Ratiometric fluorescent detection of acidic pH in lysosome with carbon nanodots

Article abstract of DOI:10.1016/j.cclet.2017.07.027

It is significant for cell physiology to keep the homeostasis of pH, and it is highly demanded to develop ratiometric fluorescent sensors toward pH. In this work, under mild condition, through the electrostatic interaction between carbon nanodots (CDs) and organic molecules, two novel ratiometric fluorescence hybrid nanosensors were fabricated for sensing acidic pH. These nanohybrid systems possess dual emission peaks at 455 and 527 nm under a single excitation wavelength of 380 nm in acidic pH condition. With the increasing of pH, the fluorescence of the 1,8-naphthalimide derivative completely quenches, while the blue fluorescence of CDs keeps constant. Furthermore, the CDs?organic molecular nanohybrids exhibit excellent anti-disturbance ability, reversible pH sensing ability, and a linear response range in wide pH range respectively. Besides the ability to target lysosome, with one of the nanosensor, stimulated pH change has been successfully tracked in a ratiometric manner via fluorescence imaging.

Full text of DOI:10.1016/j.cclet.2017.07.027

Accepted Manuscript
Title: Ratiometric fluorescent detection of acidic pH in
lysosome with carbon nanodots
Authors: Yangyang He, Zhanxian Li, Bingjie Shi, Hongyan
Zhang, Liuhe Wei, Mingming Yu
PII:
S1001-8417(17)30272-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.07.027
CCLET 4145
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
3-5-2017
13-7-2017
27-7-2017
Please cite this article as: Yangyang He, Zhanxian Li, Bingjie Shi,
Hongyan Zhang, Liuhe Wei, Mingming Yu, Ratiometric fluorescent detection
of acidic pH in lysosome with carbon nanodots, Chinese Chemical
Lettershttp://dx.doi.org/10.1016/j.cclet.2017.07.027
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Communication (heading)
Ratiometric fluorescent detection of acidic pH in lysosome with carbon nanodots
(Concise and informative. Titles are often used in information-retrieval systems. Avoid abbreviations and formulae where possible.)
Yangyang Hea, Zhanxian Lia,*, Qingyan Jiab, Bingjie Shia, Hongyan Zhangb,*, Liuhe Weia, Mingming Yua,*
aCollege of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, 450001, China.
bKey Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, Beijing, 100190, China.
*Corresponding author: Tel./fax: +86-371-67781205. E-mail address: lizx@zzu.edu.cn (Z. Li) or yumm@zzu.edu.cn (M. Yu).
Graphical Abstract
Under mild condition, through the electrostatic interaction between carbon nanodots (CDs) and organic molecules, two novel
ratiometric fluorescence hybrid nanosensors were fabricated for sensing acidic pH. The ability to target lysosome, with one of
the nanosensor, stimulated pH change has been successfully tracked in a ratiometric manner via fluorescence imaging.
This part will be used for Graphical Contents.
ABSTRACT
It is significant for cell physiology to keep the homeostasis of pH, and it is highly demanded to develop ratiometric fluorescent sensors toward pH.
In this work, under mild condition, through the electrostatic interaction between carbon nanodots (CDs) and organic molecules, two novel ratiometric
fluorescence hybrid nanosensors were fabricated for sensing acidic pH. These nanohybrid systems possess dual emission peaks at 455 and 527 nm
under a single excitation wavelength of 380 nm in acidic pH condition. Increasing of pH results in complete fluorescence quenching of the 1,8-
naphthalimide derivative, while the blue fluorescence of CDs keeps constant. Furthermore, the CDs−organic molecular nanohybrids exhibit excellent
anti-disturbance ability, reversible pH sensing ability, and a linear response range in wide pH range respectively. Besides the ability to target
lysosome, with one of the nanosensor, stimulated pH change has been successfully tracked in a ratiometric manner via fluorescence imaging.
Keywords:
Water Acidic pH;
Fluorescence;
Carbon nanodots;
Hybrid nanosensor;
Lysosome-targeted

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Intracellular pH (pHi), as a significant physiological parameter, plays a critical role in living cells [1–3], which can be influenced by
variety of physiological and pathological processes. Quantitatively measuring pH is vital for cellular analysis or diagnosis. For
instance, the pH of 4.5 to 5.5 within lysosomes provides the basic requirement for the action of lysosomal hydrolases to degrade
proteins, DNA, RNA, polysaccharides, and lipids [4]. Disruptive variation in the lysosomal pH causes defects in lysosomal function
and consequently leads to many lysosomal storage diseases [5]. Therefore, it is important to track lysosomal pH in living cells to
understand its physiological and pathological processes.
Fluorescence imaging has become an essential tool in the study of biological molecules, pathways, and processes in living cells
because of its high spatial and temporal resolution [6–11]. Particularly, because of its ability of providing quantitative measurements,
the ratiometric fluorescence technique is regarded as an ideal method for accessing intracellular pH [12]. Although considerable efforts
have been devoted to the development of pH-responsive ratiometric sensors based on organic molecules [13–17] and nanophosphors
[18–24], only a limited number of them have been applied to subcellular labeling and pH sensing. Thus, it is still challenging to
fabricate ratiometric fluorescent sensors for subcellular pH quantification.
Our previous studies demonstrated that 1,8-naphthalimide derivatives are useful fluorophores to design colorimetric and fluorescent
probes because the photophysical properties are easily influenced by reagents [25–27]. Based on photo-induced electron transfer (PET)
mechanism, We recently reported a class of 4-substituted 1,8-naphthalimide-based fluorescent off-on probes [28,29]. These
observations encouraged us to make use of such PET process to design pH-responsive fluorescent sensors based on 1,8-naphthalimide
derivatives. However, the off-on mode is not ideal for the cellular application.
On the other hand, due to their small size, tunable surface functionalities and good photostability, carbon nanodots (CDs) have many
excellent properties such as good water solubility, biocompatibility, excellent cell membrane permeability and low cost [30–37]. Such
features make CDs especially useful for fluorescent biosensing or imaging [38–44]. However, exploration of CDs as fluorescent
sensors to monitor and imagine an analyte in living cells or biological process still remains at an early stage and very few reports about
CDs fluorescent sensors toward pH has been reported.
In this paper, based on 1,8-naphthalimide derivative, CDs, and lysosome-targeted unit (morpholine), two ratiometric fluorescent
nanosensor toward pH was fabricated. In these sensors, the CDs not only serve as fluorescence out mode, but also as the anchoring site
for the sensor, a 1,8-naphthalimide derivative and the lysosome-targeted unit. The schematic illustration for the sensing of pH by the
nanosensors is shown in Scheme 1. The sensors based on this CDs-organic molecule architecture demonstrates several advantages:
First, they are ratiometric fluorescent sensors toward pH which can ensure more accurate detection especially in biosystems; Second,
the nanosensors exhibit excellent anti-disturbance ability, reversible pH sensing ability, and a linear response range in a wide pH range;
lastly, one of the two sensors is able to target lysosome and it is also capable of permeating the cell membrane and realizing pH
imaging in living cells.
2. Experiment
2.1. Materials and methods
4-Bromo-1,8-naphthalic anhydride, ethanol, monoethanolamine, piperazine, 2-methoxyethanol, propargyl bromide, K2CO3, CH3CN,
toluene, succinic anhydride, His, lle, Val, Asn, Thr, Ser, Trp, Glc, Pro, Tyr, Leu, Lys, GSH, Glu, Gly, Met, Ala, Phe, Cys, NaCl,
MgCl2, Al(NO3)3, KCl, CaCl2, CrCl3, MnCl2, FeCl3, Co(NO3)2, Ni(NO3)2,CuCl2, ZnCl2, Cd(NO3)2, HgCl2, Pb(NO3)2, Pt2Cl4, RuCl3,
RhCl3, CH3COONa, Na2SO4, Na2SO3, NaHS, Na2S2O3, Na4P2O7, KNO3, Na2N3, NaHSO4, NaHSO3, Na3PO4, Na2HPO4, NaH2PO4,
Na2SiO3, Na2CO3, NaHCO3, NaF, NaClO4, NaClO3, NaF, NaCl, NaBr, NaI, and Na2C2O4. All commercial grade chemicals and
solvents were purchased and were used without further purification.
1
Mass spectra were obtained on high resolution mass spectrometer (IonSpec4.7 Tesla FTMS-MALDI/DHB). H and 13C NMR
spectra were recorded on a Bruker 400 NMR spectrometer. Chemical shifts were reported in parts per million using tetramethylsilane
(TMS) as the internal standard. Fourier transform infrared spectroscopy (FTIR) was performed on a NEXUS-470 spectrometer at
frequencies ranging from 400 to 4,000 cm−1. Transmission electron microscopy (TEM) was performed on a Philips CM200 electron
microscope. Zeta potential was recorded on Zetasizer 3000 HS(Malvern, UK).
All spectral characterizations were carried out in HPLC-grade solvents at 20°C within a 10 mm quartz cell. UV-vis absorption spectra
were measured with a TU-1901 double-beam UV-vis spectrophotometer. Fluorescence spectroscopy was determined on a Hitachi F-
4600 spectrometer.
Sensor 1 was synthesized according to Scheme S1 and the recently reported method [45]. Compounds 4, 3 and 2 have ever been
synthesized by Yu et al [28].
2.2. Synthesis of sensor 1
Compound 2 (0.1987 g, 0.547 mmol) and succinic anhydride (0.1646 g, 1.645 mmol) were dissolved in 20 mL toluene, and the mixture
was heated to reflux and 25 μL triethylamine was added to the mixture. After 13 hours of refluxing, the mixture was cooled to room
temperature and 10 mL 0°C water was poured into the reaction solution. The crude product was obtained after filtration and the final
product (250 mg, yield: 68.9%) was obtained after vacuum drying. Characterization of 1: HRMS (EI) m/z: calcd for C24H30IN2O2
1
[M+H], 464.1743; 464.1822. H NMR (400 MHz, DMSO-d6, TMS): δH 12.161 (s, 1H), 8.44 (m, 3H), 7.82 (t, 1H), 7.34 (d, 1H), 5.76
(s, 1H), 4.30 (t, 4H), 3.43 (s, 2H), 3.26 (t, 4H), 2.80 (t, 4H), 2.42(t, 4H). 13C NMR (100 MHz, DMSO-d6): δC 173.71, 172.48, 164.19,
163.62, 156.14, 132.75, 131.20,131.11, 129.67, 126.55, 125.75, 122.88, 115.84,115.58, 79.69, 76.49, 61.63, 55.37, 52.93, 51.61, 46.44,
38.79, 29.17, and 29.10.
2.3. Preparation and characterizations of the fluorescent CDs

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CDs were prepared by hydrothermal treatment of citric acid and ethylenediamine following the reported method [46–48], which was
characterized by FTIR (Fig. S1 in supporting information) and TEM (Fig. 1).
2.4. Preparation of 1-CDs and 1-CDs-Lyso
To obtain a ratiometric fluorescence sensor with an appropriate color variation during sensing, we prepared a mixture solution
containing CDs (0.02 mg/mL) and sensor 1 (30 μM) as the sensing solutions (1-CDs) for pH sensing. In addition, in order to introduce
a lysosome-targeted unit, a mixture solution of CDs (0.02 mg/mL), sensor 1 (30 μM) and morpholine (30 μM) was also prepared as a
lysosome-targeted pH sensing system (1-CDs-Lyso).
2.5. Cell incubation and imaging
HeLa cells were seeded in a 12-well plate in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum and 1% penicillin. The cells were incubated under an atmosphere of 5% CO2 and 95% air at 37°C for 24 h before the cell
imaging experiments. The cells were washed three times with PBS buffer before used. Luminescence imaging experiments in Living
HeLa cells were operated with Nikon A1Rmp-PicoQuant FLIM. 405 nm and 630 nm semiconductor laser was used for luminescence
imaging experiments.
3. Results and discussion
3.1. Synthesis of sensor 1 and preparation of CDs, 1-CDs and 1-CDs-Lyso
For the fabrication of the sensing system, first we synthesized amino and carboxyl coated CDs by a simple hydrothermal method using
citric acid and ethylenediamine as the precursors. Fig. 1 shows the TEM image of CDs, revealing that the nanoparticles have a
diameter ranging from 3 to 4 nm. FT-IR spectrum (Fig. S1 in the Supporting Information) reveal that the CDs mainly contain carbon,
nitrogen, oxygen, and the surfaces of the CDs are functionalized with amino, hydroxyl, and carboxylic/carbonyl moieties. Fig. S2
shows the TEM images of 1-CDs and 1-CDs-Lyso, revealing that there is agglomeration behavior in the hybrid nanomaterials. The
Zeta Potential of CDs is -10.4 mV, which is ascribed to the surface negative electricity of the CDs. Further experiments demonstrate
that The Zeta Potentials of 1-CDs and 1-CDs-lyso are -7.16 mV and 3.59 mV respectively. The Zeta Potential difference of the three
kinds of nanomaterials may be ascribed to the interaction between the CDs and organic molecules.
1,8-naphthalimide derivative 1 was selected as the sensing unit, which was synthesized in a high yield starting from 4-bromo-1,8-
naphthalic anhydride through four steps. The reaction of 4-bromo-1,8-naphthalic anhydride and monoethanolamine gives the product 4
in a 69.2% yield. Compound 3 was obtained with a yield of 73.9% by the reaction of compound 4 and piperazine. The reaction of 3 and
propargyl bromide gives the product 2 in a yield of 76.5%. Sensor 1 was obtained by the reaction of compound 2 and succinic
1
anhydride in a yield of 68.9%, which was characterized by H NMR, 13C NMR and HRMS (Figures S3, S4, and S5 in Supporting
Information).
3.2. pH sensing properties
As shown in Fig. 2a, with pH increasing of the medium from 4.0 to 8.0 and upon excitation at 380 nm, the emission peak at 527 nm
decreased gradually, which was due to the molecular change from 1H to 1 (Scheme 1). In the same condition, no obvious absorption
spectral change was found (Fig. 2b), indicating that pH change cannot result in the excitation state change. However, the PL spectrum
of the CDs without (Fig. 2c) or with lysosome-targeted unit (Fig. 2d) hardly changes with pH changing. Besides, there is a good
overlap between the absorption spectrum of sensor 1 and the PL spectrum of CDs (Fig. 2b). Considering the results of figures 2a, 2b,
2c, and 2d, it is easy to conclude that the PL spectrum of CDs can be used as a reference if we use the hybrid material of compound 1,
and CDs as a pH-sensor (1-CDs). Therefore, it is possible to select a 380 nm wavelength laser to excite CDs and compound 1 in the
hybrid sensor. Under the excitation at 380 nm and in acidic condition, the emission of CDs was partially absorbed by compound 1 and
the nanohybrid system exhibits a very strong emission peak at 527 nm and a very weak emission peak at 450 nm. However, under the
same excitation and in neutral condition, no emission at 527 nm was found yet the emission at 450 nm still existed. If a lysosome-
targeted unit such as morpholine was introduced to the above system, a lysosome-targeted pH hybrid sensor (1-CDs-Lyso) could be
fabricated.
With pHapp increasing from 4.0 to 8.0 and upon excitation at 380 nm, the emission at 527 nm of 1-CDs (Fig. 3a) and 1-CDs-lyso
(Fig. 3b) decreased and that at 455 nm hardly changed in intensity with increasing pHapp. The emission intensity increase at 527 nm in
acidic solution can be ascribed to the structural transformation from compound 1 to 1H (Scheme 1) and the inhibition of PET process
[49]. The apparent pKa' was determined to be 6.47±0.22 and 6.28 ± 0.17 for 1-CDs and 1-CDs-lyso respectively via fitting the pH
titration profile based on the normalized I455/I527 (see Figures S6 and S7 in the Supporting Information) [50]. The linear ranges for the
ratiometric response from emission spectra of 1-CDs and 1-CDs-lyso are pHapp of 5.6 to 7.2 (Fig. 2c) and 5.6 to 7.4 (Fig. 2d)
respectively. Fig. 3e demonstrates the fluorescence color change of 1-CDs-lyso in different pH solutions. In addition, the enhanced
I455/I527 value of 1-CDs and 1-CDs-lyso at pHapp 7.8 and 7.4 can be recovered to the solution original I455/I527 value at pHapp 5.6 and 5.0
respectively, and this reversible ratiometric pH sensing ability can be retained for at least 10 cycles (Figures 4a and 4b).
3.3. The anti-disturbance effect study of sensors 1-CDs and 1-CDs-Lyso for pH sensing
To further assess the utility of 1-CDs and 1-CDs-Lyso as pH ratiometric hybrid nanosensors, their emission spectal response to pH 7.4
in the presence of other species including biological molecules, metal ions, and anions (see figures 5, S8, S9, S10, S11 and S12 in
supporting information) was also tested. The results demonstrated that all of the selected species have no interference in the sensing of
pHapp. This result strongly indicates that 1-CDs and 1-CDs-Lyso could be excellent ratiometric fluorescent sensors toward pH with
strong anti-interference ability.
3.4. Thermal stability and photostability study of sensor 1

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An ideal sensor ought to have good thermal stability and photostability. As shown in Fig. 6a, with irradiation (λex = 380 nm) time
extending, the emission at 527 nm of compound 1 only increased, indicating that compound 1 don’t have good photostability. With
temperature increasing from 30 to 48°C, the emission of compound 1 hardly changes (Fig. 6b), indicating good thermal stability. The
photostability and thermal experiments of the nanohybrid sensors 1-CDs (Figures 6c and 6d) and 1-CDs-lyso (Figures 6e and 6f) were
also operated, an excellent result was obtained under the same condition as that of compound 1, the emission intensity ratio I455/I527
doesn't change with time extending or temperature changing. The results demonstrate that the nanohybrid materials as pH sensors have
good thermal stability and photostability.
3.5. Ratiometric pHlyso imaging behaviour of 1-CDs-Lyso
The cytotoxicity of sensors 1-CDs and 1-CDs-Lyso toward Hela and Hepatocyte cells were measured using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetraziliumbromide (MTT) assay to evaluate the potential application of these two sensors in living cell imagining. The
cellular viability was estimated to be greater than 95% after 1 h at the sensing concentration for the two sensors, suggesting the low
cytotoxicity of 1-CDs and 1-CDs-Lyso (Fig. S13).
The intrinsic ability of 1-CDs-Lyso to target lysosome was investigated in living Hela cells. The cells stained with 1-CDs-Lyso (3.0
× 10−5 M, 0.02 mg/mL of CDs, and 3.0 × 10−5 M of morpholine, 30 min, 25°C) were co-stained further with the commercially available
lysosome-Tracker Cys deep red (1 mM, 30 min) in the culture medium. The imaging results show that the green image for 1-CDs-Lyso
channel obtained upon excitation at 630 nm is almost identical to the red image for the lysosome-Tracker channel obtained upon
excitation at 633 nm (Fig. 7). The overlay between the fluorescence images of 1-CDs-Lyso and lysosome-Tracker discloses a
Pearson's correction coefficient of 0.75, suggesting the ability of 1-CDs-Lyso to target lysosome.
The practical ratiometric pHi imaging ability of 1-CDs-Lyso was further applied in monitoring acidic pHi upon different stimulation
(Fig. 8, Fig. S14 in supporting information). In this study, the intracellular pH calibration was carried out in living HeLa cells using a
standard procedure followed by a further incubation with high K+ buffers of different pH, also containing 1 mM nigericin. As shown in
Fig. 8, the ratio of Iblue to Igreen gradually increased with pH changing from 4.0 to 7.0. The change in the ratio of Iblue to Igreen shows the
ability of sensor 1-CDs-Lyso to measure a pH-dependent signal change. The ratio value of Iblue/Igreen is 1.97, 8.19, 34.67 and 51 from
pH 4 to pH 7.
4. Conclusions
Two ratiometric fluorescent CDs-organic molecule hybrid nanosensors with single excitation wavelength was synthesized for sensing
acidic pH with strong anti-disturbance ability, good photostability and thermal stability. The linear ratiometric pH response range in a
wide range, the reversible pH sensing ability, the lysosome-targeted capacity and the cell membrane permeability make this sensor
especially suitable for the practical tracking of acidic pHi in living cells via ratiometric imaging.
Acknowledgments
We are grateful for the financial supports from National Natural Science Foundation of China (21601158, U1504203 and J1210060),
Key Laboratory of Photochemical Conversion and Optoelectronic Materials and Zhengzhou University.
Supplementary material
Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/

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Fig. 1. TEM images of as-prepared CDs.
Fig. 2. (a) Fluorescence spectra of sensor 1 (3.0 × 10−5 M) in DMSO–HEPES buffer solutions (1:100, v/v) of different pHapp values determined upon excitation
at 380 nm. (b) Absorption spectra of sensor 1 (3.0 × 10−5 M) in 0.01 M different pH HEPES solution (red, green, and blue lines) and luminescence spectra of
CDs (0.02 mg/mL) with excitation at 380 nm (black line) in pH 7.4 HEPES solution. (c) Fluorescence spectral change of CDs (0.02 mg/mL) in HEPES buffer
solutions of different pHapp values determined upon excitation at 380 nm. (d) Fluorescence spectral change of CDs (0.02 mg/mL) with 3.0 × 10−5 M of
morpholine in HEPES buffer solutions of different pHapp values determined upon excitation at 380 nm.

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Fig. 3. (a) Fluorescence spectra of sensor 1-CDs (3.0 × 10−5 M of 1 and 0.02 mg/mL of CDs) in DMSO–HEPES buffer solutions (1:100, v/v) of different pHapp
values determined upon excitation at 380 nm. (b) Fluorescence spectra of sensor 1-CDs-Lyso (3.0 × 10−5 M of 1, 0.02 mg/mL of CDs, and 3.0 × 10−5 M of
morpholine) in DMSO–HEPES buffer solutions (1:100, v/v) of different pHapp values determined upon excitation at 380 nm. (c) The linear relationship for the
ratiometric response from emission spectra of 1-CDs (c) and 1-CDs-Lyso (d) to different pHapp. Fluorescent photographs of sensor 1-CDs-Lyso (3.0 × 10−5 M of
1, 0.02 mg/mL of CDs, and 3.0 × 10−5 M of morpholine) in DMSO–HEPES buffer solutions (1:100, v/v) of different pHapp values determined upon excitation at
365 nm from a portable lamp. The pH values from left to right are 4.0, 4.4, 4.8, 5.2, 5.6, 6.0, 6.4, 6.8, 7.2, 7.6, and 8.0.
Fig. 4. Emission intensity ratio I455/I527 of 1-CDs (3.0 × 10−5 M of 1 and 0.02 mg/mL of CDs) (a) and 1-CDs-Lyso (3.0 × 10−5 M of 1, 0.02 mg/mL of CDs, and
3.0 × 10−5 M of morpholine) (b) in the same medium determined over consecutive pHapp cycles.

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Fig. 5. Emission spectral response of 1-CDs-Lyso (3.0 × 10−5 M of 1, 0.02 mg/mL of CDs, and 3.0 × 10−5 M of morpholine) in DMSO–HEPES buffer solution
(1:100, v/v, pHapp = 5) upon addition of different species (3 equiv of species relative to 1), the excitation wavelength was 380 nm. I455 and I527 represent the
emission intensity at 455 and 527 nm. The species used were blank, Cys, Hcy, Pro, Trp, Val, Ser, Leu, Phe, lle, Glu, Thr, Gly, Ala, Met, H2O2, Asp, GSH, Tyr,
and Gin.

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Fig. 6. Emission of compound 1 in DMSO–HEPES buffer solution (1:100, v/v, pHapp = 7.4, 3.0 × 10−5 M) with irradiation (λex = 380 nm) time extending (a) and
temperature changing (b). Emission intensity ratio I455/I527 of 1-CDs (3.0 × 10−5 M of 1 and 0.02 mg/mL of CDs) in DMSO–HEPES buffer solution (1:100, v/v,
pHapp = 7.4) with irradiation time extending (λex = 380 nm) (c) and temperature changing (d). Emission intensity ratio I455/I527 of 1-CDs-Lyso (3.0 × 10−5 M of 1,
0.02 mg/mL of CDs, and 3.0 × 10−5 M of morpholine) in DMSO–HEPES buffer solution (1:100, v/v, pHapp = 5) with irradiation (λex = 380 nm) time extending (e)
and temperature changing (f).

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Fig. 7. Confocal fluorescence images of Hela cells stained with 1-CDs-Lyso (3.0 × 10−5 M of 1, 0.02 mg/mL of CDs, and 3.0 × 10−5 M of morpholine) for 15
min (a) and lysosome-Tracker (2.0 mm) for 15 min (b). (c) Merged image of (a) and (b). (d) Bright-field image. (e) Intensity profile of regions of interest (ROIs)
across Hela cells. (f) Correlation plot of the intensities of 1-CDs-Lyso and lysosome-Tracker (Rr = 0.75).
Fig. 8. Confocal microscopy images of 1-CDs-Lyso (3.0 × 10−5 M, 0.02 mg/mL of CDs, and 3.0 × 10−5 M of morpholine) in living HeLa cells clamped at pH 4
(a1–a4), 5 (b1–b4), 6 (c1–c4), and 7 (d1–d4). The excitation wavelength was 405 nm. The images were collected at blue channel with 482/35 filter (first row), green
channel with 525/50 filter (second row) and overlay of first row and second row (third row). Ratio images obtained from the two above channels (fourth row).
The ratio value of Iblue/Igreen is 1.97, 8.19, 34.67 and 51 from pH 4 to pH 7.
Scheme 1 Schematic illustration for the sensing of pH by the hybrid nanosensors.