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Graphene Quantum Dots Integrated in Ionophorebased Fluorescent Nanosensors for Na and K +
+
Renjie Wang, Xinfeng Du, Yaotian Wu, Jingying Zhai, and Xiaojiang Xie ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00918 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 4, 2018
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Graphene Quantum Dots Integrated in Ionophore-based Fluorescent Nanosensors for Na+ and K+ Renjie Wang, Xinfeng Du, Yaotian Wu, Jingying Zhai, and Xiaojiang Xie* Department of Chemistry, Southern University of Science and Technology, Shenzhen, 518055, P. R. China Email:
[email protected] Abstract. To enrich the recipes of ion-selective nanosensors, graphene quantum dots (GQDs) were integrated into ionophore-based fluorescent nanosensors with exquisite selectivity and high sensitivity for Na+ and K+. The unique property of GQDs gave the nanosensors ultrasmall size (ca. 10 nm), high brightness, good biocompatibility and potential pH sensing possibility. At pH 7.4, the sensors exhibited a detection range from 0.1 mM to 1 M for Na+ and from 3 µM to 1 mM for K+. The nanosensors were successfully applied to blood serum and urine samples. Chemically induced intracellular sodium concentration change in HeLa cells was also qualitatively monitored. Key words: graphene quantum dots, nanosensors, ion-selective, sodium, potassium Fluorescent ion sensors are known to have high sensitivity and
probes. In addition, diffusion limited response time of the
could provide high spatial and temporal resolution of ion
nanosensors is much faster than some ion-selective biosensors
+
+
concentrations. Among various ions, Na and K are the most
based on nucleic acids.11
common alkali metal ions in the human body fluids. The Na+
Previous
concentration in extracellular fluid could reach ca. 440 mM for
pioneered by the groups of Bakker, Hall, Clark, and
+
Michalska.12-18 The common ground of these nanosensors is the
concentration is typically around 400 mM and 139 mM,
underlying classical ion-selective optode principle.19 However,
respectively.1 The appropriate intracellular and extracellular
the nanosensors have been built on various materials which
invertebrates and ca. 145 mM for vertebrates while cytosolic K
+
+
research
on
ionophore-based
nanosensors
was
help maintain the proper
mostly are optically inert. Apart from a few examples, the
membrane potential which is vital to the regulation of a number
intracellular applications of the nanosensors is also very limited.
concentrations of Na
and K
2
of signal transduction pathways. Imbalance of the electrolytes
A paradigm was demonstrated earlier by Clark and co-workers
concentrations is also related to a number of diseases including
where they successfully utilized plasticized poly (vinyl chloride)
congestive heart failure, kidney malfunction, severe dehydration,
sodium selective nanosensor to record sodium dynamics in
bulimia, and cancer.
3
isolated cardiomyocytes.16 More recently, we reported optical +
nanosensors for Li+, Na+, and K+ based on organosilica
and K+ is quite small.4-5 Commercial synthetic probes for Na+
nanoparticles.20 The crosslinked organosilica particles could be
and K+ such as SBFI and PBFI suffer from short excitation
readily taken into HeLa cells and provided high robustness
However, the palette of fluorescent sensors and probes for Na
wavelength, poor selectivity and sensitivity.
6-7
Over the past few
against dilution and cell metabolism compared with the
decades, different synthetic probes have been reported with
self-assembled nanosensors.
improved characteristics, some also compatible with two-photon
In this work, we present for the first time, ion-selective
microscopy and near infrared spectroscopy.
8-9
Nonetheless,
nanosensors based on graphene quantum dots (GQDs)
small molecular probes still suffer from problems such as
containing highly selective ionophores. While extending the
cellular sequestration and fast leakage after loading. In contrast,
variety of ionophore-based nanosensors, the results also lead to
ion-selective optical nanosensors become advantageous because
an optically active and readily functionalized host material. The
molecular probes are well protected by the matrix host material
surface of GQDs was modified with acetylene groups and
of the nanosensors, preventing the probes from involving in
crosslinked using azide-functionalized poly (ethylene oxide)
10
Nanosensors
through Cu+-catalyzed click reaction. The strongly green
therefore, provides high brightness and stability, and could
fluorescent GQDs was easily traceable under fluorescence
remain in cells for longer time compared with small molecular
microscopy and allowed signal interrogation at multiple
direct metabolic and other interactive processes.
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wavelengths. In recent years, the functionalization of GQDs for
washed 3 times with THF by centrifuging at 7000 rpm for 3 min
chemical sensing has received extensive attention and several
to remove any unreacted molecules. The THF residue after
groups reported graphene-based materials for the detection of
centrifugation was removed using compressed air. The solids
+
2+
3+
- 21-24
Ag , Hg , Fe , and I .
However, GQD-based nanosensors +
for alkali metal ions are very rare. Here, Na -selective and +
K -selective GQD-based nanosensors were prepared by using +
different ionophores. The Na nanosensors exhibited exquisite +
selectivity over K , rendering them an attractive sensing tool for +
were re-suspended in 1 mL deionized water containing 1.2 mg polyoxyethylene bis(azide), 3.2 mg L-ascorbic acid, and 4.2 mg CuSO4·5H2O and shaken at room temperature for 6 h. The products of the reaction were centrifuged at 7000 rpm for 3 min and washed with deionized water for at least 3 times. The
intracellular experiments. Cytosolic Na level change in HeLa
product PEG-GQDs was suspended in 1 mL deionized water
cells
and stored in refrigerator for further use.
induced
by
gramicidin
and
carbonyl
cyanide
3-chlorophenylhydrazone (CCCP) was successfully observed
Nanosensor preparation For Na+-selective nanosensors, 0.5
under fluorescence microscope with the Na+ nansoensors. The
mg Ox R, 1.5 mg TFPB, 2.5 mg NaX, 4.8 mg DOS were
nanosensors were also applied in human urine and blood serum
dissolved in 2.4 mL of methanol to form a homogeneous
samples.
cocktail solution. A volume of 80 µL PEG-GQDs stock solution
EXPERIMENTAL SECTION
and 25 µL of above-mentioned methanol cocktail were mixed and added into 4 mL of Tris-HCl solution (10 mM, pH 7.4) on a
Reagents Graphene quantum dots (GQDs), polyoxyethylene bis(azide) (MW 5000), L-ascorbic acid, copper(II) sulfate pentahydrate, sodium
ionophore
X
(NaX),
potassium
tetrakis-[3,5-bis(trifluoromethyl)-phenyl]
or
borate
sodium (TFPB),
potassium ionophore I (valinomycin), methanol, tetrahydrofuran (THF),
bis(2-ethylhexyl)
sebacate
(DOS),
2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris), citric acid, boric acid and sodium dihydrogen phosphate were purchased from Sigma-Aldrich. Propargyl bromide was purchased from J&K
Scientific
Ltd.
in
China.
synthesized according to literature.25
All
Ox
R
were
solutions
were
prepared by dissolving appropriate salts into deionized water purified by Milli-Q Integral 5. Dulbecco's modified eagle medium (DMEM), heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin solution (100X) were purchased from Corning. Cell Counting Kit-8 (CCK-8) was obtained from MedChem Express in China. Low autofluorescence cell culture medium TransDetect BrightFluore DMEM was purchased from Beijing TransGen Biotech Co., Ltd. Gramicidin was purchased from Shanghai Jingdu Biological Technology Co., Ltd. Carbonyl
cyanide
3-chlorophenylhydrazone
(CCCP)
was
purchased from Alfa Aesar. Urine sample was obtained from a volunteer. Blood serum sample was provided by Dr. Fanxin Zeng from the Department of Clinic Medical Center, Dazhou Central Hospital, China. Modification of GQDs. 1mg GQDs and 100 µL propargyl bromide were added in a flask and ultrasonically dispersed, and then heated to 60 ℃ for 2 h. The resulting precipitates were
vortexer at 1000 rpm. Methanol was removed by blowing compressed air to the surface of the resulting solution for at least 1 h. Similarly, for the preparation of K+-selective nanosensors, a 2.4 mL of methanol cocktail 0.5 mg Ox R, 1.5 mg TFPB, 2.8 mg valinomycin, 4.8 mg DOS were prepared. Instrumentation and Measurements. The morphology and size of the nanosensors were characterized by transmission electron
microscopy
(TEM,
HT-7700,
Hitachi)
operated at an acceleration voltage of 100 kV. To prepare the samples for TEM characterization, a drop of the nanosensor suspension was dropped onto a copper grid with a carbon support film, and dried in air. Infrared spectra were recorded on an FT-IR spectrometer with an ATR accessory (Nicolet iS10, Thermo Scientific) at room temperature. Fluorescence spectra was measured on a fluorescence spectrometer (Fluorolog-3, Horiba Jobin Yvon). Absorption spectra were measured using an ultraviolet−visible (UV-vis) absorption spectrometer (Evolution 220, Thermo Fisher Scientific). The pH response of PEG-GQDs were evaluated in buffer solutions with 2.5 mM of citric acid, boric acid, and NaH2PO4 adjusted to the required pH. The fluorescence response of ion-selective nanosensors with at different ion concentrations was recorded in pH 7.4 Tris-HCl buffer with stepwise addition of stock solutions. The ratio of the fluorescence intensity at 522 nm and 602 nm were used to for sensor calibration curves. Furthermore, K+ concentration in the urine sample was measure after 100x dilution with the abovementioned process by using the K+-selective nanosensors in Tris-HCl solution containing 1 mM sodium background. The K+ level in the urine sample was separately determined using
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ion-selective electrodes in potentiometry. The ion-selective
OH
pH Sensitive Region
Legends:
electrode membrane containing valinomycin as the ionophore 26
was prepared according to the literature.
All measurements
Poly (ethylene oxide) + H+
- H+
Graphene Quantum Dots
O-
Ion-Exchanger (TFPB)
were conducted in triplicated.
Chromoionophore (HInd+, Ind)
Cell culturing and cytotoxicity evaluation. Hela cells were
Na+/K+ Selective Ionophore (L)
seeded
in
DMEM
culture
supplemented
with
10%
heat-inactivated FBS and 1% penicillin-streptomycin, and incubated at 37 ℃ with humidified air containing 5% CO2. Cytotoxicity of the nanosensors was assayed using CCK-8 according to the manual. Briefly, after Hela cells were cultured in a 96-well plate (100 µL, 104 cells per well) for 24 h, different amounts of nanosensor stock solution (10, 30, and 50 µL) were added into experiment group wells and co-incubated for 24 h. The wells without nanosensors were set as the control group. 10 µL of CCK-8 reagents was introduced into each well, and the absorbance at 450 nm was recorded after 2 h on a microplate reader (cytation5, BioTek). Confocal fluorescence imaging. For fluorescence imaging, Hela cells were incubated in confocal dishes with the nanosensors for 3 h, washed carefully 3 times with phosphate buffer (10 mM, pH 7.4). Afterwards, the cells were cultured in TransDetect BrightFluore DMEM with low autofluorescence and imaged using confocal laser scanning microscopy (A1R, Nikon). Laserlines at 488 nm and 561 nm were used as the excitation together with the standard FITC and TRITC filter cubes. Chemically induced cytosolic sodium level change. Hela cells loaded with Na+ nanosensors were firstly cultured in TransDetect BrightFluore DMEM medium. Gramicidin and CCCP was added to the medium to reach final concentrations of 20 µM and 5 µM, respectively. NaCl was also added to the culture medium reach 100 mM concentration. The fluorescence intensities at 522 nm and 602 nm were continuously recorded with the plate reader. Images of the cells before and after perturbation were also captured with Cytation 5 using with colored CCD camera and LED excitation at 469±15 nm and 586±15 nm.
HInd+ TFPB + M+ (aq) L (org)
Ind TFPB + H+ (aq) LM+ (org)
Ion-Selective Region
Figure 1. Schematic illustration of the proposed structure of the ionophore-based nanosensor incorporating GQDs and the mechanism of the optical responses for pH and ions. graphene with the periphery rich in hydroxyl groups and possess strong quantum confinement and edge effects. Therefore, they typically exhibit high dispersity in aqueous environments and their photoluminescence spectra could be tailored by the size, shape, and defects.27 The nanosensors contain pH sensitive regions because of the surface hydroxy groups in the GQDs. It is more challenging, however, to obtain the ion-selective region, which required surface modification with polyethylene oxides. Although GQDs contained a large number of sp2 carbons, they did not possess interface with high enough hydrophobicity to directly adsorb the sensing optode components. In our initial experiments, we attempted to directly mix the sensing ingredients with GQDs. However, the resulting suspension did not show any fluorescence response to sample ion concentration change. To integrate the green fluorescent GQDs into ionophore-based nanosensors, the hydroxyl groups of the GQDs were reacted with propargyl bromide to add carbon-carbon triple bonds to the periphery. The reactant and product of the reaction was compared in Fourier transform infrared spectroscopy (FT-IR). As shown in Figure 2a, the asymmetric carbon-carbon stretching at 2125 cm-1, the C-H stretching at 3300 cm-1, 2930 cm-1, and 2844 cm-1 of the propargyl group confirmed the successful modification. Then, polyoxyethylene bis(azide) was used to crosslink the propargylated GQDs in the presence of Cu+.
After
crosslinking,
the
product
PEG-GQDs
was
centrifuged and washed with water. Note that neither the naked GQDs nor the propargylated GQDs could be collected through
RESULTS AND DISCUSSION
centrifugation in the same conditions, providing another
Figure 1 shows a schematic illustration of the proposed structure
evidence for the crosslinking reaction. An increase in the
of the nanosensors where PEGylated GQDs acted as a host for
hydrodynamic size of the PEG-GQDs nanosensors to around
the ion-selective optode components, i.e., ionophores (L),
150 nm due to aggregation was also observed with dynamic
ion-exchanger (TFPB) and the chromoionophore (Hind+, Ind).
light scattering (DLS, see size distribution and correlation curve
The plasticizer DOS, represented in gray, served to solubilize
in Figure S1). As shown in Figure 2b, the aqueous suspension of
the optode components. GQDs are sheets of few-layered
the PEG-GQDs was also slightly more turbid than the free
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Figure 3. (a) Fluorescence spectra of the Na+ nanosensors with various Na+ concentrations in pH 7.4 Tris-HCl buffer. (b) Selectivity pattern of the Na+ nanosensors. (c) Fluorescence spectra of the K+ nanosensors with various K+ concentrations in Figure 2. (a) FT-IR spectra of free GQDs, the reactant propargyl
pH 7.4 Tris-HCl buffer. (d) Selectivity pattern of the K+
bromide, and the purified product PEG-GQDs. (b) Pictures of
nanosensors.
the aqueous suspension of the free GQDs (1), PEG-QGDs (2),
compounds, leaving some of the hydroxy groups still exposed to
and the nanosensors with Na+ selective components (3). (c)
the aqueous solution (the pH sensitive region in Figure 1).
TEM images of the well separated nanosensors (1) and
The nanosensors were characterized with transmission electron
aggregates (2 and 3) with different magnification.
microscopy (TEM). As shown in Figure 2c, the majority of the
GQDs. The third image of Figure 2b was from a Na+ nanosensor suspension. After the abovementioned modifications, a more amphiphilic interface was obtained for the incorporation of the sensing ingredient. Here, we used the solvent displacement method to prepare the ion-selective nanosensors.28 Briefly, all the sensing components including PEG-GQDs, cation-exchanger (TFPB), chromoionophore, and ionophore were dissolved in methanol and mixed with buffer solutions (see experimental section for detailed preparation). Hydrophobic interactions drive the sensing ingredient into the crosslinked PEG-GQDs as methanol was evaporated, forming the ion-selective region shown in Figure 1. Previously, different surfactants were used to stabilize various types of nanosensors.14,
16
Here, since poly (ethylene
oxide) was already linked to GQDs, no additional surfactant was required. No sedimentation or flocculation was observed for the nanosensor suspension over an observation period of three
resulting Na+ nanosensors produced TEM images as shown in Figure 2c1 with an average diameter of ca. 10 nm, which is slightly bigger than the original GQDs (< 5 nm). Since poly (ethylene oxide) chains could not be seen under TEM, the contrast should come from the few-layered graphene sheets. The result indicates that GQDs in the nanosensors were probably crosslinked between the layers (as shown in Figure 1) due to the stabilizing π-π interaction between the GQD planes. However, in some area, aggregates of several particles as shown in Figure 2c2 and 2c3 were also observed. Comparing to the length of the polyethylene oxide chains, the possibility for the formation of inter-particular crosslinking can be precluded. Images of the K+ nanosensors were also obtained (Figure S2a). The size and the shape of individual particles was very similar. Aggregation may be formed during the TEM sample preparation. TEM image of the starting GQDs with the same sample preparation was shown in Figure S2b for comparison.
weeks. However, the surface of the GQDs was not completely
The Na+-selective nanosensors were prepared with sodium
covered with polyethylene oxide chains and the hydrophobic
ionophore X (NaX, a calix[4]arene derivative), TFPB, and a H+
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turn-on chromoionophore, Ox R, (see Figure S3 in the
even above 0.1 M.
supporting information). This chromoionophore has been
Likewise, K+-selective nanosensors were prepared by replacing
25, 28-29
As
the NaX with valinomycin (potassium ionophore I). As shown
+
in Figure 3c and 3d, the nanosensor showed a highly selective
was observed owing to the good overlap between the emission
response to K+. Notice that the relative fluorescence intensity
of GQDs and the excitation of Ox R. As Na+ concentration
difference of the two peaks between Figure 3a and 3c was
increased, Ox R emission (around 602 nm) decreased because of
simply a result of different doping amount of Ox R. The
the deprotonation of OX R while GQD emission showed an
emission intensity of Ox R showed a decrease as K+
increase, which is a result of both the energy transfer and
concentration went from 1 µM to 1 mM. However, only a
secondary filtration effect between Ox R and GQDs.
slight increase in the GQD emission was observed, indicating a
Importantly,
the
less efficient energy transfer between Ox R and GQDs. We infer
chromoionophore. Without additional chromoionophore, the
that the distribution of the sensing components in the
previously used in several ionophore-based sensors.
shown in Figure 3a, a ratiometric fluorescence response to Na
GQDs
themselves
could
not
act
as
+
suspension of GQDs alone showed very poor response to Na or K
+
(Figure S4). For the PEG-GQD particle suspension
nanosensors could be different and changing with environment. For instance, the adsorption of proteins could influence the
incorporating only the chromoionophore Ox R, only emission
intensity of GQD emission.
from GQDs were fluorescent since the deprotonated from of Ox
Moreover, according to previous studies, energy transfer within
R was nonfluorescent (Figure S5). Here, the nanosensors
the nanosensors is every sensitive to intermolecular distance,
functioned on the basis of ion-exchange theory. Therefore,
which makes the distribution of the sensing components quite
changing the doping level and ratio between the sensing
important.30-31
components could indeed alter the shape of the optical response
The freedom in the choice of the sensing components and their
curve (Figure S6). Therefore, the doping amount of the sensing
ratio is one of the advantages of ionophore-based ion-selective
components was kept consistence throughout all experiments.
optodes. Here, the chromoionophore Ox R was firstly chosen
In pH 7.4 Tris-HCl buffered solutions, the nanosensors showed
because of the spectral overlap with GQDs for ratiometric
+
a Na response from 0.1 mM to 1 M and a good selectivity over +
+
2+
K , Li , Mg , and Ca
2+
(Figure 3b). These interference ions
measurements at constant sample pH. On the other hand, we noted a highly sensitive pH response of the modified GQDs in
were evaluated because of their common presence in
the
intracellular media and other body fluids. Concentrations above
chromoionophore in nansensors containing Ox R, TFPB, and
1 M were not evaluated due to solubility. Notably, K+ ions, the
NaX could enable the dual sensing for e.g., pH and Na+. For this
most abundant intracellular metal ion, caused little interference
purpose,
fluorescence
the
mode
energy
(Figure
transfer
S7).
between
Changing
GQDs
and
the
the
chromoionophore should be reduced. Here, a different chromoionophore (Ox B) was assessed..25 As shown in Figure S3, Ox B is structurally similar compared with Ox R, but has much more red-shifted absorption, and thus much less energy transfer with GQDs. When excited at 460 nm (Figure 4a), the GQD emission around 522 nm was indeed pH sensitive with an apparent pKa around 6.0, which makes these nanosensors potentially useful for observing the cellular acidification processes. Upon excitation at 630 nm (Figure 4b), sample Na+ concentration change was successfully reflected on the decrease of the fluorescence emission around 700 nm. A small emission peak from Ox B was also observed, indicating that there may Figure 4. (a) Fluorescence pH response of GQD-based
still be some energy transfer from GQDs to Ox B. To
nanosensors
independently
containing
Ox
B,
TFPB,
+
and
NaX.
(b)
measure
sample
pH,
excitation
of
the
Fluorescence Na response of the GQD-based nanosensors at
chromoionophore should have minimum overlap with the GQD
various Na+ concentration as indicated.
emission, which was not yet fulfilled with Ox R and Ox B
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(Figure S8).
of components over time.
The short response time of the nanoscale sensors is an attractive
On the other hand, the small size and the excellent selectivity
feature. Ion-selective nanosensors with fast response have been
rendered the nanosensor very attractive for intracellular
demonstrated in several cases and modeling for the response
measurements. Therefore, the potential application of the Na+
time also has been described.14-15, 32-34 The color change upon
nanosensors was attempted in HeLa cells. The nanosensors were
changing the analyte concentration appeared instantaneous to
introduced into the cells through endocytosis by incubating the
the eyes. To obtain quantitative values, we attempted
cells in culture medium containing the nanosensors. The culture
stopped-flow methods. However, the experiment was limited by
medium was washed away before fluorescence microscopic
the mixing time of the samples and the results were not
imaging. Further reducing the size and some surface
accurate.
modification could help make the cell uptake more efficient.
As preliminary applications, the K+ concentration in a human +
Laser scanning confocal fluorescence microscopy was used to
urine sample was successfully measured with the K nanosensor.
study the intracellular distribution of the Na+ nanosensors
The results (20.1±0.2 mM) was found very close to the value
containing Ox R. As shown in Figure 5, the green color was
+
obtained from potentiometric measurements with K -selective
from the GQD emission while the red from Ox R. The
electrodes (20.4±0.1 mM, external calibration method). The
colocalization analysis confirmed that the leakage of GQDs was
calibration curves were shown in Figure S9a,b in the supporting
negligible. In addition, GQDs were known as a biocompatible
information. We also attempted to apply the nanosensors in
material.27 The cytotoxicity was also evaluated with the
blood serum samples. In absorption mode, blood sodium levels
commercial CCK-8 assay to confirm the biocompatibility of the
were successfully measured (152±3 mM) using the maximum
nanosensors. As shown in Figure 5e, the results indicated very
absorbance of Ox R for calibration (Figure S9c,d). The results
little toxicity after 24 hours of incubation with various amount
were also close to the ones from ISEs (149±1 mM). In
of nanosensors.
fluorescence mode, however, the GQD emission was easily
In order to evaluate whether the nanosensors respond to
influenced by the addition of blood serum, causing a decrease in
intracellular Na+ concentration changes, gramicidin (20µM)
intensity. (Figure S10) The nanosensors remained functional
was added into the culture medium together with CCCP (5µM)
after storing in fridge for 30 days. Figure S11 shows that the
while the extracellular Na+ concentration was changed to 100
response curves were quite similar, with a little change in the
mM. This protocol has been used in previous reports to allow
shape of response curve, which might indicate some degradation
the entry of sodium ions through the cell membrane in which gramicidin
forms
equilibration
of
sodium
channels
intracellular
and
sodium
CCCP to
allows
extracellular
concentrations.35 Since the typical cytosolic Na+ concentration for mammalian cells is around 12 mM,1 such experimental perturbation should cause an increase of the cytosolic Na+ concentration, which in turn causes a decrease in the chromoionophore emission intensity of the nanosensors. Images of the nanosensors-loaded cells in Figure 6a were recorded before adding gramicidin and CCCP while the ones in Figure 6b were recorded 20 minutes after adding gramicidin and CCCP to the cells. The fluorescence intensity of Ox R in the red channel Figure 5. Confocal fluorescence microscopy imaging of HeLa
clearly experienced a decrease, which indeed reflected an
cells loaded with the Na+ nanosensors. (a) FITC channel, (b)
increase of intracellular Na+ concentration. In the meantime, the
TRITC channel, (c) overlay. (d) Fluorescence intensity
fluorescence in the green channel showed no notable change,
distribution along the blue line in Part (a), (b), and (c). Scale bar:
indicating no dramatic intracellular pH change after treating the
25 µm. (e) Cytotoxicity evaluation of the Na+ nanosensors with
cells with gramicidin and CCCP. The dynamic fluorescence
the CCK-8 assay.
signal change was also continuously recorded on a plate reader. As shown in Figure 6c, 6d and 6e, the Ox R emission intensity
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ACS Sensors
Figure 6. Bright field and fluorescence microscopy images of the Na+ nanosensor-loaded HeLa cells before (a) and 20 min after the addition of gramicidin (20µM) and CCCP (5µM) to induce intracellular Na+ level change. Scale bar: 50 µm. The time evolution of the fluorescence intensity at 522 nm (c), 602 nm (d), and their ratio (e) upon addition of gramicidin and CCCP. Data was averaged from observations in 5 parallel wells. of emission at 602 nm gradually decreased for about ca. 20%
nanosensors to measure sample pH. The Na+ nanosensors were
and there was no dramatic change in the emission from the
readily introduced into HeLa cells to monitor the cytosolic Na+
GQDs at 522 nm. The control experiment where the cells were
increase induced by treating the cells with gramicidin and CCCP.
monitored without addition of gramicidin and CCCP also
Preliminary application of the K+ nanosensors also allowed the
confirmed that the reduction of the fluorescence intensity at 602
successful determination of potassium level in a human urine
nm was not a consequence of photobleaching. The fluorescence
sample. This ionophore-based sensing principle could in
in the green channel did not increase as much as observed in in
principle be extended to other ionophores and lead to different
vitro calibrations. As observed for the blood serum sample, the
selectivity. The results here lay the foundation for a highly
GQD emission could be sensitive to intracellular proteins as
valuable optical ion sensing platform to diagnostic and
well.
biological applications.
For quantitative concentration analysis, since in vitro
calibrations could not be used directly, a protocol to calibrate
ASSOCIATED CONTENT
the sodium levels inside the cells is being pursued in the lab.
Supporting Information
CONCLUSIONS
Additional information as noted in the text include: chemical
To summarize, GQDs were successfully modified and integrated
structures of the sensing components, supplementary TEM, the
into ionophore-based optical nanosensors for Na+ and K+. The
pH response of PEG-GQDs, spectral overlap information, and
nanosensors exhibited excellent selectivity, small individual size
calibration curves. This material is available free of charge via
(ca. 10 nm in diameter), ratiometric optical response, fast
the Internet at http://pubs.acs.org.
response, and good biocompatibility. The use of GQDs with the
AUTHOR INFORMATION
proper choice of chromoionophore might also enable the
Corresponding Author
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* Email:
[email protected] Sensing. PNAS 2015, 112, 5903-5908.
ORCID
12.
Xiaojiang Xie: 0000-0003-2629-8362
ion-selective nanospheres with voltage-sensitive dyes. J. Am.
ACKNOWLEDGEMENTS
Chem. Soc. 2014, 136 (47), 16465-8.
The authors thank the National Natural Science Foundation of
13.
China, the Thousand Talents Program of China, and the
to the nanoscale. Anal. Bioanal. Chem. 2015, 407 (14),
Shenzhen Municipal Science and Technology Innovation
3899-910.
Council for financial support.
14.
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