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Electro-responsive supramolecular graphene oxide hydrogels for active bacteria adsorption and removal Bin Xue, Meng Qin, Junhua Wu, Dongjun Luo, Qing Jiang, Ying Li, Yi Cao, and Wei Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04338 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 1, 2016
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Electro-responsive Supramolecular Graphene Oxide Hydrogels for Active Bacteria Adsorption and Removal Bin Xue, † Meng Qin, † Junhua Wu, ‡ Dongjun Luo, ⊥ Qing Jiang, ‡ Ying Li*, ∥ Yi Cao*, † and Wei Wang* † †
National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University
22 Hankou Road, Nanjing, Jiangsu, China, 210093 Fax: (+86)83595535 Email:
[email protected],
[email protected] or
[email protected] ‡
Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, 22
Hankou Road, Nanjing, Jiangsu, 210093, P.R. China ∥
Jiangsu Engineering Technology Research Centre of Environmental Cleaning Materials,
Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, Jiangsu Joint Laboratory of Atmospheric Pollution Control, Collaborative Innovation Center of Atmospheric Environment and Equipment Technology, School of Environmental Science and Engineering, Nanjing University of Information Science & Technology, 219 Ningliu Road, Nanjing, Jiangsu, 210044, P.R. China
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⊥
Department of Hepatobiliary Surgery, Nanjing Drum Tower Hospital Clinical College of
Nanjing Medical University, 321 Zhong Shan North Road, Nanjing, Jiangsu, 210008, P.R. China KEYWORDS： electro-responsive, supramolecular hydrogel, graphene oxide, bacteria adsorption, water sterilization
ABSTRACT: Bacteria contamination in drinking water and medical products can cause severe health problems. However, currently available sterilization methods, mainly based on the sizeexclusion mechanism, are typically slow and require the entire contaminated water to pass through the filter. Here, we present an electro-responsive hydrogel based approach for bacteria adsorption and removal. We successfully engineered a series of graphene oxide hydrogels using redox-active ruthenium complexes as non-covalent cross-linkers. The resulting hydrogels can reversibly switch their physical properties in response to the applied electric field along with the changes of oxidation states of the ruthenium ions. The hydrogels display strong bacteria adsorbing capability. A hydrogel of 1 cm3 can adsorb a maximum of 1×108 E. coli. The adsorbed bacteria in the hydrogels can then be inactivated by a high voltage electric pulse and removed from the hydrogels subsequently. Owing to the high bacteria removal rate, reusability, and low production cost, these hydrogels represent promising candidates for the emergent sterilization of medical products or large-scale purification of drinking water.
1. INTRODUCTION
Bacteria are major contaminations in drinking water worldwide, especially in developing countries. Bacterial contamination may cause severe health problems, including food poisoning and diseases. According to the water.org, a non-profit organization, one out of ten people in the
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world lack access to safe water. More than one million people die from water-related disease annually. Current water sterilization and purification methods to remove contaminated bacteria are mainly based on the size-exclusion mechanism. 1-5 In order to completely remove bacteria in water, pore sizes of the membranes or cartilages should be comparable to the size of bacteria, which inevitably leads to high cross-membrane water pressure and slow purification speed. Moreover, the membranes can easily get clogged. Therefore, it is highly demanded to develop efficient methods and novel materials for point-of-use water purification. Recently, Cui and coworkers have introduced a bacteria inactivation method with high efficiency and fast purification speed based on a kind of complex materials made of silver nanofibers, carbon nanotubes and cotton, operated in an electric field. 6-10 Similar ideas were also used by Vectitis et al. for virus inactivation. 11-12 With the help of electric field, the bacteria can be efficiently killed when passing through the membrane even though the pore sizes are larger than that of bacteria. Inspired by their work, here we report a proof-of-principle demonstration of bacteria removal using electro-responsive hydrogels based on a novel adsorption-deactivation mechanism. Responsive hydrogels have recently attracted great attention because of their broad applications as smart materials and devices in drug delivery, tissue engineering, sensors, actuators, and energy storage. 13-15 Electro-responsive hydrogels, as one of the attractive smart hydrogels, can change their physical properties, such as mechanical strength, volume, conductivity, and optical transparency in the presence of electric fields. 16-24 Such electrically controlled switch may lead to distinct bacteria adsorption/desorption behaviors of the hydrogels. Therefore, it is possible to use such electro-responsive hydrogels as novel bacteria removal materials. To this end, a series of electro-responsive hydrogels were engineered with graphene oxide (GO) as the backbone and redox-active ruthenium complexes as non-covalent cross-
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linkers. The resulting hydrogels can reversibly switch their physical properties in response to the applied electric field along with changes of the oxidation states of the ruthenium ions. The hydrogels display strong bacteria adsorbing capability. A hydrogel of 1 cm3 can adsorb a maximum of 1×108 E. coli or 3×108 staphylococcus aureus (SAU). The adsorbed bacteria in the hydrogels can then be inactivated by a high voltage electric pulse, desorbed and removed from the hydrogels subsequently. Owing to the high bacteria removal rate, reusability, and low production cost, these hydrogels represent promising candidates for the emergent sterilization of healthcare products or large-scale point-of-use purification of drinking water.
2. EXPERIMENTAL SECTION Synthesis of GO/Ru hydrogel and electrochemical switching between Ru2+ and Ru3+: Stable dispersions of GO in water were prepared at different concentrations with the aid of ultrasound. To prepare the GO/Ru hydrogel, the Ru complex solution was quickly added into the GO aqueous suspension. The hydrogel was centrifuged at 13800 rcf for 5 min to solidify the hydrogel. Typically, only 75% of the total water was trapped in the hydrogel and the supernatant was removed with pipettes. The conversion between Ru2+ and Ru3+ was completed by electrochemistry as shown in Figure S1. In a typical electrooxidation process, the GO/Ru(II) hydrogel without removing the supernatant was placed at the anode. At the cathode, 0.4 mM H 2 SO 4 was used as the electrolyte. The salt bridge was made of 3 % (w/v) agar gel containing 3 M KCl. The redox electrolysis was conducted under an electric potential of 2 V for 0.5 h until the current became stable. The color of the hydrogel turned lighter when the electrooxidation
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was completed. For a typical electroreduction, the current was reversed and the GO/Ru(III) hydrogel was in the cathode under a potential of 1.2 V for 0.5 h. The volumes of the hydrogels were calculated by subtracting the volume of water from the total volume of the GO solution and the ruthenium solution. All the experiments were undertaken at room temperature and the temperature of the system remained unchanged during the electrochemical processes. The adsorption of the bacteria using GO/Ru hydrogel: The bacteria was separated from the LB solution by centrifugation and suspended in PBS buffer. The OD at 600 nm of the bacteria was controlled at 0.6. The hydrogel (400 μL, containing 0.75 mg GO and 0.1 mg Ru(II)) was added into the bacteria solution (400 μL) and the adsorption progress was undertaken in an orbital shaker at room temperature and the OD at 600 nm of the supernatant was constantly measured. The maximum of adsorbed bacteria number was calculated by converting the OD at 600 nm to the concentration of cells with a cell counting chamber operating under a microscope. The catch-and-release of bacteria using GO/Ru(II) hydrogel and regeneration of the hydrogels: In a typical bacteria catching and releasing experiment, the bacteria adsorption was undertaken as described above. Then the same solution with the bacteria-adsorbed hydrogel was placed at the anode of an electrolytic cell as shown in Figure S1. Electrically deactivation of the adsorbed bacteria was achieved by applying a voltage of +15 V for 15 min and then a voltage of -15V for another 15 min across the cell electrodes. Although such a voltage may lead to electrolysis of water, because the current is very low during the electric treatment, the total amount of water decomposed is ~ 9 μL, less than 1.8 % of the total volume of the hydrogel. Then the supernatant containing deactivated and desorbed bacteria was removed and the hydrogel was washed three times with 100 μL PBS. The total volume of water to wash the gel was 300 μL.
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The measurement of the hydrogel conductivity: 400 μL hydrogel are formed in a cube and an multimeter was used to measure the conductivities. The circuit is shown in figure S18D. For GO and Ru(II) sample, the volume of the solution used in the measurement was also 400 μL. The measurement of the fluorescence spectroscopy: Spectrofluorometric experiments were performed using an FP-6500 (JASCO Inc., Japan). The emission spectra were measured using an excitation wavelength of 365 nm. Mechanical Measurements: The hydrogels were carefully transferred to the rheometer plate of the Thermo Scientific Haake RheoStress 6000 with a spatula prior to the measurement. The rheology experiments were then carried out using a strain-sweep mode with a strain amplitude range of 0.01 % to 100 % at 1 Hz and a frequency-sweep mode with frequency of 0.01 Hz to 100 Hz at 0.1 % strain (geometry: 1°/20 mm of cone and plate; gap: 0.37 mm; temperature: 20 °C). The variable temperature experiments were carried out in the temperature mode at stain of 0.1 % and frequency of 1 Hz. The measurement of zeta potentials: The zeta potential measurements were conducted in PBS buffer, the same buffer as we used for bacteria catch and release experiments, to minimize the effect of pH, ionic strength and other factors on the measured zeta potentials. Typically, 100 uL of hydrogels were suspended in 900 uL of PBS buffer and fractured by a vortex mixer (Scientific Instruments, USA) for 15 min at its maximum power. Then the zeta potential of the hydrogel suspension was measured by the Zetasizer Nano ZS (Malvern, UK). The same hydrogels were measured for at least three times to guarantee the reproducibility.
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3. RESULTS AND DISCUSSION The design of the electro-responsive hydrogels is illustrated in Figure 1. GO sheets are noncovalently cross-linked by various ruthenium complexes through π-π stacking and hydrophobic interactions with the ligands. The GO hydrogels can provide high adsorption surface areas as compared with other polymer-based hydrogels. 25-28 Although a few GO-based hydrogels have been engineered, the ones that can be reversibly modulated by electric fields have not yet been reported. 29-32 Ruthenium complexes are widely used in material design because of their unique electrochemical and physical properties. 33-35 Xu and coworkers have demonstrated that ruthenium complexes can be used as redox responsive linkers to engineer redox-responsive hydrogels. 13-14 Moreover, it was reported that the mechanical stability of Ru2+ and Ru3+ coordination complexes is distinct. 36 Since the coordination strength and density of ruthenium complexes can be modulated by changing the valence of ruthenium through electrochemistry, the modulation of the oxidation states of ruthenium complexes on the molecular level is expected to result in fast and reversible changes of the macroscopic properties of the hydrogels. The hydrogels were prepared by mixing solutions of GO and the ruthenium complex, tris(2,2′bipyridyl) dichloro-ruthenium(II) (denoted as Ru(II) hereafter). The final concentrations of GO and Ru(II) were 2.5 mg mL-1 and 0.25 mg mL-1, respectively. Upon mixing with Ru(II), the GO suspension immediately became a solid gel (denoted as the GO/Ru(II) gel), which could stably stay on the bottom of the centrifuge tube without dropping when the tube was flipped bottom-up (Figure 2A and B). Oxidation of Ru2+ to Ru3+ in the gel was accomplished by applying a +2 V potential through a platinum electrode in an aqueous solution and the resulting gel was denoted
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as the GO/Ru(III) gel (Figure S1). The reverse reaction was achieved by applying a -1.5 V potential to the GO/Ru(III) hydrogel. Upon electrical oxidation of Ru2+ in the complex to Ru3+, the hydrogel remained in the gel form while its color became much lighter (Figure 2C). The rheological properties of the GO/Ru(II) and the GO/Ru(III) hydrogels were also characterized. As shown in Figure 2D, the storage modulus, G’, was almost 10 times higher than the loss modulus, G”, over a broad angular frequency range from 1 Hz to 100 Hz for both gels, indicating solid rather than viscous property of the gels. Moreover, the G’ of the GO/Ru(II) gel was ~0.12 MPa, which is higher than that of most supramolecular hydrogels reported so far. 37-40 The oxidation of Ru2+ to Ru3+ dramatically softened the hydrogel and the G’ dropped almost 30 %. Such a substantial decrease of mechanical properties of the hydrogel upon electrochemical conversion indicated that changes of the microscopic chemical property of the cross-linker could be effectively exemplified at the macroscopic level. Notably, the G’ and G” of both the GO/Ru(II) and the GO/Ru(III) supramolecular hydrogels gradually increased from room temperature to ~ 60 °C (Figure 2E). The increase of the rigidity of the hydrogel at elevated temperature is consistent with some reported GO-based hydrogels, 26 probably derived from the thermally induced extension of the coiled graphene sheets and thus increasing their entanglement in the gels. The GO/Ru(II) and GO/Ru(III) gels were mechanically stable up to 2 % strain (Figure S2A and B). In addition, the hydrogels were stable in a wide pH range from 2 to 14 (Figure S3) and became less stable at lower GO and Ru concentrations (Figure S4). All these indicate that the ruthenium complex serve as an efficient non-covalent cross-linker for the construction of physically and chemically stable supramolecular GO hydrogels, whose properties can be switched between different oxidation states. It was also tested whether different ligands (Figure S5) could lead to different physical properties. It was found that the mechanical stability
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of the hydrogel decreased with the decrease of the ligand size (Figure 2F and Figure S6). This is because smaller ligands interact less effectively with GO, and thus they are poorer cross-linkers than bulky ligands. The interactions between GO and the ligands of ruthenium complexes in the hydrogels were further evidenced by Fourier transform infrared spectroscopy (FT-IR) (Figure S7). It is worth mentioning that ruthenium ions alone cannot cross-link GO sheets because the interactions between Ru2+ and GO are weak, so that the mixture of GO and ruthenium ions remained in the solution and the G’ was similar to the G’’ (Figure 2F). Taken together, these results indicate that the π-π stacking and hydrophobic interactions between the ligands and GO are critical for the formation of the cross-linked hydrogel network. In principle, other transition metal complexes can also be used to replace the ruthenium complexes in the hydrogel. As illustrated in Figure S8, a copper complex was also used to form hydrogel with GO. Despite that the hydrogels are formed by non-covalent interactions, they are very chemically and mechanically stable. When the hydrogels were immersed in 8 volumes of PBS (100 mM, pH 7.4) buffer or water for 60 h, less than 5 % of ruthenium complexes diffused from the hydrogel to the solution and the G’ of the hydrogels only decreased by less than 20 %, while the changes for the GO/Ru(III) hydrogels immersed in PBS or water were more predominant than the GO/Ru(II) hydrogels (Figure S9). Next, a series of experiments were performed to investigate whether the ruthenium ions in the hydrogels were indeed reversibly changed between +2 and +3 states during the electrochemical treatment of the hydrogels. Ru(II) shows characteristic fluorescence at ~550-650 nm under an excitation wavelength of ~450 nm while Ru(III) is not fluorescent. The fluorescence spectra of the as-prepared GO/Ru(II) gel, the gel after electrochemical oxidation (GO/Ru(III) gel), and the GO/Ru(II) gel subjected to an oxidation-reduction cycle (GO/Ru(II)* gel) were shown in Figure
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3A. Clearly, oxidation of Ru(II) to Ru(III) diminished the fluorescence, while reduction of Ru(III) to Ru(II) restored the fluorescence. Notably, the fluorescence spectrum of the hydrogel after an oxidation-reduction cycle was similar to that of the freshly prepared hydrogel, indicating that the electrochemical cycle is reversible. The change of the fluorescent properties of the GO/Ru(II) and GO/Ru(III) hydrogels could also be directly viewed under UV excitation (Figure 3B and C). The complete darkness of the GO/Ru(III) hydrogel suggested that the conversion of Ru(II) to Ru(III) was 100 %. This was also confirmed by cyclic voltammetry (Figure S10). The electric properties of GO did not change too much when subject to electrochemical cycles. Chronoamperometry and chronocoulometry were also used to directly monitor the redox of the gels. As shown in Figure 3D and E, the oxidation process is much faster than the reduction process in that a high voltage was used to accelerate the oxidation without producing any by products. Nonetheless, thanks to the high conductivity of the GO/Ru(III) hydrogel, the reduction can be completed within 1 h, which is significantly faster than other electro-responsive polymeric gels reported in literatures. 22-23 The total amount of electrons involved in the oxidation processes was similar to that in the reduction. This further confirmed the complete and reversible electrochemical switch of ruthenium between +2 and +3 states in the hydrogels. Next, the changes of physical properties of the hydrogels upon electrochemical switch were studied. Figure 4A and B are the scanning electron microscope (SEM) images of the GO/Ru(II) and the GO/Ru(III) hydrogels, respectively. Clearly, the GO sheets in the GO/Ru(II) gels were more wrinkled than those in the GO/Ru(III) gels. Macroscopically, this led to ~38% volume increase of the hydrogels upon oxidation (Figure 4C). Because Ru(III) complexes is mechanically less stable than the Ru(II) complexes, 41 we hypothesized that the volume changes of the hydrogel upon oxidation might be associated with partial decomposition of the
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mechanically labile Ru(III) complexes. Figure 4D summarized the volume and storage modulus changes of the hydrogels over 3 electrochemical oxidation/reduction cycles. The changes of these properties were reversible, indicating that the hydrogels can be electrochemically manipulated for many cycles. The volume of the hydrogel may depend on both the repulsion forces among GO sheets and the cross-linking strength of the ruthenium complex. The hydrogels of GO with other ruthenium complexes showed similar reversible mechanical switches upon electrochemical oxidation/reduction (Figure S11). However, the reversibility of the hydrogel decreases as the ligand of the ruthenium complex becomes larger. It seems that weak and dynamic cross-linking could facilitate the structural change of the hydrogels at the microscopic level and therefore accelerate the electro-responsiveness. The conductivity of Ru(II) solution is less than 1 S m-1 and that of GO solution is less than 2 S m-1 while the conductivity is 2.2 S m-1 for GO/Ru(II) and 6.1 S m-1 for GO/Ru(III) hydrogels, making the hydrogels suitable for electrochemical modulation (Figure 4E). The high conductivity of the hydrogels is derived from the formation of three-dimensional network of GO and Ruthenium complexes in the hydrogel. Such high conductivity is a combined contribution from electrons and ions, and is distinct from the conductivity of GO in the solid form. In all these measurements, the salt concentrations were kept the same so that the conductivity was not affected by the salt concentrations. It is worth mentioning that typically the conductivity of salt solution of ~ 100 ppm alone is less than 1 S m-1, much lower than the conductivity of the GO/Ru hydrogels. The zeta potentials of the hydrogels also changed dramatically at different states (Figure 4F). The zeta potentials of the GO, GO/Ru(II) and GO/Ru(III) are -39.5 mV, -30.9 mV, and -11.3 mV, respectively. The positive charges of the ruthenium complexes could neutralize the charges on the GO surface, leading to the decrease of the zeta potential of the hydrogels.
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Owing to their impressive switchable physical properties, there are many possible applications of the hydrogels as electrochemical activators, controllable resistors (Figure S12), and sensors. 42-46 As a proof-of-principle demonstration, the application of such gels for bacteria adsorption and removal was illustrated. When placed in a solution containing Escherichia coli (E. coli, XL1Blue, OD 600 nm = 0.6), the GO/Ru(II) and GO/Ru(III) hydrogels exhibited distinct bacteria adsorption behaviors. The SEM images in Figure 5A and B show that there were more ellipsoidal dots on the GO sheets in the GO/Ru(II) gels than those in the GO/Ru(III) gels. These ellipsoidal dots represented the adsorbed E. coli in the hydrogels with typical sizes of ~1-2 μm (Figure S13A). Clearly, there were more E. coli preferentially adsorbed into the GO/Ru(II) gels than the GO/Ru(III) gels. To further quantify the adsorption kinetics, the amount of bacteria remaining in the solution was measured. Because PBS and water is nutrition deficient, the growth of bacteria in them is negligible (Figure S14C). Therefore, the amount of adsorbed bacteria roughly equals the total amount of bacteria minus the amount of remaining bacteria in the solution. As shown in Figure 5E and Figure S15, the amount of E. coli adsorbed in the GO/Ru(II) hydrogels increased gradually in a period of 2 h, while no appreciable adsorption in the GO/Ru(III) hydrogels could be detected. Accordingly, it is possible to use the GO/Ru(II) hydrogel to “catch” E. coli in polluted water, electrochemically switch it to the GO/Ru(III) form with low bacteria adsorption ability, and meanwhile eliminate the bacteria in the gel. When the hydrogel is switched back to the GO/Ru(II) form, the E. coli capturing ability can be restored. Based on the measurement in Figure 5E, 1 cm3 of the GO/Ru(II) gel can adsorb a maximum of 1×108 E. coli. Such a high E. coli capturing ability makes the GO/Ru(II) gels competitive candidates for water treatment.
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The adsorption behavior of the GO/Ru(II) and GO/Ru(III) hydrogels towards SAU, the most dangerous of all the common staphylococcal bacteria, was also studied. As illustrated in the SEM images in Figure 5C and D, a large amount of SAU (round shape, Figure S13B) appeared on the surface of both hydrogels after being soaked in the solutions containing SAU (ATCC6538， OD 600nm = 0.6), indicating their high adsorption ability to SAU. The adsorption kinetics for SAU is shown in Figure 5F. Both gels showed extremely fast adsorption kinetics. Almost 100% of SAU in the solution were captured in the gels within 5 min. The SAU adsorption ability for both gels was significantly higher than that of E. coli. However, the adsorption did not show any bacteria selectivity towards different oxidation states of the gels. We proposed that the adsorption capability and selectivity might rise from the unique microstructures and surface properties of the hydrogels. Furthermore, the zeta potential of the hydrogels may have great effects on the bacteria adsorption behavior (Figure S16). Bacteria might prefer the habitat with similar zeta potentials. The detailed mechanism is worth further investigation. Although the hydrogels do not show consistent adsorption properties to these two kinds of bacteria at certain states, it was found that the dead bacteria cannot attach to any hydrogel because dead bacteria typically cannot retain the same surface properties as live bacteria and gradually get decomposed. This also rules out the possibility of the adsorption from gravity settling. Therefore, it is possible to use electric field to kill and remove the attached bacteria regardless the electrochemical states of the hydrogels. To this end, a “catch and release” cycle for multiple runs of water purification was designed using the same gel. As shown in Figure 5G, a pulsed electric field was introduced to kill and desorb the adsorbed bacteria for hydrogel regeneration. Before the electric field was applied, the bacteria being captured in the hydrogel remained live as confirmed by their regrowth curves.
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However, after the electric treatment (15 V for 15 min and then -15 V for 15 min), nearly all the bacteria lost their activities (Figure S14A and B). The power consumption for the deactivation process is as low as 0.15 W. As PBS buffer is a nutrition-deficient medium, the growth of bacteria during the electric treatment is neglectable (Figure S14C). To further confirm the bacteria sterilization efficiency, the solutions containing electrically treated and untreated bacteria were inoculated on agar plates (Figure S17). The amount of colonies on the agar plates with electrically untreated bacteria was numerous. By contrast, no viable colony was on the agar plates for the electrically treated bacteria, indicating both E. coli and SAU were completely inactivated. The dead bacteria were easily removed from the hydrogels by rinsing the gel with PBS for a few times (Figure S14D). About 90 % of the bacteria could be removed after 3 times of washing. Then the regenerated hydrogel was ready for next bacteria capturing cycle. As shown in Figure 5H, the adsorption ability of the GO/Ru(II) hydrogel for E. coli decreased gradually, but was still above 55 % after three cycles. For SAU, the adsorption ability of the GO/Ru(II) hydrogel also reduced after each cycle and remained 58 % after three cycles. Moreover, the accumulated bacteria removal efficiency after three runs of purification for the same bacteria solution was as high as ~96 % for water samples of high bacteria concentrations (Figure S18A). Even though the population of bacteria in the purified water based on OD measurement remained quite high, the aerobic bacterial count of the treated water was less than 100 cfu g-1 (mL), which meets the standard of drinking water (Figure S17). The stability of the hydrogels during the purification process was also measured (Figure S18B and C). The mechanical strength and conductivity of the hydrogel decreased slightly. However, even after ten purification cycles, the mechanical strength and conductivity were still ~ 50~60 % and 70 % of that for the original gel, respectively, indicating great durability of the hydrogel.
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In our experiments, in order to illustrate high bacteria adsorption capacity of the hydrogel, water containing high concentrations of bacteria (OD 600 = 0.6) was used, in which the bacteria concentrations were much higher than that of real contaminated water in nature. Therefore, the volume of water rinsing the gel was comparable with that of the purified water. To demonstrate that the hydrogel is of practical application, we diluted the bacteria in the initial water by 1000 times to make the bacteria concentration close to that in real contaminated water. Although we enlarged the volume of contaminated water, the purification efficacy remained the same as indicated in the aerobic plate count experiments (Figure S17). In this case, the volume of water rinsing the gel was 1000 times less than the purified water. The major advantage of this method is that we can first direct adsorb bacteria in the contaminated water to the hydrogel and then deactivate the adsorbed bacteria by applying electricity to the hydrogel only. Due to the relatively high conductivity of the hydrogel, only low voltage is required to deactivate the bacteria. However, if the electricity is applied to water directly for sterilization, a much higher voltage is required, because the conductivity of water is low. Such a high voltage can cause electrolysis of water. To understand the bacteria deactivation mechanism, the intrinsic toxicity of the ruthenium complexes and GO to E. coli and SAU was measured (Figure S19). The concentration of Ru(II) that can potentially release during the “catch and release” cycles is no more than 3 μM while no GO is released (Figure S9). At these concentrations, the ruthenium complex and GO are almost nontoxic to bacteria in the “catch and release” experiments. To further confirm that the bacteria were deactivated by the electric treatments, a lactate dehydrogenase (LDH) test was used. The LDH test measures the released LDH enzyme from the damaged bacteria. As shown in Figure S19C, adsorption of bacteria in the hydrogel did not cause the release of LDH. However, about
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70 % of the total LDH were released after one cycle of the electric pulse treatment, indicating that the bacteria-killing effect is a direct consequence of electrical pulses. Toxicity of the hydrogels is also a concern for their application for water purification. Therefore, the cytotoxicity of ruthenium complexes and GO to mammalian cells were also estimated. Although the hydrogels can catch mammalian cells from the solution (Figure S20), Ru(II) and GO showed almost no cytotoxicity to both LX2 (human hepatic stellate cell) and 3T3 (mouse embryonic stem cell) cell lines at the concentrations used for the “catch and release” experiments (Figure S21). As trace amount of Ru(II) may diffuse into water during water purification process, the acute toxicity of Ru(II) to mice was also studied (Figure S22). Even at a Ru(II) dose of 1 g/kg body weight (BW), all mice fed with the Ru(II) solutions survived in the experiments for more than 7 days. The administration of Ru(II) solution did not cause any noticeable effects to the performance and weight of the mice. Moreover, Ru(II) also did not lead to any appreciable change to the internal organs of the mice. These results indicate the low toxicity of the hydrogels. 4. CONCLUSIONS In summary, here we reported the preparation and application of novel electro-responsive supramolecular hydrogels based on graphene oxide and ruthenium complexes. Although the hydrogel is self-assembled through non-covalent interactions, it possesses high mechanical and chemical stability and can be reversibly cycled between the oxidized and reduced states. Moreover, the microscopic changes of the chemical properties of the ruthenium complexes through electric stimuli could cause dramatic changes of the overall physical properties of the resulting hydrogels, leading to rich functionalities. Owing to such unique physical properties and extremely low toxicity, they can be used as bacteria absorbers for fast and efficient water
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sterilization as demonstrated in this work. We expect that such electro-responsive hydrogels could also be used in other fields such as artificial muscle or catalysts. 47-48
FIGURES:
Figure 1. Schematic illustration of the electro-responsive supramolecular hydrogel made of ruthenium complexes cross-linked GO sheets. The size of the GO sheet and the ruthenium complex is not to scale. The reverse oxidation and reduction of Ru2+ and Ru3+ in the gel was accomplished by applying different potentials. The Ru(III) complexes is mechanically labile and can be partially decomposed, leading to lower cross-linking density and expansion of the hydrogel volume. The decomposition of the Ru(III) complexes does not cause the release of the hydrophobic ligands to the solution, because they interact strongly with GO sheets through π-π stacking.
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Figure 2. The mechanical properties of the GO/Ru hydrogels. Optical photographs of GO solution (A), GO/Ru(II) hydrogel (B) and GO/Ru(III) hydrogel (C). (D) The rheological properties of GO/Ru(II) and GO/Ru(III) at a strain of 0.1% in the frequency range of 0~100 Hz. (E) Storage (G’) and loss (G’’) moduli of the gels at different temperature at 0.1% of strain and 1 Hz of frequency. (F) The mechanical properties of GO hydrogels with different ruthenium complexes as cross-linkers at 0.1 % of strain and 1 Hz of frequency at r.t.. Ru(II): tris(2,2’bipyridyl) ruthenium(II), Ru’(II): tris(1,10-phenanthroline) ruthenium(II), Ru’’(II): tris(4,7diphenyl-1,10-phenanthroline) ruthenium(II).
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Figure 3. Electrochemically cycling of ruthenium between +2 and +3 states in the hydrogels. (A) Fluorescence spectra for the leachates of the as-prepared GO/Ru(II) hydrogel, GO/Ru(III) hydrogel and GO/Ru(II) hydrogel after one electrochemical redox cycle (indicated as GO/Ru(II)*) with excitation wavelength at 450 nm. (B) and (C) Optical photographs of GO/Ru(II) and GO/Ru(III) under UV light at 360 nm, respectively. (D) and (E) Representative chronoamperometry and chronocoulometry for the electrochemical redox conversion. Black curves correspond to the accumulated charge and red curves correspond to the current in the
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electrochemical redox cycle. The electric field was fixed at +2 V for the oxidation process and 1.5 V for the reduction process.
Figure 4. Electrochemical modulation of the physical and chemical properties of GO/Ru hydrogel. (A-B) SEM images of GO/Ru(II) and GO/Ru(III) respectively. (C) Optical photographs indicating the volume changes of the same hydrogel of GO/Ru at the initial state
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(left), oxidized state (center) and reduced state (right) by roughly measuring the width of the hydrogels. (D) The changes of G’ and volumes of the hydrogels subject to different numbers of oxidation and reduction cycles. (E) The conductivities for Ru(II) solution, GO solution, GO/Ru(II) hydrogel and GO/Ru(III) hydrogel. (F) The zeta potentials for GO solution, GO/Ru(II) hydrogel and GO/Ru(III) hydrogel.
Figure 5. GO/Ru hydrogels for bacteria adsorption. (A and B) SEM images of the GO/Ru(II) and GO/Ru(III) hydrogels with adsorbed E. coli (ellipsoidal). (C and D) The SEM images of the GO/Ru(II) and GO/Ru(III) hydrogels with adsorbed SAU (round). (E) The E. coli adsorption
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kinetics for GO/Ru(II) and GO/Ru(III) hydrogels monitored by OD 600 of the solution. (F) The SAU adsorption kinetics for GO/Ru(II) and GO/Ru(III) hydrogels monitored by OD 600 of the solution. (G) The schematic of bacteria catch and release as well as the hydrogel regeneration. (H) The E. coli and SAU adsorption percentage of GO/Ru(II) hydrogel in different rounds. ASSOCIATED CONTENT Supporting Information. Experimental details and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author:
[email protected],
[email protected] or
[email protected] Funding Sources Six talent peaks project in Jiangsu Province; National Natural Science Foundation of China (Nos. 21522402, 11304156, 11334004, 31170813, 81421091 and 91127026); 973 Program of China (No. 2012CB921801 and 2013CB834100); Priority Academic Program Development of Jiangsu Higher Education, Jiangsu PhD Gathering Scheme; Technology Foundation for Selected Overseas Chinese Scholar; Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT
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We thank Prof. Shuhong Yu and Prof. Huaiping Cong for generously providing the GO samples. This work is funded by Six talent peaks project in Jiangsu Province, the National Natural Science Foundation of China (Nos. 21522402, 11304156, 11334004, 31170813, 81421091 and 91127026), the 973 Program of China (No. 2012CB921801 and 2013CB834100), the Priority Academic Program Development of Jiangsu Higher Education, Jiangsu PhD Gathering Scheme, the Technology Foundation for Selected Overseas Chinese Scholar, and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China. ABBREVIATIONS GO, graphene oxide; CV, cyclic voltammetry; SEM, scanning electron microscope; TEM, transmission electron microscopy; PBS, phosphate buffer; FT-IR, fourier transform infrared spectroscopy; UV-Vis, ultraviolet visible; LDH, lactate dehydrogenase
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Table of Contents Graphic and Synopsis
Electro-responsive hydrogels with switchable physical and chemical properties upon external electric field are engineered based on graphene oxide and redox-active ruthenium complexes. Bacteria can be actively adsorbed into the hydrogels, deactivated by a high voltage electric pulse, and removed. The fast sterilization speed, low cost, and recyclability makes such hydrogels of great potential for emergent water sterilization.
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