Silica-Coated S2–-Enriched Manganese-Doped ZnS Quantum Dots

ARTICLE pubs.acs.org/ac

Silica-Coated S2
-Enriched Manganese-Doped ZnS Quantum Dots as a Photoluminescence Probe for Imaging Intracellular Zn2+ Ions Hu-Bo Ren,† Bo-Yue Wu,† Jia-Tong Chen,‡ and Xiu-Ping Yan*,† † ‡

Research Center for Analytical Sciences, College of Chemistry, and State Key Laboratory of Medicinal Chemical Biology, and Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China

bS Supporting Information ABSTRACT: Detection of intracellular Zn2+ has gained great attention because of its biological significances. Here we show the fabrication of silica-coated S2
-enriched Mn-doped ZnS quantum dots (SiO2
S
Mn
ZnS QDs) by enriching S2
with a silica shell on the surface of Mn-doped ZnS QDs via a sol
gel process for imaging intracellular Zn2+ ions. The developed probe gave a good linearity for the calibration plot (the recovered PL intensity of the SiO2
S
Mn
ZnS QDs against the concentration of Zn2+ from 0.3 to 15.0 μM), excellent reproducibility (1.2% relative standard deviation for 11 replicate measurements of Zn2+ at 3 μM), and low detection limit (3s; 80 nM Zn2+). The SiO2
S
Mn
ZnS QDs showed negligible cytotoxicity, good sensitivity, and selectivity for Zn2+ in a photoluminescence turn-on mode, being a promising probe for photoluminescence imaging of intracellular Zn2+.

Z

inc as the second most abundant transition metal is wellknown to play diverse significant roles in biological systems.1
4 Trace zinc is essential, while excessive zinc is toxic. Disrupted patterns of intracellular Zn2+ accumulation have been found in the patients with Alzheimer’s desease, diabetes, and cancer.5
7 Due to the biological significances, detection of intracellular Zn2+ has gained great attention.8
15 Although the typically used destructive methods such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry, and ion chromatography have been widely employed to detect Zn2+,16
18 nondestructive analytical and in situ methods are of great significance for monitoring intracellular Zn2+. The photoluminescence (PL) imaging of Zn2+ is such an approach due to its high selectivity and spatial resolution via photoluminescence microscopy.19
23 However, Zn2+ does not give any spectroscopic signals due to the 3d104s2 electronic configuration. Thus, various small molecule fluorescence-based probes have been synthesized for PL imaging of Zn2+.8
15,19
23 Some of these probe molecules can be used to visualize Zn2+ in living cells and to clarify some of the biological functions of Zn2+ especially in neuronal cells with a high concentration of free Zn2+.8
15,19
23 Nevertheless, it is still highly desirable to fabricate simple PL probes for imaging intracellular Zn2+. Among the typically used PL probes, quantum dots (QDs) have been investigated extensively due to their bright photoluminescence, broad ultraviolet (UV), narrow emission, and high photostability.24
29 Recently, CdTe QDs were reported as the fluorescence probe for detecting Cd2+ or Zn2+ in water samples on the basis of the effective quenching of the initial bright fluorescence of CdTe QDs by adding S2
solution and subsequent recovering of the quenched fluorescence in the presence of Zn2+ (or Cd2+).30 Although the probe of CdTe QDs with such a procedure is applicable for detecting Cd2+ or Zn2+ in water samples, it is not suitable for imaging intracellular Zn2+, because not only the r 2011 American Chemical Society

probe possesses potential toxicity from toxic Cd2+ leached from the CdTe QDs but also such a procedure is difficult to apply to intracellular imaging. Here we report the fabrication of a simple silica-coated S2
enriched Mn-doped ZnS QDs (SiO2
S
Mn
ZnS QDs) probe for imaging intracellular Zn2+. Mn-doped ZnS QDs were chosen because of excellent PL properties and a nontoxic nature.31
35 The prepared probe offers high sensitivity and selectivity for Zn2+ at physiological pH by taking advantage of the surface defects of the SiO2
S
Mn
ZnS QDs. To the best of our knowledge, no work on the utilization of the surface defects of QDs for imaging metal ions has been reported so far. The developed SiO2
S
Mn
ZnS QDs not only give negligible cytotoxicity but also offer a low fluorescence background. In addition, the silica-coating on the enriched S2
on the surface of Mn-doped ZnS QDs effectively avoids the leakage of S2
, enhances the biocompatibility and stability, and stimulates the cell endocytosis of the probe. All of the above characteristics make the probe promising for imaging intracellular Zn2+ with a low detection limit.

’ EXPERIMENTAL SECTION Materials and Reagents. All reagents used were of at least analytical grade. Ultrapure water (18.2 MΩ cm) obtained from a WaterPro water purification system (Labconco Corp., Kansas City, MO, USA) was used throughout. 3-Mercaptopropionic acid (MPA, 99%) was purchased from Aladdin Co. (Shanghai, China). Zinc acetate dihydrate (Zn(CH3COO)2 3 2H2O) and zinc sulfate (ZnSO4 3 7H2O) were purchased from Tianjin Kaitong Chemicals Co. (Tianjin, China). Manganese(II) chloride Received: August 2, 2011 Accepted: September 13, 2011 Published: September 13, 2011 8239

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Analytical Chemistry tetrahydrate (MnCl2 3 4H2O) was purchased from the Second Chemicals Co. of Shenyang (Shenyang, China). Sodium sulfide nonahydrate (Na2S 3 9H2O) was purchased from Tianjin Sitong Chemicals Co. (Tianjin, China). Sodium phosphate dibasic anhydrous (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), ethyl orthosilicate (TEOS (C2H5O)4Si), CH3COOH, CH3COONa, and tris(hydroxymethyl)aminomethane (Tris) were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Tris-HCl buffer solutions (0.01 M Tris, pH from 7.0 to 10.0) and CH3COOH
CH3COONa buffer solutions (0.01 M, pH from 4.0 to 6.0) were used in the experiments. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma (St. Louis, MO, USA). Instrumentation. The morphology and microstructure of the QDs were characterized by a Philips Tecnai G2 F20 (Philips, Eindhoven, The Netherlands) field emission high-resolution transmission electron spectroscopy (HRTEM). The samples for HRTEM were obtained by drying sample droplets from water dispersion onto a 300-mesh Cu grid coated with a lacey carbon film, which was then allowed to dry prior to imaging. The X-ray diffraction (XRD) spectra were collected on a Rigaku D/max2500 X-ray diffractometer (Rigaku, Tokyo, Japan) with Cu Kα radiation. The X-ray photoelectron spectroscopy (XPS) measurements were carried out on a Kratos Axis Ultra DLD spectrometer fitted with a monochromated Al Kα X-ray source (hν = 1486.6 ev), hybrid (magnetic/electrostatic) optics, and a multichannel plate and delay line detector. Absorption spectra were recorded on a UV-3600 UV
vis
near-IR spectrophotometer (Shimadzu, Kyoto, Japan). The photoluminescence measurements were performed on an F-4500 spectrofluorometer (Hitachi, Tokyo, Japan) equipped with a plotter unit and a quartz cell (1 cm  1 cm). Photoluminescent measurements were taken at an excitation wavelength of 316 nm. The slit width was 10 and 20 nm for excitation and emission, respectively. The photomultiplier tube (PMT) voltage was set at 700 V. In vitro cytotoxicity of the QDs was assessed using the MTT assay; the absorbance was measured at 570 nm using the Multiskan Ascent (Thermo Labsystems, Franklin, MA, USA). The images were obtained with an OLYMPUS BX-41 fluorescence microscope system (Olympus Optical Co. Ltd., Tokyo, Japan) with blue excitation sources. Synthesis of Manganese-Doped ZnS QDs. The highly luminescent Mn-doped ZnS QDs were synthesized in an aqueous solution using MPA as the stabilizer on the basis of a published procedure with minor modification.36 Briefly, 0.174 mL of MPA, 5 mL of 0.1 M ZnSO4, and 2 mL of 0.01 M MnCl2 were added into a 100 mL three-necked flask, and the final volume of the mixture was adjusted to 50 mL with ultrapure water. The mixed solution was adjusted to pH 10 with 1.5 M NaOH and stirred under argon at room temperature for 30 min. After stirring, 7.5 mL of 0.1 M Na2S 3 9H2O was quickly injected into the mixture solution and stirred for 20 min. The solution was aged at 50 °C under air for 2 h to form MPA-capped Mn-doped ZnS QDs. For purification, the obtained Mn-doped ZnS QDs was precipitated with 50 mL of ethanol, separated by centrifuging, and dried under vacuum at room temperature. The highly soluble Mn-doped ZnS QDs were obtained. Synthesis of SiO2
S
Mn
ZnS QDs. The weakly luminescent SiO2
S
Mn
ZnS QDs were prepared by enriching S2
with a silica shell on the surface of Mn-doped ZnS QDs via the sol
gel reaction of TEOS37
39 to avoid the leakage of the S2
‑ during purification, to enhance the biocompatibility and stability,

ARTICLE

and to stimulate the cell endocytosis of QDs.40,41 Typically, 30 mg of Mn-doped ZnS QDs was dispersed in 25 mL of ultrapure water in a three-necked flask, then 2 mL of 0.1 M Na2S 3 9H2O was added, the mixture (pH 11) was stirred for 30 min at ambient temperature, and 0.5 mL of TEOS was then added under stirring. The suspension was kept stirred for 12 h. The resultant mixture was transferred into 25 mL of ethanol and centrifuged. The collected solid was washed with ethanol/water and dried with argon to the SiO2
S
Mn
ZnS QDs. The final SiO2
S
Mn
ZnS QDs was protected with argon and stored at 4 °C. Cytotoxicity Assay. The MTT assay was used to assess the cytotoxicity of SiO2
S
Mn
ZnS QDs.42 In principle, MTT can be reduced by mitochondrial dehydrogenases in living cells to purple formazan precipitates. The absorption of dissolved formazan in the visible region correlates with the number of intact alive cells. Cytotoxic compounds are able to damage and destroy cells and thus decrease the reduction of MTT to formazan. To measure the cytotoxicity of SiO2
S
Mn
ZnS QDs, the human hepatocellular liver carcinoma (HepG2) cell line and mouse embryo fibroblast (Balb/3T3 ClA31-1) cell line were respectively cultured in 96-well plates with a concentration of 3000 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine plasma in 5% CO2 at 37 °C overnight to get a suitable density (70
80% confluence). Then, the old DMEM medium was replaced by a fresh DMEM medium in the presence of different concentrations of SiO2
S
Mn
ZnS QDs or in the absence of SiO2
S
Mn
ZnS QDs (for control experiments) to incubate the cell lines for 24 h. A 1 mg mL
1 amount of MTT in phosphate-buffered saline (PBS; 0.01 M, pH 7.40) was mixed with the above incubated cells in the wells with a resulting dilution factor of 1:4, and the mixture was incubated for 4 h under cell culture conditions (37 °C, 5% CO2). The medium was then aspirated from each well, and then 150 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the purple formazan crystals, followed by a 10 min incubation. The absorbance of the formed formazan (I) was measured on an ELISA plate reader at a wavelength of 570 nm. The cell viability was defined as the ratio of the absorbance in the presence of SiO2
S
Mn
ZnS QDs to that in the absence of SiO2
S
Mn
ZnS QDs (control; Isample/Icontrol). Procedures for Cell Imaging. The HepG2 cell line suspensions were plated at a density of 4  104 cells mL
1 on 20  20 mm2 glass coverslips and cultured in 6-well plates in DMEM with 10% fetal bovine plasma in 5% CO2 at 37 °C overnight to get a suitable density. A 100 μM amount of Zn(CH3COO)2 was added to four of six wells, and then the cells were incubated in the presence of Zn2+ for 20 h to prepare Zn2+-uptaken HepG2 cells. The cells in the other two wells in the absence of Zn2+ were also incubated for 20 h for comparison. Before in vitro imaging experiments, the old DMEM medium was replaced by a fresh serum-free medium containing 0.8 g L
1 SiO2
S
Mn
ZnS QDs to incubate the cells under the condition of serum starvation for 4 h. The DMEM with 20% fetal bovine plasma was added to the cells for additional 12 h incubation. The resulting cells were washed with PBS buffer (0.01 M, pH 7.40) three times before imaging. To illustrate the serum starvation stimulated the cell endocytosis of SiO2
S
Mn
ZnS QDs, the Zn2+-uptaken HepG2 cells treated with SiO2
S
Mn
ZnS QDs without serum starvation were also prepared by replacing the old DMEM medium with a fresh DMEM medium containing 10% fetal bovine plasma and 0.8 g L
1 SiO2
S
Mn
ZnS QDs to incubate the cells for 16 h. 8240

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Figure 1. (A) XRD patterns of Mn-doped ZnS QDs (curve 1) and SiO2
S
Mn
ZnS QDs (curve 2). (B) XPS spectra of Mn 2p for the Mn-doped ZnS QDs. (C) HRTEM image of Mn-doped ZnS QDs. (D) HRTEM image of SiO2
S
Mn
ZnS QDs.

Figure 2. Effect of pH on the photoluminescence intensity of SiO2
S
Mn
ZnS QDs (230 mg L
1) and the restored photoluminescence intensity (ΔI) of SiO2
S
Mn
ZnS QDs with 3 μM Zn2+.

The resulting cells were washed with PBS buffer (0.01 M, pH 7.40) three times before imaging.

’ RESULTS AND DISCUSSION The as-prepared Mn-doped ZnS QDs and SiO2
S
Mn
ZnS QDs were characterized by HRTEM, XRD and XPS. The XRD patterns of the Mn-doped ZnS QDs and SiO2
S
Mn
ZnS QDs exhibit a cubic structure (or zinc blende) with peaks for (111), (220), and (311) planes (Figure 1A). The XPS spectra of the Mn-doped ZnS QDs show the peak of Mn 2p and suggest the successful doping of Mn in ZnS QDs (Figure 1B). The very broad XRD peak at low diffraction angle indicates amorphous silica peak39 and suggests the formation of amorphous silica on SiO2
S
Mn
ZnS QDs (Figure 1A, curve 2). The HRTEM results show the Mn-doped ZnS QDs and SiO2
S
Mn
ZnS QDs with spherical shape and almost uniform size in diameter of about 4 and about 20 nm, respectively

(Figure 1C,D). HRTEM images also indicate that the Mn-doped ZnS QDs were embedded in amorphous materials (SiO2; Figure 1D). The effect of pH (from 4.0 to 10.0) on the photoluminescence intensity of SiO2
S
Mn
ZnS QDs was investigated. The results indicated that the SiO2
S
Mn
ZnS QDs intensity was stable in the range of pH 7.0
8.5, which was suitable for further usage in the physiological environment. Furthermore, the effect of pH on the photoluminescence recovery of SiO2
S
Mn
ZnS QDs with 3 μM Zn2+ was also studied (Figure 2). The results show the best recovery of SiO2
S
Mn
ZnS QDs intensity was at pH 7.5. To make SiO2
S
Mn
ZnS QDs feasible and biocompatible for further utilization, pH 7.5 was selected. In addition, the PL intensity of the SiO2
S
Mn
ZnS QDs is stable at pH 7.5 for 16 h (Figure S1 in the Supporting Information). However, the fluorescence activation of the probe in the presence of Zn2+ shows a strong pH dependence, which may make it difficult to use the probe to accurately quantify cellular zinc concentrations if the probe is likely internalized into acidic lysosomal vesicles where pH values can be as low as pH 4.5. Schematic illustration for the fabrication and application of SiO2
S
Mn
ZnS QDs as a turn-on PL probe for Zn2+ is shown in Scheme 1. To evaluate the role of S2
in the synthesis of weakly photoluminescent SiO2
S
Mn
ZnS QDs, we tested the effect of the concentration of S2‑ on the PL of Mn-doped ZnS QDs in 10 mM Tris-HCl buffer solution at pH 7.5. The PL of Mn-doped ZnS QDs significantly decreased as the concentration of S2
increased (Figure S2 in the Supporting Information). Moreover, the PL of the Mn-doped ZnS QDs did not change upon coating of the silica shell alone, but was significantly quenched by enriching S2
(Figure S3 in the Supporting Information). The above results confirm the significant role of S2
in the PL quenching of the Mn-doped ZnS QDs because of an increase of 8241

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ARTICLE

Scheme 1. Schematic Illustration for the Fabrication and Application of SiO2
S
Mn
ZnS QDs as a Turn-on PL Probe for Zn2+

Figure 4. Effect of coexisting ions on the enhancement of photoluminescence of SiO2
S
Mn
ZnS QDs (230 mg L
1) in 10 mM Tris-HCl buffer solution at pH 7.5. From left to right: Zn2+ (3 μM) alone (control); Zn2+ (3 μM) in the presence of one of the following ions, Na+ (150 mM), K+ (150 mM), Mg2+ (5 mM), Ca2+ (5 mM), Al3+ (36 μM), Fe3+ (24 μM), Cu2+ (3 μM), Mn2+ (1 μM), Cd2+ (1 μM), Ni2+ (0.5 μM), Co2+ (0.3 μM), or Hg2+ (0.3 μM). ΔI and I0 denote the enhanced photoluminescence intensity in the presence of metal ions and the luminescence intensity of SiO2
S
Mn
ZnS QDs in the absence of metal ions, respectively.

Figure 3. (A) UV
vis absorption spectra of Mn-doped ZnS QDs (red curve), SiO2
S-Mn-ZnS QDs (black curve) and SiO2
S
Mn
ZnS QDs added Zn2+ (blue curve). (B) Decay curves of the PL emission of Mn
ZnS QDs (0.1 g L
1; blue curve), SiO2
S
Mn
ZnS QDs (2.45 g L
1) before (red curve) and after addition of 45 μM Zn2+ (black curve) in 10 mM Tris-HCl buffer solution at pH 7.5. The three averaged scans of PL emission at 595 nm (300 channels) were recorded under excitation of a N2 laser at 337 nm. (C) Effect of the concentration of Zn2+ on the PL spectra of the SiO2
S
Mn
ZnS QDs (230 mg L
1). (D) Plots of the enhanced PL at 595 nm (λex = 316 nm; ΔI) as a function of the concentration of Zn2+ (CZn) from 0.3 to 15 μM.

the dangling bonds originating from the lone pairs on surface S2
.30,43,44 Addition of Zn2+ to the solution of SiO2
S
Mn
ZnS QDs led to a red shift of the UV
vis absorption spectra (Figure 3A) and enhancement of the PL of the QDs (Figure 3C) because of the elimination of the S2
dangling bonds as a result of the adsorption of Zn2+ on the surface of the QDs. The adsorption of Zn2+ on the surface of the QDs can effectively prevent the nonradiative relaxation pathways, thereby enhancing the PL intensity.30,45,46 Comparison of the PL lifetimes of the Mn-doped ZnS QDs, the SiO2
S
Mn
ZnS QDs alone, and the SiO2
S
Mn
ZnS QDs in the presence of Zn2+ reveals significant changes of the decay curves of the emission at 595 nm. The much shorter average PL lifetime of the SiO2
S
Mn
ZnS QDs (250 μs) than the Mn-doped ZnS QDs (510 μs; Figure 3B, Table S1 in the Supporting Information) indicates that the addition of S2
resulted in an increased nonradiative decay of

the QDs. Furthermore, the addition of Zn2+ increased the average PL lifetime of the SiO2
S
Mn
ZnS QDs up to 420 μs (Figure 3B, Table S1), thus activating some quenched PL centers. Addition of higher concentration of Zn2+ to the solution of SiO2
S
Mn
ZnS QDs did not induce the aggregation of SiO2
S
Mn
ZnS QDs (Figure S4 in the Supporting Information). The fabricated SiO2
S
Mn
ZnS QDs show good selectivity as a turn-on PL probe for Zn2+ (Figure 4). The Zn2+ (3 μM)-induced PL enhancement of the SiO2
S
Mn
ZnS QDs was not affected by Na+ (150 mM), K+ (150 mM), Mg2+ (5 mM), Ca2+ (5 mM), Al3+ (36 μM), Fe3+ (24 μM), Cu2+ (3 μM), Mn2+ (1 μM), Cd2+ (1 μM), Ni2+ (0.5 μM), Co2+ (0.3 μM), and Hg2+ (0.3 μM), even though the tested concentrations of these ions are much higher than their average levels in cells (Table S2 in the Supporting Information).47
50 In addition, under the optimal condition (pH 7.5; Figure 2), the developed probe gave a good linearity for the calibration plot (the recovered PL intensity of the SiO2
S
Mn
ZnS QDs against the concentration of Zn2+ from 0.3 to 15.0 μM with a correlation coefficient R2 = 0.998; Figure 3C,D), excellent reproducibility (1.2% relative standard deviation for 11 replicate measurements of Zn2+ at 3 μM), and low detection limit (3s; 80 nM Zn2+). The detection limit is better than or comparable to those of some previous fluorescent probes for Zn2+ (Table S3 in the Supporting Information). To demonstrate the availability of the prepared SiO2
S
Mn
ZnS QDs for bioimaging applications, the SiO2
S
Mn
ZnS QDs were employed to monitor the intracellular Zn2+ in HepG2 cells. Fluorescence microscopic studies showed that incubation of the HepG2 cells without uptaking Zn2+ and the SiO2
S
Mn
ZnS QDs (0.8 g L
1) with 4 h serum starvation led to very weak intracellular photoluminescence (Figure 5A), whereas incubation of the Zn2+ (100 μM)-uptaken HepG2 cells and the SiO2
S
Mn
ZnS QDs (0.8 g L
1) with 4 h serum starvation gave a significant increase in the photoluminescence (Figure 5C). The corresponding bright-field measurements after treatment with Zn2+ and SiO2
S
Mn
ZnS QDs confirm that the photoluminescence was enhanced in the intracellular area (Figure 5C0 ), indicating the Zn2+ was internalized into the living 8242

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the cytotoxicity of SiO2
S
Mn
ZnS QDs is negligible as confirmed by the facts that no significant changes in cell morphology (Figure 5C0 ) and cell viability after the treatments of HepG2 cells with SiO2
S
Mn
ZnS QDs were observed (Figure 6).

’ CONCLUSIONS In summary, we have fabricated SiO2
S
Mn
ZnS QDs as a PL probe for imaging intracellular Zn2+. The developed PL probe gives good biocompatibility and selectivity, enhanced cell endocytosis, and negligible cytotoxicity, being promising as a PL probe for imaging intracellular Zn2+. ’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Fax: (86) 22-23506075. E-mail: [email protected].

Figure 5. Fluorescence images (A
C) and their corresponding brightfield transmission images (A0
C0 ; 400): (A, A0 ) HepG2 cells were incubated with SiO2
S
Mn
ZnS QDs (0.8 g L
1) in a fresh serumfree medium (i.e., serum starvation condition) for 4 h and then in a fresh serum-containing medium (i.e., without serum starvation) for 12 h. (B, B0 ) HepG2 cells were incubated with Zn2+ (100 μM) in a fresh serum-containing medium for 20 h; the collected Zn2+-uptaken HepG2 cells were then incubated with SiO2
S
Mn
ZnS QDs (0.8 g L
1) in a fresh serum-containing medium for 16 h. (C, C0 ) HepG2 cells were incubated with Zn2+ (100 μM) in a fresh serum-containing medium for 20 h; the collected Zn2+-uptaken HepG2 cells were then incubated with SiO2
S
Mn
ZnS QDs (0.8 g L
1) in a fresh serum-free medium (i.e., serum starvation condition) for 4 h and then in a fresh serumcontaining medium (i.e., without serum starvation) for 12 h.

Figure 6. Cell viability in the presence of different concentrations of SiO2
S
Mn
ZnS QDs: (A) 3T3 cells; (B) HepG2 cells.

cells from the growth medium (Table S4 in the Supporting Information).51,52 In contrast, incubation of the SiO2
S
Mn
ZnS QDs and the Zn2+ (100 μM)-uptaken HepG2 cells without serum starvation (Figure 5B,B0 ) showed much weaker PL than the counterparts with 4 h serum starvation (Figure 5C,C0 ). The above results indicate that the serum starvation stimulated the cell endocytosis of the SiO2
S
Mn
ZnS QDs. Furthermore,

’ ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (Grant 2011CB707703), the National Natural Science Foundation of China (Grant 20935001), and the Tianjin Natural Science Foundation (Grant 10JCZDJC16300). ’ REFERENCES (1) Berg, J. M.; Shi, Y. Science 1996, 271, 1081–1085. (2) Takeda, A. Brain Res. Rev. 2000, 34, 137–148. (3) Wang, Z. Y.; Stoltenberg, M.; Jo, S. M.; Huang, L.; Larsen, A.; Dahlstr€om, A.; Danscher, G. NeuroReport 2004, 15, 1801–1804. (4) Frederickson, C. J.; Koh, J. Y.; Bush, A. I. Nat. Rev. Neurosci. 2005, 1–14. (5) Suh, S. W.; Jensen, K. B.; Jensen, M. S.; Silva, D. S.; Kesslak, P. J.; Danscher, G.; Frederickson, C. J. Brain Res. 2000, 852, 274–278. (6) Chausmer, A. B. J. Am. Coll. Nutr. 1998, 17, 109–115. (7) Henshall, S. M.; Afar, D. E. H.; Rasiah, K. K.; Horvath, L. G.; Gish, K.; Caras, I.; Ramakrishnan, V.; Wong, M.; Jeffry, U.; Kench, J. G.; Quinn, D. I.; Turner, J. J.; Delprado, W.; Lee, C.-S.; Golovsky, D.; Brenner, P. C.; O’Neill, G. F.; Kooner, R.; Stricker, P. D.; Grygiel, J. J.; Mack, D. H.; Sutherland, R. L. Oncogene 2003, 22, 6005–6012. (8) Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2000, 122, 12399–12400. (9) Komatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 10197–10204. (10) Kikuchi, K.; Komatsu, K.; Nagano, T. Curr. Opin. Chem. Biol. 2004, 8, 182–191. (11) Nagano, T. Proc. Jpn. Acad., Ser. B 2010, 86, 837–847. (12) Chang, C. J.; Jaworski, J.; Nolan, E. M.; Sheng, M.; Lippard, S. J. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 1129–1134. (13) Chang, C. J.; Nolan, E. M.; Jaworski, J.; Okamoto, K.
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