Fluorescent Silicon Nanorods-Based Ratiometric Sensors for Long

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Fluorescent Silicon Nanorods-Based Ratiometric Sensors for Longterm and Real-time Measurements of Intracellular pH in Live Cells Binbin Chu, Bin Song, Xiaoyuan Ji, Yuanyuan Su, Houyu Wang, and Yao He Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02791 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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Analytical Chemistry

Binbin Chu,‡ Bin Song, ‡ Xiaoyuan Ji, Yuanyuan Su, Houyu Wang* and Yao He* Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano & Soft Materials (FUNSOM), and Collaborative Innovation Center of Suzhou Nano Science and Technology (NANO-CIC), Soochow University, Suzhou, Jiangsu 215123, China *E-mail: [email protected]; [email protected]; Fax: 86-512-65880946.

ABSTRACT: Long-term and real-time investigation of dynamic process of pHi changes is critically significant for understanding related pathogenesis of diseases and the design of intracellular drug delivery systems. Herein, we present a one-step synthetic strategy to construct ratiometric pH sensors, which are made of europium (Eu) doped one-dimensional silicon nanorods (Eu@SiNRs). The as-prepared Eu@SiNRs has distinct emission maxima peaks at 470 nm and 620 nm under 405-nm excitation. Of particular note, the fluorescence emission intensity at 470 nm decreases along with the increase of pH, while the one at 620 nm is nearly unaffected by pH changes, making Eu@SiNRs a feasible probe for pH sensing ratiometrically. Moreover, Eu@SiNRs are found to be responsive to a broad pH range (ca. 3-9), biocompatible (e.g., ~100% of cell viability during 24-h treatment) and photostable (e.g., ~10% loss of intensity after 40-min continuous UV irradiation). Taking advantages of these merits, we employ Eu@SiNRs for the visualization of cytoplasmic alkalization process mediated by nigericin in living cells for around 30 min without interruption, revealing important information for understanding the dynamic process of pHi fluctuations.

INTRODUCTION SECTION

photostability, low fluorescence efficiency, rapid release from cells, making them hardly for continuous monitoring of pH.1115 Additionally, the close distance between the two emission bands results in a narrow pH detection range (pKa ± 1), such as Oregon green dyes (pH 4.2-5.7), fluorescein (pH 6-7.2), and SNARF (pH 6-8), limiting their widespread application in biological systems.12,16,17 Besides, the biological interactions of these fluorophores with proteins and other biomolecules may bring negative effect on their optical properties, causing improper pH measurement.16,17

In highly compartmentalized cells, each compartment has its intrinsic pH, being strongly associated with cellular metabolism processes and pathological conditions.1-4 For instance, apoptotic cell death is related with the acidification of mitochondria;5 the degradation of internalized ligands is correlated with the acidification of endosomes;6 the glycosylation of proteins is involved with the acidification of Golgi apparatus;7 and the abnormally acid microenvironment (pH 90% cell viability during 24-h treatment), strong fluorescence coupled with good photostability (ca. 10% loss of intensity after 40-min continuous UV irradiation). Taking advantages of these merits, the developed sensors are demonstrated to be high-efficacy for interrogating cytoplasmic pH fluctuations mediated by nigericin in living cells for ca. 30 min without interruption, presenting the whole dynamic changes of pHi in quantitative and long-term manners.

Fluorescence measurement of Eu@SiNRs in vitro. A series of standard pH buffers were prepared by mixing 0.2 M Na2HPO4 and 0.2 M NaH2PO4 at varied volume ratios. A Delta 320 pH-meter was used for the determination of pH value. The PBS buffer containing 500 μg/mL Eu@SiNRs in a quartz cell with a 1 cm of optical length was employed for the fluorescence measurement of Eu@SiNRs in vitro under the excitation of 405 nm. The titration curve of ratiometric signals (R = I470/I620) versus pH values was plotted. In order to evaluate the reversibility of the resultant Eu@SiNRs, the pH of solution containing 500 μg/mL Eu@SiNRs is adjusted by 1 M HCl or NaOH from pH 3 to pH 9 back and forth, followed by the measurement of emission intensities under 405-nm excitation.

EXPERIMENTAL SECTION One-Step fabrication of Eu@SiNRs-based ratiometric sensors. Eu@SiNRs were prepared via a one-pot microwave irradiation-assisted synthetic method. Briefly, the precursor solution containing APTMS, trisodium citrate, milk and EuCl3 with a mass ratio of 100:75:20:1, was transferred to a 5 mL exclusive vitreous vessel in the microwave equipment. The

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Figure 2. Characterizations of Eu@SiNRs. (A) TEM and HRTEM (insert I) images of Eu@SiNRs. (B) The length distribution, Gaussian distribution (green line) and DLS of Eu@SiNRs. (C) and (D) EDX spectra of Eu@SiNRs. (E-J) HADDF-STEM mapping of Eu@SiNRs. (K) PL and (L) absorption spectra of the SiNRs (blue line) and Eu@SiNRs (red line). NaH2PO4, 5 mM glucose, 20 mM HEPES and 10 μM nigericin. Meanwhile, the treated cell lines were immediately observed by the microscopy under continuous UV irradiation. The corresponding calibration curves of pH values against time were plotted.

Fluorescence cellular imaging. Human cervical cells (HeLa cells) and Human breast adenocarcinoma cells (MCF-7 cells) were incubated with 500 μg/mL Eu@SiNRs for 12 h at 37 °C, respectively, followed by the elimination of excess Eu@SiNRs by using PBS buffer (pH 7.4) to wash cells for three times. The Eu@SiNRs-treated cells were then incubated with the buffer at various pH values at 37 °C for 30 min. Such buffer contains 30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES and 10 μM nigericin. A confocal laser microscope (Leica, TCS-SP5 II) was employed for the monitoring the cellular fluorescence with 30% power of diode laser to avoid cell damage induced by high-power and long-term irradiation (ca. 40 min). In addition, the offset of the microscope was set as 2% to reduce the interference of self-fluorescence from biological species. Two channels of 450-520 nm and 580-650 nm were selected to collect the fluorescence emissions of Eu@SiNRs. The commercial image software (Leica LAS AF Lite) was used for the analysis of region of interest (ROI). All fluorescence images were recorded under the identical optical conditions (e.g., the same brightness, the same contrast).

RESULTS AND DISCUSSION One-Step Fabrication of Eu@SiNRs. Microwave irradiationassisted synthesis has been extensively exploited for largescale and facile preparation of zero/one-dimensional fluorescent silicon nanomaterials ascribed to its good reaction selectivity, rapid and homogeneous heating.31-33 On the other hand, lanthanide (e.g., europium (Eu) (III)) has been widely used as the doped agents to construct fluorescent probes and labels in optics and bioanalysis due to their fascinating emission and narrow bandwidths.34-37 Therefore, based on our previously reported method for fabricating fluorescent SiNRs,31 we herein further introduce a one-pot synthetic strategy for the preparation of Eu-doped SiNRs, in which the doped Eu can be effectively sensitized by the surface ligands of SiNRs, displaying prominent fluorescence, as schematically illustrated in Figure 1A (Please see the details in Experimental Section). In this system, the surface ligands (e.g., carboxyl groups) of SiNRs can provide plenty of reaction sites ready for chelating Eu3+ ions, ultimately realizing the integration of blue and red emission into a single entity (As well known, lanthanide ions can generate intense visible fluorescence emission tuned by suitable surface ligands under UV light, which is arising from f-f or f-d energy transfer.34,38).

Long-term and real-time measurement of cytoplasmic pH in live cells. To test the ability of the developed probe for long-term and real-time measurement of pH changes in live cells, the visualization of cytoplasmic alkalization process mediated by nigericin in living cells for around 30 min without interruption was realized by using Eu@SiNRs. In details, both HeLa cells and MCF-7 cells were loaded with 500 μg/mL Eu@SiNRs for 12 h at 37 °C, and then the treated cells were incubated with buffers (pH 9.0), which is consisted of 30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM

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As previously reported, the fluorescence of SiNRs is originated from the involvement of surface energy traps and delocalization of electrons.31 In principle, the pH change of surrounding environment would lead to protonation and deprotonation process of surface groups (e.g., amine groups or carboxyl groups), and further induce electrostatic charging and Fermi level shift of the Eu@SiNRs probes. In this case, the surface defects of SiNRs would be passivated and recreated, thus leading to the changes of fluorescence emission. Therefore, once pH changing from acidic to basic, the ionization of surface group would result in fluorescence quenching (maximum emission wavelength: ~470 nm) of SiNRs. Meanwhile, the fluorescence (maximum emission wavelength: ~620 nm) of Eu (III) contained in the Eu@SiNRs remains unchanged despite change of pH values.38-40 As a result, based on calculation of the intensities ratio of 470 nm and 620 nm (R = I470/I620), such two fluorescence emission peaks (~470 nm peak is sensitive to pH, while ~620 nm peak is inert to pH) simultaneously observed in Eu@SiNRs can be utilized for the measurement of ratiometric signals, readily enabling the ratiometric detection of pH changes (Figure 1B). In addition, the as-prepared Eu@SiNRs can readily enter into live cells by active endocytosis, firstly producing Eu@SiNRscontaining endosome, and then being distributed in cytoplasm, as shown in Figure 1C.41

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nm emission band is assigned to SiNRs and 580, 620 nm emission bands belong to the characteristic transitions of Eu3+, also demonstrating the successful doping of Eu in SiNRs system. As shown in Figure 2L, either Eu@SiNRs or SiNRs exhibit broad UV-vis absorption at around 320 nm. Furthermore, the resultant Eu@SiNRs preserve strong fluorescence during 30-d storage without additional special protection, indicating good storage stability of the Eu@SiNRs (Figure S4). Features of Eu@SiNRs-based pH sensors. The atomic ratio of Eu to Si in precursor is a key factor for a fold of ratiometric enhancement between the two emission peaks of as-prepared Eu@SiNRs. Figure S5 shows the fluorescence spectra of Eu (III) in Eu@SiNRs in PBS buffer (pH 7.0), which is prepared with different atomic ratio (0.1%-2.0%) of Eu to Si in precursor. The strongest fluorescence intensity of Eu (III) in Eu@SiNRs (about 4-fold ratiometric enhancement between the two emission peaks) is achieved when the atomic ratio of Eu to Si in precursor is 1.0%. As such, the optimal atomic ratio (1.0%) of Eu to Si in precursor is employed in the following experiments unless otherwise noted. Next, a series of PL spectra of Eu@SiNRs when excited at 405 nm in PBS buffers with different pH values (pH 3-9) are recorded in Figure 3A. Specifically, the fluorescence intensity at 470 nm assigned to SiNRs present a distinct pH response, decreasing with increasing pH. In contrast, the emission intensity at 620 nm assigned to Eu3+ remained unaffected (Inset in Figure 3A). Such two fluorescence emission peaks observed in Eu@SiNRs, where one is sensitive to pH and another is inert to pH, can be utilized for achieving ratiometric signals of the ratio of intensities at 470 nm and 620 nm (I470/I620). Meanwhile, the pKa (the pH at which the measured property is half its maximum) is calculated as 5.5 for the Eu@SiNRs probe (Figure S6). The corresponding histograms of ratiometric signals versus pH values are displayed in Figure 3B. As revealed, ratiometric signals gradually decrease along with the increase of pH values. And a good linearity in vitro between pH and ratiometric signals is obtained responding to a wide pH range of 3.0-8.0 (Figure 3C). The corresponding regression equation is R = 10.36-0.91 pH with the good correlation coefficient r2 = 0.993. Afterwards, the reversibility of the developed pH sensors is evaluated. The pH of solution is varied between 3 and 9 in a cyclic manner and the corresponding intensity ratios (I470/I620) are calculated. As shown in Figure 3D, the ratiometric values for the two pH are reduplicative even after ten cycles, indicating the good reversibility of Eu@SiNRs against extreme pH fluctuations. To further test the selectivity of Eu@SiNRs, different cations, anions, and amine acids are selected as interference reagents. As displayed in Figures S7 and S8, the interference reagents scarcely influence the fluorescence intensity ratios of Eu@SiNRs, indicating the good selectivity of the developed ratiometric pH sensors.

The morphologies and sizes of the as-prepared Eu@SiNRs are systematically characterized using TEM, high-resolution TEM (HRTEM) and dynamic light scattering (DLS). As shown in Figure 2A, the resultant Eu@SiNRs appears as onedimensional rod-shaped structure with good monodispersibility. Inset in Figure 2A presents the HRTEM image of single Eu@SiNRs, and its morphology is similar to that of pure SiNRs (Figure S1). Typically, the average length of Eu@SiNRs is 142.5 nm, calculated by measuring more than 200 particles (Figure 2B), close to ca. 135.1 nm of pure SiNRs (Figure S1). The hydrodynamic diameter of Eu@SiNRs is ca. 164.2 nm measured by DLS (Figure 2B), also close to the result of 163.9 nm of pure SiNRs (Figure S1). Meanwhile, as shown in Figure S2, when Eu (III) ions are doped onto the surface of SiNRs, the zeta potential decreases from neutral charge (ca. -2.72 mV) to negative charge (ca. -10.71 mV). Next, to further analyze the chemical compositions of Eu@SiNRs, energy dispersive X-ray spectroscopy (EDX) and high-angle annular dark filed scanning TEM (HADDF-STEM) mapping are performed. As revealed in Figure 2C and D, the element components semi-quantitatively determined by EDX are ~16.08% Eu and ~63.43% Si in weight ratio; ~2.71% Eu and ~57.93% Si in atomic ratio, confirming the presence of Eu element doped into SiNRs. The HADDF-STEM image in Figure 2E-J demonstrates the uniform distribution of Si, Eu, O, P, and C elements existed in the prepared composite nanostructure. The Fourier transform infrared (FTIR) spectra of Eu@SiNRs and SiNRs in Figure S3 exhibit the characteristic Si-O-Si asymmetric stretch vibrations at 10001200 cm-1 and O-Si-O bending vibrations at 400-800 cm-1. As for PL spectra of Eu@SiNRs and SiNRs, under 405 nm UV irradiation, there are three typical emission peaks centered at 470, 580, and 620 nm respectively in Eu@SiNRs, while only one peak at 470 nm in pure SiNRs (Figure 2K). The 470

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Analytical Chemistry fluorescence of the Eu@SiNRs in live cells reach the strongest intensity at this time, facilitating the achievement of favorable specificity and accuracy in quantitative measurements of intracellular pH. Next, co-localization images are captured by CLSM to investigate the intracellular distribution of Eu@SiNRs. As presented in Figure 5A and 5B, in both HeLa and MCF-7 cells, blue signals (collected from 450-520 nm) are well co-localized with red signals (collected from 580-650 nm), producing pink signals in the merged images, which certifies that Eu@SiNRs is very stable in the cellular environment for at least 12h. Moreover, as shown in Figure 5C and 5D, there exists a significant difference in intensity profiles of blue and red signals within the ROI (region of interest, green line in Insets) between nucleus and cytoplasm. As further revealed in corresponding histograms of fluorescence intensity, both blue and red fluorescence distributed in cytoplasm is much stronger than that distributed in nucleus, indicating the resultant Eu@SiNRs mostly locate in cellular cytoplasm, rather than distribute in nuclei.

Figure 3. (A) PL spectra of 500 μg/mL Eu@SiNRs in PBS buffers with different pH values under 405 nm excitation. (B) Corresponding histograms and (C) calibration curve of ratiometric signals R (I470 /I620) versus pH values. (D) pH reversibility assess of the Eu@SiNRs between pH 3 and 9. The cycles were repeated for 10 times Before biological application of Eu@SiNRs, its cytotoxicity on HeLa and MCF-7 cells is evaluated by measuring the cellular mitochondrial activity via MTT assay. As shown in Figure 4A and 4B, compared to the viability in the control groups, the viability of experimental groups treated by Eu@SiNRs with different concentrations for 24 hours remains above 100%, similar to other kinds of nanomaterials (e.g., fullerene nanomaterials, graphene oxide, and cerium oxide nanoparticles) reported previously,42,43 the Eu@SiNRs might serve as free radical scavengers to remove reactive oxygen species, inhibiting cell apoptosis to promote cell proliferation to some extent. Furthermore, it is observed that there is no significant difference in morphologies of HeLa and MCF-7 cells between control and experimental groups treated for 12 and 24 h, as displayed in Figure 4C and 4D. All cytotoxicity data demonstrate Eu@SiNRs are low- or non-cytotoxic, which therefore could be further employed for following cellular studies.

Figure 4. Cell viability of HeLa (A) and MCF-7 (B) cells treated with Eu@SiNRs with different concentrations for various incubation time by using MTT assay. Morphology of HeLa (C) and MCF-7 (D) cells treated with Eu@SiNRs of different concentrations (31.25, 62.5, 125, 250, 500 μg/mL) for 12 (I-VI) and 24 h (VII-XII).

We next testify whether Eu@SiNRs entering into cells is driven by active endocytosis or not, the accumulation of Eu@SiNRs within the cells under different temperatures is observed by confocal microscopy (CLSM). As observed in Figure S9, for both HeLa and MCF-7 cells, obvious blue and red signals of Eu@SiNRs are detectable in the cytoplasm when the cells are incubated with Eu@SiNRs at 37 oC for 6 h. On the contrary, neither blue nor red signals of Eu@SiNRs could be clearly observed in the cytoplasm when incubated at a non-physilogogic temperature of 4 oC for 6 h. Such temperature-dependent cellular internalization process suggests Eu@SiNRs enters into cells based on energydependent endocytosis. Then we further optimize incubation time. As exhibited in Figure S10, as incubation time prolongs, blue and red fluorescence signals are gradually enhanced in cellular cytoplasm, and reach the maximum intensity at 12 h. As thus, 12 h is selected as the optimal incubation time since

Long-term and real-time measurement of cytoplasmic pH in live cells. Finally, to explore ratiometric intracellular pH sensing capabilities of the synthesized Eu@SiNRs, cells loaded with Eu@SiNRs are incubated with high K+ buffer with different pH values (ranging from 3 to 9) containing a K+/H+ antiporter (10.0 μM of nigericin), which has been widely employed for regulation of pHi. The nigericin can effectively regulate the movement of K+/H+ along or against electrochemical proton gradient, modulating transmembrane pH gradient as well as membrane potential.1-4 After that, the cytoplasmic pH can be homogenized to the culture medium. As expected, both HeLa and MCF-7 cells at lower cytosolic pH values display brighter blue fluorescence compared to cells

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at higher cytosolic pH values, while red fluorescence from the cells is observed similar for all samples and is unaffected by the changing pH (Figures 6A and 6C). Next, the corresponding fluorescence intensity for each image is quantified by using Image J and the ratiometric signals are obtained. As shown in Figures 6B and 6D, the titration curve of intensity ratios versus pH values is plotted, yielding a linear response to a wide cytoplasmic pH range (pH: 4-9). In HeLa cells, the linear regression equation is R=3.95-0.42 pH with a correlation coefficient r2 = 0.990. In MCF-7 cells, the linear equation is R=1.33-0.14 pH with a correlation coefficient r2 = 0.992.

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buffer (pH 7.4) keep stable under 40-min continual UV irradiation (e.g., intensity decreases by ~5.7% during 40-min continual UV irradiation), suggesting the strong stability of Eu@SiNRs in live cells during long-term UV irradiation. To visualize the cytoplasmic alkalization process assisted by Eu@SiNRs, the cell lines loaded with Eu@SiNRs are treated with high K+ buffer (pH 9.0) containing 10.0 μM of nigericin. Afterwards, the treated cells are immediately observed under the microscope with the continuous 405-nm excitation up to 1920 sec. As revealed in Figure 7B, distinguished from nearly unchanged red fluorescence (580-650 nm) in live MCF-7 cells, the blue fluorescence (450-520 nm) gradually quenches when prolonging incubation time. To gain more quantitative information on the pH dynamic changes mediated by nigericin, the calibration curve of ratiometric signals (R=I470/I620) versus corresponding cytoplasmic pH values is plotted. As displayed in the fitting lines in Figure 7C, the ratiometric signals (R) changes a little in MCF-7 cells treated with K+ buffer (pH 7.4) during the whole process (red line). On the contrary, the R value in MCF7 cells treated with K+ buffer (pH 9.0) fluctuates with a relatively low amplitudes in the first ~930 s, and sharply decreases in the following ~990 sec (blue line). Accordingly, as presented in Figure 7D, the initial cytoplasmic pH values of MCF-7 cells are calculated to be 7.12. Typically, the cytoplasmic pH of MCF-7 cells raises slowly from 7.12 to 7.44 in MCF-7 cells treated with pH 7.4 K+ buffer (red line). While, the cytoplasmic pH dramatically raises to 8.55 treated by high K+ buffer (pH 9.0) for ~30 min (green line). Specifically, in the beginning ~930 s, pH growth rate (K1=△pH/△time) is relatively low (2.15 × 10-4 units/sec), reflecting the low rate of K+/H+ exchange mediated by nigericin in the initial phase, which preserves the membrane potential and transmembrane pH gradient. In the following ~990 sec, the pH growth rate (K2=△pH/△time) is up to 1.21× 10-3 units/sec, revealing the enhanced effect of nigericin on the regulation of electrochemical proton gradient, which results in a high K+/H+ transporting rate. Ultimately, the cytoplasmic pH reaches to ~8.55, close to the pH value of external medium, indicating the equilibrium between cytoplasm and surrounding medium. Similarly, in HeLa cells, the same cytoplasmic alkalization process is observed (Figure S11). The long-term pH changes monitored by Eu@SiNRs in different cell lines verifies the mechanism of K+/H+ antiporters-induced permeabilization of cell membrane,3,4,14 which is vital for understanding the key role of antiporter in the regulation of pHi.

Figure 5. Intracellular distribution of Eu@SiNRs. Confocal images of Eu@SiNRs in live HeLa (A) and MCF-7 cells (B). The cell lines are loaded with 500 μg/mL Eu@SiNRs at 37 oC for 12 h. Scale bars, 25 μm. Inset images (I-IV) represent the enlarged single HeLa cell and MCF-7 cell. Scale bars, 10 μm. The distribution profiles of fluorescence intensity of Eu@SiNRs along the diameter of a single HeLa cell (C) and MCF-7 cell (D). Insert I presents single HeLa cell, and Insert II presents corresponding histograms of mean fluorescence intensity in nucleus and cytoplasm.

CONCLUSIONS

It is still a great challenge for unraveling the detailed dynamic process of pH change in live cells, mainly due to the lack of a suitable pH sensor, simultaneously featuring singlewavelength excitation and multi-wavelength emission, favorable fluorescence coupled with robust photostability, minimal toxicity, as well as wide-pH range response. Of particular note, the newly developed Eu@SiNRs-based pH sensors fulfill all the above-mentioned requirements. As shown in Figure 7A, both blue and red fluorescent signals stemmed from Eu@SiNRs in MCF-7 cells treated with K+

In summary, we present a kind of silicon-based ratiometric pH sensors, which is made of europium (Eu) doped onedimensional silicon nanorods (Eu@SiNRs), suitable for longterm and real-time measurements of pHi in live cells. The asprepared Eu@SiNRs display distinct emission peaks at 470 nm, 620 nm when excited by 405 nm.

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Figure 6. Intracellular pH calibration via Eu@SiNRs. CLSM images of Eu@SiNRs in HeLa cells (A) and MCF-7 (C) cells at pH values of 3.0 (I), 4.0 (II), 5.0 (III), 6.0 (IV), 7.0 (V), 8.0 (VI) and 9.0 (VII), respectively. The Eu@SiNRs (500 μg/mL)-treated cells were respectively incubated with high K+ buffer containing 10 μM nigericin with different pH values at 37 °C for 30 min. The bottom color strip represents the pseudocolor changing with pH. Scale bars, 25 μm. Corresponding histograms of the fluorescence intensity ratio (R=I470 /I620) versus cytoplasmic pH values ranging from 3 to 9 in HeLa cells (B) and MCF-7 cells (D). Inset presents the linear relationship between R and pH values in the range of 4 to 9. The error bars show the standard deviation determined from three independent measurements. producing ratiometric signals (I470/I620). Besides, the developed pH sensors feature favorable photostability, low cytotoxicity and broad pH detection range (ca. 3-9), allowing long-term visualization of pH dynamic changes in living cells.

The Supporting Information is available free of charge on the ACS Publications website at DOI:... Characterization (e.g., TEM and HRTEM images, size distribution, and DLS) of pure SiNRs (Figure S1), Zeta potentials of deionized water, SiNRs, and Eu@SiNRs (Figure S2), FTIR spectra of Eu@SiNRs and SiNRs (Figure S3), the storage stability of Eu@SiNRs during one month storage (Figure S4), PL spectra of Eu@SiNRs (Figure S5), the pKa of Eu@SiNRs (Figure S6), the effects of different ions and species on the fluorescence intensity ratio of Eu@SiNRs (Figure S7 and Figure S8), energy-dependent endocytosis of Eu@SiNRs (Figure S9), intracellular localization of Eu@SiNRs for different incubation times (Figure S10), long-term and real-time measurement of cytoplasmic pH in HeLa cells (Figure S11).

Figure 7. Snapshot images of Eu@SiNRs in MCF-7 cells treated with pH 7.4 (A) and 9.0 (B) PBS buffer clamped at different time. The images of Eu@SiNRs are respectively collected at 450-520 nm and 580-650 nm with excitation at 405 nm. Scale bars, 10 μm. Scatter plots of fluorescence intensity ratio (R= I470/I620) (C) and cytoplasmic pH values (D) in MCF-7 cells versus time.

* [email protected] (Houyu Wang) * [email protected] (Yao He)

Notably, the emission peak at 470 nm is found to be pH sensitive and the one at 620 nm is pH insensitive, thus

‡These authors contributed equally.

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The authors declare no competing financial interest.

We express our grateful thanks to Prof. Shuit-Tong Lee's general help and valuable suggestion. The authors appreciate financial support from National Basic Research Program of China (973 Program 2013CB934400, 2012CB932400), the National Natural Science Foundation of China (61361160412, 31400860, 21575096, and 21605109), 111 Project and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and China Postdoctoral Science Foundation (7131701914).

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