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Ratiometric Fluorescent Silicon Quantum Dots−Ce6 Complex Probe for the Live Cell Imaging of Highly Reactive Oxygen Species Qianqian Zhao, Ren Zhang, Daixin Ye, Song Zhang, Hui Chen,* and Jilie Kong* Department of Chemistry, Fudan University, Shanghai 200433, P. R. China S Supporting Information *
ABSTRACT: The monitoring of reactive oxygen species (ROS) in living cells remains challenging because of the complexity, short half-life, and autofluorescence of biological samples. In this work, we designed a ratiometric fluorescent probe for the detection and imaging of ROS, which was constructed from silicon quantum dots (Si QDs) with chlorin e6 (Ce6) through electrostatic attraction and showed well-resolved dual fluorescence emission signals (490 and 660 nm). Sensitive and selective biosensing of hydroxyl radical (•OH) was demonstrated on the basis of fluorescence quenching of the Si QDs and Ce6 as an internal reference to avoid environmental interference, with a detection limit of ∼0.97 μM. The endogenous release of •OH was also monitored and imaged in living cells. KEYWORDS: silicon quantum dots, chlorin e6 (Ce6), ratiometric fluorescence probe, highly reactive oxygen species, hydroxyl radical (•OH), cell imaging
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INTRODUCTION Radical oxygen species (ROS) play an important role in regulating a wide range of physiological functions such as cell signal transduction, aging, and cancer, among others.1 ROS include singlet oxygen (1O2), hydrogen peroxide (H2O2), superoxide (O2−), hydroxyl radical (•OH), hypochlorite (ClO−), and peroxynitrite (ONOO−). The ROS are always generated from metabolic processes,2 and their overaccumulation can result in oxidative stresses that are involved in the pathogenesis of many diseases, such as cardiovascular disease, cancer, and neurological disorders.2−4The hydroxyl radical (•OH) belongs to the most highly reactive oxygen species (hROS) and has a short half-life (∼1 ns). It has a high reactivity with many different biological species, and thus, it is regarded as the most aggressive free radical. There is much evidence that • OH can damage DNA and mediate the redox alteration of cellmembrane Ca2+ channels. Therefore, an analytical method with extremely high sensitivity is urgently needed for probing •OH, specifically in biological systems. In the past decades, several methods have been used to determine •OH, such as electron spin resonance (ESR), highperformance liquid chromatography (HPLC), fluorescence spectroscopy, and electrochemical methods, among others.5−10 © 2016 American Chemical Society
Among these, fluorescence spectroscopy provides many advantages, such as high sensitivity, simplicity, and real-time monitoring in living cells, tissues, and animals. Several organic fluorescent probes have been designed for the detection and in vivo imaging of ROS in multiple living cellular models, such as inflammation and mitochondrial stress, and in intact live zebrafish.8,11,12 However, these applications of organic probes in real living cell or animal systems remain limited by their potential photobleaching, biotoxicity, and spontaneous autooxidation stress. Therefore, it remains a great challenge to develop sensitive, selective, stable, biocompatible, and convenient fluorescent probes and methods for probing ROS and uncovering the roles of ROS in health and disease states. Recently, luminescent silicon quantum dots (Si QDs) have attracted great interest in many fields for myriad applications due to their many merits (e.g., low cost, high quantum yield, size-controlled fluorescence, high stability against photobleaching, and excellent biocompatibility).13−19 Many biological application studies have focused on long-term imaging with Received: September 23, 2016 Accepted: December 27, 2016 Published: December 27, 2016 2052
DOI: 10.1021/acsami.6b12047 ACS Appl. Mater. Interfaces 2017, 9, 2052−2058
Research Article
ACS Applied Materials & Interfaces Si QDs in living cells and animals,20−26 which could significantly impact clinical diagnosis and therapies. Additionally, due to the efficient quenching by some specific molecules of the luminescence of Si QDs, Si QDs have also been applied in the detection of small molecules directly and indirectly, such as Cu2+, glucose, and high-energy compounds (2,4,6trinitrotoluene(TNT), dinitrotoluene (DNT), etc.).27−32 However, to the best of our knowledge, few research studies have used the luminescent response of SiQDs to biological signaling molecules (hROS, pH, Cu2+, etc.) for biosensors, mainly because of the complexity of the living cells and animals. To solve this problem, the ratiometric sensor strategy has recently attracted significant attention because of its sensitivity and built-in correction for avoiding environmental effects as compared to first-generation single-intensity probes. For the monitoring and in vivo imaging of ROS in living cells, several dual-emission, ratiometric, fluorescent nanocomplexes have been designed on the basis of the fluorescence quenching of gold nanoclusters or a specific organic molecule (2-[6-(4′hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoicacid, HPF) by the hROS and a stable dye encapsulated in silica particles acting as an internal reference.33−36 However, this type of nanocomplex design normally requires tedious chemical covalent bonding or surface modification steps. Herein, we report for the first time a dual-emission, ratiometric, fluorescent silicon quantum dot-Ce6 nanocomplex for detecting and monitoring the hydroxyl radical (•OH) in living cells. The basic working principles of the Si−Ce6 nanocomplex are shown in Scheme 1. In this method, the
prepared with ultrapure water. Furthermore, the ultrafiltration devices (MW:3000) were purchased from Pall Reagent Co., Ltd. (Shanghai, China). HepG-2 cells (human hepatoma cancer cell line) were obtained from the cell bank of the Shanghai Science Academy. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin− streptomycin were purchased from Gibco (Thermo Fisher Scientific, U.S.A.). The cell culture dishes for cell imaging and 96-well plates for testing cell viability were purchased from NEST. Apparatus. UV−vis absorption instrument (Agilent 8453, U.S.A.) was used to measure the optical absorption spectrum with a 1 cm path length. The fluorescence spectrum (Hitachi F7000, Japan) was tested at an excitation wavelength of 410 nm, and the slits for excitation were set at 2 nm and those for emission were set at 5 nm. Field emission transmission electron microscopy (FETEM, Tecncai G2 F20S-Twin) was used to observe the morphology of the Si QDs−Ce6, operating at 200 kV. To identify the groups of the Si QDs, Ce6, and Si QDs− Ce6, Fourier transform infrared spectroscopy (FT-IR, Avatar360, U.S.A.) was used. Systematic characterizations of the Si QDs, including X-ray photoelectron spectroscopy (XPS, PHI 5000C ESCA) equipped with a Mg Kα source (1253.6 eV photons),were performed. In addition, the zeta potentials of the Si QDs and SiQDs−Ce6 were measured in water solutions using a Malvern Zeta sizer (ZEN 3600, Worcestershire). The viability of HepG-2 cells incubated with different concentrations of Si QDs−Ce6 was obtained from an enzyme-labeling instrument (SPR-960, China). Moreover, a confocal laser microscope (Leica TCS SP8, Germany) was used to collect cell images with an excitation wavelength of 405 nm. Preparation of the Si QDs and Si QDs−Ce6 Complex. This work is based on our previous research, in which the preparation of silicon quantum dots (Si QDs) and the method of synthesis has been reported.37 APTES was mixed with a 0.1 M NaAA solution under stirring at room temperature and ambient pressure. After 4 h, we obtained water-dispersible SiQDs. Here, we selected Ce6 as the functional molecule because it has three carboxyl groups that can be deprotonated to COO− at pH higher than 5 and has negative charges. Therefore, Ce6 molecules can attach on the surface of Si QDs by electrostatic force. We combined 1.9 mL of freshly prepared Si QDs solution with 0.1 mL of 50 μM Ce6 and let it stand for 48 h at room temperature under dark conditions. A 3K ultrafiltration device was used three times to exclude impurity influences, such as free APTES and Ce6 in the solution. Fluorescence Spectroscopy. In the fluorescence assay, a quartz cuvette with an optical path length of 1 cm was used. The sample was excited at 410 nm, the emission wavelength was collected from 430 to 750 nm, and each experiment for • OH detection was carried out three times. The •OH was generated through the Fenton reaction (Fe2+ + H2O2 = Fe3+ + OH− + •OH) by different amounts of Fe2+ and H2O2. After incubation with the probe for 1 h at 37 °C, the fluorescence spectrum was obtained. In the interference experiment, ClO− was provided by NaClO (200 μM). ONOO− (200 μM) was generated from the chemical reaction between H2O2 (200 μM) and NaNO2 (200 μM). 1O2 was produced by H2O2 with the catalyzing of Na2MoO4 at basic condition. MTT Assay. First, HepG-2 cells at a density of 5 × 103 cells per well in 96-well plates were cultured (37 °C, 5% CO2) in DMEM medium containing 10% FBS and antibiotics (100 μg/ mL penicillin and 100 units/mL streptomycin) overnight.
Scheme 1. Working Principles of the Ratiometric Fluorescent Si QDs−Ce6 Nanocomplex for the Detection and Imaging of •OH
SiQDs−Ce6 complex was formed through the electrostatic interaction between the SiQDs and Ce6. The fluorescence of SiQDs remains was quenched by •OH, while that of Ce6 remained constant. The ratio of the fluorescent intensities of the Si QDs and Ce6 at two wavelengths is measured as a builtin correction to avoid environmental interference so the probe can be applied in bioassays with high sensitivity.
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EXPERIMENTAL SECTION Materials. (3-Aminopropyl)triethoxysilane (APTES, 99%), ascorbic acid sodium salt (NaAA), dimethyl sulfoxide (DMSO), hydrogen peroxide and sodium hypochlorite solution (H2O2, 30%), and sodium hydroxide (NaOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Metal ion solutions were prepared from their chloride salts. Chlorin e6 (Ce6) was purchased from Frontier Scientific (Logan, UT, U.S.A.). Reduced glutathione, lipopolysaccharides (LPS, from Escherichia coli) and 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. All chemical reagents were analytical grade and were used without further purification. All solutions were 2053
DOI: 10.1021/acsami.6b12047 ACS Appl. Mater. Interfaces 2017, 9, 2052−2058
Research Article
ACS Applied Materials & Interfaces Then, Si QDs−Ce6 were dispersed in DMEM at different concentrations (200, 100, 50, 20, 10, or 5 μg/mL). After 24 h, we washed the cells with 1× PBS buffer three times. After that, MTT (20 μL, 5 mg/mL) and 80 μL of DMEM were added into each well and incubated for 4 h to allow the formation of formazan dye. Subsequently, 150 μL of DMSO was added into each well and followed with gentle shaking for 10 min. Cell viability was evaluated by the MTT colorimetric procedure. Using an enzyme-labeling instrument to measure the absorbance at 492 nm in quintuplicate, the cell viability values were determined according to the following formula: cell viability (%) = (the absorbance of experimental group − the absorbance of blank group)/(the absorbance of blank control group − the absorbance of blank group) × 100%. Intracellular Biosensing and Imaging. We used glassbottom cell dishes for cell attachment, where 100 μg/mL Si QDs−Ce6 dispersed in DMEM were added to the cell dishes, which were maintained at 37 °C for 2 h for cell uptake. After that, we used 1× PBS to wash the cells three times, followed by adding 10 μg/mL LPS into two cell dishes to probe the •OH in cells16−19 incubated separately for 1 and 2 h. At the same time, 0.1% DMSO and 10 μg/mL LPS were added to the third cell dish for 2 h of incubation. After the different methods to prepare the HepG-2 cells, PBS buffer was used to wash the cells three times. In addition, 1 mL of 4% paraformaldehyde solution was added to every cell culture dish, which were then maintained at 37 °C for cell fixing for 15 min and washed three times. The samples can be stored at 4 °C for 2 weeks. The cell images were taken on the Leica TCS SP8 with the laser at 405 nm and equipped with an oil immersion 63× objective. The fluorescence images of Si QDs were obtained from480 to 530 nm, and the Ce6 window was from 640 to 680 nm.
Figure 1. (a) TEM image of SiQDs−Ce6. (b),(c) Characteristics of Si QDs with XPS. (d) FTIR spectra of Si QDs, Ce6, and SiQDs−Ce6.
which shows the characteristic peaks of −COOH groups. For the Si QDs−Ce6 complex, all the strong transmission peaks of −COOH, N−H, and Si−O bonds appeared in the FTIR spectrum and confirmed that Ce6 was attached on Si QD surfaces. Three carboxyl groups of Ce6 (Figure 2a) are
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RESULTS AND DISCUSSION Preparation and Characterization of Si QDs−Ce6. Si QDs were synthesized simply by the facile mixing and stirring of APTES and NaAA at room temperature for 30 min as described in our previous report.37 The Si QDs−Ce6 were prepared through the electrostatic interaction between Si QDs with a positive charge and Ce6 with a negative charge. From the TEM image (Figure 1a), we can determine that the average size of the Si QDs−Ce6 is ∼2 nm and that they are monodispersed. Their small size and excellent hydrophilicity offer great advantages for in vivo bioimaging because particles with a diameter
