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Mimicking Horseradish Peroxidase and NADH Peroxidase by Heterogeneous Cu -Modified Graphene Oxide Nanoparticles 2+

Shan Wang, Rémi Cazelles, Wei-Ching Liao, Margarita VázquezGonzález, Amani Zoabi, Raed Abu-Reziq, and Itamar Willner Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b00093 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 12, 2017

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Mimicking Horseradish Peroxidase and NADH Peroxidase by Heterogeneous Cu2+-Modified Graphene Oxide Nanoparticles Shan Wang, Rémi Cazelles, Wei-Ching Liao, Margarita Vázquez-González, Amani Zoabi, Raed Abu-Reziq and Itamar Willner* Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

ABSTRACT: Cu2+-ion-modified graphene oxide nanoparticles, Cu2+-GO NPs, act as a heterogeneous catalyst mimicking functions of horseradish peroxidase, HRP, and of NADH peroxidase. The Cu2+-GO NPs catalyze the oxidation of dopamine to aminochrome by H2O2 and catalyze the generation of chemiluminescence in the presence of luminol and H2O2. The Cu2+-GO NPs provide an active material for the chemiluminescence detection of H2O2, and allow the probing of the activity of H2O2-generating oxidases and the detection of their substrates. This is exemplified with detecting of glucose by the aerobic oxidation of glucose by glucose oxidase, and the Cu2+-GO NPs-stimulated chemiluminescence intensity generated by the H2O2 product. Similarly, the Cu2+-GO NPs catalyze the H2O2 oxidation of NADH to the biologically-active NAD+ cofactor. This catalytic system allows its conjugation to biocatalytic transformations involving NAD+-dependent enzyme, as exemplified for the alcohol dehydrogenase-catalyzed

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oxidation of benzyl alcohol to benzoic acid through the Cu2+-GO NPs catalyzed regeneration of NAD+.

KEYWORDS: Catalysis, dopamine, chemiluminescence, nanotechnology, biocatalysis Substantial research efforts are directed toward the development of homogeneous or heterogeneous catalysts that mimic horseradish peroxidase, HRP, or other peroxidases.1–3 For example, hemin-functionalized peptides such as microperoxidase-11,4 Fe(III)-porphyrin-modified de novo proteins,5–8 and transition metal compounds such as Cu(II) or Ni(II) ion complexes9–11 and metal oxide nanoparticles12,13 were reported to mimic the functions of HRP. Also, hemin/Gquadruplex structures were reported to act as HRP mimicking catalysts14,15 or NADH peroxidase mimicking DNAzymes.16 For example, similar to HRP, the hemin/G-quadruplex catalyzed the oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) dianion, ABTS2–, to ABTS•– by H2O217–19 or the generation of chemiluminescence in the presence of luminol/H2O2.20–22 Also, heterogeneous nanomaterials, such as iron oxide,23,24 Au25,26 or Cu27 nanoparticles were reported to reveal HRP mimicking activities. The broad interest in HRP mimicking catalysts rests on the possible use of the homogeneous or heterogeneous catalysts as a substitute for the native enzyme. Indeed, similarly to the application of HRP as amplifying label for many biosensing platforms, the hemin/G-quadruplex or microperoxidase-11 were applied as amplifying labels for many electrochemical28–31 or optical32–36 sensors (e.g., DNA sensors, aptasensors or immunosensors). Also, in analogy to HRP-catalyzed chemical transformations, HRP-mimicking catalysts were reported to induce similar processes. For example, hemin/G-quadruplex was reported to catalyze the oxidation of aniline by H2O2 to yield polyaniline37–39 or to mediate the H2O2 oxidation of dopamine to aminochrome.40,41 Here we wish to report on the synthesis of Cu2+-modified graphene


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oxide nanoparticles (Cu2+-GO NPs) acting as heterogeneous catalysts mimicking HRP and NADH peroxidase. We demonstrate that the Cu2+-GO NPs catalyze the generation of chemiluminescence in the presence of luminol/H2O2 and catalyze the oxidation of dopamine to aminochrome by H2O2. We further demonstrate that the Cu2+-GO NPs can be applied to follow the activities of oxidases (e.g., glucose oxidase), and we demonstrate the catalyzed oxidation of NADH to NAD+ by H2O2 in the presence of the Cu2+-GO NPs. The graphene oxide (GO) NPs (~6 nm) were prepared using the modified Hummers method42 followed by the solvothermal treatment of the material with dimethylformamide43 that yields nitrogen doped graphene nanoparticles.44 The nanoparticles were reacted with CuCl2 to yield the Cu2+-GO NPs (For a detailed procedure to prepare the Cu2+-GO NPs, see supporting information). (For further structural and spectroscopic characterizations of the Cu2+-GO NPs, see Figure S1–S4, supporting information). Recently, the preparation of Cu2+-modified graphene oxide sheets45 were used to develop electrochemical H2O2 sensors by the electrocatalyzed reduction of H2O2,46,47 and the Cu2+-functionalized graphene oxide sheets were used as catalysts for the epoxidation of styrene.48 Previous studies demonstrated that the GO NPs include surface functionalities such as hydroxyl, carboxylic acid, ether, carbonyl and nitrogen functionalities. Although the precise nature of the binding modes of Cu2+ to the surface of the GO NPs is unknown, from the FTIR spectra of the Cu2+-GO NPs as compared to the unmodified GO NPs, we suggest that Cu2+-carboxylate or mixed Cu2+-carboxylate/amine complexes are associated with the Cu2+-GO surface (see Figure S5). X-ray photoelectron spectroscopy (XPS) measurements indicate that Cu2+ ions are associated with the GO particles (atomic concentration of constituents corresponding to Cu-4.8%, O-22.7%, N-14.7%, C-57.8%) (see Figure S6) and inductively coupled plasma atomic emission spectroscopy


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(ICP-OES) analysis indicated a coverage of Cu2+ ions corresponding to 0.21 mg per milligram of GO NPs. The Cu2+-GO NPs catalyze the oxidation of dopamine (1) to aminochrome (2) in the presence of H2O2, Figure 1(A). The time-dependent increase of the absorption spectra of aminochrome (2) upon the oxidation of dopamine (1) are depicted in Figure 1(B). The rates of dopamine oxidation to aminochrome in the presence of variable concentrations of dopamine are depicted in Figure 1(C), and inset. As the concentrations of dopamine increases the reaction rate is enhanced, reaching a saturation rate at a dopamine concentration of ca. 4.0 mM. Figure 1(D) and S7 depict the oxidation rate of dopamine, 1 mM, in the presence of variable concentrations of the Cu2+-GO NPs. (For the rate of dopamine oxidation in the presence of a fixed concentration of Cu2+-GO NPs, 10 μg mL–1, and dopamine, 10 mM, and variable concentrations of H2O2 see Figure S8). Furthermore, we find that the catalytic activity of the heterogeneous Cu2+-GO NPs catalyst is controlled by the loading of the Cu2+-ions on the GO NPs (Figure S9). As the content of Cu2+-ions increases, the catalytic oxidation of dopamine to aminochrome is enhanced and it reaches a saturation value corresponding to a loading of 0.21 mg of Cu2+ per 1 mg of GO NPs. Accordingly, these particles were applied for all subsequent experiments. The oxidation of dopamine to aminochrome is specific to Cu2+-GO NPs and other metal ion-modified GO NPs (Ni2+, Co2+, Pd2+, Cd2+) did not show any catalytic activities. Also, control experiments revealed that unmodified GO NPs did not show any catalytic activity towards the oxidation of dopamine, and the Cu2+ ions (without the GO NPs matrix), at the same content associated with the Cu2+-GO NPs, showed an inefficient catalytic oxidation of dopamine (ca. 10% of the Cu2+-GO NPs activity), see Figure S10. It should be noted that previous studies have demonstrated that hemin and hemin deposited on a GO matrix act as horseradish peroxidase mimicking catalysts.49 Accordingly, we deposited hemin on the GO NPs


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and compared the activity of hemin-GO NPs (at the same molar ratio of Cu2+ ions on the GO NPs) to that of the Cu2+-GO NPs toward to catalyzed oxidation of dopamine to aminochrome (Figure S11). We find that the Cu2+-GO NPs reveal a 3.3-fold enhanced catalytic performance, as compared to the hemin-GO NPs, toward the oxidation of dopamine to aminochrome. Similarly, the Cu2+-GO NPs catalyze the generation of chemiluminescence in the presence of luminol/H2O2, Figure 2(A). The chemiluminescence spectra generated in the presence of variable concentrations of Cu2+-GO NPs (fixed concentrations of luminol/H2O2) are shown in Figure 2(B). Similarly, Figure 2(C) depicts the chemiluminesece spectra generated by the Cu2+-GO NPs, 10 μg mL–1, and luminol, 0.5 mM, and variable concentrations of H2O2. As the concentration of H2O2 increases, the chemiluminescence is intensified. These results suggest that the Cu2+-GO NPs, and the resulting chemiluminescence might provide a sensor for the detection of H2O2. Figure 2(C) inset shows the calibration curve corresponding to the chemiluminescence intensities generated in the presence of H2O2. It should be noted that not all of the characteristic reactions of HRP could be stimulated by Cu2+-GO NPs. For example, the oxidation of ABTS2– by H2O2 to form the colored product, ABTS•–, was not catalyzed by the Cu2+-GO NPs. Presumably, the absorption of ABTS2– on the Cu2+-GO NPs through π-π interactions blocked the catalytic Cu2+ sites. Unmodified GO NPs did not show any HRP activity and did not yield any chemiluminescence in the presence of luminol/H2O2 (see Figure S12). These results indicate that the Cu2+ ions functionalizing the GO NPs provide the catalytic sites for mimicking HRP. Realizing that the Cu2+-GO NPs sense quantitatively H2O2, the NPs were applied to follow the activities of glucose oxidase, GOx, as a model system for probing H2O2-generating oxidases. Since GOx catalyzes the aerobic oxidation of glucose to gluconic acid, we applied the Cu 2+-GO NPs as heterogeneous catalysts for probing the activity of GOx. Figure 2(D) depicts the


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chemiluminescence spectra observed upon analyzing different concentrations of glucose (fixed GOx concentration, 0.4 U mL–1, and fixed time-interval of aerobic oxidation, 0.5 h). As the concentration of glucose increases, the chemiluminescence spectra are intensified, consistent with the higher concentration of H2O2 generated by the biocatalytic system. We then attempted to apply the Cu2+-GO NPs as heterogeneous catalysts for mimicking the NADH peroxidase, biocatalytic system, Figure 3(A). In this system, the catalyzed H2O2 oxidation of NADH to NAD+ proceeds. Figure 3(B) depicts the time-dependent conversion of NADH to NAD+ in the presence of different concentrations of H2O2 (followed by λ = 340 nm). As the concentration of H2O2 is higher, the rate of oxidation of NADH is enhanced. The rates of NADH oxidation to NAD+ in the presence of variable concentrations of NADH are depicted in Figure 3(C), and inset. Control experiments revealed that no oxidation of NADH to NAD+ proceeds in the presence of the unmodified GO NPs and H2O2. Similarly, an inefficient catalytic oxidation of NADH (ca. 12% of the Cu2+-GO NPs activity) occurs in the presence of the Cu2+ ions (without the GO NPs matrix) at the same content associated with the Cu2+-GO NPs (see Figure S13). Also, other metal ion-functionalized GO NPs (Pd2+, Ni2+, Co2+) did not affect the oxidation of NADH. The results imply that the Cu2+ ions associated with the GO NPs catalyze the oxidation of NADH. One of the major issues relates, however, to the confirmation that NADH is oxidized to the biologically-active NAD+ cofactor (two-electron oxidation process, rather than the one-electron oxidation to the radical that forms the biologically-inactive NAD dimer). Toward this end, we applied the Cu2+-GO NPs catalyst for the regeneration of NAD+ in a biocatalytic cycle. The Cu2+GO NPs were applied for the H2O2 oxidation of NADH at a relatively low concentration, 50 μM, in the presence of an hydride acceptor, e.g. benzyl alcohol (6), and alcohol dehydrogenase, AlcDH, as biocatalyst, Figure 3(A). That is, the AlcDH-catalyzed reduction of NAD+ formed upon the


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Cu2+-GO NPs-catalyzed oxidation of NADH by the benzyl alcohol recycles the NADH cofactor. This process proceeds only upon generating the bioactive cofactor NAD+ by the Cu2+-GO NPs (and does not proceed if the NAD-dimer is formed). Interestingly, we find that the oxidation product of benzyl alcohol in the presence of AlcDH/NAD+ is benzoic acid (rather than benzaldehyde). The benzoic acid product is well documented in the literature50 and originated from the secondary AlcDH/NAD+ oxidation of the hydrated form of benzaldehyde. We find that, within a time-interval of 5 hour at 25 °C, we generated a concentration of ca. 229.8 μM of the product benzoic acid, while the initial concentration of the NADH cofactor corresponded to 50 μM. That is, the bioactive cofactor NAD+ was regenerated 4.6 times in the system implying that the heterogeneous catalyst recycled the NAD+ cofactor in the system. Although this process was not optimized and the long term stability of the NADH/NAD+ system was not evaluated, the Cu2+-GO NPs/H2O2 system provides a new NAD+ regeneration process that could be implemented to drive biocatalytic transformations. Finally, the catalytic stability of the systems presented in this study, and the possibility to recycle the catalyst were examined. We find that the Cu2+-GO NPs catalyze, as a competitive path, the decomposition of H2O2, even without dopamine or NADH. Therefore, we used an excess of H2O2 to stimulate the oxidation of dopamine to aminochrome or of NADH to NAD+. The possibilities to retain the catalytic functions of the Cu2+-GO NPs for two processes were demonstrated as depicted in Figure S14. The Cu2+-GO NPs-catalyzed oxidation revealed a saturation absorbance value for aminochrome after ca. 30 minutes. Accordingly, after completion of the first cycle of oxidation of dopamine to aminochrome for 30 minutes, a second cycle was initiated by the addition of dopamine/H2O2. The Cu2+-GO NPs-catalyzed oxidation of dopamine retained ca. 90% of the first catalytic recycle, implying that the catalyst can be recycled (see Figure S14(A)). Similarly,


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The Cu2+-GO NPs catalyzed oxidation of NADH to NAD+ revealed ca. 100% recyclability of the catalyst (see Figure S14(B)). For the effect of the solvothermal generation of the 6 nm-sized Cu2+GO NPs on the catalytic oxidation of dopamine by other Cu2+ modified GO matrices see supporting information, Figure S15, and accompanying discussion. In conclusion, the present study has introduced Cu2+-ion-modified graphene oxide nanoparticles, Cu2+-GO NPs, as a heterogeneous catalyst mimicking horseradish peroxidase (HRP) and NADH peroxidase functionalities. The Cu2+-GO NPs acted as a heterogeneous catalyst for the H2O2 oxidation of dopamine to aminochrome and for the catalyzed generation of chemiluminescence, in the presence of luminol/H2O2, characteristic functions of HRP. The generation of chemiluminescence by the Cu2+-GO NPs/luminol/H2O2 was applied to sense H2O2 and to probe H2O2-generating oxidases and their substrates. Specifically, the probing of glucose oxidase and its substrate glucose were demonstrated by the aerobic oxidation of glucose, in the presence of glucose oxidase, and the subsequent chemiluminescence detection of the resulting H2O2. Thus, the Cu2+-GO NPs could be applied to analyze other oxidases and their enzyme such as lactate oxidase/lactate or choline oxidase/choline. Also, it was demonstrated that Cu2+-GO NPs mimic the functions of NADH peroxidase and the heterogeneous catalytic NPs catalyzed the oxidation of NADH to NAD+. This allowed the coupling of the Cu2+-GO NPs to NAD+-dependent enzyme through the catalytic regeneration of the biologically-active NAD+ cofactor. This was exemplified with the biocatalyzed oxidation of benzyl alcohol to benzoic acid, in the presence of alcohol dehydrogenase, AlcDH and the H2O2/Cu2+-GO NPs regeneration system. The catalytic NAD+ cofactor regeneration system may be coupled to other NAD+-dependent enzymes such as alanine dehydrogenase or lactate dehydrogenase, thus paving the way for various biotechnological applications. Also, a further interesting path to follow involves the examination of the cytotoxicity


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of Cu2+-GO NPs and the possible application of the catalyst for monitoring intracellular processes. Furthermore, the application of other carbon-based, or carbon-like, nanomaterials as supports of metal ions acting as heterogeneous catalysts should be possible.

Figure 1. (A) The Cu2+-GO NPs-catalyzed oxidation of dopamine (1) to aminochrome (2) by H2O2. (B) Time-dependent absorbance spectra of aminochrome (2) generated upon the oxidation of dopamine (1) 10 mM, in the presence of Cu2+-GO NPs 10 μg mL–1 and H2O2 10 mM: 0 ~ 60 min, every six minutes, the total volume of the reaction mixture: 150 μL. (C) Time-dependent absorbance changes upon the oxidation of variable concentrations of dopamine (1) with Cu2+-GO NPs 10 μg mL–1 and H2O2 10 mM: (i) 0, (ii) 0.005, (iii) 0.025, (iv) 0.1, (v) 0.5, (vi) 1, (vii) 5, (viii) 10 mM, the total volume of the reaction mixture: 100 μL. Inset: The rates of dopamine (1) oxidation by H2O2 at variable concentrations of dopamine. (D) Time-dependent absorbance spectra upon oxidation of dopamine (1) to aminochrome (2) by H2O2 using variable concentrations


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of Cu2+-GO NPs in the presence of dopamine 1 mM and H2O2 10 mM: (i) 0, (ii) 0.1, (iii) 0.2, (iv) 0.5, (v) 1, (vi) 2, (vii) 5, (viii) 10 μg mL–1, the total volume of the reaction mixture: 100 μL. All the experiments were conducted in MES buffer 10 mM, including 0.4 mM MgCl2 and 2 mM KCl, pH = 5.5.

Figure 2. (A) The Cu2+-GO NPs-catalyzed oxidation of luminol (3) to aminophthalate (4) by H2O2 and the concomitant generation of chemiluminescence. (B) Chemiluminescence spectra generated upon the oxidation of luminol (3) using variable concentrations of Cu2+-GO NPs in the presence of luminol 0.5 mM and H2O2 1 mM: (i) 0, (ii) 0.5, (iii) 1, (iv) 2, (v) 5, (vi) 10 μg mL–1, the total volume of the reaction mixture: 150 μL. (C) Chemiluminescence spectra generated upon the oxidation of luminol (3) by variable concentrations of H2O2 in the presence of luminol 0.5 mM and Cu2+-GO NPs 10 μg mL–1: (i) 0, (ii) 0.05, (iii) 0.1, (iv) 0.25, (v) 0.5, (vi) 1, (vii) 1.5 mM, the total volume of the reaction mixture: 150 μL. Inset: Derived calibration curve corresponding to the


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chemiluminescence intensity at λ = 425 nm, generated by the oxidation of luminol with variable concentrations of H2O2. (D) Chemiluminescence spectra generated upon the oxidation of luminol (3) with the H2O2 produced by the GOx-catalyzed aerobic oxidation of variable concentrations of glucose in the presence of luminol 0.5 mM and Cu2+-GO NPs 10 μg mL–1: (i) 0, (ii) 0.1, (iii) 0.25, (iv) 0.5, (v) 1, (vi) 2.5 mM, the total volume of the reaction mixture: 150 μL. Inset: Derived calibration curve corresponding to the chemiluminescence intensity at λ = 425 nm, generated by the oxidation of luminol with the H2O2 produced by the GOx-catalyzed aerobic oxidation of variable concentrations of glucose. All experiments were conducted in PB buffer 280 mM, pH = 9.0.

Figure 3. (A) Cu2+-GO NPs for the regeneration of the NAD+ cofactor. Frame I: Cu2+-GO NPscatalyzed oxidation of NADH (5) to NAD+ (6) by H2O2. Frame II: Regeneration of the NAD+


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cofactor by the coupled Cu2+-GO NPs/alcohol dehydrogenase/benzyl alcohol system. (B) Timedependent absorbance changes upon the oxidation of NADH (5) to NAD+ (6) in the presence of variable concentrations of H2O2, NADH 1 mM and Cu2+-GO NPs 10 μg mL–1: (i) 0, (ii) 0.1, (iii) 0.2, (iv) 0.5, (v) 1, (vi) 2, (vii) 5, (viii) 10 mM, the total volume of the reaction mixture: 100 μL. Inset: The calibration curve corresponding to the rates of NADH oxidation as a function of H 2O2 concentration. (C) Time-dependent absorbance changes upon oxidation of variable concentrations NADH (5) in the presence of Cu2+-GO NPs 10 μg mL–1 and H2O2 2 mM: (i) 0, (ii) 0.005, (iii) 0.0125, (iv) 0.025, (v) 0.05, (vi) 0.125, (vii) 0.25, (viii) 1 mM, the total volume of the reaction mixture: 100 μL. Inset: The calibration curve corresponding to the rates of NADH oxidation as a function of NADH concentration. All the experiments were performed in 17.5 mM HEPES buffer solution that included 7 mM MgCl2 and 17.5 mM NaCl, pH = 7.2. ASSOCIATED CONTENT Supporting Information. Materials, methods, and additional ﬁgures. (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: 972-2-6585272. Fax: 972-2-6527715. Author Contributions S. W. planed and performed the experiments and analyzed the results, and participated in formulation of the paper. R. C., W-C. L. and M. V-G. assisted in the experimental work. A. Z. and R. A-R. performed the HPLC and IR analysis. I. W. guided the project and participated in the formulation of the paper.


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Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENT This research is supported by the German-Israeli Program (DIP). REFERENCES (1) Wei, H.; Wang, E. Chem. Soc. Rev. 2013, 42, 6060–6093. (2) Golub, E.; Lu, C. H.; Willner, I. J. Porphyrins Phthalocyanines 2015, 19, 65–91. (3) Ragg, R.; Tahir, M. N.; Tremel, W. Eur. J. Inorg. Chem. 2016, 1906–1915. (4) Marques, H. M. Dalton Trans. 2007, 4371–4385. (5) Rau, H. K.; Haehnel, W. J. Am. Chem. Soc. 1998, 120, 468–476. (6) Katz, E.; Heleg-Shabtai, V.; Willner, I.; Rau, H. K.; Haehnel, W. Angew. Chem., Int. Ed. 1998, 37, 3253–3256. (7) Feiters, M. C.; Rowan, A. E.; Nolte, R. J. M. Chem. Soc. Rev. 2000, 29, 375–384. (8) Das, A.; Hecht, M. H. J Inorg. Biochem. 2007, 101, 1820–1826. (9) Xianyu, Y.; Zhu, K.; Chen, W.; Wang, X.; Zhao, H.; Sun, J.; Wang, Z.; Jiang, X. Anal. Chem. 2013, 85, 7029−7032. (10) Somturk, B.; Hancer, M.; Ocsoy, I.; Özdemir, N. Dalton Trans. 2015, 44, 13845–1385. (11) Organo, V. G.; Filatov, A. S.; Quartararo, J. S.; Friedman, Z. M.; Rybak-Akimova, E. V. Inorg. Chem. 2009, 48, 8456–8468. (12) Chen, J.; Patil, S.; Seal, S.; Mcginnis, J. F. Nat. Nanotechnol. 2006, 1, 142–150. (13) Natalio, F.; André, R.; Hartog, A. F.; Stoll, B.; Jochum, K. P.; Wever, R.; Tremel, W. Nat.


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Mimicking Horseradish Peroxidase and NADH ... - ACS Publications

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