Entrapping of Fullerenes, Nanotubes, and Inorganic Nanoparticles by

Mar 13, 2013 - Guancheng Jiang , Shuanglei Peng , Xinliang Li , Lili Yang , João B.P. ... Fan Liu , Tou-Jun Zou , Zhi-Lin Tan , Guo-Ping Yan , Jun-Fa...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/est

Entrapping of Fullerenes, Nanotubes, and Inorganic Nanoparticles by a DNA−Chitosan Complex: A Method for Nanomaterials Removal Anatoly A. Zinchenko,* Noriko Maeda, Shengyan Pu, and Shizuaki Murata Graduate School of Environmental Studies, Nagoya University, Chikusa, Nagoya 464-8601, Japan S Supporting Information *

ABSTRACT: We report a protocol for entrapping of various water-dispersed nanomaterials: fullerenes, multiwall carbon nanotubes, quantum dots (semiconductor nanoparticles), and gold nanorods, into a DNA−chitosan complex. In contrast to small-size nanomaterial particles, the bulky DNA−chitosan interpolyelectrolyte complex incorporating the dispersed nanomaterials can be easily separated from aqueous media by centrifugation, filtration, or decantation. While the removal of nanoparticles by centrifugation is equally efficient for every type of nanoparticles and reaches 100%, the higher efficiency of the nanomaterials removal by other two methods is favored by larger size of nanoparticles. The application of this entrapping protocol for removal of nanomaterials from water is discussed.

■

INTRODUCTION

quantum dots, and nanorods, and describe the influence of environmentally relevant conditions on the removal efficiency.

During the past decade, developing of nanotechnologies and nanomaterials brought a great number of scientific advantages in almost every area of human life because of their superior properties in comparison to the materials of common scale. Caused by recent growth of nanomaterials’ production and broadening of their applications,1 environmental impacts of the nanomaterials are being increasingly concerned.2−6 Small nanometer size of the nanomaterials plays an important role in their increased mobility through water and soil7 and bioaccumulation.8,9 On the other hand, nanosized materials can be inhaled, ingested, absorbed through skin by living organisms,10 and being carried by body liquids, they can easily reach living cells,11 penetrate through a cell membrane,12 and induce cell damage. At the molecular level, light-activated nanomaterials may cause significant damage of important biological molecules, such as DNA13−15 and proteins.16,17 Therefore, certain types of nanomaterials may represent obvious environmental and health risk in a coming future.18,19 Until present, toxicity assessments for some important classes of nanomaterials have been carried out;20−23 however, further efforts are still necessary to understand nanomaterials pollution and transport in the environment,7,24−27 as well as to establish methodologies for removal of nanomaterials in case of potential environment nanocontamination.28,29 Importantly, the approaches to the latter problem have not been developed until now with a few exceptions. Motivated by the importance of the above issue, we propose a facile, efficient, and universal (independent of nanomaterial structure) method for entrapping nanoparticles in aqueous solutions by complexation with two natural polyelectrolytes, DNA, and chitosan. In this report, we demonstrate successful realization of this protocol for removal of water-soluble fullerenes, nanotubes, semiconductor © 2013 American Chemical Society

■

EXPERIMENTAL SECTION Materials and Methods. Sodium salt of DNA (∼9000 bp, 90% purity, A260 A280 = 1.75) was a gift from Maruha Nichiro Holdings, Inc. (Japan). Chitosan 50 (Mw = 6100−8050 g/mol, deacetylation degree 80 mol %) was purchased from Wako Pure Chemical Industries Ltd. (Japan). Hydroxyfullerene (C60(OH)n, n = 6−12, Nanom spectra D100) was purchased from Frontier Carbon, Japan. Semiconductor nanoparticles (Qdot 565 ITK Amino (PEG) and Qdot 705 ITK Amino (PEG) quantum dots) were obtained from Invitrogen (Japan). Amine conjugated gold nanorods particles were custom-made by Nanopartz (U.S.A.). Multiwall carbon nanotubes NC7000 were purchased from Nanocyl (Belgium). Table 1 summarizes the nanomaterials used in the current study together with their geometrical and charge characteristics. Preparation of Stock Solutions. DNA. Five millimolar DNA stock solution was prepared by dissolution of DNA sodium salt in pure Milli-Q water, and the concentration of DNA phosphate groups was confirmed by UV absorbance spectroscopy at 260 nm based on double stranded DNA absorbance coefficient 6600 L·mol−1·cm−1. Chitosan. Because chitosan is not soluble under neutral pH conditions, 1 mM and 5 mM chitosan stock solutions were prepared by dissolution of chitosan in 10 mM HCl solution followed by the dilution with 40 mM Tris-HCl (pH 6.8). The concentration of chitosan monomer units was calculated based Received: Revised: Accepted: Published: 4489

June 18, 2012 January 20, 2013 March 13, 2013 March 13, 2013 dx.doi.org/10.1021/es302441c | Environ. Sci. Technol. 2013, 47, 4489−4496

Environmental Science & Technology

Article

Table 1. General Characteristics of Investigated Nanomaterials

a

nanomaterial

Qdot565

Qdot705

hydroxyfullerenes

MWCNTb

gold nanorodsc

size (nm) ξ potential (mV)a concentration optimal chitosan to DNA ratioa mixing order

5 (TEM) −11 ± 5 20 nM (∼20 mg/L)d 1.6 1. chitosan 2. DNA

12 (TEM) −11 ± 5 20 nM (∼250 mg/L)d 1.6 1. chitosan 2. DNA

1 (theoretical) −20 ± 8 1.9 mg/L 1.5 1. chitosan 2. DNA

20 × 300 (TEM) +28 ± 9 26 mg/L 1.5 1. DNA 2. chitosan

10 × 30 (TEM) +10 ± 4 0.43 nM (∼20 mg/L)d 1.5 1. DNA 2. chitosan

Under neutral pH condition. bAmine-conjugated. cDispersed with cetyltrimethylammonium bromide. dRough estimation.

Experiments at various pH were performed by adjusting the solution pH by either 0.1 M HCl or 0.1 M NaOH solutions. The pH of the samples was measured by Horiba B-212 pH meter at room temperature. The data on the dependence of nanomaterial concentration in solution with varied concentrations and pH (Figure 1 and 2) are based on a single experiment. For statistical analysis of the

on the 80% deacetylation degree data provided by manufacturer. Hydroxyfullerene. Hydroxyfullerene (C60(OH)n) was dispersed in water at 0.01% concentration and sonicated for 15 min. Insoluble fullerene was separated by centrifugation at 15 000 rpm for 15 min. To determine the concentration of the fullerene in solution, a small amount of hydroxyfullerene was completely dissolved in 1,4-dioxane and the solution was diluted by water 50 times, upon which no precipitation was observed. The absorbance at 500 nm was measured by UV−vis spectroscopy and the extinction coefficient of hydroxyfullerene was determined. The concentration of the fullerene in the water stock solution used for further experiments was found to be 3.2 mg/L. MWCNT. Multiwall carbon nanotubes NC7000 were dispersed into water by mixing 2 mg/L MWCNT with 1 wt % cetyltrimethylammonium chloride, (CTAB, Wako Pure Chemical Industries, Ltd., Japan), sonication at 20 kHz for 10 min, and separated from nondispersed fractions by centrifugation at 5000 rpm for 10 min. The solution with dispersed nanotubes was further purified from a large initial excess of CTAB by three successive rounds of high-speed centrifugation (22 000 rpm, 30 min), removal of mother liquid containing no nanotubes (colorless liquid), and addition of the same amount of Milli-Q water. The concentration of MWCNT in the resulted solution was measured by UV−vis spectrometry based on extinction coefficient at λ = 500 nm equal to 0.024 L·g−1·cm−1. Gold Nanorods. Solution of gold nanorods was prepared by dilution of concentrated stock solution provided by a manufacture with Milli-Q water. General Protocol for the Removal of Nanomaterials. To aqueous solution of dispersed nanomaterials with the concentrations shown in Table 1, DNA and chitosan solutions were successively added with the interval of 15 min and gently mixed. The order of DNA and chitosan mixing was chosen to first add the like-charged component to nanomaterials and then the oppositely charged polyelectrolyte. The resulted solution was allowed to stay over 1 h, after which the formed precipitates were separated by one of the following methods: (i) samples were centrifuged at 10 000 rpm for 15 min using ultracentrifuge (model 7780, Kubota, Japan); (2) samples of nanomaterial−DNA−chitosan three-component system in 2.0 mL test microtube were allowed to stay at room temperature for additional 24 h, after which around 90% of the supernatant above precipitate was carefully removed and subjected to analysis; and (iii) samples were filtrated through a cellulose acetate membrane filter (Advantec, Japan) with 0.20 μm pore size. After removal of each nanomaterial using one of the above method, the concentration of nanomaterial in the supernatant or filtrate was determined by UV−vis spectroscopy.

Figure 1. Removal efficiency of various nanomaterials by the mixture of DNA and chitosan from aqueous solutions. (A, control) Dependence of a relative amount of precipitated DNA on the ratio of DNA to chitosan monomer units after separation of insoluble precipitate by centrifugation. The amount of DNA was detected by UV−vis spectroscopy at a wavelength 260 nm. (B−F) Dependences of a relative amount of fullerene (B), Qdot 565 (C), Qdot 705 (D), MWCNT (E), and gold nanorods (F) removed from solution on the ratio of DNA to chitosan monomer units after addition of DNA and chitosan and separation of insoluble precipitate by centrifugation. Concentrations of DNA is 300 μM for removal of Qdots 565 and 705, and 100 μM for other nanomaterials and in the control experiment (A). The amount of nanomaterials was detected by UV−vis spectroscopy at 330 nm for Qdots, MWCNT, and fullerenes, and at 820 nm for gold nanorods. 4490

dx.doi.org/10.1021/es302441c | Environ. Sci. Technol. 2013, 47, 4489−4496

Environmental Science & Technology

Article

aqueous solutions of various pHs at the concentrations shown in Table 1 at room temperature. Transmission Electronic Microscopy (TEM). TEM observations were performed at room temperature using a HITACHI H-800 microscope (Japan) at 200 kV acceleration voltage. For TEM measurements, a drop of the solution containing nanomaterial, DNA, and chitosan, was placed immediately after addition of the last component (to avoid aggregation of the complex into a large precipitate) onto a 3 mm copper grid covered with a collodion film. Blotted solution was removed after 3 min with a filter paper, and the sample was dried at room temperature before observation.

removal efficiency of nanomaterials by methods i−iii above, each removal experiment was repeated three times.

■

RESULTS AND DISCUSSION DNA−Chitosan Complex Formation in Solutions with Various pH. It is widely known that mixing of oppositely charged water-soluble polyelectrolytes results in formation of polymer-colloid complexes.30,31 A 100 μM DNA (∼9000 bp, concentration is given in phosphate units) solution was treated with chitosan (M w = 6100−8050 g/mol), and after centrifugation (10 000 rpm for 15 min) DNA concentration in supernatant was analyzed by UV−vis spectroscopy at 260 nm. The relation between DNA to chitosan monomer units ratio and the percentage of precipitated DNA, calculated based on DNA absorbance intensity before and after centrifugation, is shown in Figure 1A. Addition of chitosan to the DNA solution from [chitosan]/[DNA] = 0 to 1.5 results in a gradual decrease of DNA amount in solution until a complete DNA precipitation at the ratio 1.5. When [chitosan]/[DNA] ratio was further increased above 1.5, the amount of DNA remained in solution after centrifugation increased again, indicating the formation of nonstoichiometric chitosan-rich water-soluble cationic complexes. Because the ionization degree of DNA and chitosan polyelectrolytes depends on the pH of solution, the degree of nanomaterial removal as well as the optimal chitosan/DNA ratio to achieve the maximal removal of nanomaterial is influenced by the acidity of media. Figure 2A shows three precipitation curves of DNA by chitosan at pH 3.3, 6.8, and 10.8, respectively. The character of the curves, that is, DNA precipitation at lower concentrations of chitosan and redispersion into solution at higher concentrations, resembles the trends in Figure 1A, obtained under neutral conditions, but differs in [chitosan]/[DNA] ratios, at which the nanomaterials’ removal is the most complete. Under acidic condition, amino groups of deacetylated chitosan are well protonated, while the negative charge of DNA phosphates is suppressed; therefore, charge-stoichiometric, insoluble complex is formed at lower [chitosan]/[DNA] (∼0.75 at pH 3.3) in comparison to complex obtained at neutral pH. The opposite tendency was observed under basic conditions at pH 10.8, at which the higher concentrations of chitosan were necessary to achieve the maximal removal of complex. It is worth mentioning that chitosan is poorly soluble under basic conditions because of amines deprotonation and the observed results at pH 10.8 (Figure 2A) should be interpreted as a mixed effect of phase separation (precipitation) of chitosan itself in basic solutions and the formation of polyelectrolyte complex with DNA. Removal of Nanomaterials from Aqueous Solutions by Entrapping Them into a DNA−Chitosan Complex. It was suggested that the interpolyelectrolyte DNA−chitosan complex formed in a solution containing particles of nanomaterials may entrap the nanoparticles into the water-insoluble

Figure 2. Effect of solution pH on the efficiency of nanomaterials removal. (A) Dependence of a relative amount of DNA remained in solution of DNA and chitosan mixture on the ratio of DNA to chitosan monomer units at pH = 3.3, 6.8, and 10.8 after separation of insoluble precipitate by centrifugation. (B, C) Dependence of a relative amount of Qdot 705 (B) and fullerene (C) removed from solution on the ratio of DNA to chitosan monomer units in solutions of acidic, neutral, and alkaline pHs after addition of DNA and chitosan and separation of insoluble precipitate by centrifugation. Concentrations of DNA is 300 μM for removal of Qdot and 100 μM for removal of fullerene.

UV−Vis Spectroscopy. UV−vis spectra of DNA and nanomaterials were recorded on a Jasco J-550 spectrophotometer in a 1.0 cm × 1.0 cm × 5.0 cm quartz cells at a room temperature. The amount of nanomaterials was detected by UV−vis spectroscopy at the wavelengths that are not influenced by DNA absorbance: at λ = 330 nm for Qdots, MWCNT, and fullerenes, and at λ = 820 nm for gold nanorods, supposing that the absorbance of the nanomaterials is linearly dependent on the concentration of nanomaterial in solution. The detection limit of spectrometer is 0.001 OD, which corresponds to maximum 1% experimental error of removal percentage measurements. ζ-Potential Analysis. The ζ potential of nanomaterials was measured by a Zetasizer Nano ZS (Malvern, England) in 4491

dx.doi.org/10.1021/es302441c | Environ. Sci. Technol. 2013, 47, 4489−4496

Environmental Science & Technology

Article

can be considerably removed even at ratios much lower than 1.5 with the efficiency around 80%. In contrast, small semiconductor nanoparticles (Qdots) of 5 or 12 nm diameter can be efficiently removed only at the ratios near 1.5. The nanoscale carbon materials represent an intermediate case; their removal efficiency gradually increased from 40% to 100% at 1.5 polyelectrolyte’s ratio. Higher efficiency of the removal of larger particles can be explained by both larger contact surface between nanoparticles and polyelectrolyte molecules as well as by the possibility of precipitation upon centrifugation of even noncharge-stoichiometric complexes, where cationic and anionic charges are not compensated, due to a loading with large (heavy) nanoparticles. Next, we studied the influence of pH on the removal of nanomaterials by polyion complex. Figure 2B and C show spectroscopic data on removal efficiencies of Qdot 705 and fullerenes, respectively, by DNA and chitosan under basic, neutral, and acidic pH of the original solution with dispersed nanomaterial as a function of [chitosan]/[DNA] ratio. According to zeta potential measurements (Supporting Information available), the average change on fullerene particles was around −25 mV and this value was not significantly influenced by the change of the pH. In contrast, zeta potential of Qdots having amino groups on the surface was highly dependent on the proton concentration and changed from ∼+20 mV at acidic pH to ∼−20 mV at alkali pH, respectively, apparently because of the protonation of the amino groups at a high proton concentration. The correlation between [chitosan]/[DNA] ratios for the maximal removal efficiency of Qdot 705 (Figure 2B) and [chitosan]/[DNA] ratios at which DNA precipitates completely (Figure 2A) is affected by solution pH. At neutral pH, when Qdot particles are weakly charged, [chitosan]/[DNA] ratio at which the best removal results were obtained (1.6) was the same as the [chitosan]/[DNA] ratio at which the most complete DNA precipitation took place. At acidic pHs, where Qdots are positively charged (ξ ≈ +20 mV), less positively charged chitosan should be added for complete removal of Qdots in comparison to the corresponding chitosan-DNA system without nanomaterials. On the contrary, at alkali pH, Qdots are negatively charged (ξ ≈ −20 mV), therefore, larger amount of cationic chitosan is necessary to achieve the maximal removal. These tendencies make it clear that the efficient removal of nanomaterials requires the formation of neutral complex with compensated charges. It is noteworthy, however, that at any pH there was no significant decrease in the removal percentage of Qdot, which emphasizes the applicability of the method to be used in a broad range of solution pHs. In contrast to Qdots, fullerene has only nonionogenic hydroxyl surface groups and the electrostatic surface charge of fullerenes is almost not affected by the pH change of solution (see Supporting Information); therefore, the ratios of [chitosan]/[DNA] for optimal removal of fullerenes (Figure 2C) are exactly the same as the ratios at which the most complete precipitation of DNA by chitosan occurs (Figure 2A) under each studied pH. The above results revealed the importance of solution pH for selection of proper chitosan−DNA ratios to entrap and remove nanomaterials. Under optimal conditions the nanomaterials can be removed by the proposed method with over 95% efficiency in a broad pH range. Beside pH change, another important factor affecting DNA− chitosan complex stability is the ionic strength of solution. In

complex, and this can be used as a strategy for removal of nanomaterial from water dispersions. For our study, we chose four widely used and well-described nanomaterials of various chemical composition, size, shape, and charge: hydroxyfullerenes, multiwalled carbon nanotubes, semiconductor nanoparticles (Qdots), and gold nanorods. The main characteristics of studied nanomaterials are summarized in Table 1. Removal of nanomaterials from water solutions was performed by successive addition of chitosan and DNA solutions into solution with dispersed nanomaterials and separation by centrifugation at 10 000 rpm. First, we tested separately the effects of DNA, chitosan and their mixture on removal of negatively charged fullerenes (ζ ≈ −20 mV) by monitoring the residual amount of fullerene in supernatant by UV−vis spectroscopy after centrifugation. Addition of only DNA (100 μM) to fullerene (1.9 mg/L) solution and centrifugation had no effect (0% removal) on the fullerene contents in solution due to no complexation between likecharged components. Addition of chitosan (150 μM) into the same solution of fullerene and centrifugation resulted in about 45% removal of fullerene from solution, obviously because of the formation of electrostatic complex between positively charged chitosan and negatively charged fullerene nanoparticles. When the same amounts of polyanion and polycation were successively added into solution of fullerenes, the complete 100% removal of fullerenes was achieved, indicating that the mixture of oppositely charged polyelectrolytes is promising for a highly efficient removal of dispersed nanomaterials from aqueous solutions. The idea to use a pair of polyanion and polycation was applied to nanomaterials of various nature, and Figure 1B−F summarizes the results of UV−vis spectroscopy measurements of nanomaterials’ removal by DNA and chitosan from solutions with pH 6.8 at different [chitosan]/[DNA] ratios. After centrifugation, the residual amount of nanomaterial in the supernatant was analyzed by UV−vis spectroscopy at λ = 330 nm for all nanomaterials except gold nanorods monitored at λ = 850 nm. During preliminary tests it was found that several percents higher removal efficiency was achieved upon first addition of like-charged polyelectrolyte to nanomaterial and further precipitation with oppositely charged component; therefore, the order of mixing was chosen depending on the charge on nanomaterial (Table 1, mixing order). The similar tendency was found for each nanomaterial: the increase of the amount of chitosan until [chitosan]/[DNA] = 1.5 caused the decrease of nanomaterial in solution, indicating that nanomaterials were gradually entrapped into the insoluble complex with DNA and chitosan, but at the ratios higher than 1.5 the efficiency of nanomaterials’ removal decreased again as a result of a formation of water-soluble complexes enriched with positively charged chitosan resembling the experimental data in Figure 1A. With no regard to the size or charge on nanomaterials, the maximum removal efficiency of each nanomaterial reached the value near 100% at the [chitosan]/ [DNA] = 1.5, that is, when the precipitation of DNA by chitosan without nanomaterials in solution (Figure 1A) was the most complete. It is also important to mention that 10 000 rpm centrifugation of each type of free nanomaterial in the absence of polyions for 15 min could not remove more than 1% of the dispersed nanomaterial. Comparing the removal efficiency of five types of nanomaterials (Figure 1B−F), it can be concluded that larger particles such as gold nanorods of 30 nm length and 10 nm thickness 4492

dx.doi.org/10.1021/es302441c | Environ. Sci. Technol. 2013, 47, 4489−4496

Environmental Science & Technology

Article

Filtration and settling of complexes with relatively heavy carbon nanotubes were characterized by rather high removal efficiencies (88% and 96%, respectively). Removal efficiency of small quantum dots decreased to about 70−80%. Interestingly, the best removal efficiency method between filtration and settling can vary for the same nanomaterial (quantum dots) of different size, i.e., filtration is better for complexes with larger Qdot 705, but settling shows better results for smaller Qdot 565. Comparison of three methods of removal in Figure 3 clearly shows that centrifugation is the best method, but filtration and settling methods work satisfactorily only for relatively large nanomaterials and these two methods show similar percentage of nanomaterial removal. According to the above data (Figure 3) the removal efficiencies of various nanomaterials depends strongly on the size of the nanomaterial, therefore, the removal of neutral small nanoparticle such as fullerene is the most challenging. Therefore, in order to increase the efficiency of nanomaterial removal, one may consider repeating the removal protocol to achieve a better performance. To examine the removal efficiency at lower nanomaterials concentration, we tested the removal of 4 nM Qdot 705 by DNA and chitosan in a similar manner. It was found that at 5 times lower concentration of Qdots, the percentage of nanomaterial removal changed to 95% (10 000 rpm centrifugation), 92% (filtration), and 79% (settling), being of the same order with that of 20 nM nanomaterial removal efficiency, i.e., 100%, 85%, and 69%, respectively. The estimated residual amount of nanoparticles after two runs of removal reaches around 95% even using the least efficient settling protocol. Thus, the removal efficiency of small size nanomaterials can be amplified by multiple repeating of the removal protocol. Microscopic Observations of DNA−Chitosan−Fullerene Complexes. To get deeper insight into morphology of DNA−chitosan complex with the entrapped nanomaterial we performed transmission electron microscopy (TEM) observations of carbon nanotubes entrapped into the polyelectrolyte complex. A typical TEM image of MWCNT entrapped into DNA−chitosan complex prepared according to general removal protocol is shown in Figure 4. The complexes are large aggregates of several micrometers size with a branched structure. The enlarged image in Figure 4B clearly shows the existence of nanotubes (indicated by arrows) incorporated into the pale matrix of DNA−chitosan complex. Importantly, nanotubes included in the complex are not aggregated, pointing out on the fact that the mechanism of carbon nanotubes removal is not related to the aggregation of nanomaterials together caused by polyelectrolyte addition, but rather it is an entrapping of the nanomaterial into the DNA−chitosan complex.

order to make clear the effect of NaCl on nanomaterial removal by DNA−chitosan complexes, we varied NaCl concentration from 0 to 3 M and studied the percentage of precipitated DNA (Supporting Information available). It was found that DNA− chitosan complexes form and precipitate completely at [chitosan]/[DNA] = 1.5 below 1 M NaCl concentration, while at higher ionic strength only partial precipitation of interpolyelectrolyte complex was observed. Therefore, the scope of this method is limited by low and moderate salt concentrations below ∼1 M of monovalent salt, yet broad enough for treatment of a majority of environmentally relevant samples. Influence of Separation Method on the Removal Efficiency. The efficiency of nanomaterial separation by entrapping into interpolyelectrolyte complex is influenced by the centrifugation rate: for instance, removal efficiency for Qdots 705 by centrifugation of DNA−chitosan−Qdot complex at [chitosan]/[DNA] = 1.6 is also satisfactory at 5000 rpm, but it decreases down to 92% and 85% at 3000 and 1000 rpm, respectively. However, although it is very efficient, the separation of DNA−chitosan−nanomaterial complexes by ultracentrifugation is not practical for large-scale applications. Therefore, we tested the removal efficiency of the nanomaterials-containing complexes by more practical methods: filtration through 0.20 μm pore size filter or by natural precipitation (settling) over 24 h. Removal efficiencies of each studied nanomaterial performed by these three methods are summarized in Figure 3. To compare these separation methods we chose neutral pH solution and ratio of [chitosan]/[DNA] corresponding to the maximum nanomaterial removal.

Figure 3. Efficiency of fullerene, Qdots, MWCNT, and nanorods removal by DNA and chitosan mixtures using centrifugation at 10 000 rpm, filtration through 0.20 μm filter, and settling for 24 h. The experimental conditions correspond to the composition of solutions in Figure 1 at which the maximal removal efficiency was observed. Concentrations of DNA was 300 μM for removal of Qdots and 100 μM for others nanomaterials. Error bars indicate the standard statistical deviation of the average value based on data obtained in three independent experiments. Taking into account the detection limit of spectroscopic analysis (see Experimental Section), 100% removal efficiency in graph above corresponds to less than 1% residual nanomaterial in solution.

■

DISCUSSION We developed a simple procedure for the removal of widely used classes of nanomaterials by a pair of oppositely charged polyelectrolytes to be used as a decontamination protocol for nanomaterials-polluted water. Mixing of DNA and chitosan solutions at the ratios near the charge equivalence results in formation of complexes that entrap the nanomaterials dispersed in solution. The method is robust and works equally well under acidic and basic conditions and ionic strengths below 1 M of monovalent salt. The efficiency of this method generally depends on the protocol used for separation of DNA− chitosan−nanomaterial complex, which reaches 100% when

Contrary to 100% separation by 10 000 rpm centrifugation, when other two methods were applied the efficiency of the removal decreased, and the efficiency of removal again correlated with the size of nanomaterials. Large gold nanorods (10 nm × 30 nm) were separated with almost 100% efficiency by either method. On the other hand, the removal efficiency of the smallest hydroxyfullerenes (∼1 nm) decreased significantly down to ∼60% in the case of filtration or settling/decantation. 4493

dx.doi.org/10.1021/es302441c | Environ. Sci. Technol. 2013, 47, 4489−4496

Environmental Science & Technology

Article

Figure 4. Typical transmission electron microscopy images of DNA−chitosan−MWCNT complex formed at DNA concentration 100 μM and [chitosan]/[DNA] ratio equals 1.5. Dashed square on image A is the enlarged area on B. Arrows on image B indicate single MWCNTs.

Removal of nanomaterials by chitosan (Figure 5A−C) is possible only if nanomaterials are negatively charged, and the efficient removal can be achieved only at the point of charge equivalence (Figure 5B). The important merit to use a pair of polyelectrolytes is its universality and good performance toward nanoparticles of different charge and size, that is, cationic, anionic, and neutral nanosized colloids can be removed using the same protocol and the same ratio of polyanion to polycation at constant pH of solution. This universality is based on the mechanism, according to which nanomaterial binds to the oppositely charged component of polyelectrolytes, either DNA or chitosan, while the like-charged polyelectrolyte is used to neutralize uncompensated charges to form hydrophobic insoluble complex. (Figure 5B′) As shown in Figure 1, the efficiency of DNA and chitosan pair for removal of nanomaterial is higher than that of oppositely charged polyelectrolyte alone. Although this phenomenon requires a more in-depth investigation, we speculate that interaction of colloidal particles and oppositely charged polyelectrolyte is likely to give soluble complexes32 because it contains uncompensated, either cationic or anionic, charges (Figure 5A′ or C′) and such complexes possess a high colloidal stability in solution. In contrast, the presence of both polyanion and polycation at a ratio when cationic and anionic charges are compensated (i.e., at optimal [chitosan]/[DNA] ratio for nanomaterial removal) assures that even in solution of either negatively or positively charged colloids the complex with a high degree of charge compensation is formed as shown in Figure 5A′−C′. Because of the bulkiness, hydrophobicity, and low electrostatic charge, these three-component complexes have a poor colloidal stability and easily precipitate. DNA and chitosan used in this method are natural polyelectrolytes that are available at a reasonable cost from waste products of fishery industry. The application of these biopolymers for environmental treatment is attractive from the viewpoint of DNA and chitosan reuse, as well as their biocompatibility, biodegradability, and nontoxic character. Although in our study we demonstrated only prove-of-concept experiments based on DNA and chitosan, mainly aimed at their reuse from marine waste, the utilization of other combinations of low-cost natural (e.g., alginic acid, chondroitin sulfate, etc.) or synthetic (e.g., poly(acrylic acid), carboxymethyl cellulose,

complexes were separated by centrifugation, and varies from 60% to 100% when either filtration or settling procedures were used. Disregarding the separation protocol used, better results were obtained for nanomaterials of a larger size, while for small size nanomaterials a multistep removal procedure can be recommended to achieve over 95% removal efficiency. The mechanism of removal is mainly based on electrostatic interaction between charged colloids and polymers as was demonstrated by the experiments with negatively charged fullerene that removal was possible only by cationic chitosan or the pair of DNA and chitosan, but not by anionic DNA. Figure 5 schematically illustrates removal of negatively charged nanomaterials by only chitosan (Figure 5A−C) and by the pair of DNA and chitosan (Figure 5A′−C′) at different charge ratios of anionic nanomaterial to cationic polyelectrolyte.

Figure 5. Schematic representation of two scenarios of anionic nanoparticles removal either by only oppositely charged cationic polyelectrolyte (A−C) or by a mixture of cationic and anionic polyelectrolytes (A′−C′) at different charge ratios between anionic NP and cationic polyelectrolyte. A and A′: Low concentrations of nanomaterials. B and B′: Total charge equivalence between NP and polycation. C and C′: Excess of nanoparticles. 4494

dx.doi.org/10.1021/es302441c | Environ. Sci. Technol. 2013, 47, 4489−4496

Environmental Science & Technology

Article

effects of functional nanomaterials delivered to various cell lines. J. Appl. Toxicol. 2010, 30 (1), 74−83. (12) Leroueil, P. R.; Hong, S.; Mecke, A.; Baker, J. R.; Orr, B. G.; Banaszak Holl, M. M. Nanoparticle interaction with biological membranes: does nanotechnology present a Janus face? Acc. Chem. Res. 2007, 40 (5), 335−42. (13) Lu, Z. X.; Zhang, Z. L.; Zhang, M. X.; Xie, H. Y.; Tian, Z. Q.; Chen, P.; Huang, H.; Pang, D. W. Core/shell quantum-dotphotosensitized nano-TiO2 films: Fabrication and application to the damage of cells and DNA. J. Phys. Chem. B 2005, 109 (47), 22663− 22666. (14) Petersen, E. J.; Nelson, B. C. Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA. Anal. Bioanal. Chem. 2010, 398 (2), 613−650. (15) Yamazaki, Y.; Zinchenko, A. A.; Murata, S. A facile method for the assessment of DNA damage induced by UV-activated nanomaterials. Nanoscale 2011, 3 (7), 2909−2915. (16) Deng, Z. J.; Liang, M. T.; Monteiro, M.; Toth, I.; Minchin, R. F. Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation. Nat. Nanotechnol. 2011, 6 (1), 39−44. (17) Wang, J.; Ding, N.; Zhang, Z. H.; Guo, Y.; Wang, S. X.; Xu, R.; Zhang, X. D. Investigation on damage of bovine serum albumin (BSA) catalyzed by nano-sized silicon dioxide (SiO2) under ultrasonic irradiation using spectral methods. Spectrosc. Spectral Anal. 2009, 29 (4), 1069−1073. (18) Handy, R. D.; Owen, R.; Valsami-Jones, E. The ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs. Ecotoxicology 2008, 17 (5), 315−325. (19) Ray, P. C.; Yu, H. T.; Fu, P. P. Toxicity and environmental risks of nanomaterials: Challenges and future needs. J. Environ. Sci. Health C 2009, 27 (1), 1−35. (20) Jones, C. F.; Grainger, D. W. In vitro assessments of nanomaterial toxicity. Adv. Drug Delivery Rev. 2009, 61 (6), 438−456. (21) Gao, J.; Youn, S.; Hovsepyan, A.; Llaneza, V. L.; Wang, Y.; Bitton, G.; Bonzongo, J. C. J. Dispersion and toxicity of selected manufactured nanomaterials in natural river water samples: Effects of water chemical composition. Environ. Sci. Technol. 2009, 43 (9), 3322−3328. (22) Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M. L.; Stroeve, P.; Mahmoudi, M. Toxicity of nanomaterials. Chem. Soc. Rev. 2012, 41 (6), 2323−2343. (23) Dhawan, A.; Pandey, A.; Sharma, V. Toxicity assessment of engineered nanomaterials: Resolving the challenges. J. Biomed. Nanotechnol. 2011, 7 (1), 6−7. (24) Jaisi, D. P.; Saleh, N. B.; Blake, R. E.; Elimelech, M. Transport of single-walled carbon nanotubes in porous media: filtration mechanisms and reversibility. Environ. Sci. Technol. 2008, 42 (22), 8317−23. (25) Chen, G. X.; Liu, X. Y.; Su, C. M. Transport and retention of TiO2 rutile nanoparticles in saturated porous media under low-ionicstrength conditions: Measurements and mechanisms. Langmuir 2011, 27 (9), 5393−5402. (26) Kiser, M. A.; Ladner, D. A.; Hristovski, K. D.; Westerhoff, P. K. Nanomaterial transformation and association with fresh and freezedried wastewater activated sludge: Implications for testing protocol and environmental fate. Environ. Sci. Technol. 2012, 46 (13), 7046− 7053. (27) Chowdhury, I.; Cwiertny, D. M.; Walker, S. L. Combined factors influencing the aggregation and deposition of nano-TiO2 in the presence of humic acid and bacteria. Environ. Sci. Technol. 2012, 46 (13), 6968−6976. (28) Limbach, L. K.; Bereiter, R.; Mueller, E.; Krebs, R.; Gaelli, R.; Stark, W. J. Removal of oxide nanoparticles in a model wastewater treatment plant: Influence of agglomeration and surfactants on clearing efficiency. Environ. Sci. Technol. 2008, 42 (15), 5828−5833. (29) Kiser, M. A.; Westerhoff, P.; Benn, T.; Wang, Y.; Perez-Rivera, J.; Hristovski, K. Titanium nanomaterial removal and release from wastewater treatment plants. Environ. Sci. Technol. 2009, 43 (17), 6757−6763.

etc.) polyelectrolytes is equally promising and can be similarly used for decontamination from nanomaterials. Our choice to use DNA as a polyanion was also reasoned by DNA’s macromolecular stiffness to facilitate assembling of nanoparticles on DNA chain rather than DNA wrapping around nanoparticles, typical mechanism for commonly used flexible polyelectrolytes, to gain higher loading degrees of polyelectrolyte complexes with nanoparticles.

■

ASSOCIATED CONTENT

* Supporting Information S

ζ-potential of fullerenes and Qdots 705 at various pHs, and spectroscopic data on DNA precipitation by chitosan at various NaCl concentrations. This information is available free of charge via the Internet at http://pubs.acs.org/.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Maruha Nichiro Holdings, Inc., Japan, for free DNA samples from salmon milt. We gratefully acknowledge the High Voltage Electron Microscope Laboratory at EcoTopia Science Institute, Nagoya University, for assistance with TEM observations.

■

REFERENCES

(1) Edwards, M. F.; Instone, T. Particulate products their manufacture and use. Powder Technol. 2001, 119 (1), 9−13. (2) Nel, A.; Xia, T.; Madler, L.; Li, N. Toxic potential of materials at the nanolevel. Science 2006, 311 (5761), 622−627. (3) Jiang, G. X.; Shen, Z. Y.; Niu, J. F.; Zhuang, L. P.; He, T. D. Nanotoxicity of engineered nanomaterials in the environment. Prog. Chem. 2011, 23 (8), 1769−1781. (4) Gottschalk, F.; Nowack, B. The release of engineered nanomaterials to the environment. J. Environ. Monitor. 2011, 13 (5), 1145−1155. (5) Peralta-Videa, J. R.; Zhao, L. J.; Lopez-Moreno, M. L.; de la Rosa, G.; Hong, J.; Gardea-Torresdey, J. L. Nanomaterials and the environment: A review for the biennium 2008−2010. J. Hazard. Mater. 2011, 186 (1), 1−15. (6) Thomas, C. R.; George, S.; Horst, A. M.; Ji, Z. X.; Miller, R. J.; Peralta-Videa, J. R.; Xia, T. A.; Pokhrel, S.; Madler, L.; GardeaTorresdey, J. L.; Holden, P. A.; Keller, A. A.; Lenihan, H. S.; Nel, A. E.; Zink, J. I. Nanomaterials in the environment: From materials to highthroughput screening to organisms. ACS Nano 2011, 5 (1), 13−20. (7) Lecoanet, H. F.; Bottero, J. Y.; Wiesner, M. R. Laboratory assessment of the mobility of nanomaterials in porous media. Environ. Sci. Technol. 2004, 38 (19), 5164−5169. (8) Judy, J. D.; Unrine, J. M.; Bertsch, P. M. Evidence for biomagnification of gold nanoparticles within a terrestrial food chain. Environ. Sci. Technol. 2011, 45 (2), 776−781. (9) Rico, C. M.; Majumdar, S.; Duarte-Gardea, M.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Interaction of nanoparticles with edible plants and their possible implications in the food chain. J. Agric. Food Chem. 2011, 59 (8), 3485−3498. (10) Oberdorster, G.; Oberdorster, E.; Oberdorster, J. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 2005, 113 (7), 823−39. (11) Mahmood, M.; Casciano, D. A.; Mocan, T.; Iancu, C.; Xu, Y.; Mocan, L.; Iancu, D. T.; Dervishi, E.; Li, Z. R.; Abdalmuhsen, M.; Biris, A. R.; Ali, N.; Howard, P.; Biris, A. S. Cytotoxicity and biological 4495

dx.doi.org/10.1021/es302441c | Environ. Sci. Technol. 2013, 47, 4489−4496

Environmental Science & Technology

Article

(30) Philipp, B.; Dautzenberg, H.; Linow, K. J.; Kotz, J.; Dawydoff, W. Poly-electrolyte complexes recent developments and open problems. Prog. Polym. Sci. 1989, 14 (1), 91−172. (31) Thunemann, A. F.; Muller, M.; Dautzenberg, H.; Joanny, J. F. O.; Lowen, H. Polyelectrolyte complexes. Polyelectrolytes with Defined Molecular Architecture, Vol. II; Schmidt, M., Ed.; Springer: Berlin, 2004; pp 113−171. (32) Zinchenko, A. A.; Sakaue, T.; Araki, S.; Yoshikawa, K.; Baigl, D. Single-chain compaction of long duplex DNA by cationic nanoparticles: Modes of interaction and comparison with chromatin. J. Phys. Chem. B 2007, 111 (11), 3019−3031.

4496

dx.doi.org/10.1021/es302441c | Environ. Sci. Technol. 2013, 47, 4489−4496