Article pubs.acs.org/ac
Thiols-Induced Rapid Photoluminescent Enhancement of Glutathione-Capped Gold Nanoparticles for Intracellular Thiols Imaging Applications Xiaoqing Su, Hui Jiang,* and Xuemei Wang* State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, People’s Republic of China S Supporting Information *
ABSTRACT: The rapid detection and imaging of intracellular thiols is of great importance during the occurrence and development of some chronic diseases. Here we demonstrate the rapid thiols-induced photoluminescence (PL) enhancement of the low luminescent glutathione (GSH) stabilized Au nanoparticles, AuGSH (low). The dynamic PL investigation reveals that the PL enhancement fits a first-order reaction model. The X-ray photoelectron spectroscopic and mass spectroscopic results indicate that AuGSH (low) are mainly comprised of “thiols-insufficient” Au species and the additional thiols can efficiently attach to the “unsaturated” surface of Au nanoparticles, accompanied by significant PL enhancement. The noncytotoxic AuGSH (low) probe can be successfully applied for imaging of intracellular thiols. Generally, this work illustrates the great prospects of facile-prepared AuGSH (low) as a candidate for thiols labeling and imaging.
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(Cys) could induce core-etching of lysozyme VI stabilized Au8 NCs and quench their fluorescence, allowing the selective detection for thiols.18 In another work, the red-emitted fluorescence by GSH protected Ag NCs was found to be selectively inhibited by Cys owing to the specific thiol−silver interaction.19 A turn-off thiols sensor is also constructed based on thiols promoted agglomeration of fluorescent polyethylenimine-capped Ag NCs to larger nonfluorescent Ag nanoparticles.20 To obtain enhancement-type sensors of thiols, the metal NCs are first dynamically quenched by heavy metal ions and the further addition of thiols may recover the fluorescence of NCs through their higher affinity to these metal ions. By using bovine serum albumin (BSA) stabilized Au NCs synthesized by a microwave-assisted heat route, Tian et al.21 developed a turnon fluorescent sensor for GSH in both living cells and human blood samples. The presence of Hg (II) leads to a severe quenching (98%) of fluorescence by Au NCs because of the strong metallophilic d10−d10 interaction between Hg(II) and Au(I) on the surface of Au NCs. After introducing GSH to the system, the complexation of GSH and Hg(II) triggers fluorescent enhancement. The sensor shows good selectivity to GSH over Cys. In a very similar work, Park and co-workers22 also used Hg quenched BSA stabilized Au NCs prepared at 37 °C for thiols detection. However, the system showed no GSH selectivity at the same concentration of GSH and Cys. These contradictory results indicate that the preparation conditions of
hiols are essential in various biological processes, especially for maintenance of reductive environments through scavenging intracellular reactive oxygen species during oxidative stress, since unbalanced environments may cause damage to cells and induce undesired cell death.1 As known, the abnormal changes in glutathione (GSH) appear during occurrence of diabetes and HIV infection,2 while Nacetylcysteine (NAC) is relevant to neurodegenerative diseases such as Parkinson’s3 and Alzheimer’s4 disease. Thus, the rapid detection and imaging of biothiols in psychological fluids or cells is of great importance for early monitoring of important diseases. A large amount of methods are proposed for the detection of biothiols, including electrochemical, colorimetric, mass spectroscopic, and fluorescent methods.5 Recently, fluorescent detection has attracted considerable interest.6−8 Conventional thiol probes are organic molecules, which can react with thiol group through nucleophilic substitution,9 disulfide bond cleavage,10 and native chemical ligations.11 Although these reported probes may exhibit good detection selectivity and sensitivity, the fluorophores typically have small Stokes shifts, which is liable to serious excitation background interference.12 Moreover, most organic dyes are sensitive to illumination, which is disadvantageous for long-term observation. To overcome these shortcomings, inorganic probes based on metal−thiol interactions were designed.13,14 Among these probes the luminescent metal nanoparticles, or so-called metal nanoclusters (NCs) are excellent probe candidates, due to their larger Stokes shifts as well as antiphotobleaching ability.15−17 Typically, the metal−thiol interaction leads to efficient fluorescence quench. For examples, GSH and cysteine © XXXX American Chemical Society
Received: April 9, 2015 Accepted: September 14, 2015
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Analytical Chemistry Scheme 1. Illustration of Thiols-Induced Formation of High-Luminescence AuGSH from Low-Luminescence AuGSH
on a FLS920 fluoremeter (Edinburgh, Livingston, U.K.). The X-ray photoelectron spectroscopic (XPS) data were collected on a PHI 5000 VersaProbe X-ray photoelectron spectrometer with an Al Kα = 280.00 eV excitation source. The transmission electron microscopic (TEM) images were obtained on a JEM2100 TEM (JEOL, Tokyo, Japan) with an accelerating voltage of 200 kV. The mass spectroscopy (MS) was measured on a MALDI TOF-TOF 4800 plus mass spectrometer (AB Sciex, Framingham, MA, USA). The MTT assays were performed on a microplate reader (Thermo Scientific). The confocal microscopic imaging was shot on a Zeiss 710 confocal microscopy (Zeiss, Germany). Images were acquired using a 63× oil immersion lens with argon laser set at 488 nm. Preparation and Separation of AuGSH Samples. The precursors for AuGSH (low) samples were prepared by mixing HAuCl4 of 5 mL and GSH of 5 mL at a molar ratio of 1:1 (final concentration, 5.0 mM) in a 15 mL sterile centrifuge tube. After the mixture turned almost colorless in ∼10 min, it was heated at 80 °C for 2 h, without any stirring. The yellow solution was cooled and filtered through a 0.22 μm membrane to remove the possible bulk gold. Then the same volume of absolute ethanol was added to the filtrate. The obtained turbid solution was precipitated by 8000 rpm for 15 min and dried in a desiccator overnight. The weight of the dried sample [AuGSH (low)] was ∼12 mg (yield, ∼ 47%). The AuGSH (low) sample can be stored at 4 °C for at least 6 months. It is not well dispersed in deionized water, but well soluble in 5 mM PBS, pH 7.0. After further addition of GSH, Cys, NAC, and CyA, the obtained samples are denoted as AuGSH (high), AuGSH (Cys), AuGSH (NAC), and AuGSH (CyA), respectively. For time-resolved fluorescence, XPS, TEM, and MS characterizations, all samples were put in the ultrafilters (Pall, molecular weight cutoff (MWCO) of 3 kDa) and centrifuged at 8000 rpm for 15 min to remove the residual small molecules. The samples (MW > 3 kDa) were diluted with deionized water and subjected to further ultrafiltration for three times. Unless
NCs (microwave heating vs 37 °C incubation) may cause great variances in detection performances. Although GSH stabilized Au nanoparticles are intensively studied, their complicated formation processes are still incompletely known. This work proposed a facile and costeffective method for thiols assay. We first prepared a kind of low luminescent glutathione stabilized Au nanoparticles, namely, AuGSH (low). Interestingly, the photoluminescence (PL) of AuGSH (low) was found to increase significantly after further addition of thiols (Scheme 1). The mass spectroscopic results indicated that AuGSH (low) samples are mainly comprised of thiols-insufficient Au species, and the additional thiol ligands can efficiently attach to the surface of Au nanoparticles and induce PL enhancement. The AuGSH (low) probe can be successfully applied for intracellular imaging of biothiols, with great prospects as biomedical labels.
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MATERIALS AND METHODS Reagents and Instruments. Glutathione, Cys, NAC, cysteamine hydrochloride (CyA), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), N-methyl maleimide (NMM), bovine serum albumin (BSA), horseradish peroxidase (HRP), superoxide dismutase (SOD), and urease were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade from Sinoreagent, Shanghai, China. Phosphate buffer (pH 7.0, 10 mM) was prepared by mixing Na2HPO4 and NaH2PO4. The human cervical cancer HeLa cells and human glioma U-87 cells were purchased from Shanghai Institute of Biological Sciences, Chinese Academy of Science. The fetal bovine serum (FBS, Gibco) and Dulbecco’s modified Eagle’s medium (DMEM, high glucose, Hyclone) were filtrated through a 0.22 μm sterile membrane before use. UV−vis spectra were recorded on a Biomate 3S spectrometer (Thermo Scientific Co. Ltd., Waltham, MA, USA). PL spectra were recorded on an RF-5301PC fluoremeter (Shimadzu, Kyoto, Japan). The time-resolved fluorescence was measured B
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Analytical Chemistry specific indication otherwise, all experiments are performed at room temperature. In Vitro Cytotoxicity Assays. The cell viability was evaluated by the conventional MTT assays. The HeLa cells and U-87 cells were trypsinized in the log phase, seeded in 96well plates at a concentration of 1.0 × 104 cells/well, and maintained overnight at 37 °C in an incubator containing a 5% CO2 humidified environment. The AuGSH (low) or AuGSH (high) samples were premixed in DMEM containing 10% (v/v) FBS and streptomycin (0.1 mg/mL)/penicillin (100 U/mL). The cells were treated with different concentrations of the AuGSH samples for 24 h. The control groups were cultivated under the same conditions without AuGSH. After incubation, 20 μL of MTT stock solution of 5 mg/mL was added and incubated for additional 4 h. Subsequently, the supernatant in the wells was discarded, followed by further addition of 150 μL of DMSO into each well and gentle shaking at 37 °C for 10 min. Eventually, the optical density (OD) at 492 nm was scanned by the microplate reader. The cell viability was determined by the following equation:
Figure 1. PL spectra of AuGSH (low) and AuGSH added with GSH, Cys, NAC, or CyA, respectively. Inset: photograph of samples under illumination with a 365 nm UV lamp. From left to right: AuGSH (low), AuGSH (high), AuGSH (Cys), AuGSH (NAC), and AuGSH (CyA), respectively.
We further investigated the PL behaviors of AuGSH (low) after addition of more thiols. To probe the possible influence from groups other than -SH, especially carboxyl and amine groups, four kinds of thiols were used. GSH and Cys have similar main chains, while the amine group of Cys is acetylated in NAC and the carboxyl group of Cys is lost in CyA. Interestingly, after addition of GSH, Cys, NAC, or CyA, the PL increases rapidly, with 5−9-fold enhancement in 10 min, which can be easily distinguished from AuGSH (low) sample under illumination of a UV lamp at 365 nm (Figure 1 inset). There are no significant changes in UV−vis spectra after addition of the thiol compounds, with only a little decrease in absorbance below 450 nm (SI Figure S2). Meanwhile, the effect of molar ratios of precursors (AuCl4−:GSH) on PL were checked (SI Figure S3). After 2 h reaction at 80 °C, the PL appeared at a molar ratio lower than 1:0.8. Similarly, significant PL enhancement was observed at the ratio lower than 1:0.8. Therefore, the formation of low luminescent AuGSH species is dependent on the molar ratios of precursors. In the following experiments, we only use the molar ratio of 1:1 unless specified. The PL enhancement dynamics are recorded at different concentrations of thiols (Figure 2). Addition of all thiols give similar adsorption patterns, implying that thiol groups are responsible for the PL enhancement. For thiols between 0.1 and 0.5 mM, the enhancement ratio, denoted as the ratio between PL intensity after and before addition of thiols, may reach ∼6 for GSH and Cys and 4−5 for NAC and CyA, suggesting that the modification of functional groups may affect the enhancement efficiency. To confirm the key role of the -SH group, the oxidized GSH dimer, namely, GSSG, and another amino acid, phenylamine, were added to AuGSH (low) (SI Figure S4A). Since proteins can induce 3−6-fold PL enhancement of dihydrolipoic acid coated Au nanoparticles due to protein adsorption,27 PL changes by common proteins including BSA, HRP, SOD, and urease, were also investigated here (SI Figure S4B). In all of the preceding cases, little PL changes were observed. Considering that the sizes of most proteins, such as a 3-D size of 14.0 nm × 4.0 nm × 4.0 nm for BSA,28 are larger than that of single Au NC (around 2 nm), its -SH group can hardly bind to Au NCs due to steric hindrance, indicating the insignificant interference from protein thiol groups. Therefore, it can be concluded that AuGSH (low) was
cell viability/% = ([A]test − [A]blank ) /([A]control − [A]blank ) × 100
where [A] represents the absorbance at 492 nm. Confocal Microscopic Imaging. The U87 and HeLa cells were seeded in a 6-wells plate with coverslips and incubated at 37 °C for 10 h. AuGSH (low) samples were added to each well, with a final 100 μg/mL and incubated for an additional 6 h. Then the medium was removed, and the cells were washed three times with PBS (pH 7.4). The cells were immobilized with 500 μL of 4% paraformaldehyde in PBS for 15 min. The excess paraformaldehyde was discarded, and the coverslips were treated with ethanol of 75% for 10 min. The coverslips were maintained at 4 °C in the dark before microscopic observation. The procedures were generally the same for thiol-removing experiments by using NMM, except that the cells were first treated with NMM solution (0.1 mM) for 1 h before loading of AuGSH (low) samples. The gray scale values for individual cells were collected by ImageJ software (1.49v).
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RESULTS AND DISCUSSION
PL Enhancement of AuGSH (Low) by Additional Thiols. The preparation of high-luminescence AuGSH nanoparticles have been well reported in the recent decade. For examples, Zheng and co-workers incubated the mixture of HAuCl4 and GSH at room temperature for 2 weeks to obtain GSH coated Au nanoclusters (NCs).23 Jin’s group used borohydride to accelerate the formation of GSH coated Au NCs.24 Considering that external heat can also accelerate the nucleation of the luminescent AuGSH sample,25,26 here we investigated the possible rapid synthesis of AuGSH at lower temperatures. Weak but distinguishable PL emission only emerges for the mixture of HAuCl4 and GSH (molar ratio of AuCl4−:GSH at 1:1) at a temperature higher than 60 °C for 2 h (Figure S1, Supporting Information (SI)). The AuGSH sample prepared at 80 °C (i.e., AuGSH (low)) still shows low PL, with optimized excitation and emission wavelengths at ∼450 and 570 nm, respectively (black curve, Figure 1). The UV−vis spectrum of the AuGSH (low) sample shows absorption below 450 nm, but no well defined adsorption peaks are observed (black curve, SI Figure S2). C
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Figure 2. Time dependent PL curves by addition of different concentrations of GSH (A), Cys (B), NAC (C), or CyA (D) to AuGSH (low) samples. The solid curves show the fitted results by the first-order model.
CyA, corresponding to the overall elevation in UV−vis absorbance (cyan curve, SI Figure S2). This is most probably caused by the electrostatic interactions between the positively charged CyA (note we used cysteamine hydrochloride) and negatively charged Au NCs, resulting in the aggregationinduced emission (AIE) known for Au NCs.26 Mechanistic Investigation of PL Enhancement. To determine whether the PL enhancements were caused by the generation of highly luminescent AuGSH species (static mode) or by collision between thiol molecules and AuGSH (low) (dynamic mode), the free thiols in AuGSH (high), AuGSH (Cys), AuGSH (NAC), and AuGSH (CyA) were measured by Ellman’s method, a typical colormetric method for detection of thiols, respectively (see the SI). The results show a recovery of 73−86% for the added thiols (SI Table S2). The loss in free thiols indicates that a considerable part of the added thiols did participate in the reactions with AuGSH (low). The above AuGSH samples are further purified by ultrafiltration (MWCO, 3 kDa) and washed repeatedly to remove the possible unreacted precursors and small byproducts. All luminescent species are found to be retentive by the membrane and no PL was observed for the filtrates, demonstrating that all AuGSH samples have a MW over 3 kDa (SI Figure S5). The XPS data for AuGSH (low) and AuGSH (high) show similar results, i.e., two well distinguished peaks at 87.0 eV (Au 4f7/2) and 83.8 eV (Au 4f5/2) (Figure 3A and SI Figure S6). As known, Au (I) species are considered to be a key element for fluorescent emissions by GSH coated Au NCs. However, the Au(I) binding energy (BE) above 84.0 eV is not significant in our case, indicating the low Au(I) components. The BE at 83.8 eV hardly changes (difference less than 0.1 eV) after addition of
stable enough, while the free -SH group is indispensable for the PL enhancement. Several works have concerned the thiols’ effect on PL. Thiols, especially GSH, are known to be capable of etching large Ag29 or Pt30 nanoparticles to form luminescent nanoparticles. The etching steps are usually time-consuming (days to weeks) or require high etching temperature (for example, 65 °C). Considering the fast PL enhancement processes in our work, this mechanism seems unrealistic. We also note a recent work that described the PL enhancement of gold−histidine complexes by GSH.31 In that case, other thiols including Cys show no enhancement at all and the enhancement effect was ascribed to the significant steric hindrance by GSH. Since no further evidence was provided, the underlying mechanism was not well elucidated. It has also been reported that Cys and GSH can induce edge-to-edge coupling of plasmonic Au nanocubes (40 nm in side length), rendering a band-selective enhancement of two-photon PL.32 This work exhibits some evidence resembling our results. However, the proposed mechanism for the PL enhancement was based on the plasmonic effects, which is completely absent in our case. Therefore, the thiols-induced PL enhancement requires further investigation. According to the deduction for reaction of thiols and AuGSH (low), the dynamics of PL enhancement has been fitted by a common first-order model (detailed in the SI) and the fitted parameters are listed in SI Table S1. In most cases, the model may fit the experimental data well, with a square correlation coefficient (R2) of 0.97−0.99 (solid curves, Figure 2). However, the fitting results are undesired for CyA (Figure 2D). The PL reaches stability in 2−3 min for CyA, while it takes almost 10 min for the other three thiol compounds. The formation of large aggregates (even visible) is observable after addition of D
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Figure 4. MS analysis of AuGSH (low), AuGSH (high), and AuGSH (NAC).
[Au19(SG)20]8− to [Au19(SG)22]8−. Another feature is that the species with lower molecular weights (MWs) generally tend to generate species with higher MWs. To clarify this fact, the MS intensity ratio of two relevant peaks is used as a criteria. For Au15 species, the intensity ratio of of peak 4 (m/z 1504.9, [Au15(SG)10]4−) to peak 1 (m/z 1198.6, [Au15(SG)6]4−) and peak 3 (m/z 1428.0, [Au15(SG)9]4−) in AuGSH (high) is 3.5fold and 1.6-fold more than those in AuGSH (low), respectively. For Au19 species, the intensity ratio of peak 7 (m/z 1930.1, [Au19(SG)13]4−) to peak 5 (m/z 1700.6, [Au19(SG)10]4−) in AuGSH (high) is 4.2-fold higher than that in AuGSH (low). These results reveal that a large amount of AuGSH (high) is formed by addition of GSH to AuGSH (low). Similarly, after addition of NAC, the disappearance of peak 2 (m/z 1234.3) is accompanied by the generation of peak 13 (m/ z 1719.5), which can be attributed to the transformation of [Au19(SG)20]8− to [Au19(SG)20(NAC)24]8−. Peak 1 (m/z 1198.6, [Au15(SG)6]4−), peak 3 (m/z 1428.0, [Au15(SG)9]4−), and peak 5 (m/z 1700.6, [Au19(SG)10]4−) are also inhibited, while the appearance of peak 11 (m/z 1361.2, [Au15(SG)6(NAC)4]4−), peak 12 (m/z 1590.9, [Au 15 (SG) 9 (NAC) 4 ] 4− ), and peak 14 (m/z 1863.3, [Au19(SG)10(NAC)4]4−) can be used to explain the reaction steps reasonably. These results clearly show that NAC can successfully attack AuGSH (low) to form dual ligands (NAC and GSH) stabilized nanoparticles, as described in the aforementioned hypothesis. Besides, a series of [AuGSH + nNa+ − nH+] peaks, with an even m/z distance of 22.0, are also observed for peak 13 (13A to 13C, n = 1−3), peak 14 (14A to 14E, n = 1−5), and a peak with an m/z of 2007.0 ([Au19(SG)14]4−) (SI Figure S8). Generally, the MS analysis validates that the as-prepared AuGSH (low) samples are mainly comprised of “GSHinsufficient” Au species, which form defect-like structures and impede the efficient PL emission. Once the “vacant” sites are taken up by additional thiol ligands, the PL emissions will be significantly enhanced. Confocal Microscopic Imaging of Intracellular Thiols. The application of the AuGSH (low) sample as a PL probe for thiol detection was attempted by addition of different concentrations of GSH (0−5 mM) to AuGSH (low) solution of 0.5 mg/mL (SI Figure S9). All PL emissions were measured
Figure 3. XPS (A) and time-resolved fluorescence (B) of AuGSH (low, black) and AuGSH (high, red).
thiols. Therefore, no reduction processes occur during addition of thiols, the common reducing agents, to AuGSH (low) sample. This result differs from the presence of about half of the Au(I) in the AuGSH sample during 2 week incubation at room temperature,23 possibly due to complete reduction of gold precursors at higher temperatures. The time-resolved fluorescence measurement (Figure 3B) reveals that the AuGSH (low) sample has two fitted lifetimes of 1.77 ± 0.03 μs (46.15%) and 10.06 ± 0.12 μs (53.85%), while the corresponding lifetime of AuGSH (high) increased to 2.54 ± 0.05 μs (32.55%) and 10.44 ± 0.09 μs (67.45%), respectively. The morphological characterizations by TEM imaging verified that both samples are nanoparticles (SI Figure S7). The mean sizes are 2.1 ± 0.3 nm for AuGSH (low) and 2.6 ± 0.4 nm for AuGSH (high), according to the size statistics from 139 and 126 individual nanoparticles, respectively. The prolonged fluorescence lifetime and different average sizes also indicates the possible formation of new AuGSH species after addition of thiols. The MALDI-TOF MS may unearth more detailed information at sub-nanometer levels. We investigated the MS for AuGSH (low), AuGSH (high), and AuGSH (NAC), respectively (Figure 4). Abundant characteristic peaks, labeled from peak 1 to peak 14, are present within m/z from 1000 to 2000. These peaks have been identified to explore the possible reactions after addition of GSH or NAC to AuGSH (low) sample. Interestingly, almost all AuGSH species during this m/z range contain Au15 or Au19 cores. Compared with AuGSH (low), most MS peaks are available in AuGSH (high) (SI Table S3). A significant difference may be the disappearance of peak 2 (m/z 1234.3) and the simultaneous appearance of peak 9 (m/z 1309.3), which corresponds to the transformation of E
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Analytical Chemistry after 10 min static incubation. The PL increases linearly with the added GSH from 0.1 to 5.0 mM (R = 0.998), which can be visibly evaluated under 365 nm illumination. The PL emissions also increase linearly for both Cys and NAC, with a range of 0.1−1.0 mM (R = 0.989 and 0.990). Considering that the AuGSH (low) sample shows specific and rapid PL response to external thiols, we wonder whether it can act as a candidate for evaluation of intracellular thiols (usually at a concentration level of millimolar). To probe the possible bioimaging applications, the cytotoxicity of AuGSH samples was first inspected. The excellent biocompatibility of luminescent Au nanoparticles has been well reported by in vitro or in vivo experiments. 33 Here we used U87 and HeLa cells as models. The MTT experiments demonstrated that no significant decreases in cell viability were observed for both AuGSH (low) and AuGSH (high) samples, with above 80% cell viability maintained even at a relatively high concentration of 250 μg/mL (Figure 5 and SI Figure S10). Therefore, the materials and their products are almost non-cytotoxic, allowing the following thiols imaging applications.
Figure 6. Imaging of thiols in HeLa (A−C) cells incubated without (A) or with (C) AuGSH (low) sample (0.1 mg/mL) for 6 h. (B) Cells pretreated with 1 mM NMM before incubation of AuGSH (low). (D) Comparison of gray scale values per unit area obtained from individual cells in control (A, black), NMM pretreated (B, olive), and AuGSH (low) treated (C, dark yellow) group. Scale bar: 50 μm.
average gray scale values per unit area are 3.83 ± 0.95 and 8.88 ± 2.57 for NMM pretreated and AuGSH (low) group, respectively (Figure 6D). Using a threshold of 1.38 ± 0.17 for the control group, the gray scale value per unit area for NMM treated group is only ∼32.7% of that for the AuGSH (low) group, demonstrating that AuGSH (low) can be used as a potential agent for intracellular thiols imaging. Compared with the traditional organic dyes for thiols imaging, the preparation of AuGSH (low) is time-saving, without tedious synthetic and purification procedures. Considering the raw materials are common and commercially available, the overall cost is also competitive. So it is believed to be a promising probe candidate for future personalized biomedical diagnosis.
Figure 5. Cytotoxicity of U87 cells after treatment with AuGSH (low, blue) or AuGSH (high, red) for 24 h.
The intracellular thiols imaging was investigated with confocal fluorescent microscopy. The incubation time of AuGSH (low) was initially optimized (SI Figure S11). The luminescence is negligible in 2 h, but gradually appears after incubation of 4 h and reaches a significant level at 6 h. Hence 6 h was selected as the sequent incubation time. After 6 h incubation, the AuGSH (low) treated groups show significant PL in HeLa (Figure 6C) and U87 cells (SI Figure S12C), while almost no luminescence was observed for the control group, i.e., HeLa (Figure 6A) and U87 cells (SI Figure S12A) without treatment of AuGSH (low). The PL mainly locates outside the nuclear regions, corresponding to the distribution of thiols in the cytoplasm. To validate that the PL actually originated from the high luminescent AuGSH species, we used NMM, a known thiols extinguisher, to pretreat the cells before further incubation of AuGSH (low). The PL was largely inhibited by the introduction of 1 mM NMM for 1 h (Figure 6B and SI Figure S12B), confirming that the observed luminescence is triggered by high-luminescence AuGSH generated from internalized AuGSH (low). To offer some quantitative information, the gray scale values were calculated for individual cells in the rectangle regions (SI Figure S13). The
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CONCLUSION In summary, we have demonstrated the thiols-induced fast PL enhancement of AuGSH (low) because of the attachment of thiols to ligand-insufficient AuGSH (low) species. Based on this principle, a biocompatible and low-cost probe is proposed for intracellular imaging of biothiols, indicating great prospects as biomedical labels. Besides, this work also offers a facile strategy for preparation of dual ligands (i.e., GSH and other thiols) costabilized Au nanoparticles, which can provide versatile conjugation sites as efficient luminescent labels.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02559. Optimizations of synthetic conditions, XPS spectra, TEM images, assays of thiols, MTT assays, imaging experF
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imental details, PL dynamics fitted model, determination of free thiols recovery, identification of MS peaks (PDF)
(24) Wu, Z.; Chen, J.; Jin, R. Adv. Funct. Mater. 2011, 21, 177−183. (25) Wang, M. L.; Wu, H.; Chi, Y. W.; Chen, G. N. Microchim. Acta 2014, 181, 1573−1580. (26) Luo, Z. T.; Yuan, X.; Yu, Y.; Zhang, Q. B.; Leong, D. T.; Lee, J. Y.; Xie, J. P. J. Am. Chem. Soc. 2012, 134, 16662−16670. (27) Shang, L.; Brandholt, S.; Stockmar, F.; Trouillet, V.; Bruns, M.; Nienhaus, G. U. Small 2012, 8, 661−665. (28) Wright, A. K.; Thompson, M. R. Biophys. J. 1975, 15, 137−141. (29) Le Guevel, X.; Spies, C.; Daum, N.; Jung, G.; Schneider, M. Nano Res. 2012, 5, 379−387. (30) Le Guevel, X.; Trouillet, V.; Spies, C.; Jung, G.; Schneider, M. J. Phys. Chem. C 2012, 116, 6047−6051. (31) Zhang, X.; Wu, F.-G.; Liu, P.; Gu, N.; Chen, Z. Small 2014, 10, 5170−5177. (32) Guan, Z. P.; Li, S.; Cheng, P. B. S.; Zhou, N.; Gao, N. Y.; Xu, Q. H. ACS Appl. Mater. Interfaces 2012, 4, 5711−5716. (33) Wang, J.; Ye, J.; Jiang, H.; Gao, S.; Ge, W.; Chen, Y.; Liu, C.; Amatore, C.; Wang, X. RSC Adv. 2014, 4, 37790−37795.
AUTHOR INFORMATION
Corresponding Authors
*(H.J.) Phone/Fax: +86-25-83792177. E-mail:
[email protected]. cn. *(X.W.) Phone/Fax: +86-25-83792177. E-mail: xuewang@seu. edu.cn. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is financially supported by 863 programs (Grant 2015AA020502) and the National Natural Science Foundation of China (Grants 81325011, 21327902, and 21175020). We truly thank Dr. Dechen Jiang at Nanjing University for his kind help in the MS characterization.
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DOI: 10.1021/acs.analchem.5b02559 Anal. Chem. XXXX, XXX, XXX−XXX
