. This is an
outstanding point as it clearly evidences the robustness and
reliability of thermal measurements performed on the basis of
the analysis of the Ag2S NCs fluorescence lifetime, since their
sensitivity does not critically depend on their surface pro-
perties. This would be a great advantage over other lumine-
scence nanothermometers whose thermal sensitivity has been
found strongly dependent on the environment and surface
properties.24,39,40 In addition, data shown in Fig. 5(a) reveal
Fig. 5 (a) Temperature dependence of the relative lifetime based
thermal sensitivity of both DDT and PEG coated Ag2S nanocrystals.
Symbols are the experimental data obtained from the analysis of data
included in Fig. 4(a) and solid lines are guides for the eye. (b) Lifetime
based thermal sensitivity of different luminescent nanothermometers
and their spectral operating ranges.25,41,42,48–52
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that Ag2S NCs provide a lifetime based thermal sensitivity that
depends only slightly on temperature in the 20–42 °C range. In
this temperature range Ag2S NCs provide a lifetime thermal
sensitivity as large as 3–4% °C−1 that is above most of the life-
time thermal sensitivities previously reported in the literature
(see the comparative graph in Fig. 5(b)). Only polymeric nano-
structures have provided a larger lifetime thermal sensitivity
but with the drawbacks of a much reduced temperature
operation range (30–36 °C instead of 20–50 °C for our
Ag2S NCs).41,42
The clear advantage of Ag2S NCs over the already developed
LT-LNThs is their spectral operating range. From Fig. 5(b) it is
clear that Ag2S NCs are the only LT-LNThs that operate in the
second biological window and, thus, they are the only
LT-LNThs potentially capable of subtissue lifetime based
thermal sensing. In order to provide a proof of concept of the
potential application of our Ag2S NCs for subtissue lifetime
based thermal sensing, a simple experiment was designed (see
schematic representation in Fig. 6(a)). These experiments were
designed and conducted with the final objective of determin-
ing the magnitude of the differences between inner and
surface temperatures of a tissue undergoing localized heating.
This is an open question of great relevance especially when
dealing with photothermal therapies of subcutaneous tumors.
In these cases detailed knowledge of the relationship between
the skin and inner tumor’s temperature is essential in order to
understand heat dissipation in treated tumors as well as to
achieve easy control over therapy by simple surface (skin)
thermal measurements.43 As schematically indicated in
Fig. 6(a), in our case the point heating source is constituted by
a high resistive Kanthal wire (0.2 mm radius) that is placed
under chicken breast tissue. This wire was connected to a
voltage supply in order to control the magnitude of heating.
The surface temperature of the tissue was measured by using
an infrared thermal camera (FLIR E40). In order to simul-
taneously detect the subtissue temperature (Tst, which is the
temperature of the resistive wire) a thin film of Ag2S NCs was
placed between the heating wire and the tissue. This thin film
provides the same thermal sensitivity of Ag2S NCs dispersed in
either water or toluene (results not shown for the sake of
brevity). Optical excitation of the sensing film was provided by
using an 80 ps 450 nm pulse diode laser. The time decay curve
of the Ag2S NCs in the film was obtained by using the same
experimental set-up described in Section B. Fig. 6(b) and (c)
show optical images of the system constituted by the heating
wire and sensing film with and without a 3 mm thick tissue.
Fig. 6(d) shows the infrared thermal images obtained when a
tissue of thickness equal to 1, 3 and 5 mm was placed on top
of the heating wire (images were obtained when an electrical
current of 0.6 A was established). From these thermal images
we have extracted the maximum surface temperature (Tsf ) for
different tissue thicknesses. As can be observed in Fig. 6(e),
Tsf decreases monotonically with the tissue thickness as it was
indeed expected due to the increment in the thermal impe-
dance. For each particular tissue thickness the subtissue temp-
erature (wire’s temperature) was obtained by analyzing the
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Fig. 6 (a) Schematic representation of the ex vivo experiments designed and conducted to demonstrate the ability of Ag2S NCs for lifetime based
subtissue thermal sensing. (b) Optical image of the thin film of Ag2S NCs used for subtissue lifetime thermal sensing. The heating wire is also shown.
(c) Optical picture of the thin film and wire under a 3 mm thick tissue. (d) Infrared thermal images (surface thermal images) obtained for three tissue
thicknesses. Wire’s current was set to 0.6 A. (e) Dependence of both surface and subtissue temperatures as a function of the tissue thickness.
Symbols are experimental data and dashed lines are guides for the eye. (f ) Numerical simulations of the cross sectional temperature patterns
obtained by assuming the tissue thickness of 0.5 and 5 mm. (g) Relative temperature difference between surface and subtissue temperature incre-
ments as a function of the tissue thickness. Symbols are data obtained from experimental measurements included in (e) whereas the solid line is
obtained from numerical simulations.

fluorescence decay curves of the Ag2S NC thin film. The vari-
ation of this subtissue temperature (Tst) with the tissue thick-
ness is also shown in Fig. 6(e). At variance with the surface
temperature, Tst has been found to be almost independent of
the tissue thickness. Fig. 6(g) shows the relative difference
between the subtissue and surface temperature (ΔTR = (Tst −
Tsf )/ΔTst, where ΔTst is the absolute increment in the subtissue
temperature) as a function of tissue thickness as calculated
from the data shown in Fig. 6(e). Note that ΔTR becomes more
relevant as the tissue thickness is increased. Indeed, for a
tissue thickness of 5 mm, we have found ΔTR ≈ 80%, so that
for such a thickness the “apparent” surface temperature incre-
ment is only 20% of the actual subtissue temperature incre-
ment. This evidences the critical importance of achieving sub-
tissue thermal reading in bio-applications where surface
measurements (achieved by using infrared thermal cameras)
could lead to a drastic underestimation of actual inner temp-
erature. The existence of large differences between subtissue
and surface temperatures has been previously evidenced in
in vivo experiments. For instance, during photothermal thera-
pies of cancer tumors these temperature differences have been
reported to be as large as 57%, 40%, and 20% for thermal
therapies based on carbon coated nanoparticles, quantum
dots and rare earth doped nanoparticles, respectively.43–45
Thus, in vivo experimental data reveal, on average, a discre-
pancy between surface and intratumoral temperatures close to
40% for a subtissue depth close to 2 mm (which is the average
tumor radius used in animal models). This is, indeed, in good
accordance with the experimental data shown in Fig. 6(e) that
predict, for this thickness, a relative temperature difference of
50%. In order to provide further support to our experimental
measurements we have conducted numerical simulations of
the temperature patterns created in the tissue by the heating
wire. Fig. 6(f ) shows two characteristic thermal patterns calcu-
lated by assuming a tissue thickness of 0.5 and 5 mm, respect-
ively. From these simulations the existence of relevant temp-
erature differences between the surface and heating source
(wire, in our case) are evidenced, which are increased with the
tissue thickness. By performing simulations with different
tissue thicknesses we were able to compute the dependence of
ΔTR on the tissue thickness. Results obtained from numerical
simulations are shown as the solid line in Fig. 6(g) from which

This journal is © The Royal Society of Chemistry 2017 Nanoscale, 2017, 9, 2505–2513 | 2511

Paper
an excellent agreement between experimental data and simu-
lations is obtained, which demonstrates the suitability of Ag2S
NCs for subtissue lifetime thermal sensing. Furthermore,
temperature induced lifetime reduction is accompanied by a
remarkable intensity reduction as well as by a substantial
change is the spectra shape, which makes Ag2S NCs very prom-
ising candidates for fast and high precision multiparameter
Published on 11 January 2017. Downloaded by Universidad Autonoma de Madrid on 04/10/2017 12:44:43.
temperature sensing via neuron network reconstruction, a
possibility that has already been demonstrated based on other
semiconductor nanocrystals.46,47
Finally, we should note that the use of pulsed laser sources
with longer wavelengths instead of a 450 nm laser used here
would result in an improved penetration depth. For instance, a
subtissue penetration length of 808 nm excited Ag2S has been
experimentally found in our laboratory to be more than two
times larger than those reported here under 450 nm laser exci-
tation. Nevertheless, the use of the 450 nm laser (lying out of
the biological windows) does not affect the validity of the main
conclusion of this work, which is the great potential of Ag2S
NCs for lifetime based thermometry.
D. Conclusions
In summary, we report a systematic investigation of the
fluorescence dynamics of colloidal Ag2S NCs. It has been
found that decay curves are strongly dependent on the coup-
ling with the surrounding medium. Ag2S NCs stable in water
(PEG coated) have been found to follow faster dynamics with
respect to those NCs dispersed in toluene (DDT coated) due to
the activation of surface related de-excitation processes,
while keeping the bulk recombination dynamics unaltered.
Fluorescence decay curves obtained at different emission
wavelengths have suggested the absence of quantum
confinement effects for ca. 4 nm diameter Ag2S NCs due to
their significantly larger size in comparison to their Bohr
exciton radius. Fluorescence dynamics have also been found to
be strongly temperature dependent. It is reported here that the
fluorescence decay time associated with bulk transitions
presents a marked decrease in the temperature range of
interest in biomedicine (0–50 °C). We have, indeed, found that
Ag2S NCs are among the most sensitive lifetime based
luminescent nanothermometers with the outstanding
advantage of operating in the second biological window that
makes them particularly promising for biological applications.
As a matter of fact, this work also includes ex vivo experiments
and numerical simulations in which the potential capacity of
Ag2S NCs for subtissue accurate thermal reading is
demonstrated.
We are firmly convinced that the unique combination of
properties of Ag2S NCs (brightness, biocompatibility, stability
and passivation with different capping shells, operating spec-
tral range) together with the results shown here would make
them relevant agents in future for in vivo applications in which
multimodal imaging and multiparameter sensing would be
required.
2512 | Nanoscale, 2017, 9, 2505–2513
View Article Online
Nanoscale
Acknowledgements
This work was supported by different Brazilian Agencies:
FINEP (Financiadora de Estudos e Projetos) through the grants
INFRAPESQ-11 and INFRAPESQ-12; CNPq (Conselho Nacional
de Desenvolvimento Cientıfico e Tecnologico) Grants INCT
NANO(BIO)SIMES and Project Universal nr. 483238/2013-9;
CAPES (Coordenadoria de Aperfeiçoamento de Pessoal de
Ensino Superior) by means of the Project PVE nr. A077/2013,
an International project between Universidad Autónoma de
Madrid and Universidade Federal de Alagoas (Spain-Brazil).
Prof. Dr Daniel Jaque is the PVE (Pesquisador Visitante
Especial) of this project. H. D. A. Santos is supported by a PhD
scholarship from CAPES. This work was also supported by
Comunidad Autónoma de Madrid (S2013/MIT-2740), by the
Spanish Ministry of Economy and Competitiveness (MAT2013-
47395-C4-3-R, FIS2015-67367-C2-1-P, MAT2013-40823-R and
MAT2016-75362-C3-1-R). Diego Ruiz acknowledges the Spanish
Ministry of Economy and Competitiveness for funding by FSE
and IEJ in the frame of the SNGJ.
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