Environ. Sci. Technol. 2009, 43, 1598–1604
Engineered Nanomaterial Transformation under Oxidative Environmental Conditions: Development of an in vitro Biomimetic Assay K E V I N M . M E T Z , †,| ANDREW N. MANGHAM,‡ MATTHEW J. BIERMAN,‡ SONG JIN,‡ ROBERT J. HAMERS,‡ AND J O E L A . P E D E R S E N * ,†,§ Environmental Chemistry and Technology Program, Department of Chemistry, and Department of Soil Science, University of Wisconsin, Madison, Wisconsin 53706
Received August 7, 2008. Revised manuscript received December 9, 2008. Accepted December 17, 2008.
Once released into the environment, engineered nanomaterials may be transformed by microbially mediated redox processes altering their toxicity and fate. Little information currently exists on engineered nanomaterial transformation under environmentally relevant conditions. Here, we report the development of an in vitro biomimetic assay for investigation of nanomaterial transformationundersimulatedoxidativeenvironmentalconditions. The assay is based on the extracellular hydroquinone-driven Fenton’s reaction used by lignolytic fungi. We demonstrate the utility of the assay using CdSecore/ZnSshell quantum dots (QDs) functionalized with poly(ethylene glycol). QD transformation was assessed by UV-visible spectroscopy, inductively coupled plasma-optical emission spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDX). QDs were readily degraded under simulated oxidative environmental conditions: the ZnS shell eroded and cadmium was released from the QD core. TEM, electron diffraction analysis, and EDX of transformed QDs revealed formation of amorphous Se aggregates. The biomimetic hydroquinone-driven Fenton’s reaction degraded QDs to a larger extent than did H2O2 and classical Fenton’s reagent (H2O2 + Fe2+). This assay provides a new method to characterize transformations of nanoscale materials expected to occur under oxidative environmental conditions.
Introduction As nanomaterial production and use rise, introduction of engineered nanoparticles into the environment becomes inevitable. Inadvertent release of these materials into the environment may occur at numerous points during a product’s life cycle including production, transport, manufacturing, consumer use, recycling, and disposal (1, 2). Release * Corresponding author phone: (608) 263-4971; fax: (608) 2652595; e-mail:
[email protected]. † Environmental Chemistry and Technology Program. ‡ Department of Chemistry. § Department of Soil Science. | Present address: Department of Chemistry, Albion College, Albion, MI. 1598
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 5, 2009
to the environment during recycling and disposal is of particular concern for nanoparticles incorporated into limited use and/or disposable products. Environmental redox processes may induce breakdown of organic compounds and can also result in dissolution or precipitation of inorganic species. Microorganisms mediate many environmental redox reactions either directly through synthesis of enzymes or indirectly via biogenic oxidant/ reductant production (3). Understanding possible transformations of engineered nanomaterials is essential for evaluating their potential environmental impact. Few systematic studies on the environmental fate of nanomaterials have been published. The stability of some nanomaterials in laboratory settings has been reported (4-8), but conditions employed were not representative of natural environments. Understanding environmental transformations and fate of engineered nanomaterials will enable design and development of environmentally benign nanomaterials, as well as their use as environmental tracers, in environmental sensing, and in contaminant remediation. For example, functionalized Fe3O4 magnetic nanoparticles have been proposed as sorbents for removing toxic metals from natural waters and biological fluids (9). Their chemical stability was assessed by monitoring Fe release into various matrices after 2-h exposure (9). Mechanisms responsible for Fe release, possible changes in nanoparticle surface chemistry, and particle dissolution over longer exposure periods were not discussed. Thus, transformation of such nanoparticles in the environment and concomitant impacts on metal sorption remain unclear. Assessment of the potential environmental transformation of engineered nanomaterials would be facilitated by the development of test systems designed to represent specific environmental conditions. Such test systems for engineered nanomaterials are currently lacking. The objective of this study was to develop a simple, yet robust, assay for investigating potential transformation of engineered nanomaterials under environmentally relevant oxidative conditions. To accomplish this, we developed an assay mimicking the extracellular chemistry of lignolytic fungi. To demonstrate the utility of the assay, we employed functionalized CdSecore/ZnSshell semiconductor nanocrystals or quantum dots (QDs) as prototypical nanoparticles and examined their transformation. We selected QDs because their optical properties provided a convenient means to qualitatively and quantitatively assess changes induced by the biomimetic oxidative assay. QDs cores are often composed of toxic heavy metal chalcogenides encapsulated in a shell composed of a wider band gap material. Passivation by such shells improves optical emission (10), and is reported to protect the QD core from degradation (11). Extensive study of CdSe QDs in laboratory settings has yielded data on alterations to physical structure induced by severe oxidative treatments (viz. photo-oxidation, exposure to high H2O2 concentrations), and strongly acidic environments. These data can aid in understanding the extent of transformation induced by the biomimetic assay (4-7, 10).
Materials and Methods Quantum Dot Preparation. Synthesis and functionalization of CdSecore/ZnSshell QDs are described in the Supporting Information (SI). Biomimetic Assay Design. The biomimetic oxidative assay was modeled after the extracellular chemistry generated by the lignolytic brown rot fungus Gloeophyllum trabeum. Brown rot fungi produce reactive oxygen species in soil environments to initiate decay of woody plant debris (12, 13). One 10.1021/es802217y CCC: $40.75
2009 American Chemical Society
Published on Web 01/28/2009
demonstrated system for extracellular ROS production by lignolytic fungi is a dimethoxyhydroquinone-driven Fenton’s reaction (12-15). In this system, fungi produce H2O2 and organic reductants, and use iron in the environment to sustain a Fenton’s reaction, producing hydroxyl radicals (12-15). In Fenton’s reaction, Fe2+ promotes H2O2 decomposition to produce hydroxyl radicals and Fe3+ (i.e., H2O2 + Fe2+ f • OH + OH- + Fe3+). Brown rot fungi produce organic reductants (e.g., 2,5-dimethoxyhydroquinone, 4,5-dimethoxycatechol) to reduce Fe3+ to Fe2+, thus sustaining the reaction (13, 14, 16). Fenton’s reaction proceeds optimally at low pH (17) consistent with proton activities typical of extracellular environments of lignolytic fungi (viz. pH 3-5) (16). Laboratory G. trabeum cultures exhibit extracellular pH values between 4.3 and 4.7 (15) and extracellular H2O2 concentrations between 50 and 300 µM (13), while iron and 2,5-dimethoxyhydroquinone concentrations each range between 10 and 50 µM (12, 13, 15). To mimic these conditions, solutions consisting of 200 µM H2O2, 20 µM Fe2+, 20 µM of an organic reductant, and 2 µM QDs in 10 mM buffer were prepared. Concentrations of assay components were environmentally relevant, and approach optimal H2O2:Fe molar ratios used in advanced oxidation processes based on Fenton’s chemistry (17, 18). Although G. trabeum produces 2,5-dimethoxyhydroquinone as a reductant, this compound is not currently commercially available and its use would require synthesis and purification (13). We therefore tested two commercially available reductants to sustain the Fenton reaction: methoxyhydroquinone (MHQ) and L-ascorbic acid (AA). In principle, any reductant capable of rapidly reducing Fe3+ to Fe2+ could be used. To achieve a proton activity similar to that present in the vicinity of lignolytic fungal hyphae, we buffered reaction mixtures to pH 4.1 with 10 mM acetate. To provide a point of reference, we also exposed QDs to classical Fenton’s reagent using the same H2O2 and Fe2+ concentrations. As controls, we exposed QDs to each single assay constituent (viz. H2O2, MHQ, AA, Fe2+; Figure S6), as well as to every binary combination of constituents (e.g., H2O2 + MHQ, Fe2+ + AA; Figures S7 and S8) to verify that observed changes resulted from catalytically produced oxidants and not other assay components. All experiments and controls were conducted in triplicate and each experiment was repeated at least three times. The SI contains additional details on assay protocols. Reactant and Product Characterization. UV-visible spectrophotometry, X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), fluorescence emission spectroscopy, and inductively coupled plasma-optical emission spectroscopy (ICP-OES) were used to characterize QDs and changes induced by exposure to the assay (see the SI for details).
Results and Discussion Characterization of As-Synthesized QDs. The quantum dots investigated were poly(ethylene glycol) (PEG)-functionalized CdSecore/ZnSshell QDs (Figure 1a). The presence of the ZnS shell was verified by XPS (Figure S1). Shell thickness did not exceed 1 nm. Quantum dots were functionalized with methoxy-terminated PEG-thiol (either PEG350 or PEG5000) via interaction of the terminal thiol with Zn using a ligand exchange procedure (19). PEG renders QDs soluble and stable in aqueous solutions (19) and is biologically inert (20), making it a useful surface coating for biological applications of QDs (11) and facilitating toxicity assessment of parent and transformed species. Furthermore, PEGthiols can serve as a model for the stability of other thiol ligand-exchanged QDs that are often further functionalized to produce bioconjugates (11).
FIGURE 1. (a) Illustration of PEGylated CdSecore/ZnSshell quantum dot (n ) 6 or 100 in this study). (b) UV-visible absorption (solid line) and fluorescence emission (dotted line) spectra of water-stable PEG5000-QD in unbuffered ddH2O (pH 8). (c) High-resolution transmission electron micrographs of CdSecore/ZnSshell QDs on a lacey carbon-coated Cu grid, showing lattice-resolved images in the inserts (the smallest insert is a 5 nm × 5 nm box). Atomic spacing was 3.6 Å, sonsistent with wurtzite CdSe structures.(d) FTIR spectra of QDs ligand exchanged with PEG5000 (top, black) and PEG350 (bottom, orange). No evidence of coordinating ligands used in QD synthesis was observed by FTIR following ligand exchange with PEG. VOL. 43, NO. 5, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
1599
Figure 1b shows typical UV-visible and fluorescence emission spectra for the water-stable PEG5000-QDs conjugate stock suspension (unbuffered distilled and deionized water (ddH2O), pH 8). The UV-visible spectrum exhibits a steady increase in absorbance as wavelength decreases from 600 nm to a strong absorbance peak centered at 525 nm, caused by the first exciton of the CdSe core; absorbance increases nearly linearly as wavelength decreases further. Similar spectra were obtained from aqueous suspensions of PEG350QD conjugates (data not shown). QD core diameter and number concentration can be determined from first exciton peak position and absorbance in the UV-visible spectrum (21). QDs used in this study had first exciton peaks centered between 525 and 530 nm, corresponding to core diameters of 2.6 ( 0.1 nm (21). While the accuracy of UV-visible spectroscopy methods for determining absolute measures of core diameter and QD concentration has recently received criticism (22), UV-visible spectra retain utility for comparative analyses (e.g., examining changes induced by exposure to oxidative conditions). We employed TEM to independently verify QD size and observed crystalline nanoparticles with diameters of 3-4 nm (Figure 1c), in good agreement with the size of the core-shell QDs estimated from UV-visible and XPS data (viz. 3.5-4.5 nm). FTIR spectra of QD-PEG conjugates in water exhibited at least eight clearly identifiable peaks (Figure 1d), all attributable to PEG (see the SI for peak assignments) Using an empirical relationship between effective diameter and molecular mass of free PEG molecules (23), we estimated the length of attached PEG350 and PEG5000 molecules to be 1 and 4.5 nm. Theoretical diameters of individual PEGylated QDs (based on estimated contributions of the CdSe core, ZnS shell, and extended PEG ligands) were ∼6.5 nm for PEG350-QDs and ∼14 nm for PEG5000-QDs. Dynamic light scattering measurements, using number averaging techniques, revealed that in ddH2O PEG350- and PEG5000-QDs had hydrodynamic diameters of 26 ( 3 nm and 23 ( 1 nm, suggesting PEG5000-QDs formed very small aggregates, while PEG350-QDs aggregated to a larger extent. In 10 mM Na acetate, hydrodynamic diameters of PEG350and PEG5000-QDs (29 ( 4 nm and 24 ( 1 nm) were statistically indistinguishable from those in ddH2O. Transformation of PEG5000-QDs. Transformations of PEG5000-QDs conjugates induced by exposure to oxidizing solutions were monitored by UV-visible absorption spectroscopy, fluorescence emission spectroscopy, DLS, TEM, EDX, and ICP-OES. Exposure of PEG5000-QDs to MHQFenton’s reaction at pH 6, to examine the effect of raising assay pH on QD integrity, is reported in the SI. Optical Characterization of Transformed QDs. UV-visible spectra and fluorescence emission spectra (see the SI) were collected from exposed QD suspensions. UV-visible absorption spectroscopy allows simultaneous monitoring of changes in the physical size of QD cores and changes in QD number concentration. Fluorescence emission spectra of QDs also respond to changes in the physical size of the core, but are sensitive to many other factors (e.g., trapped states, shell condition, surface functionalization, solution chemistry). This complicates interpretation of changes in QD fluorescence emission spectra. UV-visible absorbance spectra of QDs in acetate buffer exhibited first exciton peaks centered near 530 nm, comparable to stock solutions in ddH2O (Figure 2a). Solution spectra of H2O2-exposed QDs exhibited shapes similar to those of buffer controls, but first exciton peak position was blue-shifted to ∼515 nm (Figure 2a), indicating a ∼0.2-nm reduction in core diameter, and peak height declined by ∼8%. After exposure to the methoxyhydroquinone-driven Fenton’s (MHQ-Fenton’s) reaction, PEG5000-QD UV-visible spectra increased monotonically as wavelength declined; the characteristic first exciton peak was absent from these spectra. 1600
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 5, 2009
FIGURE 2. (a) UV-visible absorption spectra of PEG5000-QDs exposed to methoxyhydroquinone-driven Fenton’s reaction. All reactions were conducted in 10 mM acetate buffer (pH 4.1). (b, c) Transmission electron micrographs of PEG5000-CdSecore/ZnSshell QDs after overnight suspension in 10 mM acetate (pH 4.1) and deposition on an amorphous carbon background. (d) Nanoparticle aggregates obtained after overnight exposure of PEG5000-CdSecore/ZnSshell QDs to the MHQ-driven Fenton’s reaction. The dotted circle in (d) represents the area used in analysis by selected area electron diffraction (inset) and energy dispersive X-ray spectroscopy (e). The copper signal in (e) is due to the TEM grid. Compared to buffer and H2O2 controls, MHQ-Fenton’s spectra showed higher absorbance at λ < 525 nm. Methoxyhydroquinone in its reduced or oxidized states does not absorb strongly over the wavelength ranged shown in Figure 2a and therefore did not contribute significantly to the observed monotonically increasing absorbance (see the SI). In most experiments, differences in position and amplitude of the first exciton peak of QDs exposed to H2O2 or classical Fenton’s reaction (H2O2 + Fe2+) barely exceeded measurement uncertainty ((1 nm wavelength, ( 0.01 absorbance; see the SI). The similarities in first exciton peak positions and absorbances for QDs exposed to H2O2 alone and to classical Fenton’s reagent indicated the amounts of • OH produced by classical Fenton’s reaction were insufficient
to degrade PEG5000-QDs to a larger extent than did H2O2 by the end of the 16- to 18-h exposure period. Differences in UV-visible spectra for QDs exposed to classical Fenton’s conditions and to ascorbic acid-driven Fenton’s reaction (AAFenton) also fell within experimental error, indicating AA failed to enhance •OH production to an extent that affected PEG5000-QD size or concentration. This was unexpected as AA was previously used to mimic the extracellular chemistry of brown rot fungi to study phenethyl polyacrylate oxidation (12). In contrast, lack of the first exciton peak in spectra of QDs exposed to MHQ-Fenton’s reaction indicated substantial alteration to QD cores and that methoxyhydroquinone enhanced •OH production in a manner strongly impacting PEG5000-QD integrity. Exposure of CdSecore/ZnSshell QDs to H2O2 induced a blue shift in first exciton peak position similar to that observed upon ∼26 h exposure to high intensity UV light, while exposure to the MHQ-Fenton’s reaction produced changes analogous to sustained photo-oxidation of CdSe QDs (9). These data differ from the photo-oxidation experiments in that we employed a ZnS shell on the QDs. Zinc sulfide shells reportedly act as protective barriers to oxidation of the core, preventing release of Cd and Se (11). Contrary to these reports, the ZnS layer of our QDs did not prevent oxidation of the CdSe core by H2O2 or •OH produced by Fenton’s reagent. Microscopic Investigation of Transformed QDs. To further examine changes to PEGylated QDs induced by exposure to H2O2 and (reductant-assisted) Fenton’s reaction, we separated particles with diameters J3 nm from solution with a 10-kDa nominal MWCO centrifugal concentrator. Given the particle sizes described above, unless disaggregated and severely degraded, QDs were not expected to pass the filter. In one experiment, nanoparticles retained by the centrifugal filter were examined by TEM and EDX. Unaltered individual QDs from aqueous suspensions (10 mM acetate) had diameters
