Fluorescent and Photostable Silicon Nanoparticles Sensors for Real


Aug 19, 2016 - Fluorescent sensors suitable for dynamic measurement of intracellular pH (pHi) fluctuations should feature the following properties: fe...
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Fluorescent and Photostable Silicon Nanoparticles Sensors for RealTime and Long-Term Intracellular pH Measurement in Live Cells Binbin Chu,† Houyu Wang,† Bin Song, Fei Peng, Yuanyuan Su, 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 S Supporting Information *

ABSTRACT: Fluorescent sensors suitable for dynamic measurement of intracellular pH (pHi) fluctuations should feature the following properties: feeble cytotoxicity, wide-pH range response, and strong fluorescence coupled with good photostability, which are still remaining scanty to date. Herein, by functionalizing fluorescent silicon nanoparticles (SiNPs) with pH-sensitive dopamine (DA) and pH-insensitive rhodamine B isothiocyanate (RBITC), we present the first demonstration of fluorescent SiNPs-based sensors, simultaneously exhibiting minimal toxicity (cell viability of treated cells remains above 95% during 24-h treatment), sensitive fluorescent response to a broad pH range (∼4−10), and bright fluorescence coupled with robust photostability (∼9% loss of fluorescence intensity after 40 min continuous excitation; in contrast, fluorescence of Lyso-tracker is rapidly quenched in 5 min under the same experiment conditions). Taking advantage of these merits, we further employ the resultant fluorescent SiNPs sensors for the detection of lysosomal pH change mediated by nigericin in live HeLa and MCF-7 cells in long-term (e.g., 30 min) manners. Interestingly, two consecutive stages, i.e., alkalization lag phase and logarithmic growth phase, are observed based on recording the whole process of pH change, offering important information for understanding the dynamic process of pHi fluctuations.

T

interest, these significant progresses may intrigue new opportunities for designing novel kinds of fluorescent SiNPs sensors featuring unique optical properties and benign biocompatibility, which nevertheless remain vacant up to the present. Herein, we introduce a kind of fluorescent SiNPs-based pH sensors, in which one single SiNP is conjugated with pHsensitive dopamine (DA) and pH-insensitive rhodamine B isothiocyanate (RBITC) molecules, simultaneously featuring wide-pH range response, strong fluorescence, excellent photostability, and feeble cytotoxicity, especially capable for the ratiometric measurement of pHi changes in long-term and realtime manners. Typically, the as-prepared dual-modified SiNPs (DMSiNPs) sensors manifest distinct fluorescent changes in response to a wide pH range (∼4−10). In addition, the fluorescent DMSiNPs sensors show noninvasiveness (>95% of cell viability), lysosome-targeting ability (0.90 of Pearson’s colocalization coefficient), and remarkable photostability under long-term UV irradiation (∼9% loss of intensity after 40 min continual UV irradiation). Taking advantage of these unique merits, the developed sensors allow real-time investigation of lysosomal pH fluctuations mediated by nigericin in live cells for

he crucial parameter of intracellular pH (pHi) associated with cell viability and metabolism process is mainly regulated by the activities of integral membrane protein-based antiporters, including Na+/H+ (e.g., NhaA), Ca2+/H+ and K+/ H+ antiporters (e.g., nigericin) in virtually all cells.1−9 Longterm and real-time investigation of dynamic process of pHi changes is critically significant for understanding related pathogenesis of diseases.6−9 To achieve this goal, high-quality fluorescent sensors are of particular importance, which should essentially satisfy the following merits: (i) minimal toxicity, (ii) wide-pH range response, and (iii) strong fluorescence coupled with robust photostability.10−29 Despite sufficient progress on the development of various pH-sensitive sensors (please see detailed information in Table 1),10−29 there still remains urgent demand for designing new sensing devices which fully satisfy the above-mentioned requirements. On the other aspect, tremendous efforts recently have been devoted to developing luminescent nanoparticles for biological and biomedical applications. Typically, fluorescent silicon nanoparticles (SiNPs) with relatively high photoluminescence quantum yield (PLQY) (∼15−25%), robust photostability, and low- or non-toxicity have attracted great attention for a myriad of optical applications.30−39 To date, the SiNPs conjugated with biomolecules, such as immunoglobulin G (IgG), doxorubicin (DOX), and peptide containing arginine-glycine-aspartic acid (RGD) sequence, have been demonstrated to be highly efficacious for long-time cellular imaging.35−37 Of our particular © 2016 American Chemical Society

Received: June 29, 2016 Accepted: August 19, 2016 Published: August 19, 2016 9235

DOI: 10.1021/acs.analchem.6b02488 Anal. Chem. 2016, 88, 9235−9242

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Analytical Chemistry

solution was incubated with 100 μL of EDC (50 mg/mL) and 25 μL of NHS (50 mg/mL) at 25 °C for 15 min in sequence, respectively, to fully activate the carboxylic acid groups of SiNPs. Finally, such mixture was incubated with DA with slow stir for 4 h at 25 °C in the dark. Of note, excess free or unreacted DA were removed using Nanosep centrifugal devices (MW cutoff, 10 kDa; Millipore) through centrifugation at 5500 rpm/min for 10 min. The product was collected and washed with PBS buffer (10 mM). Note that corresponding absorption intensities of DMSiNPs should be corrected by eliminating the scattering background from pure SiNPs. The final DMSiNPs conjugates were diluted with PBS buffer (10 mM) to the desired concentrations and stored at 4 °C in the dark for the following experiments. Fluorescence Measurement of DMSiNPs Sensors in pH Buffers. A series of standard pH buffers were prepared by mixing 0.2 M Na2HPO4 and 0.2 M NaH2PO4 at varied volume ratios. The pH value was measured by Delta 320 pH-meter. Then, 1 mL of the standard pH buffer containing 0.48 mg/mL DMSiNPs was transferred to a quartz cell of 1 cm optical length to measure fluorescence spectra at the excitation wavelength of 405 nm (λex = 405 nm) and the excitation wavelength of 543 nm (λex = 543 nm), respectively. The ratio signal (R = I465/I575) was calculated from the emission intensities at 465 and 575 nm. A pH calibration curve was obtained by plotting R as a function of pH. The pH of DMSiNPs solution (0.48 mg/mL) between pH 4 and 10 was adjusted back and forth by 1 M HCl or NaOH and then measured by a pH-meter. The precise concentration of NaOH was titrated by potassium hydrogen phthalate, and the precise concentration of HCl was titrated by sodium tetraborate. The fluorescence spectra were recorded with λex = 405 and 543 nm. Cell Culture and Fluorescence Imaging. Human cervical cells (HeLa cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM). Human breast adenocarcinoma cells (MCF-7 cells) were cultured in RPMI-1640 medium. Both medium were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and relevant antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin). Both cell lines were cultured at 37 °C in a 5% CO2 incubator with humidified atmosphere. For fluorescence imaging, the two kinds of cells were incubated with DMSiNPs (0.48 mg/mL) for 24 h at 37 °C. Before use, the treated cells were washed with PBS (pH 7.4) for three times to remove the excess DMSiNPs. Fluorescence imaging experiments were performed on a confocal laser microscope (Leica, TCS-SP5 II) with 30% power of diode laser to avoid high power laser-induced cell damage during long-term detection (∼40 min). The interference of selffluorescence of cells was reduced by setting the offset as −2%. The processing and analysis of region of interest (ROI) was performed by the commercial image analysis software (Leica Application Suite Advanced Fluorescence Lite (LAS AF Lite)). The total fluorescence intensity of ROI was obtained from corresponding fluorescence images by summing the fluorescence intensities of all the pixels within a single cell. All the fluorescence images were taken under exactly the same optical conditions, and the same brightness and contrast was applied to the images by the microscope automatically. Intracellular pH Calibration. The HeLa or MCF-7 cells were incubated with 0.48 mg/mL DMSiNPs for 24 h at 37 °C followed by washing and resuspending with PBS buffer to avoid autofluorescence coming from the culture medium. The treated

Table 1. Comparison of Currently Established Fluorescent Sensors for Monitoring Intracellular pH fluorescent pH sensorsa organic dyes fluorescent proteins organic dyesnanomaterials composites II−VI QDs SiNPs

without heavy metal

wide-pH range responseb

strong fluorescence coupled with good photostabilityc

√ √

× ×

× ×

12−14 11, 15



×



14, 16

× √

√ √

√ √

23, 24 this work

refs

Organic dyes indicate fluorescein isothiocyanate (FITC), Rhodamine, DND-160, etc.; fluorescent proteins refer to HdeA derivants, SsaM/ SpiC/SsaL, etc.; organic dyes-nanomaterials composites involve FITCnanoscale metal organic frameworks (FITC-NMOFS), carboxyfluorescein (FAM)-DNA nanoribbons, etc.; II−VI QDs represent CdSe/ ZnS core−shell QDs. bWide pH-range response indicates sensors manifest distinct fluorescent changes in response to a wide pH range (∼5−9), covering four units. cStrong fluorescence indicates ∼15% of quantum yield of sensors and good photostability refers to ∼9% loss of fluorescence intensity of sensors after 40 min continual high-power UV irradiation. a

∼30 min. As thus, a correlation between lysosomal pH changes and incubation time can be quantitatively identified, revealing two consecutive stages, i.e., alkalization lag phase and logarithmic growth phase, during the dynamic process of pH change.



EXPERIMENTAL SECTION Preparation of NH2-SiNPs. The amino groups-coated SiNPs (NH2-SiNPs) were first prepared via one-pot microwave irradiation-assisted synthetic method, as described in our previous reports.35,36 In brief, the reaction precursor solution was composed of (3-aminopropyl)-trimethoxysilane, trisodium citrate dehydrate, and deionized water in a ratio of 1:4:25, saturated with nitrogen. Then, the resultant precursor solution was transferred into the exclusive vitreous vessel, followed by microwave irradiation for 15 min under 160 °C, finally producing fluorescent NH2-SiNPs with maximum emission at ∼470 nm. Next, the as-prepared NH2-SiNPs were purified by dialysis (1 kDa) to remove residual reagents. The purified SiNPs solution with strong blue luminescence was quantified as 2.44 mg/mL via UV absorption method at 350 nm.35,36 Fabrication of DMSiNPs Sensors. The NH2-SiNPs solution prepared above was mixed with RBITC dissolved in 0.1 M NaHCO3 solution at room temperature overnight to obtain RBITC modified SiNPs (RBITC-SiNPs). Then, the reaction solution was filtered by using Nanosep centrifugal devices (MW cutoff, 10 kDa; Millipore) through centrifugation at 5500 rpm/min for 10 min to remove unreacted RBITC.29 The as-prepared RBITC-SiNPs were then dissolved in the acetic solution (0.7%, v/v, pH = 3.7), followed by the gradual addition of glyoxylic acid until the pH of mixture reached at 2.7. The mixture was stirring for 90 min at 25 °C. The imine groups were formed when the pH of mixture was adjusted as 4.5 by the slow addition of 1 M NaOH with slow stir for 45 min. Then, the imine groups coated SiNPs were subsequently reduced as carboxyl groups by adding 5% (w/v) aqueous solution of sodium borohydride to the mixture dropwise with slow stirring for 60 min at 25 °C.40 Afterward, 200 μL of COOH-SiNPs 9236

DOI: 10.1021/acs.analchem.6b02488 Anal. Chem. 2016, 88, 9235−9242

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Figure 1. Construction of dual-modified SiNPs (DMSiNPs)-based pH sensor. (A) Schematic illustration of the synthesis of DMSiNPs. (B) Photos of SiNPs (1), DA (2), RBITC (3), and DMSiNPs (4) in ambient environment and under UV irradiation (λex = 360 nm) as well as TEM image of DMSiNPs. Inset shows the enlarged HRTEM image of a single DMSiNPs. (C) Schematic illustration of cellular internalizations of DMSiNPs. Inset in part C presents charge-transfer mechanism of DMSiNPs at acidic or basic conditions.



cells then were incubated with the buffer containing 10 μM nigericin, 30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, and 20 mM HEPES with various pH values at 37 °C for 30 min. Fluorescence cellular imaging experiments were performed at 37 °C on a confocal laser microscope with 30% power of diode laser with two excitations of 405 nm for SiNPs and 543 nm for RBITC through a 63× 1.4 NA objective; fluorescence emissions were collected through the following channels of 425−500 nm for SiNPs and 570−625 nm for RBITC. Then, the fluorescence images were recorded, and the pH calibration curve was constructed with Leica software. Long-Term and Real-Time Measurement of Lysosomal pH in Live Cells. To verify the feasibility of DMSiNPs for long-term and real-time measurement of pH changes in live cells, a time relapse experiment at a time interval of 20 s was designed to visualize the lysosomal alkalization process when the DMSiNPs loaded cell lines were, respectively, incubated with high K+ buffer (pH 9.0) in the presence of 10.0 μM of nigericin12,13 and the total time lasted for 30 min. In detail, the HeLa or MCF-7 cells were incubated with 0.48 mg/mL DMSiNPs for 24 h at 37 °C followed by washing and resuspending with PBS buffer. The resultant cells then were incubated with the buffer containing 10 μM nigericin, 30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, and 20 mM HEPES with pH 9.0 at 37 °C. Afterward, the treated cell lines immediately suffer continuous UV irradiation up to 1800 s under the microscopy. The images of DMSiNPs channels were, respectively, collected at 425−500 nm with excitation at 405 nm and at 570−625 nm with excitation at 543 nm through a 63× 1.4 NA objective. The pH values of cells at various periods were quantified using the corresponding pH calibration curves (Figure 5B and Figure S11) and sequentially plotted against time (Figure 8 and Figure S13).

RESULTS AND DISCUSSION Fabrication of DMSiNPs-Based pH Sensor. The DMSiNPs-based pH sensor is fabricated as schematically illustrated in Figure 1A. First, the amino groups surfaceterminated SiNPs (NH2-SiNPs) are facilely prepared through the well-established one-pot synthesis method assisted by microwave irradiation.35,36 Second, the surface of NH2-SiNPs can be facilely modified with pH-insensitive rhodamine B isothiocyanate (RBITC) through the reaction between the amino groups of SiNPs and isothiocyanate groups of RBITC (Step 1).29 Thereafter, the remaining surface-covered amino groups are transformed to carboxylic acid groups via the classic Schiff base reaction (Step 2).40 Finally, the carboxylic acid groups coated with SiNPs readily react with the amino groups of DA via N-hydroxysuccinimide (NHS) and N-(3(dimethylamino)propyl)-N-ethylcarbodiimde hydrochloride (EDC) as zero length cross-linkers (Step 3),41 finally yielding the dual-functionalized SiNPs with strong fluorescence, distinct pH-responsive ability, negligible cytotoxicity, good lysosometargeting ability, and strong photostability. As shown in Figure 1B, the aqueous solution of DMSiNPs exhibits bright blue fluorescence at pH 7.4 (λex = 360 nm). Compared with PLQY of pure SiNPs at pH 7.4 (e.g., ∼21%), the PLQY of DMSiNPs at the same conditions (e.g., ∼19%) does not significantly change. According to the transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) characterizations (Figure 1B), the DMSiNPs appear as spherical particles with good monodispersibility. Typically, the average size of DMSiNPs is calculated as ∼8.8 nm by measuring over 200 particles. In contrast, the average size of SiNPs is ∼5.25 nm (see Figure S1 in the Supporting Information), smaller than that of DMSiNPs, providing visual evidence that SiNPs are coated with RBITC and DA molecules. The as-prepared DMSiNPs are also characterized by dynamic light scattering (DLS), Fourier transform-infrared (FT-IR) spectroscopy, UV−vis absorption spectra, and zeta potentials, 9237

DOI: 10.1021/acs.analchem.6b02488 Anal. Chem. 2016, 88, 9235−9242

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Analytical Chemistry further confirming the successful bioconjugation of SiNPs with RBITC and DA (Figures S2−S5). Notably, when RBITC and DA are conjugated to the surface of SiNPs, the zeta potential increases from negative charge (∼−15.55 mV) to positive charge (∼11.8 mV) (Figure S5). As such, the positively charged DMSiNPs can associate with the negatively charged cell membrane, then spontaneously access into cytoplasm via a ubiquitous pathway of endocytosis, and finally distribute in the endolysosome (as illustrated in Figure 1C).25,42,43 It is worth pointing out that, at different pH values of endolysosome, the fluorescence of DMSiNPs (λem = 465 nm) exhibit a characteristic pH-dependent behavior at λex = 405 nm. Particularly, at acidic pH, SiNPs serve as an electron donor, thus are able to accept electrons from catechol (reduction state of DA) and result in relatively high fluorescence intensity; while at basic pH, SiNPs act as an electron acceptor, thereby can lose electrons to quinone (oxidation state of DA) and lead to high quenching efficiency of SiNPs.23,24 In addition, we further validate the indispensable role of oxygen in pH response of DMSiNPs (Figure S6), which is similar to QDs-dopamine conjugates.23,24 Whereas, simply mixing SiNPs with DA without covalent conjugation only strongly quenches fluorescence of SiNPs.34 Meanwhile, the fluorescence of RBITC conjugated to SiNPs (λem = 575 nm) changes little at λex = 543 nm, displaying pH-insensitive behavior under the same conditions, which could be employed as reference fluorescence channel. The fluorescence intensity ratio of SiNPs at 465 nm to RBITC at 575 nm (R = I465/I575) generates a titration curve in a wide pH range, enabling quantification of intracellular pH. Furthermore, the resultant DMSiNPs maintains strong PL free of additional special protection during 2-d storage, indicating adaptable storage stability of the SiNPs-based sensors (Figure S7). Features of DMSiNPs-Based pH Sensors. The molar feed ratio of RBITC to DA is a key factor for pH response range of DMSiNPs. As shown in Figure 2A, DMSiNPs prepared with different molar feed ratios of RBITC to DA from 1:4 to 1:71 correspond to different pH-sensitive ranges. Among of these, the broadest pH range from 4 to 10 is achieved when the molar feed ratios of RBITC to DA is 1:24 (solid line). As such, the optimal molar feed ratio of RBITC to DA (1:24) is employed in the following experiments unless otherwise noted. Figure 2B shows the PL spectra of DMSiNPs in PBS buffer at various pH values. DMSiNPs present a distinct fluorescent response to pH (emission wavelength (λem) from ∼400 to ∼600 nm, maximum λem= 465 nm) at λex = 405 nm. Particularly, compared with the PL intensity at pH 7.0, the PL intensity raises dramatically when pH < 7.0 (∼1.25-time intensity enhancement at pH 4) since the electron transfer from DA to SiNPs; while the PL intensity decreases sharply when pH > 7.0 (quenching efficiency can be up to ∼50% at pH 10), because the electron transfer from SiNPs to DA.23,24 On the contrary, the fluorescence intensity of reference RBITC linked to SiNPs changes slightly at λex = 543 nm (λem from ∼570 to ∼650 nm, maximum λem of ∼575 nm) (see inset in Figure 2B). The corresponding titration curve of ratio signal R (I465/I575) is given in Figure 2C, showing a good ratio signal linearity in wide pH range 4.0−10.0 (linear equation: R = 2.27−0.17 pH, correlation coefficient r2 = 0.998). The reversibility of fluorescent SiNPs sensors is then assessed. As indicated in Figure 2D, the fluorescence intensity of sensors increases evidently with the addition of HCl, while fluorescence rapidly recoveries upon the addition of NaOH.

Figure 2. (A) Plots of the fluorescence intensity ratio (R = I465/I575) versus pH values for 0.48 mg/mL DMSiNPs prepared with different molar feed ratios from 1:4 to 1:71 (RBITC/DA). I465 is recorded with excitation at 405 nm; I575 is recorded with excitation at 543 nm.(B) PL spectra of 0.48 mg/mL DMSiNPs prepared with the molar feed ratios of 1:24 (RBITC/DA) in PBS buffers at various pH values with λex= 405 nm. Inset shows PL spectra at the same conditions with λex = 543 nm. (C) Corresponding linear relationship between R and pH values in the range of 4−10. (D) pH reversibility assess of DMSiNPs between pH 4 and 10. The cycles were repeated for six times.

The reversible cycle between pH 4 and pH 10 can be repeated 6 times under the same conditions. The good selectivity of fluorescent SiNPs sensors against intracellular species (Figure S8) and redox substances (Figure S9) is also examined. As depicted in Figures S8 and S9, both intracellular species and redox substances scarcely influence the fluorescence intensity ratio R, suggesting the effects of these substances on the developed pH sensor can be neglected. We next evaluate the cytotoxicity of DMSiNPs. As revealed in Figure 3, no obvious morphological change of HeLa or MCF-7 cells is observed after incubated with various concentrations of DMSiNPs for 24 h. The cell viability of DMSiNPs-treated HeLa or MCF-7 cells determined by CCK-8 (cell counting kit-8) assay remains above 95%, indicating DMSiNPs are noncytotoxic or low-cytotoxic, which is ascribed to favorable biocompatibility of silicon.30−39 To further interrogate the intracellular distribution of DMSiNPs, DMSiNPs loaded cell lines are labeled with Lysotracker Green DND-26, a specific commercial lysosome dye with green fluorescence. On the basis of direct visual colocalization images presented in Figure 4, blue (SiNPs) and red (RBITC) fluorescence of DMSiNPs is observed within the cell, respectively, both of which are well overlapped with green fluorescence originated from Lyso-tracker with a high Pearson’s colocalization coefficient (Rr) of ∼0.90. This phenomenon clearly demonstrates Lyso-tracker could specifically assess into lysosome, indicating high specificity of the DMSiNPs. These attractive features including wide linear range, excellent reversibility, good selectivity, negligible cytotoxicity, and lysosome-targeting ability, provide feasibility of fluorescent SiNPs sensors for real-time sensing dynamic changes of lysosomal pH values in live cells. Long-Term and Real-Time Measurement of Lysosomal pH in Live Cells. In this section, we further employ the resultant fluorescent SiNPs sensors for quantitatively determining lysosomal pH values in live cells via the calibration 9238

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Figure 3. Cytotoxicity assessment of DMSiNPs. Morphology of HeLa (A) and MCF-7 (B) cells treated with DMSiNPs of different concentrations (0.03, 0.06, 0.12, 0.24, 0.48 mg/mL) for 0.5, 1, 3, 6, 12, and 24 h. (C) Corresponding cell viability of HeLa and MCF-7 cells treated with DMSiNPs for 24 h. The viability of the control cells was considered 100%. All error bars represent the standard deviation determined from three independent assays.

experiments. Typically, the lysosomal pH is homogenized to culture medium according to a well-established protocol.10−13 As depicted in Figure 5A for live cell imaging, the red fluorescence intensity of RBITC channel (first row, λex = 543 nm, λem = 570−625 nm) is nearly unchanged in the range of pH 4.0−10.0. In sharp contrast, the blue fluorescence intensity of SiNPs channel (second row, λex = 405 nm, λem = 425−500 nm) gradually decreases along with the increase of pH value. Figure 5B shows that there exhibits a linear response to pH change in broad range of pH 5.0−10.0, which covers most physiological pH values (R = 3.66−0.36 pH, r2 = 0.997). Similar results are achieved in MCF-7 cells treated with the DMSiNPs (Figure S10), and a good linear calibration curve is obtained in the pH value from 4.0 to 9.0 (R = 2.71−0.24 pH, r2 = 0.991), as shown in Figure S11. It is worthwhile to point out that, compared to relatively poor photostability of Lyso-tracker, the as-prepared fluorescent and photostable SiNPs sensors are especially suitable for longterm monitoring dynamic change of lysosomal pH, stemmed from their strong stability against photobleaching. As presented in Figure 6, the green fluorescent signals of Lyso-tracker distributed in HeLa cells quickly quench in less than 5 min of

Figure 4. Confocal images of intracellular distribution of DMSiNPs in live HeLa and MCF-7 cells. The cell lines are loaded with 0.48 mg/mL DMSiNPs at 37 °C for 24 h, followed by incubation with Green DND26 (Lyso-tracker) (125 nM) for 20 min. Blue, DMSiNPs fluorescence collected at 425−500 nm with excitation at 405 nm; red, DMSiNPs fluorescence collected at 570−625 nm with excitation at 543 nm; green, Lyso-tracker fluorescence collected at 510−560 nm with excitation at 476 nm through a 63× 1.4 NA objective; purple, merged signal. Scale bars in HeLa cells represent 7.5 μm and scale bars in MCF-7 cells represent 10 μm.

Figure 5. Intracellular pH calibration via DMSiNPs-based pH sensors. (A) LSCFM images of DMSiNPs in HeLa cells at pH values of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. The DMSiNPs-(0.48 mg/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 red fluorescence channel is collected at 570−625 nm with excitation at 543 nm and the blue fluorescence channel is collected at 425−500 nm with excitation at 405 nm through a 63× 1.4 NA objective. Scale bars, 25 μm. (B) Corresponding histograms of the fluorescence intensity ratio (R) of SiNPs at 465 nm to RBITC at 575 nm (R = I465/I575) versus lysosomal pH values ranging from 4 to 10. Inset presents the linear relationship between R and pH values in the range of 5 to 10. The error bars show the standard deviation determined from three independent measurements. 9239

DOI: 10.1021/acs.analchem.6b02488 Anal. Chem. 2016, 88, 9235−9242

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Figure 6. Photostabilitiy comparison of Lyso-tracker and DMSiNPs in live HeLa cells under the continual excitations over 40 min. (A) Lysotracker channel (λex = 476 nm, λem = 510−560 nm). (B) Red DMSiNPs channel (λex = 543 nm, λem = 570−625 nm). (C) Blue DMSiNPs channel (λex = 405 nm, λem = 425−500 nm). Scale bars, 5 μm.

UV irradiation (e.g., the intensity decreases by ∼90% after 5 min UV irradiation); in sharp contrast, for the DMSiNPs, both red and blue fluorescent signals are well retained after 40 min continual UV irradiation under the same conditions (e.g., red fluorescence intensity loses by ∼7% after 40 min continual UV irradiation; blue fluorescence intensity drops by ∼9% after 40 min continual UV irradiation). These above merits suggest this kind of fluorescent SiNPs sensors as new high-performance sensing platform, capable of long-term measurement of lysosomal pH fluctuations in vitro. Nigericin, as a common K+/H+ antiporter in secondary active transport, has been widely used for modulation of pHi.10−13 On the basis of the generally accepted alternating-access mechanism, it allows K+/H+ to move along or against electrochemical proton gradient, which could simultaneously coordinate membrane potential and transmembrane pH gradient.6−9 However, unraveling the detailed action process is still handicapped by a lack of highquality pH sensors suitable for quantitatively detecting pHi change in real-time, long-term, and wide-range fashions. To verify the feasibility of fluorescent SiNPs sensors for dynamic observation of lysosomal pH changes, live-cell imaging is further performed to visualize the lysosomal alkalization process when the DMSiNPs-treated cell lines are incubated with high K+ buffer (pH 9.0) in the presence of 10.0 μM of nigericin.10−13 Afterward, the cell lines are subject to continuous excitation up to 30 min under the microscopy. Compared to the nearly unchanged fluorescence in RBITC channels, the fluorescence in SiNPs channels observed from snapshot images of live HeLa (Figure 7) and MCF-7 (Figure S12) cells treated with DMSiNPs gradually quenches with the time increases. To further gain deep insight into the dynamic change of nigericin-mediated pH, the fluorescence intensity ratios (R) and corresponding lysosomal pH values of cells at various periods are plotted against time. As quantitatively depicted in Figure 8, the original lysosomal pH values of HeLa cells are found to be ∼5.45, and can raise to ∼8.74 within ∼30 min treated by high K+ buffer (pH 9.0) containing nigericin, which is consistent with previously reported lysosomal alkalization process.13,20 Interestingly, the whole process can be further subdivided into two phases characterized by pH growth rates, those are alkalization lag phase (designated as Stage I), and logarithmic growth phase (designated as Stage II). Particularly, in Stage I, lysosomal pH displays random fluctuations with relatively low amplitudes during the first ∼720 s (average pH growth rate (K1

Figure 7. Snapshot images of DMSiNPs in HeLa cells clamped at different time. The images of DMSiNPs are, respectively, collected at 425−500 nm with excitation at 405 nm and at 570−625 nm with excitation at 543 nm through a 63× 1.4 NA objective. Scale bars, 10 μm.

Figure 8. Scatter plots of fluorescence intensity ratio (R) (blue) of SiNPs at 465 nm to RBITC at 575 nm (R = I465/I575) and lysosomal pH values (red) in HeLa cells versus time. The whole process can be divided into two stages: alkalization lag phase (Stage I), and logarithmic growth phase (Stage II) according to pH growth rates of K1 (ΔpH/Δtime), 9.44 × 10−4 units/s in Stage I and K2, 2.41 × 10−3 units/s in Stage II.

= ΔpH/Δtime), 9.44 × 10−4 units/s), indicating negligible effect of nigericin on electrochemical proton gradient. In Stage II, lysosomal pH progressively increases from ∼6.14 to ∼8.74 9240

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Analytical Chemistry (K2, 2.41 × 10−3 units/s), very close to the pH value of external medium. Similarly, the typical two stages of alkalization lag phase (Stage I) and logarithmic growth phase (Stage II) are also observed in MCF-7 cells (Figure S13). In Stage I, lysosomal pH changes around the original pH value of ∼4.50 within a range of ∼0.73 pH units during the first ∼870 s (K1, 8.39 × 10−4 units/s). In Stage II, lysosomal pH sharply increases from ∼5.23 to ∼8.38 in the following ∼930 s (K2, 3.40 × 10−3 units/s). Such significant pH rising is attributed to the enhanced effect of nigericin, leading to a high K+/H+ transporting rate and the destruction of the membrane potential and transmembrane pH gradient.6−9,13

ACKNOWLEDGMENTS



REFERENCES

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CONCLUSIONS In summary, we herein present a class of high-quality pH sensor constructed by using fluorescent SiNPs as new optical labels. Of particular significance, such a kind of fluorescent SiNPs-based pH sensors simultaneously feature broad-pH range response (pH value, 4−10), strong fluorescence, robust photostabiltiy, lysosome-targeting ability, and negligible cytotoxicity. Notably, the developed sensor maintains stable and strong fluorescence during 40 min continual observation under UV irradiation, which is in sharp contrast to rapid fluorescence quenching of Lyso-tracker in 5 min under the same experimental conditions. Therefore, dynamic process of lysosomal pH fluctuations can be sensitively and specifically recorded in real-time and longterm manners to take advantage of these unique merits of SiNPs-based sensors, revealing two consecutive stages (i.e., alkalization lag phase and logarithmic growth phase) during the kinetic action process of K+/H+ antiporter. Our findings suggest this kind of fluorescent and photostable SiNPs-based sensor as novel high-performance sensing platform and provide invaluable information for understanding intracellular behaviors. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02488. TEM image and corresponding size distribution of SiNPs (Figure S1), DLS of SiNPs and DMSiNPs (Figure S2); FT-IR spectra of DA, RBITC, SiNPs, and DMSiNPs (Figure S3); UV−vis absorption spectra of SiNPs, RBITC, DA, and DMSiNPs (Figure S4); zeta potentials of SiNPs, RBITC, DA, and DMSiNPs (Figure S5); effects of oxygen on pH response of DMSiNPs (Figure S6); storage stability of DMSiNPs (Figure S7); interference study on intracellular species (Figure S8) and redox substances (Figure S9); LSCFM images of DMSiNPs in MCF-7 cells at different pH values (Figure S10) and corresponding pH calibration (Figure S11), and long-term real-time lysosomal pH measurement in live MCF-7 cells (Figures S12 and S13) (PDF)





This work was supported by National Basic Research Program of China (973 Program Grants 2013CB934400 and 2012CB932400), NSFC (Grants 61361160412, 51072126, and 21575096), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and China Postdoctoral Science Foundation (Grants 7131701914), as well as Collaborative Innovation Center of Suzhou Nano Science and Technology (NANO−CIC).





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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Fax: 86-512-65880946. Author Contributions †

These authors B.C. and H.W. contributed equally.

Notes

The authors declare no competing financial interest. 9241

DOI: 10.1021/acs.analchem.6b02488 Anal. Chem. 2016, 88, 9235−9242

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DOI: 10.1021/acs.analchem.6b02488 Anal. Chem. 2016, 88, 9235−9242