Biocompatible ZnS:Mn quantum dots for reactive oxygen generation and detection in aqueous media
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We report here the versatility of Mn-doped ZnS quantum dots (ZnS:Mn QDs) synthesized in aqueous medium for generating reactive oxygen species and for detecting cells. Our experiments provide evidence leading to the elimination of Cd-based cores in CdSe/ZnS systems by substitution of Mn-doped ZnS. Advanced electron microscopy, X-ray diffraction, and optical spectroscopy were applied to elucidate the formation, morphology, and dispersion of the products. We study for the first time the ability of ZnS:Mn QDs to act as immobilizing agents for Tyrosinase (Tyr) enzyme. It was found that ZnS:Mn QDs show no deactivation of Tyr enzyme, which efficiently catalyzed the hydrogen peroxide (H2O2) oxidation and its eventual reduction (−0.063 V vs. Ag/AgCl) on the biosensor surface. The biosensor showed a linear response in the range of 12 μmol/L–0.1 mmol/L at low operation potential. Our observations are explained in terms of a catalase-cycled kinetic mechanism based on the binding of H2O2 to the axial position of one of the active copper sites of the oxy-Tyr during the catalase cycle to produce deoxy-Tyr. A singlet oxygen quantum yield of 0.62 in buffer and 0.54 in water was found when ZnS:Mn QDs were employed as a photosensitizer in the presence of a chemical scavenger and a standard dye. These results are consistent with a chemical trapping energy transfer mechanism. Our results also indicate that ZnS:Mn QDs are well tolerated by HeLa Cells reaching cell viabilities as high as 88 % at 300 µg/mL of QDs for 24 h of incubation. The ability of ZnS:Mn QDs as luminescent nanoprobes for bioimaging is also discussed.
KeywordsZnS:Mn Biosensing Photodynamic therapy Quantum dots Singlet oxygen Tyrosinase
Quantum dots (QDs) are recently being envisioned as potential photosensitizers (PS) for photodynamic therapy (PDT) to treat cancer (Thakor and Gambhir 2013; Allison and Moghissi 2013; Chen et al. 2013), due to their flexible bioconjugation, biological targeting efficiency, high quantum yield (QY), broad absorption with relatively narrow, symmetric emission, and long-term stability (Sotelo-Gonzalez et al. 2013; Zhang et al. 2011; Zhuang et al. 2003; Shen et al. 2012). The availability of QDs whose optical properties can be tuned by size, composition, and doping has brought forth new pathways to produce reactive oxygen species (ROS), in particular singlet oxygen (1O2), by photochemical methods. As a proof of concept, many QDs have been tested, but a strong focus has been placed on CdSe and CdTe QDs. For instance, CdSe QDs can be used to sensitize molecular oxygen through a triplet energy transfer mechanism. They can produce a 1O2 QY of ~5 % in toluene (cf. 43 % in Pc4 PS used in clinical trials), and their optical properties can be tuned to treat both shallow- and deep-seated tumors (Samia et al. 2003). Shi et al. working with CdTe QDs electrostatically bound to meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (PS) obtained a 1O2 QY of ca. 43 % (being ≥40 % reported for traditional PSs) (Shi et al. 2006). Nonetheless, they did not fully quantify their results. Still, the cytotoxicity and solubility in water of Cd-based QDs (despite extended efforts to modify their surface) remain a significant concern. In this regard, Hsiech et al. overcoated CdSe cores with ZnS, being then attached to a cyclometalated Ir complex-type sensitizer (Hsieh et al. 2006). Although the 1O2 QY in benzene was improved, it was the Ir complex that was directly excited instead of the QDs. It has been reported that ZnS shells greatly reduce the toxic effects of Cd-based QDs in live cells and provide an improved 1O2 QY (as high as 31 %) (Tsay et al. 2007; Kirchner et al. 2007). Nevertheless, they are still tested in non-aqueous solutions due mainly to the surface chemistry of the core material. We have thus investigated the possibility to omit the use of Cd-based cores and substitute their optical contribution by Mn doping in ZnS (Bhargava and Gallaguer 1994). Recently, Zhou et al. reported on the sensitizing effect of cysteine-capped Mn-doped ZnS (ZnS:Mn)/Si QDs via the 1O2 quenching method (Zhou et al. 2011), but the findings of this exploratory work need further confirmation. The omission of the Si coating and evaluation of the quantification process of 1O2 using ZnS:Mn QDs via photooxidation are still a challenge.
Due to the biocompatibility and high QY (compared to Cd-based QDs) of ZnS:Mn QDs, they are also employed as nanoprobes in order to better understand their main “nano-bio” interaction with cells and bacteria. It has been reported that ZnS:Mn QDs offer improved ability to image single cancer cells and colonies without causing any effect on their metabolic activity and morphology (Mathew et al. 2010; Manzoor et al. 2009). Correlated reports have shown that ZnS:Mn capped with different ligands possesses a great capability to image Staphylococcus aureus, exceptional potential to be used for a rapid screening of metal-accumulating Lysinibacillus fusiformis cells, and intrinsic inhibiting effect on the growth of Escherichia coli (Sharma et al. 2010; Sajimol et al. 2013; Kong et al. 2011; Baruah et al. 2012; Li et al. 2004). As a result, ZnS:Mn QDs can be effectively employed to detect cells, especially which are associated to the formation of biofilms. The identification of P. aeruginosa cells has aroused a great deal of interest to the scientific community due to their adherence to surfaces (forming biofilms) of nosocomial elements (e.g., catheters, heart valves, and prostheses), and their potential pathogenesis in patients suffering from cystic fibrosis, both constituting a critical health-related issue in hospitals (Cornelis 2008). However, no reports on labeling P. aeruginosa cells using biocompatible ZnS:Mn QDs have appeared in the literature to date.
ZnS:Mn QDs exhibiting highly active surface areas with an isoelectric point (IEP) of 7.2 are also attractive as innovative matrixes to immobilize enzymes with low IEP, since their interaction is mainly electrostatic. When immobilizing such large biomolecules on solid substrates, it is desirable to retain their electroactive response on the modified electrode surface, and avoid any process related to irreversible denaturalization. Tyrosinase (Tyr) is one of the most used enzymes for the modification of metal electrodes due to its intrinsic enzyme specificity and ability to catalyze the oxidation of many substrates by phenols (Jang et al. 2010). The effective immobilization of Tyr by ZnS:Mn QDs would thereby be a promising alternative given their excellent mechanical, chemical, and thermal stability over prolonged periods (Chauhan et al. 2011). The assembly of Tyr/ZnS:Mn-based amperometric biosensors to detect ROS (such as hydrogen peroxide, H2O2) may facilitate a more effective enzymatic binding, improve the properties of the bioactive layer associated with the transducer, and lead to greater efficiency in terms of sensitivity, selectivity, stability, and simplicity (Vreeke et al. 1992; Garguilo et al. 1993). Although some nanostructured immobilizing matrixes have been proposed to enhance the electron transfer rate (Jang et al. 2010; Liu et al. 2011; Zhou et al. 2010; Song et al. 2010); most of them still show low reusability and storage stability, and poor long-term stability due to the large electrochemical prosthetic groups deeply embedded into the structure of the enzyme (Chauhan et al. 2011). In order to increase the electron transfer efficiency of redox enzymes, QDs have recently been proposed due to their inherent large surface-to-volume ratio, high surface reaction activity, and strong absorption ability, which increases the binding site on the electrode surface (Chauhan et al. 2011; Çevik et al. 2012, Xia et al. 2014). Specifically, inexpensive and environmentally benign metal sulfide QDs with sizes similar to those of the working enzymes enable excellent interaction with the active centers buried deep within the protein shells. Recently, Chauhan et al. detected organophosphorus insecticides based on ZnS-immobilized rat brain acetylcholinesterase (Chauhan et al. 2011). In the presence of acetylthiocholine chloride, ZnS QDs promoted electron transfer reactions at low working potential, catalyzed electrochemical oxidation of enzymatically formed thiocholine increasing the detection sensitivity, and exhibited long-term storage stability. Nonetheless, no further reports confirming the immobilizing enzyme characteristics of ZnS QDs have appeared in the literature. The evaluation of new electron transfer paths employing more stable surface-passivated ZnS:Mn QDs is still a challenge, for instance, for the detection of H2O2 at low concentrations.
We report here on the versatility of luminescent water-soluble ZnS:Mn QDs for Tyr immobilization and multiple biological detection. The capability of ZnS:Mn QDs to produce 1O2 in the presence of 1,3-diphenylisobenzofuran (DPBS, 1O2 sensor) is presented. The ZnS:Mn QDs are also employed as nano-probes for imaging P. aeruginosa cells, and intended as immobilizing matrixes for Tyr enzyme by cross-linking on modified Pt electrode to detect H2O2.
Materials and methods
Synthesis of ZnS:Mn quantum dots
Mn-doped ZnS QDs capped with 3-mercaptopropionic acid (MPA, ≥99 %, Sigma Aldrich, USA) were prepared by an inorganic wet chemical approach reported elsewhere (Beltran-Huarac et al. 2013a, b). Briefly, 1.705 g of ZnSO4·H2O (≥99.9 %, Sigma Aldrich, USA), 0.085 g MnSO4·H2O (≥ 99 %, Sigma Aldrich, USA), and 2.61 mL MPA were dissolved into 50-mL three-neck round-bottom flask using high-purity deionized water (HPDW), resulting in a 5 at.% Mn doping. The pH of the mixed solution was adjusted to 11 using 1 M NaOH (99.99 %Sigma Aldrich, USA). After argon purging, 50 mL of 0.2 M aqueous solution of Na2S (Sigma Aldrich, USA) was gradually added. The mixture was stirred at room temperature with a controlled reflux system and then aged for 14 h at 50 °C. The flocculate was removed from the supernatant by ultracentrifugation, then copiously rinsed with HPDW, and dried at 60 °C overnight in order to eliminate any remaining by-product and adsorbate. The final products re-dispersed in HPDW rapidly produced a deep orange solution when exposed to UV light, which is a clear indicator of the compound formation, and were employed for further ex situ characterization. ZnS QDs were also prepared following the same recipe for comparison purposes. Peng et al. reported that this synthesis process yields an Mn doping level of 0.380 at.% when 5 at.% Mn is used in the synthesis process as measured by ICP (Peng et al. 2005).
The phase and crystalline structure of QDs were analyzed using an X-Ray Diffractometer (XRD), Model Siemens D5000. Raman spectra were collected via a Jobin–Yvon T64000 spectrometer (resolution ~1 cm−1) with Ar-ion laser excitation (514.5 nm), attached to an optical microscope with 80× resolution. The surface morphology, crystallite size distribution, and elemental composition were studied using a JEOL JEM-2200FS Cs-corrected high-resolution transmission electron microscope (HRTEM) operated at 200 kV. A FluoroMax-2 spectrofluorometer was employed to collect the photoluminescence (PL) spectra.
For the biosensor construction, the polycrystalline Pt electrode surface was mechanically polished with alumina paste (0.05 μm), washed with HPDW, and rinsed abundantly with anhydrous ethanol (≥99.5 %, Sigma Aldrich, USA). The active part of the electrode was a 4-mm-diameter disk, and the other parts were covered with isolating epoxy resin. The as-treated surface was submerged in a 10 mM solution composed of 4-aminothiophenol (ATP, 97 %, Sigma Aldrich, USA) diluted in anhydrous ethanol to form a self-assembled monolayer (SAM). Afterwards, 0.2 mg of MPA-capped ZnS:Mn QDs were linked onto SAM surface using a 2 mM solution of N,N’-dicyclohexylcarbodiimide (DCC, 60 wt% in xylenes, Sigma Aldrich, USA) in the presence of N,N-dimethylformamide (DMF, 99.8 %, Sigma Aldrich, USA). The Tyr enzyme from mushrooms (low isoelectric point, lyophilized powder, ≥1000 unit/mg solid, Sigma Aldrich, USA) was then immobilized on ZnS:Mn QDs via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysulfosuccinimide (EDC/Sulfo-NHS, Sigma Aldrich, USA) cross-linking. To do this, the electrode was immersed into a solution of EDC and Sulfo-NHS (1:3 wt. %) diluted in 1 mL of phosphate-buffered saline (PBS, 0.1 M and pH 7.0) for 0.5 h in order to activate the ZnS:Mn QDs. The electrode was then transferred to a solution consisting of 3 mg Tyr enzyme and 2 mL PBS for 2 h and kept at 4 °C. The electrode was next rinsed with PBS to remove any unbound enzyme.
Electrochemical responses were analyzed by current voltammetry (CV) and conducted at room temperature using an SP-150 potentiostat/galvanostat and EC-Lab Express software purchased from BioLogic Science Instruments. A three-electrode configuration was used in this study, and the electrodes were purchased from Bioanalytical Systems Inc., USA. The fabricated Pt/SAM/QD/Tyr was used as the working electrode. Ag/AgCl 3 M KCl and Pt wire were used as the reference and auxiliary electrodes, respectively. The voltammograms were collected from −0.4 to 1.2 V with a scan rate of 50 mV s−1 and at concentrations of H2O2 ranging from 0 to 100 μM.
Determination of singlet oxygen
The 1O2 generation was verified by photooxidation of 1,3-diphenylisobenzofuran (DPBF, 97 %, Sigma Aldrich, USA) and monitored by absorption. A stock solution of capped QDs at 10 mg/mL and 400 μL of 6 × 10−5 M DPBF (1O2 sensor) in ethanol were gradually mixed in 2 mL of HPDW and in 2 mL of buffer separately. The mixture was adequately transferred to a quartz cuvette, then continuous airflow supplied by a gas syringe was progressively added and homogenized by vigorous stirring. The absorbance of the samples was first recorded in the dark using an UV–Vis spectrophotometer (DU 800, Beckman Coulter), and then irradiated with a 532 nm laser (fluence ~101 mW/cm2) placed 0.4 m apart. Visible-light excitation was used to avoid the self-oxidation process of DPBF through absorption in the UV region. The absorption spectra were obtained every 2 s during approximately 16 s. Rose bengal (RB, dye content 95 %, Sigma Aldrich, USA) prepared at 1 × 10−5 M was used as control (Beltran-Huarac et al. 2010). All the experiments were done in triplicate.
Imaging P. aeruginosa cells
In order to bind the QDs to the phosphoryl and carboxyl groups present in the cellular wall for bacterial imaging applications, the QDs were positively charged by means of protonated amine groups of chitosan (high purity, M v 110–150 kDa, Sigma Aldrich, USA) following a similar approach described above by MPA.51 For batch cell cultures, the P. aeruginosa strain (pellets with a mean assay value of 1.0–9.9 × 103 CFU purchased from MicroBiologics, USA, 0353E3) with an initial concentration of 5.4 × 103 CFU/mL was diluted in 15 mL of nutrient broth and incubated at 35 °C for 48 h. The growth curves were obtained by mixing 5 mL of inoculated P. aeruginosa, 5 mL of QDs (40 mM), and 90 mL of nutrient broth, and then incubated at 37 °C under gentle shaking (110 rpm) for 3 h. For negative control, the QDs were replaced by 5 mL of HPDW. In order to monitor the agglomeration and PL of QDs dispersed in nutrient broth, 5 mL of QDs (40 mM) were added to 95 mL of nutrient broth. The absorbance for both the QDs incubated with or without bacteria was monitored and recorded using a UV–Visible spectrophotometer (Helios, 640 nm), until reaching the stationary phase in the cell population growth curve. No substantial difference was observed in the log and death phases. After incubation, the cell–QDs suspension was washed and precipitated for 30 min. Three mL of re-dispersed precipitate and supernatant were then collected for absorbance measurements. Further washing and gentle sonication were applied to the solution to separate the QDs attached to cellular membranes. For confocal microscopy imaging, 200 μL of each solution (bacteria–QDs, control and QDs) was added dropwise on a chamber-covered glass cell (Thermo Scientific, Nunc Lab-Tek II) and then excited at 405 nm. The confocal images were recorded using a Zeiss observer Z-1, a laser LSM 510 META using a magnification of 100×, and a U filter set, and were processed through ZEN Lite software developed by Carl Zeiss MicroImaging GmbH.
MTS cell viability assay
The cytotoxicity effects of ZnS:Mn QDs were performed on HeLa cells using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). HeLa cells were cultured in Eagle’s minimum essential medium supplemented with 10 % fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B (Cellgro) at 37 °C with 5 % CO2. Cells were plated at a density of ca. 2 × 104 in 96-well plates and grown until reaching 80–90 % confluence. Then, cell culture medium was removed and 100 µL of complete medium supplemented with QDs at different concentrations (ranging from 5.2 to 1000 µg/mL) was added, and three wells with only fresh complete medium were used as a positive control. After 24-h incubation, the medium was removed and a solution of fresh media containing 20 % of CellTiter 96® AQueous One Solution reagent was added. Wells with fresh complete medium and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent without cells were used as a negative control. Afterwards, cells were incubated at 37 °C for 30 min. Then, the 96-well plates were centrifuged at 2000 rpm for 10 min. The supernatant was transferred onto a new microplate and the absorbance at 490 nm was recorded using a Synergy H1 Hybrid Multi-Mode Microplate Reader. The cell viability percentage was ascertained by means of the following equation: % = [(A 490 of QD-treated cells)/(A 490 of untreated cells)] × 100. All the experiments were done in triplicate.
Results and discussion
Characterization of the ZnS:Mn quantum dots
The microstructure quality of ZnS:Mn QDs was studied by Raman scattering spectroscopy (Fig. 1b). The well-pronounced low-frequency Raman bands observed were simulated using the damped harmonic oscillator phonon model (DHOPM) (Beltran-Huarac et al. 2014). From the simulation, the Raman modes are determined to be at 261 and 342 cm−1, which can be ascribed to the characteristic transverse and longitudinal optical modes of cubic ZnS, respectively (Nilsen 1969). The corresponding fit shows that those bands are red-shifted by ~10 cm−1 and narrow (full width at half maximum, FWHM ~23 cm−1) when compared to bulk ZnS (Nilsen 1969), which clearly indicates that the zinc blende ZnS is under tensile stress as a result of the growth process. The morphology and size distribution of ZnS:Mn QDs were studied by electron microscopy. The HRTEM images depicted in Fig. 1c show well-dispersed QDs with a high degree of crystallinity whose sizes range from 2 to 7 nm. A closer look of representative QDs shows that they are almost spherical with lattice fringes readily observable and a diameter of ~4 nm. The surfaces of QDs were clean, smooth, and atomically resolved with d-spacing of 0.31 nm corresponding to the major plane (111) of ZnS, consistent with the XRD and SAED analyses. The statistical size distribution (see lower inset of Fig. 1c) obtained by image analysis further confirmed that the average size of QDs was ~4 nm in accordance with the XRD results. Taken altogether, the XRD, Raman, and HRTEM analyses indicate the formation of narrowly distributed high-quality ZnS:Mn QDs with a zinc blende structure.
Detection of reactive oxygen species (hydrogen peroxide)
Generation of reactive oxygen species (singlet oxygen)
Production of 1O2 Φ Δ for some semiconductors and metals used as PSs
Cytotoxicity and cell imaging
In summary, we have synthesized ZnS:Mn QDs in aqueous solution at room temperature for multiple types of biological detection and enzyme immobilization. Our findings indicate that ZnS:Mn-immobilized Tyr biosensor is able to detect not only phenols but also hydrogen peroxide at concentrations below 12 μM. A correlated synergistic effect was observed between the high catalytic activity of Tyr and the large surface area of QDs, which resulted in an enhanced electrochemical response. Through an indirect assay to monitor the singlet oxygen quantum yield, it was evidenced that ZnS:Mn QDs can be used as an efficient photosensitizer for photodynamic therapy exhibiting a 1O2 QY of 0.62 ± 0.02 in buffer and 0.54 ± 0.03 in water. HeLa cells being exposed to ZnS:Mn QDs for 24 h show high tolerance reaching cell viabilities as high as 88 % at 300 µg/mL. The ability of chitosan-capped ZnS:Mn QDs to penetrate microbial cells and serve as luminescent nano-probes makes them suitable to be used as phylogenetic oligonucleotide probes for single-cell detection. Our observations furnish evidence on the possibility to avoid the use of organic acid-stabilized Cd-based systems and substitute their optical contribution with Mn doping in the ZnS system for bioimaging, offering a promising biomaterial for expanding the biomedical applications of semiconducting nanocrystals, and bringing forth new arenas to manipulate their theranostic capabilities.
This work was supported in part by PR NASA EPSCoR (NASA Cooperative Agreement # NNX13AB22A) and the Institute for Functional Nanomaterials (NSF Grant 1002410). J. Gonzalez-Feliciano and D. Bracho Rincon were supported by the Molecular Sciences Research Center Fellowship. This work was also supported by PES funds from UPR to C. I. Gonzalez. We gratefully acknowledge the valuable assistance of Ms. Griselle Hernández-Cancel in the electrochemical measurements. Special thanks to Dr. Kai Griebenow for the use of his research facilities; Dr. Javier Avalos, Olga Medina, and Rafael Velazquez for the bacteria culture; Dr. Luis A. Rivera for his support in photooxidation measurements; and Dr. Liz Díaz-Vázquez for providing fruitful discussions in biosensor fabrication.
Compliance with ethical standards
Conflict of interest
The authors report no conflict of interest.
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