Versatile Dual Photoresponsive System for Precise Control of

Jun 29, 2017 - A versatile method for photoregulation of chemical reactions was developed through a combination of near-infrared (NIR) and ultraviolet...
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Versatile Dual Photoresponsive System for Precise Control of Chemical Reactions Can Xu,†,‡ Wei Bing,†,‡ Faming Wang,†,‡ Jinsong Ren,† and Xiaogang Qu*,† †

Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡ University of Chinese Academy of Sciences, Beijing 100039, China S Supporting Information *

ABSTRACT: A versatile method for photoregulation of chemical reactions was developed through a combination of near-infrared (NIR) and ultraviolet (UV) light sensitive materials. This regulatory effect was achieved through photoresponsive modulation of reaction temperature and pH values, two prominent factors influencing reaction kinetics. Photothermal nanomaterial graphene oxide (GO) and photobase reagent malachite green carbinol base (MGCB) were selected for temperature and pH regulation, respectively. Using nanocatalyst- and enzyme-mediated chemical reactions as model systems, we demonstrated the feasibility and high efficiency of this method. In addition, a photoresponsive, multifunctional “Band-aid”like hydrogel platform was presented for programmable wound healing. Overall, this simple, efficient, and reversible system was found to be effective for controlling a wide variety of chemical reactions. Our work may provide a method for remote and sustainable control over chemical reactions for industrial and biomedical applications. KEYWORDS: chemical reaction regulation, NIR, UV, enzyme, catalyst, photosensitive

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construct a temperature/pH dual photoresponsive system. Regulation of chemical reactions was realized by simple introduction of light of two different wavelengths. This remotely controlled photoswitching system may provide wide potential benefits in areas including light signal amplifiers, bioelectronics, information storage devices, biosensors, pharmaceuticals, and as model systems for naturally occurring photoactivated biomolecules.2,3,5,12 The working principle of the system is illustrated in Scheme 1. On one hand, graphene oxide (GO) was chosen as a photothermal material.12,15−17 It could absorb near-infrared (NIR) light and convert it into thermal energy with high efficiency, which in turn heats up the reaction solution. On the other hand, malachite green carbinol base (MGCB) was chosen as a pH regulation reagent.18−20 The MGCB molecule could release OH− under UV light irradiation and generate a progressive shift in pH values. With dual operation of NIR and UV light, a broad range of temperatures and pHs could be adjusted on-demand, with profound effects on reaction kinetics and catalyst activities. This approach has many advantages

egulation of chemical reactions can provide remarkable opportunities for the chemical industry and biotechnology.1−3 However, up to now, few comprehensive studies have centered on chemical reaction regulation.1,2 In the case of enzyme-mediated biochemical reactions, most of the reports have been focused on covalent enzyme modifications,4−6 which are expensive, complex, and reduce enzymatic activity. A more general, noninvasive, and reversible method for regulation of chemical reactions is therefore highly desirable. It is known that temperature and pH values are two of the most influential factors determining the rates of almost all types of reactions as well as the efficiencies of catalysts. By taking advantage of these traits, the reaction activity can be easily regulated by changing the temperature and pH conditions in a controllable manner. Among the various control methods, light represents an ideal external stimulus as it possesses several advantages over traditional modulators.7−11 First, light is clean, reliable, and inexpensive. Second, light irradiation is a noninvasive technique that results in minimal secondary perturbation of the reaction system. Most importantly, light irradiation can be easily controlled in spatial and temporal fashion. In nature, there are many sophisticated photoresponsive systems, such as photosynthesis, vision, and photomorphogenesis.12−14 In this report, we employed a photothermal nanomaterial and a photobase molecule to © 2017 American Chemical Society

Received: March 1, 2017 Accepted: June 29, 2017 Published: June 29, 2017 7770

DOI: 10.1021/acsnano.7b01450 ACS Nano 2017, 11, 7770−7780

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ACS Nano Scheme 1. Illustration of the Dual-Responsive Photoswitch Systema

a (a) Photothermal effect of graphene and light-induced pH changing effect of MGCB. (b) With dual control of NIR and UV light, precise control over a broad range of temperatures and pHs is realized.

including: (1) facile operation, label-free chemistry, and fastresponsiveness; (2) widespread application, even for systems nonresponsive to photoiridiation; and (3) the regulation is reversible, noninvasive, and allows for spatiotemporal control. In the present study, we focused on the photoregulation of nanocatalyst and enzyme-mediated reactions and explored one of its biomedical applications as an antibacterial treatment for augmented wound healing. We expect that our finding will provide a method for remote and nondestructive control of chemical reactions, confering advantages in future industrial, environmental, and medical applications.

Figure 1. (A) (a) Illustration of the photothermal effect of GO. (b) AFM height image of GO. (c) Temperature change curves of the GO solution exposed to the 808 nm NIR lasers with different power densities. Control: water exposed to 808 nm NIR (2 W/ cm2). (d) Thermal photographs of GO solution (40 μg/mL) exposed to 808 nm NIR lasers for 10 min with different power densities. (B) (a) Illustration of the light-induced pH change by MGCB. (b) Photographs. (c) UV−vis absorption spectra. (d) pH values of MGCB solution exposed to UV light for varying times: 1: 0 min, 2: 1 min, 3: 2 min, 4: 4 min, 5: 6 min, 6: 8 min, and 7: removal of UV light and placment of the sample in the dark for 40 min.

RESULTS AND DISCUSSION Graphene oxide (GO) (Figure 1A) was synthesized from graphite by modified Hummers method.21 According to AFM characterization, the obtained GO sheet was mostly singlelayered, with a topographic height of ∼1.0 nm (Figure 1Ab). The UV−vis absorption spectrum of the GO dispersion (Figure S1) showed a strong peak at 230 nm, which was assigned to the π−π* transition of aromatic CC bonds and a shoulder peak at ∼290−300 nm, which was attributed to the n−π* transition of the CO bonds.22 Through irradiation with an 808 nm NIR laser, the photothermal effect of GO was investigated.12,15−17 In marked contrast to the buffer sample, the GO solution showed a rapid increase in temperature when exposed to the laser for a short time (10 min) and exhibited an energy-dependent photothermal effect (Figure 1Ac, d). After removal of the light, the temperature could quickly returned to its initial value (Figure S2A). In consideration of reaction regulation, long-term temperature maintenance was deemed necessary. Herein, the

maintenance was also executed by NIR light at a lower power than the initial irradiation. For example, after treatment of GO solution with NIR light of 2 W/cm2, the NIR light of 1 W/cm2 was then introduced. As shown in Figure S2B, the temperature could be sustained for 50 min, and after removal of the light, it could be restored quickly. Triphenylmethane leucohydroxide derivative MGCB was used as a light-induced hydroxide ion emitter (Figure 1Ba).18−20 In the presence of 302 nm UV light, the MGCB molecule elevates to an excited state and dissociates into ion pairs, malachite green (MG) cation and hydroxide anion, resulting in an obvious color change of the solution as well as increase in pH value. When the UV light was turned off, MG cations would recombine with OH ions and return to MGCB. The pH would also recover to the initial value.18 As shown in 7771

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ACS Nano Figure 1Bb, the MGCB solution turned from colorless to deep green rapidly when exposed to a high-pressure UV lamp (500 W, 50 W/cm2), which was ascribed to the state conversion of MGCB. These changes were also reflected by UV−vis spectra. As shown in Figure 1Bc, in the absence of UV light, the solution showed weak absorption in the visible range. While under UV irradiation, a new absorption peak at 617 nm appeared, and the intensity was increased with irradiation time. In the general case, pH could be increased from 5.0 to 9.0 by irradiation for 8 min (Figure 1Bb−d, samples 1−6). After removal of UV light and placing the sample in the dark, the color and pH value of MGCB solution returned to the initial state within 40 min (Figure 1Bb−d, sample 7). Otherwise, the pH could be maintained by exposing the solution to visible light (Figure S3). The influences of NIR/UV light on the pH value and UV light on the temperature of GO solution as well as the influences of NIR/UV light on the temperature and NIR light on the pH value of MGCB solution were also investigated. The results showed no obvious influences except for an increase in temperature of GO solution (from 22 to 49 °C) when treated by UV light (Figure S4). The dual responsive system was simply constructed by mixing GO solution and MGCB solution together (defined as GO-MGCB, Figure 2A). Before investigating the NIR/UV responsive properties of the hybrid materials, the potential influence of temperature on the pH change ability of MGCB was taken into consideration. As shown in Figure S5, for temperatures below 40 °C, negligible influences were observed. However, above 50 °C, there were decreases in the rate of pH change, and after removal of UV light, the pH value recovered quickly, attributed to the rapid reformation of MG cation with hydroxide anion under high temperature. This indicated that our system was not particularly suitable for reactions requiring both high pH and high temperature (>50 °C). Also, as consistent with the GO solution, the UV light could heat the mixture of GO-MGCB from room temperature (22 °C) to 51 °C within 8 min (Figure S6A). To ensure the efficiency of pH change, the UV irradiation was conducted in a water/ice bath if the irradiation time exceeded 3 min. As shown in Figure S6B, when placed in a water bath, the increase in temperature could be reduced remarkably during the process of UV irradiation. As a result, the mixture showed linear pH increases and reached pH 9.0 after 8 min (Figure 2Ba, b), which was similar to that of MGCB alone.The photothermal properties of GO-MGCB under irradiation of NIR was also studied and showed similar results as that of GO alone (Figure 2Ba, c). The induced temperature/pH change could be maintained after appropriate treatments (Figure 2Ca, b). Expectedly, when the light was removed, the temperature/pH recovered to the original state. These processes were thus reversible and could be recycled for many times (Figures 2Ca, b and S7). In order to verify its feasibility and universality, four natural enzymes and four nanocatalysts23,24 catalyzed chemical reactions were chosen as model systems. The natural enzymes included acid phosphatase (ACP), alkaline phosphatase (ALP), cellulase, and amylase. The nanocatalysts included Fe3O4 nanoparticles (Fe 3 O 4 NPs), 2 5 gold nanoparticles (AuNPs), 2 6 − 2 9 cerium oxide nanoparticles (CeO 2 NPs),23,30−32 and platinum nanoparticles (PtNPs).33 The activities for these catalysts were measured by the method described in the Experimental Section. The reactions catalyzed by these catalysts were illustrated in Figures S8 and S9, and the

Figure 2. (A) (a) Illustration of the photothermal and photoinduced pH change properties of GO-MGCB solution under the irradiation of NIR and UV light, respectively. (B) (a, top) Photographs and (b) pH value curves of GO-MGCB solution exposed to UV light for different times. (a, bottom) Thermal photographs and (c) temperature change curves of GO-MGCB solution exposed to 808 nm NIR laser (2 W/cm2) for different times. (C) The maintenance and cycles of (a) temperature and (b) pH values of GO-MGCB solution.

absorption and fluorescence spectra of the products were shown in Figure S10. Based on these results, the pH−activity and temperature−activity profiles were obtained and are shown in Figures 3a, b and 4a, b. The NIR (2 W/cm2) and UV (50 W/cm2) light sources were introduced to regulate the reactions. For ex situ studies, the light was introduced first, and then the catalysts and the substrates were added after the temperature/pH values of the systems stabilized. The activity maps (Figures 3c and 4c) illustrated that broad activity ranges for these catalysts could be created by varying the light irradiation time. It is worth mentioning that for natural enzyme systems, the light irradiation time was constricted within a limited range to avoid thermal- or pH-induced enzyme denaturation. As mentioned above, our system was also not suitable for reactions requiring both high pH and high temperature (>50 °C). Designating the maximum activity under optimum light 7772

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Figure 3. Light-controlled enzyme activity. (a) The illustration, (b) the pH activity and temperature activity profiles, (c) the activity maps obtained by varying the light irradiation time, and (d) remote control of the activities of (A) acid phosphatase, (B) alkaline phosphatase, (C) cellulase, and (D) amylase.

irradiation condition as the “ON” state, while the lowest activity under proper light irradiation condition as the “OFF” state, the ON/OFF regulation of enzymes/catalyst activity could be easily achieved (Figures 3d and 4d). Control experiments (Figure S11) showed that in most cases, the presence of just one component (GO or MGCB) showed diminished modulation capacity for the reaction. In a few cases, one component showed some modulation ability, yet the differences between the “ON” and “OFF” states were not as pronounced as those of the GO-MGCB system (Figures 3d and 4d). These indicated that the combination of the two materials (GO and MGCB) was necessary for efficiently modulating the reaction activity. As for certain applications, the in situ introduction of light may be needed. Different from the ex situ studies, the light was introduced directly to the whole system which included GO/ MGCB, the catalysts, and the substrates in the in situ studies. The enzyme ALP and the nanocatalyst PtNPs were chosen as models. Their activities by in situ NIR/UV irradiation at the optimal conditions (the “ON” state conditions in Figures 3d

and 4d) were investigated. As shown in Figure S12, PtNPs still maintained most of the activity, while ALP lost 33% activity. The effects of NIR/UV light alone on the activity of ALP were also considered. The results showed that the reduction of enzyme activity was mainly attributed to the irradiation of UV light (Figure S12C). As reported previously, the deactivation of enzymes under UV light can be prevented by some radical scavengers, such as threo-1,4-dimercapto-2,3-butanediol (DTT), cysteine, and ascorbic acid (AA).34 Herein, AA was selected as a UV-protector. As shown in Figure S12A, the presence of AA could maintain most of the activity of the enzyme in the process of in situ light irradiation. By taking advantage of the photoswitchable system described above, we constructed a stimuli-responsive hydrogel and explored its potential application in antibacterial treatment and wound healing. It is well-known that the wound healing process can be divided into two main phases: the inflammation phase and the cell proliferation phase.35 However, most of the commercial Band-aids are only helpful in the first phase, while they have no 7773

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Figure 4. Light-controlled nanocatalyst activity. (a) The illustration, (b) the pH−activity and temperature−activity profiles, (c) the activity maps obtained by varying the light irradiation time, and (d) remote control of the activities of (A) Fe3O4 NPs, (B) AuNPs, (C) CeO2 NPs, and (D) PtNPs.

effect or are even detrimental in the cell proliferation phase for injured tissue. This is the reason why Band-aids are only helpful in the first few days, while they must be removed to allow the tissues to recover themselves afterward. In short, the wound should be treated to heal in an orderly and timely manner.36 In consideration of this shortcoming, it is desirable to develop multifunctional “Band-aids” that have controllable properties to act in concert with the wound healing process. In the present study, a light-controlled antibacterial hydrogel (Figure 5A) was prepared by integration of GO, MGCB, and CeO2 NPs in agarose gel (Figure S13). It is remarkable that CeO2 NPs have contradictory roles as either an oxidant or antioxidant, depending on reaction conditions.23,30,37,38 In acidic environments, CeO2 NPs have strong oxidative properties and can generate reactive oxygen species (ROS) to kill bacteria.28,37,39 While in neutral/basic environments, CeO2 NPs display antioxidative properties and can promote cell proliferation.30,40,41 Since the ideal materials for tissue healing should have both antibacterial properties in the first step and promotion of cell proliferation in the following step, the CeO2

NPs are expected to be an excellent adjuvant for wound healing by changing the reaction environment procedurally. As the GOMGCB system has photoinduced pH change and photothermal properties, it may provide a suitable reaction environment for on-demand administration of CeO2 NPs. As shown in Figure 5B, similar to GO-MGCB solution, the GO-MGCB-agarose hydrogel also displayed both of the UV light-responsive pH change and NIR-responsive temperature increase properties. The agarose gel exhibited slower pH change rate as compared with solution phase, which was attributed to the decrease in UV light penetration through agarose gel relative to solution. Meanwhile, compared to the GO-MGCB solution, the hydrogel had better pH retention capacity. The pH value could be maintained for about 24 h after removal of UV light (Figure S14), while the solution could only sustain a high pH for about 6 h (data not shown). This might be attributed to the lower molecular diffusion and reaction rate due to viscosity of the hydrogel. Next, the light-controlled activity of the GO-MGCB-CeO2 gel was studied (Figure 5C). The oxidant-sensitive dye, Rhodamine B (RhB) (Figure 7774

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Figure 5. (A) Illustration of light-controlled, CeO2 NPs emdeded in a hydrogel and potential use as photosensitive Band-aids to facilitate programmed wound healing. (B) Photothermal- and light-induced pH change of GO-MGCB containing agarose gel. (a, top) Photographs and (b) pH profile of GO-MGCB gel irradiated with UV light for different times. (a, bottom) Thermal photographs and (c) temperature change curves of GO-MGCB gel exposed to NIR laser (2 W/cm2) for different times. (C) The light-controlled activity of the GO-MGCB-CeO2 containing agarose gel. Fluorescent photographs of GO-MGCB-CeO2 gel after immersion and reaction with (a) RhB (50 μM) and (b) H2O2 (120 mM)-terephthalic acid (1 mM) systems under different light-treated conditions. UV light treatment time: 4 min. NIR light (2 W/cm2) treatment time: 3 min. Control: Without pretreatment by any light. All of the photographs were taken on a UV flatbed.

5Ca),42 and an antioxidant-sensitive system, H2O2-terephthalic acid (Figure 5Cb),43 were integrated into the hydrogel, respectively. The results showed that before pretreatment by UV light, the hydrogel exhibited a high degradation effect on RhB and could accelerate the H2O2-terephthalic acid reaction. These results indicated that CeO2 NPs had oxidative activity in the absence of UV light because of the acidic environment of the hydrogel. Following pretreatment by UV light for 4 min, the hydrogel failed to degrade RhB and showed protective ability over terephthalic acid by preventing oxidative activity by H2O2. This was attributed to the pH increase of the hydrogel in the presence of UV light, which made CeO2 NPs display an antioxidative property. The hydrogel activity was also associated with NIR light. Both of the oxidant and antioxidant activities could be improved after treatment by NIR light for a short time (3 min) (Figure 5C). As bacterial infection is the main cause of wound infection and inflammation, the antibacterial activity of the photosensitive hydrogel was investigated. Escherichia coli (denoted as E. coli) was chosen as the model bacteria in the following

studies. Disc diffusion assays presented the antibacterial activities of the various hydrogels against E. coli. As shown in Figure 6, there were no apparent bacterial inhibition zones in the samples of agarose gel and GO-MGCB containing agarose gel, indicating that they had poor antibacterial effects. For GOMGCB-CeO2 containing agarose gel, without UV irradiation, the bacterial inhibition zones with large diameters were observed apparently around the gels, indicating excellent bacterial inhibition activities. In addition, NIR light also showed positive effect for the inhibition of bacteria. Following pretreatment by UV light, the gels displayed diminished antibacterial activity. Overall, the GO-MGCB-CeO2 gel with only NIR light irradiation (−UV+NIR) showed the best antibacterial activity. This was because the acidic environment of the gel without UV light promoted the activity of CeO2 NPs as strong oxidants, which could generate high concentrations of ROS to kill the bacteria. Additionally, with the help of NIR light, the temperature of the gel rose, resulting in improved oxidative and antibacterial activities because of the temperaturedependent activity of CeO2 NPs (Figure 4C). 7775

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Figure 6. Photographs of the bacterial inhibition zones of different gels under different light treatment conditions.

bacteria solution pretreated by GO-MGCB-CeO2 hydrogel (−UV+NIR), but without further treatment of the gel by UV light (−UV), the cells showed low density and shrinking morphology (Figure 7Ba). Contrastingly, with further treatment of the gel by UV light (+UV), the cells showed high density and elongated morphology, indicating high cell proliferation and viability (Figure 7Bb). First, this was because the GO-MGCB-CeO2 gel (−UV+NIR) had high antibacterial ability, which could kill most of the bacteria (>90%, Figure S14). The few remaining bacteria had negligible effect on human cell proliferation. Second, after further treatment by UV light, the high pH value in the gel enabled CeO2 NPs to exhibit remarkable antioxidative activity, which was beneficial to promoting cell growth and proliferation.30,40,41 Without UV light, the gel was acidic, under which conditions CeO2 exhibited high oxidative activity and was harmful to cells,28,37,39 resulting in poor cell proliferation and viability (Figure 7Ba). Control experiments of GO-MGCB gels without CeO2 NPs (−UV +NIR) were also carried out. The results showed that they had no stimulatory effect on cell proliferation, demonstrating that the effects were attributed to CeO2 NPs. The assays for the human embryonic kidney cells, 293T, were also carried out and showed similar results as those of 2H11 cells (Figure S19). Overall, the GO-MGCB-CeO2 gel with proper light treatment exhibited multifunctional properties, which could kill bacteria and promote cell proliferation in a spatiotemporal manner. This may provide a method for developing photosensitive “Bandaids” for wound healing.

The bacteria growth−inhibition assays in liquid medium were also carried out and showed similar results as those of disc diffusion assays (Figure S15). First, the bacteria solution was incubated with different gels and treated with different lights. After incubation, the bacterial viability was monitored by optical density at 600 nm (OD600 nm). As shown in Figure S15, the blank agarose gel and GO-MGCB gel showed poor bacteria killing activities. Conversely, the GO-MGCB-CeO2 gel pretreated by NIR light, but without pretreatment by UV light (−UV+NIR) showed the best bacteria killing effect. Fluorescence microscopy imaging assays were also carried out to monitor the bacterial viability directly. Fluorescent dye, propidium iodide (PI), was used to stain dead bacteria. The results were consistent with that of optical assays (Figures S16−18). As discussed above, after the antibacteria/inflammation stage, the next step in wound healing is to promote cell proliferation, which can facilitate the regeneration of injured tissue. In following, cell proliferation assays were then carried out. After treatment as described above, the mixture of samples containing the gel and remaining bacteria were incubated with mammalian cells for the following studies, respectively. 2H11 (SV40 transferred murine endothelial cells) and 293T (human embryonic kidney 293 cells with SV40 large T-antigen) cell lines were chosen as model mammalian cell lines. Figure 7Aa showed that the 2H11 cells alone had abundant cell density and elongated cell morphology after culture for 12 h. Following inoculation with bacteria (details in Experimental Section), the cell density was quite low and exhibited rounded/ shrinking morphology, indicating low cell proliferation and poor cell viability (Figure 7Ab). This was due to the bacteriamediated suppression of cell growth and proliferation. For the

CONCLUSION In summary, we present an example of a general method for regulating chemical reactions by simple photoswitching to 7776

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Figure 7. Fluorescence microscopy images of 2H11 cells. (A) Control experiments of (a) 2H11 cells alone and (b) 2H11 cells incubated with bacteria solution without any pretreatment. 2H11 cells incubated with bacteria solutions pretreated by (B) GO-MGCB-CeO2 gel (−UV+NIR) and (C) GO-MGCB (−UV+NIR). (a) Without (−) or (b) with (+) further treatment by UV light.

remotely modulate the temperature and pH of a reaction system. Photothermal nanomaterial (GO) and photobase molecule (MGCB) were used to construct the photoswitchable system, in which the temperature and pH could be controlled by simply introducing light of two different wavelengths. The method is label-free, noninvasive, reversible, and readily applicable for almost all types of chemical reactions. Moreover, it can be easily controlled in a spatial and temporal manner. Importantly, by taking advantage of this light-controlled system, we successfully constructed a photosensitive wound-healing hydrogel platform, which could kill bacteria efficiently and promote cell proliferation. The distinctive advantage of this platform may provide a method for developing effective “Bandaids” for wound healing. Further, this work would be highly

beneficial in future industrial, environmental, and biomedical applications.

EXPERIMENTAL SECTION Reagents and Materials. Graphite was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ncetyltrimethylammonium bromide (CTAB), cerium(III) nitrate, sodium citrate, H2PtCl6, and glucose were obtained from Alfa Aesar. Malachite green carbinol base (MGCB), glucose oxidase (GOx), horseradish peroxidase (HRP), terephthalic acid, cellulase, amylase, sodium carboxymethyl cellulose, soluble starch, and β-cyclodextrin (βCD) were purchased from Aladdin (Shanghai, China). ELF97 phosphatase substrate was obtained from Invitrogen. Alkaline phosphatase from bovine intestinal mucosa was purchased from New England Biolabs. Acid phosphatase from potato and rhodamine B (RhB), propidium iodide (PI), and acridine orange (AO) dyes were 7777

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ACS Nano purchased from Sigma-Aldrich. FeCl3·6H2O, HAuCl4, and dialysis bags (molecular weight cut off = 2000) were ordered from Shanghai Sangon Biotechnology Development Co., Ltd. E. coli (ATCC 25922) bacterial strain was obtained from Chuanxiang Biotechnology, Ltd. (Shanghai, China). Nanopure water (18.2 MΩ /cm, Millpore Co., USA) was used in all experiments to prepare all buffers. All the chemicals were used as received without further purification. General Techniques. UV−vis spectroscopy was carried out on a JASCO V-550 UV−vis spectrometer (JASCO International Co. Ltd., Easton, MD). All UV−vis spectra were measured in a 1.0 cm-pathlength cell. Fluorescence measurements were carried out on a JASCO FP-6500 spectrofluorometer. A CW laser diode (LSR808NL-2000) with wavelength of 808 nm and adjustable power was used for the NIR laser irradiation experiments. For sample irradiation, the laser head was placed above the sample, at a distance of 1.2 cm. Irradiating from the top, the laser focused on a 5 mm × 5 mm square spot (0.25 cm2 laser area) of the sample. For the NIR laser of 50, 125, 250, and 500 mW, the power density was 0.2, 0.5, 1, and 2 W/cm2, respectively. A highpressure UV lamp (500 W, 50 W/cm2, GY-500, Beijing Tianmai Henghui Lighting & Electrical Appliance Co., Ltd., Beijing, China) with single-wavelength output at 302 nm was used as the UV light source. For sample irradiation, the lamp was placed above the samples, at a distance of 20 cm. As per the manufacturer’s protocol, this distance represented a power density of 50 W/cm2. The lamp tube was about 10 cm, thus the whole cuvette could be irradiated. Photothermal Effect of Graphene Oxide. Graphene oxide (GO) was synthesized from graphite by modified Hummers method.7 For photothermal effect investigation, GO (40 μg/mL) solutions were irradiated using the NIR laser (808 nm) at a power density of 0.5, 1, 1.5, and 2 W/cm2 for 10 min, respectively. The temperature was recorded every 0.5 min from 0 to 3 min and every 1 min from 3 to 10 min. For temperature maintenance, NIR light with lower power was introduced for further use. For example, with NIR light of 2 W/cm2, the maintenance NIR light was 1 W/cm2. Light-Induced pH Changing Effect of Molecular Malachite Green Carbinol Base. In a typical procedure, stock solutions of CTAB (200 mM) and MGCB (100 mM in dimethyl sulfoxide) were mixed and diluted with water to yield final concentrations of 50 mM CTAB and 1 mM MGCB. For investigation of light-induced pH changing effect, MGCB solution samples 1−6 were irradiated using a UV lamp (50 W/cm2) for 1, 2, 3, 4, 6, and 8 min, respectively, and the pH values were recorded. The reversibility study was accomplished by irradiating the MGCB solution under UV light for 8 min and then leaving it in the dark for 40 min (sample 7). For pH maintenance, the solution was exposed to visible light. Dual Responsive System. The dual responsive system was constructed by mixing GO solution (40 μg/mL) and MGCB solution (1 mM) together. The temperature and pH changes were recorded by irradiating the mixture under NIR light (2 W/cm2) and UV light (50 W/cm2), respectively. To avoid uncontrollable temperature rise, UV light irradiation was conducted in a room-temperature water bath. The cooling process would be taken if the temperature was monitored to increase. For temperature maintenance, NIR light with lower power (1 W/cm2) was introduced for further use. For pH maintenance, the solution was exposed to visible light. Enzyme/Nanocatalyst Activity Assays. Acid phosphatase: 0.1 U/mL of acid phosphatase was incubated with 0.5 μM of ELF97 (phosphatase substrate), and the activity was monitored by the fluorescence spectrum (λex = 350 nm, λem = 517 nm). Alkaline phosphatase: 0.1 U/mL of alkaline phosphatase was incubated with 0.5 μM of ELF97 (phosphatase substrate), and the activity was monitored by the fluorescence spectrum (λex = 350 nm, λem = 517 nm). Cellulase: 1 U/mL cellulase was incubated with 1 mg/mL sodium carboxymethyl cellulose for 2 h. One of the products, glucose, was assayed by GOx (2 mg/mL)-HRP (20 ng/mL) cascade reaction in the presence of terephthalic acid (1 mM) and monitored by fluorescence spectrum (λex = 318 nm, λem = 435 nm). Amylase: 1 U/mL amylase was incubated with 1 mg/mL soluble starch for 2 h. One of the products, glucose, was assayed by glucose oxidase (GOx, 2 mg/mL)-horseradish peroxidase (HRP, 20 ng/mL) cascade reaction in the presence of

terephthalic acid (1 mM) and monitored by fluorescence spectrum (λex = 318 nm, λem = 435 nm). Fe3O4 Nanoparticles Synthesis and Catalytic Activity Measurements. The spherical magnetic Fe3O4 particles were prepared by one-pot polyol media solvothermal method.8 Typically, FeCl3·6H2O (1.5 g) was dissolved in ethylene glycol (40 mL) to form a stable orange solution. NaAc (4.0 g) was added into the above solution under vigorous magnetic stirring until completely dissolved. The obtained homogeneous solution was transferred to a Teflon-lined stainless-steel autoclave (50 mL) and sealed to heat at 200 °C. After reaction for 8 h, the autoclave was cooled to ambient temperature naturally. The obtained magnetite particles were washed with ethanol and deionized water in sequence and then dried under vacuum at 60 °C for 12 h. For Fe3O4 NP catalytic activity measurement, the as prepared Fe3O4 NPs were dissolved in solution to give a final concentration of 20 μg/mL. H2O2 (120 mM) and terephthalic acid (1 mM) were used as substrates. The catalytic activities were monitored by fluorescence spectrum (λex = 318 nm, λem = 435 nm). Gold Nanoparticles Synthesis and Catalytic Activity Measurements. AuNPs were synthesized according to the classical citrate reduction method.9 Briefly, 250 mL of 1 mM HAuCl4 was added into a 500 mL round-bottom flask equipped with a condenser, heated to boiling in an oil bath under vigorous stirring. Then, 25 mL of 38.8 mM sodium citrate was added into the mixture rapidly, resulting in a color change from pale yellow to burgundy. After 10 min, the heating mantle was removed, and the solution was stirred for 15 min. Then the sample was cooled and stored in a 4 °C refrigerator until further use. The concentration of the prepared AuNP dispersion was determined by UV−vis spectrometry as reported previously and was calculated as 17 nM. For further use, the sample was concentrated by centrifugation to about 100 nM. For catalytic studies, the as prepared AuNPs were dissolved in solution to give a final concentration of 35 nM. Then the catalyst was incubated with 50 mM of glucose for 1 h. The reaction solution was subsequently diluted with 25 mM phosphate buffer (pH 5.0). The reaction product, H2O2, was assayed by reaction with terephthalic acid (1 mM) in the presence of 10 ng/mL HPR, being monitored by fluorescence spectrum (λex = 318 nm, λem = 435 nm). Cerium Oxide Nanoparticles Synthesis and Catalytic Activity Measurements. CeO2 NPs with high water solubility were synthesized by the previously reported alkaline hydrolysis method.10 A solution containing 5.0 mL of 1.0 M cerium(III) nitrate and 10.0 mL of β-cyclodextrin (β-CD) (1.0 mM) was added dropwise to a 30.0 mL ammonium hydroxide solution and stirred for 24 h at 25 °C. The solution was then centrifuged at 4000 rpm for two 30 min cycles to settle any debris and large agglomerates. Finally, free β-CD was removed by dialysis using a 2000 MWCO cellulose membrane, and the purified solution was stored at 4 °C. For oxidation activity measurement in solution, 40 μg/mL of CeO2 NPs was incubated with 50 μM Rhodamine B (RhB). The dye degradation was monitored by the fluorescence spectrum (λex = 355 nm, λem = 582 nm). Platinum Nanoparticles Synthesis and Catalytic Activity Measurements. PtNPs were prepared according to the procedure reported previously.11 First, 1 mL of 1% H2PtCl6 aqueous solution was added into 100 mL of water and heated to boiling. Then, 3 mL of 1% sodium citrate aqueous solution was added rapidly, and the mixture was kept at a boiling temperature for about 30 min. Then the sample was placed in a 4 °C refrigerator as stock. For catalytic activity measurement, 10 μg/mL of PtNPs were incubated with 50 mM H2O2 for 2 h. Then the residual H2O2 was assayed by reaction with terephthalic acid (1 mM) in the presence of 5 nM HPR and monitored by fluorescence spectrum (λex = 318 nm, λem = 435 nm). Light-Controlled Enzyme/Catalyst Activity. For light-controlled activity studies, NIR (2 W/cm2) and UV (50 W/cm2) light sources were introduced. To avoid uncontrollable temperature rise, UV light irradiation was conducted in a water bath. For temperature maintenance, NIR light with lower power (1 W/cm2) was introduced for further use. For pH maintenance, the solution was exposed to visible light. If the reaction temperature was higher than 50 °C, UV light (50 W/cm2) was introduced at appropriate intervals to maintain the pH value. The activity maps were created by varying the light 7778

DOI: 10.1021/acsnano.7b01450 ACS Nano 2017, 11, 7770−7780

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ACS Nano

light (2 W/cm2) for 5 min every 30 min. Then the antibacterial activities of the samples were evaluated by the optical density at 600 nm (OD600 nm). Fluorescence microscopy imaging assays were also carried out to monitor the bacterial viabilities directly. Fluorescent dye, propidium iodide (PI), was used to stain dead bacteria. Cell Culture. 2H11 (SV40 transferred murine endothelial cells) and 293T (human embryonic kidney 293 cells with SV40 large Tantigen) cells were cultured in 25 cm2 flasks in Dulbecco’s Modified Eagle’s Medium DMEM (Gibco) containing 10% (v/v) fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere of 5% (v/v) CO2. The media were changed every 2 days, and the cells were passaged by trypsinization before confluence. Cell Proliferation Assay. The cells were cultured in 24-well plates for 6 h. Then the mixture of samples containing the remaining bacteria and gels (obtained as described in the Antibacterial Measurements section) were added on the plates. The cells were incubated with the mixture at 37 °C for another 6 h. For “+UV” samples, the gels were taken out from the mixture and treated by UV light (50 W/cm2) for 10 min before being added to the cell-containing plate. For “+NIR” samples, the gels were further treated by NIR light (2 W/cm2) for 5 min every 1 h. For control samples, the human cell lines were cultured in the absence or presence of bacterial solution without the gel. Fluorescence microscopy imaging assays were carried out to monitor the cell proliferation directly. Fluorescent dye, calcein, and AM were used to stain the cells.

irradiation time. For ex situ studies, the enzymes/nanocatalysts and substrates were added after removal of the high-power lights. For in situ studies, the enzymes/nanocatalysts were added before introduction of light. ALP and PtNPs were used as models for in situ studies. To investigate the influence of NIR or UV light alone on the activity of ALP, NIR light (808 nm, 2 W/cm2) or UV light (302 nm, 50 W/cm2) was introduced, respectively. For activity measurement, 0.1 U/mL of acid phosphatase was incubated with 0.5 μM of ELF97, and the activity was monitored by the fluorescence spectrum (λex = 350 nm, λem = 517 nm). The blank sample contained ELF97 alone. For enzyme activity protective assay, ascorbic acid (25 mM) was added into the reaction solution. The reaction was conducted at the optimum temperature and pH conditions. Activity Measurements. All of the activities were measured by monitoring the fluorescence intensity changes of the substrates before and after enzyme/nanoparticle-catalyzed reactions. Here, the activity (A) was defined as the catalyzed rate: A = ΔF/c × t, where c is the concentration of substrate and t is the reaction time. ΔF = F − F0, where F0 and F are the fluorescence intensity of substrate before and after reaction, respectively. As the values of c and t were all the same for reactions catalyzed by the same enzyme/nanoparticle, the activity was determined by ΔF. Defining the activity of the reaction under the optimal temperature and pH condition as Amax, the relative activity = A/Amax = ΔF/ΔFmax. For light-controlled reactions, the Amax was the reaction activity which was taken under optimal temperature and pH conditions without light. Agarose Gel Preparation. A 15% agarose gel was prepared by microwave heating method. The agarose powder was added into water and heated by microwave until completely dissolved. Then the solution was added into the round container mold and allowed to gel. For hybrid agarose gel, 60 μg/mL of GO, 2 mM of MGCB, and 100 μg/mL of CeO2 were selectively integrated into the heated agarose solution to gel. UV Light Responsive pH Change and NIR Light Responsive Temperature Change Properties of the GO-MGCB Agarose Gel. The method was the same as the solution system mentioned above. NIR light (2 W/cm2) and UV light (50 W/cm2) were introduced. Light-Controlled Activity of the GO-MGCB-CeO2 Gel. For “+ UV” samples, the GO-MGCB-CeO2 gels were pretreated by UV light (50 W/cm2) for 4 min. Then the gels immersed were immersed in dye RhB (50 μM) and H2O2 (120 mM)-terephthalic acid (1 mM) solutions for 10 min, respectively. Subsequently the gels were taken out from the solutions and reacted for 30 min in the presence or absence of NIR light (2 W/cm2). For “−UV” samples, the procedures were the same, but without UV light pretreatment. Bacterial Culture. A monocolony of E. coli on a solid Luria− Bertani (LB) agar plate was transferred to 20 mL of liquid LB culture medium and grown for 12 h at 37 °C under 180 rpm rotation. Then the bacteria were diluted with broth to 106 cfu mL−1. In all experiments, the concentrations of bacteria were determined by measuring the optical density at 600 nm (OD600 nm). Antibacterial Measurements. Disc diffusion method and bacteria growth-inhibition assays in liquid medium were applied to detect the antibacterial performance of the gels. The experimental process for disc diffusion method is described as follows: The diluted bacterial samples were spread on an agar dish and cultured for 24 h at 37 °C. The samples of blank agarose, GO-MGCB gels, and GO-MGCB-CeO2 gels were then added to the discs. For “+UV” samples, the gels were pretreated by UV light (50 W/cm2) for 10 min. For “+NIR” samples, the gels were treated by NIR light (2 W/ cm2) for 5 min every 30 min. Then the antibacterial activities of the samples were evaluated by the diameter of the intact region of the agar plate. The experimental process for bacteria growth−inhibition assays in liquid medium is as follows: The as-prepared bacteria solution (500 μL) was mixed with the samples of blank agarose, GO-MGCB gels, and GO-MGCB-CeO2 gels, respectively, and cultured at 37 °C for 12 h. For “+UV” samples, the gels were pretreated by UV light (50 W/ cm2) for 10 min. For “+NIR” samples, the gels were treated by NIR

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01450. Reagents and materials; general techniques; Figures S1− S19 (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Jinsong Ren: 0000-0002-7506-627X Xiaogang Qu: 0000-0003-2868-3205 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We acknowledge Bryant Yung for proof reading the manuscript. This work was supported by the National Basic Research Program of China (grant 2012CB720602), the National Natural Science Foundation of China (grants 21401187, 21210002, 21431007, 21533008), and the Jilin Scientific and Technological Development Program (grant 20150520005JH). REFERENCES (1) Assion, A.; Baumert, T.; Bergt, M.; Brixner, T.; Kiefer, B.; Seyfried, V.; Strehle, M.; Gerber, G. Control of Chemical Reactions by Feedback-Optimized Phase-Shaped Femtosecond Laser Pulses. Science 1998, 282, 919−922. (2) Slyadnev, M. N.; Tanaka, Y.; Tokeshi, M.; Kitamori, T. Photothermal Temperature Control of a Chemical Reaction on a Microchip Using an Infrared Diode Laser. Anal. Chem. 2001, 73, 4037−4044. (3) Willner, I.; Willner, B. Photoswitchable Biomaterials as Grounds for Optobioelectronic Devices. Bioelectrochem. Bioenerg. 1997, 42, 43− 57. (4) Shimoboji, T.; Larenas, E.; Fowler, T.; Kulkarni, S.; Hoffman, A. S.; Stayton, P. S. Photoresponsive Polymer-Enzyme Switches. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 16592−16596. 7779

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DOI: 10.1021/acsnano.7b01450 ACS Nano 2017, 11, 7770−7780