Unraveling the Enzymatic Activity of Oxygenated Carbon Nanotubes

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Unraveling the Enzymatic Activity of Oxygenated Carbon Nanotubes and Their Application in the Treatment of Bacterial Infections Huan Wang, Penghui Li, Dongqing Yu, Yan Zhang, Zhenzhen Wang, Chaoqun Liu, Hao Qiu, Zhen Liu, Jinsong Ren, and Xiaogang Qu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b05095 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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Unraveling the Enzymatic Activity of Oxygenated Carbon Nanotubes and Their Application in the Treatment of Bacterial Infections ⊥

Huan Wang,†,‡ Penghui Li, Dongqin Yu,†,‡ Yan Zhang,†,§ Zhenzhen Wang,†,§ Chaoqun Liu,†,§Hao Qiu, †,‡Zhen Liu,*,† Jinsong Ren,*,† Xiaogang Qu*,† †

State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology,

Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡

University of Science and Technology of China, Hefei,230029, P. R. China

§

Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China

⊥

MOE Key Laboratory of Green Chemistry, College of Chemistry, Sichuan University,

Chengdu,610064, P. R. China KEYWORDS: carbon nanotubes, nanozymes, oxygenated groups, competitive inhibition, bacterial infections

ACS Paragon Plus Environment

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ABSTRACT Carbon nanotubes (CNTs) and their derivatives have emerged as a series of efficient bio-catalysts to mimic the function of natural enzymes in recent years. However, the unsatisfiable enzymatic efficiency usually limits their practical usages ranging from materials science to biotechnology. Here, for the first time, we present the synthesis of several oxygenated groups-enriched carbon nanotubes (o-CNTs) via a facile but green approach, as well as their usages as high-performance peroxidase mimics for biocatalytic reaction. Exhaustive characterization of the enzymatic activity of o-CNTs have been provided by exploring the accurate effect of various oxygenated groups on their surface including carbonyl, carboxyl, and hydroxyl groups. Owing to the ‘competitive inhibition’ effect among all these oxygenated groups, the catalytic efficiency of o-CNTs is significantly enhanced by weakening the presence of non-catalytic sites. Furthermore, the admirable enzymatic activity of these o-CNTs has been successfully applied in the treatment of bacterial infections, and the results of both in vitro and in vivo nanozyme-mediated bacterial clearance clearly demonstrate the feasibility of o-CNTs as robust peroxidase mimics to effectively decrease the bacterial viability under physiological conditions. We believe that the present study will not only facilitate the construction of novel efficient nanozymes by rationally adjusting the degree of ‘competitive inhibition’ effect, but also broaden the biological usages of o-CNTs-based nanomaterials via their satisfactory enzymatic activity.


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Natural enzymes with high substrate specificity and activity are extremely efficient in catalyzing a variety of biological reaction under mild conditions.1-3 However, their practical usages are usually restricted by their intrinsic characters, such as low operational stability, high sensitivity towards harsh environments, poor recovery capacity, and high costs in preparation. To overcome these limitations, an increasing research interest has been focused on the design and synthesis of various artificial enzymes as alternatives to mimic natural enzymes.4, 5 Along with the rapid development of nanotechnology and material science, a variety of nanomaterials including nano-composite materials,6-8 noble metal/alloy nanoparticles,9-14 metal oxide-based nanospheres,15-19 and carbon-based nanostructures,20-22 have emerged as novel enzyme mimics, which can also be defined as nanozymes, and have been widely used in various biocatalytic systems. Recently, carbon nanotubes (CNTs) with negligible toxicity both in vitro and in vivo have exhibited their potential in the field of biomedicine.23-25 For instance, unique tubular structure and long range π conjugation of CNTs endow them with a facile attachment of many small-sized nanoparticles and molecular drugs.26-30 With distinctive optical properties, CNTs and their derivatives can serve as contrast agents for Raman imaging, near-infrared fluorescence imaging, and photoacoustic imaging, as well as agents for cancer phototherapies.31-38 Especially, CNTs and their derivatives have been used as nanozymes in bioanalysis and biosensing.21 However, CNTs-based nanozymes usually hold negligible biocatalytic activity in neutral environments, which thus limit their wide usages in biomedicine upon various physiological and pathological conditions.8 Accordingly, rational design and synthesis of novel CNTs-based nanozymes with superior enzymatic activity over a broad pH range is urgently needed.


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In recent years, several studies have demonstrated that carbonyl groups in nanocarbon can act as active sites during the oxidative dehydrogenation reaction.39-42 Moreover, our research has indicated that various functional groups on the surface of graphene quantum dots can play different roles in the biocatalytic process.43 Inspired by the aforementioned discoveries, we envisioned that the appropriate shielding of non-active sites and modest oxidation of CNTs could improve the biocatalytic performance of CNTs-based nanozymes. In this study, for the first time, we developed a series of oxygenated groups-enriched carbon nanotubes (o-CNTs) as highly efficient peroxidase mimics via a simple but green approach. As expected, o-CNTs prepared via a one-pot oxidation reflux exhibited excellent enzymatic activity over a broad pH range. Results of chemical titration and theoretical calculation indicated that carbonyl groups on the surface of o-CNTs were the active centers, whereas carboxyl groups and hydroxyl groups served as competitive sites and inhibited the catalytic reaction, respectively. More importantly, carboxyl groups exhibited more intense inhibition effect than hydroxyl groups due to their intrinsic hydrogen bonding interaction and high negative charges. Considering the intrinsic character of ‘competitive inhibition’ effect in nanozymes, 2-bromo-1-phenylethanone-modified o-CNTs (o-CNTs-BrPE) were further prepared via deactivating existent carboxyl groups on the surface of o-CNTs. By weakening the competitive inhibition, o-CNTs-BrPE with substantial carbonyl groups and negligible carboxyl groups held the highest peroxidase activity and biocatalytic efficiency among all the o-CNTs. Furthermore, results of both in vitro and in vivo nanozyme-mediated bacterial clearance clearly demonstrated the feasibility of o-CNTs as robust peroxidase mimics to enhance the generation of reactive oxygen species under physiological conditions, as well as protect the tissue from bacteria-triggered edema and inflammation. We expected that our study would lay the foundation for the further work to elucidate the enzymatic


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activity and related mechanism of various new nanomaterials, as well as provide valuable insight into the development of safe and efficient nanoagents to treat various degrees of bacterial infections. Considering that concentrated nitric acid as oxidizers could introduce more oxygenated groups and less foreign substance than other oxidative conditions, one-pot nitric acid-assistant reflux method by using pristine CNTs (p-CNTs) as precursors was used to prepare oxygenated groups-enriched CNTs (o-CNTs) in our present design.44-45 After successful oxidation modification, abundant oxygenated groups including carbonyl (-C=O), hydroxyl (-OH) and carboxyl (-COOH) groups occurred on the surface of o-CNTs. Subsequently, to explore the detail roles of these oxygenated groups during the biocatalytic reaction, phenylhydrazine (PH), benzoic anhydride (BA), and 2-bromo-1-phenylethanone (BrPE) were employed as high-specificity deactivating agents to react with different oxygenated groups on the surface of o-CNTs. Molecular structures and related reaction process were illustrated in Figure 1a and Figure S1. Newly developed derivatives of o-CNTs were further defined as o-CNTs-PH, o-CNTs-BA, and o-CNTs-BrPE.41 TEM images indicated that there was nearly no difference in morphology between p-CNTs and various functional CNTs including o-CNTs, o-CNTs-PH, o-CNTs-BA, as well as o-CNTs-BrPE (Figure 1b and Figure S2). All the CNTs held similar tubular structure, demonstrating that appropriate oxidation process and subsequent high-specificity surface modification could not cause serious morphology change. XPS spectra were used to explore the distribution of various oxygenated groups on the surface of these CNTs. As shown in Figure 1c, after concentrated HNO3 treatment, the oxygen content in CNTs increased from 3.65% to 12.71% calculated by XPS quantitative analysis after deconvolution of O 1s spectra. In the sample of well-prepared


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o-CNTs, the significantly increased oxygen levels mainly originated from the large amount of the newly formed oxygenated groups including hydroxyl, carbonyl, and carboxyl. The presence of N 1s peak in o-CNTs-PH could be ascribed to the formation of C=N bonds (Figure S3a-e).41 Compared with o-CNTs, decrease in the intensities of -C=O signal for o-CNTs-PH, -OH signal for o-CNTs-BA, and -COOH signal for o-CNTs-BrPE could be clearly detected. Based on the attenuated total reflection infrared spectroscopy (ATR-IR), obvious absorption peak of -C=O at 1720 cm-1 occurred in the sample of o-CNTs, further confirming more oxygenated groups on the surface of o-CNTs compared with p-CNTs (Figure S3f). Zeta potential value of o-CNTs was much lower than that of p-CNTs (Figure S4). Moreover, the removal of -COOH from o-CNTs by BrPE could lead to the formation of o-CNTs-BrPE with higher zeta potential value. All these results therefore demonstrated the successful synthesis of o-CNTs and their derivatives, and the occurrence of deactivation process of various functional groups. The oxidation of 3, 3', 5, 5'-Tetramethylbenzidine (TMB) in the presence of H2O2 was selected as a model catalytic reaction to investigate the peroxidase-like activity of o-CNTs. As shown in Figure 2a, o-CNTs exhibited extremely high peroxidase-like activity. However, p-CNTs held negligible enzymatic activity under the same experimental condition, which indicated that the introduction of various oxygenated groups played an important role during the biocatalysis. Similar

with

other

nanozymes,

the

peroxidase-like

activity

of

o-CNTs

revealed

concentration-dependent and temperature-dependent manners (Figure 2b and Figure 2c). More importantly, compared with classical peroxidase mimics that exhibited negligible biocatalytic activity at neutral condition,20-23 the o-CNTs held excellent peroxidase-like activity over a broad pH range, even 20 % of the activity was retained under neutral condition, which showed more potential for bio-related usages (Figure 2c). Terephthalic acid (TA) fluorescence experiment was


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carried out to clarify the enzymatic mechanism of o-CNTs’ peroxidase-like activity by monitoring the production of hydroxyl radicals. Considering the fluorescence quenching effect of CNTs, the fluorescence of supernatant after total removal of CNTs was collected and explored carefully. As shown in Figure S5, o-CNTs could efficiently convert TA without fluorescence into 2-hydroxyterephthalic acid (TAOH) with high fluorescence in the presence of H2O2. All these results indicated that the peroxidase-like activity of o-CNTs originated from their excellent ability to decompose H2O2 into hydrogen radicals.22 In order to understand the contribution of various oxygenated groups towards the peroxidase-like activity of o-CNTs, we further explored the biocatalytic activity of several derivatives of o-CNTs, such as o-CNTs-PH, o-CNTs-BA, and o-CNTs-BrPE. Compared with that of o-CNTs, the enzymatic efficiency of o-CNTs-PH after efficient shielding of -C=O showed a significant decrease of 85%, indicating that -C=O played a decisive and positive role in the biocatalytic process (Figure 2d). Moreover, the enzymatic activities of o-CNTs-BrPE and o-CNTs-BA were significantly higher than that of o-CNTs, demonstrating that the presence of – COOH and -OH could inhibit the peroxidase-like activity of o-CNTs more or less. Quantitative results of activation energy (Ea) based on Arrhenius formula were then used to discuss the potential of various CNTs-based biocatalytic reaction.46 ln (v)=A-Ea/R×1/T A was the frequency factor, R was the gas constant (8.314 J mol-1 K-1), and T was the absolute temperature (K). Activation energy was the energy which must be available to a chemical system with potential reactants to result in a chemical reaction. Activation energy might also be defined as the minimum energy required starting a chemical reaction. Thus, a lower Ea value could make the enzymatic reaction occur more easily. As shown in Figure 2e and Figure S6, Ea values


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could be calculated as 33.65, 37.42, 24.67, and 22.44 kJ mol-1 for o-CNTs, o-CNTs-PH, o-CNTs-BA, and o-CNTs-BrPE based on the results of the linear fitting of logarithmic plots of the reaction rates and the reciprocal of reaction temperature raging from 298.15 K to 313.15 K. o-CNTs-PH held the highest Ea value, indicating that the enzymatic reaction catalyzed by o-CNTs-PH with shielded -C=O showed the lowest activity, which verified that-C=O were the active centers in the enzymatic reaction. Compared with o-CNTs, o-CNTs-BA and o-CNTs-BrPE with shielded –C-OH and –COOH could greatly reduce the energy barrier of enzymatic reaction, indicating that the existence of -COOH and -OH could increase the Ea values of biocatalytic reaction and bring adverse effects to the enzymatic activity. Quantitatively, o-CNTs-BrPE with shielded -COOH held the lowest Ea value, verifying that the activity of the enzymatic reaction could be inhibited most effectively with the introduction of -COOH. All these results powerfully suggested that different oxygenated groups showed diverse effects on the enzymatic activity. To explore the unfavorable effect of -COOH and-OH during the biocatalytic process, a 104 carbon atom-contained carbon nanotube with a simulated size of 0.9 nm in length and 1.2 nm in diameter was constructed as a model to perform the theoretical research (Figure 3a). We further modified above model with -C=O, -OH, and –COOH at the edges to simulate various oxygenated group-modified CNTs, which were denoted as CNTs-C=O, CNTs-C-OH, and CNTs-COOH. Density functional theory (DFT) calculations were then performed on above model structure using Gaussian at the B3LYP/6-31G level of theory. The binding energies ∆G between H2O2 and CNTs-C=O, CNTs-C-OH, as well as CNTs-COOH could be calculated to be -0.064, -0.095, as well as -0.262 eV, whereas the ∆G between H2O2 and p-CNTs was 0.231 eV (Figure 3b). According to these results, the binding capacity between H2O2 and CNTs was very


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weak because the interaction between them mainly rooted from electrostatic repulsion and Van der Waals force. The unfavorable binding mode therefore hindered p-CNTs from efficient contacting with substrates. Owing to the presence of a hydrogen bond between a H2O2 molecule and -OH or -C=O, CNTs-C-OH and CNTs-C=O thus showed better binding capacity than p-CNTs. Significantly, -COOH could form two hydrogen bonds with a H2O2 molecule and lower the binding energy to -0.262 eV, indicating that CNTs-COOH held the best binding capacity among all these functional CNTs. Comprehensive insight into the enzymatic activity of oxygenated groups-enriched carbon nanotubes and related biocatalytic mechanism were summarized as follows (Figure 3c). As high-performance active center, -C=O with slight binding capacity with H2O2 could efficiently decrease the Ea value and enhance the decomposition process of H2O2. However, both -OH and –COOH with better binding capacity than -C=O could not catalyze the decomposition of substrates like the active sites. These results indicated that the existence of -OH and -COOH could compete with -C=O for the binding of H2O2, causing a decrease in enzymatic activity. Due to the better binding capacity of -COOH than that of -OH for H2O2, -COOH could suppress the biocatalytic process more effectively than -OH. Therefore, specific sites of nanozymes with excellent substrate binding capacity and negligible catalytic ability could seriously suppress the enzymatic activity to varying degrees, which could be defined as ‘competitive inhibition’ effect of nanozymes. We believed that the design of efficient biocatalytic systems with high enzymatic activity could be achieved by regulating the degree of ‘competitive inhibition’ effect of nanozymes, which could help us understand the biocatalytic activity of various nanozymes. As expected, o-CNTs-BrPE after deactivating -COOH exhibited the highest peroxidase-like activity among all these functionalized CNTs (Figure 2d).


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Because H2O2 could be catalyzed into highly toxic hydroxyl radicals during the o-CNTs-based biocatalytic process under pathological conditions, a nanozyme-mediated antibacterial platform against drug-resistant bacteria was developed to explore the feasibility of o-CNTs as robust peroxidase mimics to enhance the generation of reactive oxygen species. The concentration of H2O2 used here was much lower than that used in clinic (166 mM),22 thus avoiding unwanted side

effects

such

as

tissue

toxicity

and

nephrotoxicity.47

Gram-negative

bacteria

ampicillin-resistant E. coli and Gram-positive bacteria methicillin-resistant S. aureus, which could be defined shortly as E. coli and S. aureus, were chosen as models to explore the antibacterial ability of our antibacterial platform. As shown in Figure 4a and Figure 4b, a relative higher concentration of H2O2 (10-1 M) was needed to achieve a satisfactory antibacterial ability with a low bacterial percentage survival of 10 % in the absence of o-CNTs. However, to achieve a similar effect on both E.coli and S.aureus, the concentration of H2O2 in the sterilization significantly decreased to 10-3 M and 10-2 M with the assistance of o-CNTs, which could be attributed to the high peroxidase-like activity of o-CNTs. In addition, we found the positive correlation between antibacterial abilities and peroxidase-like activities by comparing the antibacterial performance of various functional CNTs. As expected, o-CNTs-BrPE with the highest peroxidase-like activity held the best antibacterial ability, whereas p-CNTs and o-CNTs-PH with negligible peroxidase-like activity did not show obviously antibacterial activity even compared with the group treated with H2O2 only (Figure 4c and Figure 4d). In addition, antibiotics showed negligible antibacterial ability because of the intrinsic drug-resistance of these bacteria, which were further visualized by the spread plate method (Figure 4e). Scanning electron microscope was the used to explore the morphology changes of bacteria after various treatments. As shown in Figure 4f and Figure S7, pristine S.aureus cells and S.aureus cells


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treated with o-CNTs-BrPE, p-CNTs, H2O2, H2O2+p-CNTs, as well as methicillin were typically spherical-shaped with intact and smooth cell walls, indicating that o-CNTs-BrPE revealed negligible effect on the morphology of S.aureus cells. However, once S.aureus cells were treated with o-CNTs-BrPE together with 10 mM H2O2, the cell walls became wrinkled and rough. Significantly, we could obtain similar results by using E.coli cells as typical experimental model (Figure 4g and Figure S7). All these results could be attributed to the fact that hydroxyl radicals could efficiently oxidize and destroy bacterial cells, as well as induce further death of bacteria.48 After understanding the antibacterial effect of these o-CNTs-based nanozymes in vitro, we further explored their ability against bacteria in vivo by using the model of wound infections.49, 50 Prior to the in vivo studies, we investigated the cytotoxicity of these o-CNTs-based nanozymes by using MTT assay associated with epithelial cells. All the cell viabilities were not hindered by o-CNTs and their derivatives even up to a high concentration of 100 µg mL-1, indicating that these

o-CNTs-based

nanozymes

held

negligible

cytotoxicity

(Figure

S8).

Calcein

AM/propidium iodide double staining assay additionally demonstrated that these o-CNTs-based nanozymes did not induce cellular death (Figure S9). The influence of above o-CNTs-based nanozymes on the cell migration was then explored. Cells treated with o-CNTs or their derivatives could gradually grow to the middle of the interspace and exhibit a similar appearance to those ones in the control group without any treatment, indicating that these o-CNTs-based nanozymes had negligible adverse effect on cell migration (Figure S10). With the outstanding in vitro bio-compatibility in hand, we further studied whether these nanozymes could be used to treat skin wound infections in vivo (Figure 5a). A round full-thickness cutaneous wound with a diameter of 5 mm was created on the back of each mouse. After being infected by MRSA, mice were divided into 7 groups with different treatments: untreated, p-CNTs, o-CNTs-BrPE, H2O2,


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p-CNTs+H2O2, o-CNTs-BrPE+H2O2, and methicillin. After that, above wounds were dressed with medical gauze, which were denoted as B-G, P-G, BrPE-G, H+B-G, H+P-G, H+BrPE-G, and M-G, respectively. Photos of wounds were taken from above groups at each expected time points and the whole therapeutic process was defined as 14 days. As shown in Figure 5b, in the group of H+BrPE-G, there was no edema and inflammation at the end of therapeutic process. In contrast, other groups exhibited different levels of inflammation during the wound healing process (Figure 5b). Quantitative analysis of the wound area indicated that the H+BrPE-G group held best wound healing 7 days post-wounding among all the test groups. Significantly, the wound in the H+BrPE-G group had almost completely healed 14 days post-wounding. However, the P-G had no significant effects on the wound size after treatment (Figure 5c). To fully assess the antibacterial activity of these o-CNTs-based nanozymes, wound skins from all the groups were harvested 14 days post-wounding and the numbers of bacteria extracted from skin supernatant were evaluated by using spread plate method. Bacteria from skins were cultured on agar plates overnight, and the colonies were counted for quantitative analysis. As shown in Figure 5d and Figure 5e, nearly no bacterial growth could be detected on the agar plates in the group of H+BrPE-G, indicating that o-CNTs-BrPE could convert H2O2 into highly toxic hydroxyl radicals effectively. However, a great deal of bacterial colonies on the agar plates could be found in other groups. In detail, group of H+P-G showed a similar number of bacterial colonies on the agar plates with the group of H+B-G. Importantly, the group of BrPE-G exhibited a decrease in the number of bacteria as compared with the group of B-G, which was ascribed to the generation of endogenous H2O2 molecules around the inflammatory sites.51 Moreover, the sharp decrease in the number of bacteria in the M-G group could be attributed to the ability of antibiotics, such as methicillin, to prevent the infection by exotic bacteria from the


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surroundings. Above results indicated that o-CNTs-BrPE could prevent wound infections with high performance. To investigate the wound healing process, hematoxylin-eosin (H&E) staining images of the skins were obtained from mouse wounds 7 and 14 days post-wounding. Re-epithelialization was considered as a major step during the process of wound healing.52 Because of its satisfactory anti-infection activity, H+BrPE-G group revealed elongated newly formed epithelium compared with other groups 7 and 14 days post-wounding, suggesting the enhanced wound re-epithelialization (Figure 6a). Furthermore, much less inflammatory cells could be found on the wound with H+BrPE-G treatment at 7 and 14 days post-wounding, indicating the relieved bacterial infections. Moreover, no obvious abnormal effect could be observed in the group of H+BrPE-G on 7 and 14 days compared with the healthy skin (Figure 6a and Figure S11). Immunohistochemistry experiments were further performed to assess the treatment interactions and effects. As shown in Figure 6b and Figure 6c, type-I collagen staining was used to investigate the collagen formation of the wounds. The B-G and P-G groups showed unrepaired collagen fibers, whereas the groups of BrPE-G, H+B-G, H+P-G, and M-G exhibited a certain degree of the collagen regeneration. Significantly, the group of H+BrPE-G held the highest collagen content. Meanwhile, CD31 staining indicated that the density of newly formed blood vessels in the group of H+BrPE-G was significantly increased compared with the other groups (Figure 6b and Figure 6d). All these exciting results demonstrated that this nanozyme-based antibacterial platform could effectively promote wound healing by inhibiting bacterial infections with negligible toxicity. In addition, H&E staining images of major organs from above groups 14 days post-wounding indicated that all these o-CNT-based nanozymes exhibited high biocompatibility and negligible toxicity (Figure S12). Moreover, no inflammation


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occurred in above organs after biocatalytic antibacterial treatments, promising the further practical usages of these o-CNTs-based nanozymes. In summary, for the first time, we have developed several oxygenated groups-enriched carbon nanotubes (o-CNTs) via a facile but green approach, and utilized them as efficient peroxidase mimics for biocatalytic reaction and anti-bacterial treatment. Importantly, we provided exhaustive characterization of the enzymatic activity of o-CNTs by exploring the accurate effect of various oxygenated groups on their surface. In detail, we not only confirmed the carbonyl groups were the source of the enzymatic activity of o-CNTs, but also proposed the ‘competitive inhibition’ effect on the surface of these o-CNTs-based nanozymes. By weakening the competitive inhibition, 2-bromo-1-phenylethanone-modified o-CNTs with extremely high biocatalytic efficiency were then prepared via deactivating existent carboxyl groups, which were used in nanozyme-mediated bacterial clearance. In vivo results further demonstrated that our o-CNTs-based antibacterial platform could effectively decrease bacteria-triggered edema and purulent inflammation on a mouse model of wound infections with negligible side effects. We believed that our present design not only facilitated the construction of novel nanozymes with high enzymatic activity by rationally adjusting the degree of ‘competitive inhibition’ effect, but also broadened the biological usages of o-CNTs-based nanomaterials via their admirable enzymatic activity. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Experimental method, material characterization and eight figures are provided in Supporting Information (PDF).


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AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] *[email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Financial support was provided by support the National Natural Science Foundation of China (21431007, 21533008), the Frontier Science Key Program of CAS (QYZDJ-SSW-SLH052), and the Jilin Province Science and Technology Development Plan Project (20160520129JH, 20170101184JC). REFERENCES (1) Barber, J. Chem. Soc. Rev. 2009, 38, 185-196. (2) Garcia-Viloca, M.; Gao, J.; Karplus, M.; Truhlar, D. G. Science 2004,303, 186-195. (3) Wolfenden, R.; Snider, M. J. Acc. Chem. Res. 2001, 34, 938-945. (4) Lin, Y.; Ren, J.; Qu, X. Acc. Chem. Res. 2014, 47, 1097-1105. (5) Wei, H.; Wang, E. Chem. Soc. Rev. 2013, 42, 6060-6093. (6) Guo, Y.; Deng, L.; Li, J.; Guo, S.; Wang, E.; Dong, S. ACS Nano 2011, 5, 1282-1290. (7) Xue, T.; Jiang, S.; Qu, Y.; Su, Q.; Cheng, R.; Dubin, S.; Chiu, C.-Y.; Kaner, R.; Huang, Y.; Duan, X. Angew. Chem. Int. Ed. 2012, 51, 3822-3825. (8) Tao, Y.; Lin, Y.; Huang, Z.; Ren, J.; Qu, X. Adv. Mater. 2013, 25, 2594-2599. (9) Comotti, M.; Della Pina, C.; Matarrese, R.; Rossi, M. Angew. Chem. Int. Ed. 2004, 43, 5812-5815. (10) Pengo, P.; Polizzi, S.; Pasquato, L.; Scrimin, P. J. Am. Chem. Soc. 2005, 127, 1616-1617.


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Figure 1. (a) Molecular structures of PH, BA, and BrPE. (b) TEM images of p-CNTs (b1), o-CNTs (b2), o-CNTs-PH (b3), o-CNTs-BA (b4), and o-CNTs-BrPE (b5). Scale bars were 50 nm. (c) O 1s XPS spectra of p-CNTs (c1), o-CNTs (c2), o-CNTs-PH (c3), o-CNTs-BA (c4), and o-CNTs-BrPE (c5). Inset: corresponding oxygen contents of p-CNTs, o-CNTs, o-CNTs-PH, o-CNTs-BA, and o-CNTs-BrPE.


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Figure 2. (a) Time-dependent absorbance changes at 652 nm in the presence of o-CNTs or p-CNTs in PBS (25 mM, pH 4, 37 °C). The concentrations of H2O2 and TMB were 25 mM and 1 mM. Inset: typical digital photograph of TMB solution (400 µL, 1 mM) catalyzed by o-CNTs in the presence of H2O2 in PBS. From left to right: TMB+H2O2, TMB+H2O2+p-CNTs, TMB+H2O2+o-CNTs. Control: TMB+H2O2without nanozymes. (b) Time-dependent absorbance changes at 652 nm by using different concentrations of o-CNTs as peroxidase mimics. (c) Temperature-dependent (red)/pH-dependent (blue) absorbance changes at 652 nm by using o-CNTs as peroxidase mimics. Error bars represented standard deviation from the mean (n=3). (d) Peroxidase-like activity of different functional CNTs (25 µg mL-1) in PBS (25 mM, pH 4, 37 °C). Error bars represented standard deviation from the mean (n=3). Asterisks indicated statistically significant differences (*P

Unraveling the Enzymatic Activity of Oxygenated Carbon Nanotubes

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