pubs.acs.org/Langmuir © 2010 American Chemical Society
Graphene Oxide as a Matrix for Enzyme Immobilization Jiali Zhang,†,§ Feng Zhang,‡,§ Haijun Yang,† Xuelei Huang,‡ Hui Liu,‡ Jingyan Zhang,*,‡ and Shouwu Guo*,† †
National Key Laboratory of Micro/Nano Fabrication Technology, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai, 200240 China, and ‡State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science & Technology, Shanghai, 200237 China. §These authors contributed equally to this work. Received October 16, 2009. Revised Manuscript Received January 25, 2010
Graphene oxide (GO), having a large specific surface area and abundant functional groups, provides an ideal substrate for study enzyme immobilization. We demonstrated that the enzyme immobilization on the GO sheets could take place readily without using any cross-linking reagents and additional surface modification. The atomically flat surface enabled us to observe the immobilized enzyme in the native state directly using atomic force microscopy (AFM). Combining the AFM imaging results of the immobilized enzyme molecules and their catalytic activity, we illustrated that the conformation of the immobilized enzyme is mainly determined by interactions of enzyme molecules with the functional groups of GO.
Introduction Graphene oxide (GO), as a basic material for the preparation of individual graphene sheets in bulk-quantity, has attracted great attention in recent years.1-3 In addition, the incredibly large specific surface area (two accessible sides), the abundant oxygencontaining surface functionalities, such as epoxide, hydroxyl, and carboxylic groups, and the high water solubility afford GO sheets great promise for many more applications.1,2 For instance, the GO nanosheets modified with polyethylene glycol have been employed as aqueous compatible carriers for water-insoluble drug delivery.4 The intrinsic oxygen-containing functional groups were used as initial sites for deposition of metal nanoparticles and organic macromolecules, such as porphyrin, on the GO sheets, which opened up a novel route to multifunctional nanometerscaled catalytic, magnetic, and optoelectronic materials.5-7 However, few studies about the binding of biomacromolecules, such as enzymes, to GO have been reported to date. Since the discovery of the advantageous property of immobilized enzymes, the challenges in this area have been to explore new substrate materials with appropriate structures (including the morphology and surface functionality) and compositions to deepen the understanding of enzyme immobilization and thus to improve the catalytic efficiency of the immobilized *To whom correspondence should be addressed. E-mail:
[email protected]. cn (S.G.);
[email protected] (J.Z.). (1) Li, D.; Muller, M. B.; Gilje, S.; Kaner, R. B.; Wallance, G. G. Nature Nanotechnol. 2008, 3, 101–105. (2) Park, S.; Ruoff, R. S. Nature Nanotechnol. 2009, 4, 217–223. (3) Tung, V. C.; Allen, M. J.; Yang, Y.; Kaner, R. B. Nature Nanotechnol. 2009, 4, 25–29. (4) Liu, Z.; Robinson, J. T.; Sun, X.; Dai, H. J. Am. Chem. Soc. 2008, 130, 10876–10877. (5) Lomeda, J. R.; Doyle, C. D.; Kosynkin, D. V.; Hwang, W.; Tour, J. M. J. Am. Chem. Soc. 2008, 130, 16201–16206. (6) Muszynski, R.; Seger, B.; Kamat, P. V. J. Phys. Chem. C 2008, 112, 5263– 5266. (7) Xu, Y.; Liu, Z.; Zhang, X.; Wang, Y.; Tian, J.; Huang, Y.; Ma, Y.; Zhang, X.; Chen, Y. Adv. Mater. 2009, 21, 1275–1278. (8) Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003, 42, 3336–3337. (9) Betancor, L.; Luckarift, H. R. Trends Biotechnol. 2008, 26, 566–572. (10) Badalo, A.; Gomez, J. L.; Gomez, E.; Bastida, J.; Maximo, M. F. Chemosphere 2006, 63, 626–632. (11) Chen, B.; Pernodet, N.; Rafailovich, M. H.; Bakhtina, A.; Gross, R. A. Langmuir 2008, 24, 13457–13464.
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enzymes.8-11 Recently, along with the development of nanostructured materials, a range of nanomaterials with different sizes and shapes have been utilized as the substrates for enzyme immobilization.12-14 It has been demonstrated that the enzymes immobilized on the nanostructured materials have some advantages over the bulk solid substrates.8,15 However, similar to bulk solid substrates, to efficiently immobilize enzymes on nanostructured material surfaces, in many cases, labored work was required to modify/functionalize the substrate surface.16,17 Moreover, for most of the nanostructured materials, it is hard to fully characterize their surfaces using conventional surface analytical tools. This limits the deep understanding of enzyme immobilization. Consequently, new nanostructured materials that not only can immobilize the enzyme enthusiastically but also can enable insight into the interactions between enzymes and the substrate are still in need of exploration. GO sheets should be an ideal substrate for the study of enzyme immobilization on nanostructured materials. As aforementioned, the individual GO sheet is enriched with oxygen-containing groups, which makes it possible to immobilize enzymes without any surface modification or any coupling reagents. The atomically flat surface of GO should provide a platform to characterize the immobilized enzyme using conventional surface imaging techniques, such as atomic force microscopy (AFM), and to further study the interactions between enzyme molecules and the GO surface. We describe herein the immobilization of horseradish peroxidase (HRP) and lysozyme, as model enzymes, on the GO. The enzyme immobilization was characterized in situ with AFM in a liquid cell, and the catalytic activity of the immobilized HRP was assayed using phenol and hydrogen peroxide as catalytic reaction substrates. (12) Kim, J.; Grate, J. W.; Wang, P. Chem. Eng. Sci. 2006, 61, 1017–1026. (13) Zhi, C.; Bando, Y.; Tang, C.; Golberg, D. J. Am. Chem. Soc. 2005, 127, 17144–17145. (14) Tsang, S. C.; Yu, C. H.; Gao, X.; Tam, K. J. Phys. Chem. B 2006, 110, 16914–16922. (15) Takahashi, H.; Li, B.; Sasaki, T.; Miyazaki, C.; Kajino, T.; Inagaki, S. Chem. Mater. 2000, 12, 3301–3305. (16) Lee, Y. M.; Kwon, O. Y.; Yoon, Y. J.; Ryu, K. Biotechnol. Lett. 2006, 28, 39–43. (17) Lin, Y.; Lu, F.; Tu, Y.; Ren, Z. Nano Lett. 2004, 4, 191–195.
Published on Web 03/18/2010
DOI: 10.1021/la904014z
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Experimental Section GO was prepared using natural graphite powder through a modified Hummers method.18,19 The as-obtained yellow-brown aqueous suspension of GO was stored at RT on a lab bench, and used for characterizations and enzyme immobilization. The samples for Fourier transform infrared (FT-IR) measurement were prepared by grinding the dried powder of graphene oxide with KBr together and then compressing the mixture into thin pellets (EQUINOX 55, Bruker, Germany). The specimens of transmission electron microscopy (TEM) (JEM-2010) were prepared by placing the aqueous suspension (∼0.02 mg/mL) of graphene oxide on the carbon-coated copper grids, and blotted after 30 s. AFM images of graphene oxide were taken on a MultiMode Nanoscope V scanning probe microscopy system (Veeco). The samples for AFM were prepared by dropping the aqueous suspension (∼0.02 mg/mL) of GO on a freshly cleaved mica surface. AFM images of the GO-bound enzymes were acquired in a liquid cell using tapping mode. To acquire in situ AFM images for enzyme immobilization, the liquid cell was circulated with the fresh enzyme solution during imaging.20 Enzyme immobilization was carried out by adding the desired amount of GO to 0.1 M phosphate buffer that contained the enzymes to be immobilized.21 The mixture was incubated for 30 min on ice with shaking and then centrifuged. The supernatant was used to determine the enzyme loading. The immobilized enzymes were washed three times with the same buffer to remove physical adsorbed enzymes. The resulting immobilized enzymes were then subjected to activity assay. A colorimetric assay was employed to evaluate HRP activity.22 The initial reaction rates were obtained via a linear fit of the curve of the product absorbance at 510 nm versus the reaction time (Supporting Information Figure S2).23
Figure 1. (a) Tapping mode AFM image of graphene oxide (GO) on a mica surface, (b) height profile of the AFM image, (c) TEM image of the GO, and (d) schematic model of GO.
Results and Discussion The morphology of as-prepared GO was characterized first using AFM (Figure 1a). The height of the flat GO sheet is ∼1 nm (Figure 1b), demonstrating a single atomic layer thickness structure feature. The thin nanoplate motif of the GO sheets was also confirmed by TEM (Figure 1c). The functional groups (Figure 1d) existing on the GO surface were verified by FT-IR spectroscopy (Supporting Information Figure S1). The enzyme immobilization was carried out by incubating the GO (0.5 to 1 mg/mL aqueous dispersions) with the enzymes in phosphate buffer solution at 4 °C. We found that HRP can be spontaneously immobilized on GO. Presumably, the amine groups of HRP may form amide bonds with the carboxylic groups of GO; however, without any coupling reagents, this covalent interaction usually happens very slowly.24 Therefore, the covalent bonding may not contribute to HRP-GO interaction. To elucidate the contribution of other interactions, the phosphate buffers with pH from 4.8 to 8.8, were tested. As shown in Figure 2, the loading of HRP on the GO decreases with increasing pH. HRP (pI = 7.2) has a net positive charge at pH below 7.2 and a net negative charge at pH above 7.2. The GO sheets are negatively charged in the aqueous solution with a pH range from 4 to 11 (see Supporting Information Figure S3).1-3 Thus, in the buffer solutions with a pH range from 4.8 to 7.2, the positively charged HRP interacts with the negatively charged GO by electrostatic interaction, while in the buffer solutions from pH 7.2 to 8.8, HRP and GO both are negatively charged, and will repel each other. Therefore, less HRP (18) (19) 56. (20) (21) (22) (23) (24)
Figure 2. pH influence on HRP loading. Conditions: 50 μg GO and 2 μg/mL HRP.
was loaded. Only an ∼30% enzyme loading decrease was observed when the pH of the buffer solutions increased from 4.8 to 8.8 (Figure 2), suggesting that other interactions, such as hydrogen bonding between the oxygen-containing functionalities of GO and surface amino acid residues of HRP, may contribute to GO-HRP interaction, too. Owing to the strong electrostatic interactions and hydrogen bonding, the maximum loading of HRP on GO at pH 7.0 is about 100 μg/mg of GO, which is much higher than the loadings on many reported materials.25-27 To further illustrate the electrostatic interaction between the enzymes and GO, we examined the immobilization of lysozyme, an enzyme with pI = 10.3 (positively charged at pH 7.0). The lysozyme can be spontaneously immobilized on GO, too, with the maximum loading of about 700 μg/mg of GO at pH 7.0. The positively charged surface of lysozyme apparently is favorable for its interaction with GO. The loading difference between HRP and lysozyme indicates that the interactions of substrate-enzymes are determined by the surface charges of the specified enzymes and the substrate. The high enzyme loadings reveal the exceptional potential of GO as a solid substrate for enzyme immobilization. The enzyme immobilization was monitored in situ using AFM. Figure 3a and b shows typical AFM images of the GO in a liquid
Hummers, W. S.; Offerman, R. E. J. Am. Chem. Soc. 1958, 80, 1339–1339. He, H.; Klinowski, J.; Forster, M.; Lerf, A. Chem. Phys. Lett. 1998, 287, 53– Guo, S.; Ward, M. D.; Wesson, J. A. Langmuir 2002, 18, 4284–4291. Cheng, J.; Ming, Yu, S.; Zuo, P. Water Res. 2006, 40, 283–290. Nicell, J. A.; Wright, H. Enzyme Microb. Technol. 1997, 21, 302–310. Buchanan, I. D.; Nicell, J. A. Biotechnol. Bioeng. 1997, 54, 251–261. Cao, Y.; Kyratzis, I. Bioconjugate Chem. 2008, 19, 1945–1950.
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(25) Pundir, C. S.; Malik, V.; Bhargava, A. K.; Thakur, M.; Kaliam, V.; Singh, S.; Kuchhal, N. K. J. Plant Biochem. Biotechnol. 1999, 8, 123–126. (26) Azevedo, A. M.; Vojinovic, V.; Cabral, J. M. S.; Gibson, T. D.; Fonseca, L. P. J. Mol. Catal. B: Enzym. 2004, 28, 121–128. (27) Gomez, J. L.; Bodalo, A.; Gomez, E.; Bastida, J.; Hidalgo, A. M.; Gomez, M. Enzyme Microb. Technol. 2006, 39, 1016–1022.
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Letter Table 1
Figure 3. Tapping mode AFM images of the GO-bound HRP with (a) lower and (b) higher enzyme loadings acquired in a liquid cell. (c) Schematic model of the GO-bound HRP. (d) Initial reaction rates of GO-bound HRP versus HRP concentration.
cell after being incubated together with HRP in phosphate buffer for 30 min. With a lower enzyme loading (HRP/GO = 3:500, in weight), the particles (bright spots, presumably the immobilized enzyme molecules) on the GO surface were observed (Figure 3a). The average diameter and height of the particles on the GO surface are about 140 and 15 A˚, respectively. The dimension size of the immobilized HRP molecule, 140 140 15 A˚, is roughly consistent with the dimension size of free HRP, 30 65 75 A˚3.28 This is the first picture of the native immobilized enzyme. The larger average diameter and shorter height of the immobilized HRP molecules revealed that immobilization induced some conformational changes of the HRP molecules. With a higher enzyme loading (HRP/GO = 3:50, in weight), the enzyme molecules tethered densely over all of the GO surface in the AFM image (Figure 3b). The distribution of HRP on the GO surface should be determined by the intrinsic sites of the oxygen functionalities. Except for the carboxylic groups, which are located at the periphery, others, such as hydroxyl and epoxide groups, distributed randomly over the GO surface.19 The mole ratio of C/O of the GO used in the work is about 4, and thus, HRP may densely bind on the GO surface. This is in agreement with the AFM image (Figure 3b) where we observed the increased surface coverage with higher enzyme loading. The catalytic property of the HRP immobilized on GO was investigated using phenol as a reducing substrate. We found that the initial catalytic reaction rates of the immobilized HRP were linear to the HRP loading under an excess and constant substrate concentration (Figure 3d), though they are relatively lower than that of free HRP.29 This result suggested that the voids presented between the immobilized HRP molecules are enough for the free diffusion of substrate and product into and out of the HRP active sites, though the immobilized enzymes seem crowded on the GO surface (see Figure 3b). Given the single atomic layer feature of the GO sheet, the total surface area is about 7.05 1022 A˚2/g, and assuming the average transverse area of one molecule HRP is about 3000 A˚2, the HRP molecules cover less than 50% of the surface area of GO even with the higher enzyme loading. (28) Henriksen, A.; Schuller, D. J.; Meno, K.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8504–8060. (29) Cooper, V. A.; Nicell, J. A. Water Res. 1996, 30, 954–964.
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sample
Km (mM)
Kcat (s-1)
Kcat/Km (mM-1 s-1)
Free HRP GO Immobilized HRP (lower loading) GO immobilized HRP (higher loading)
2.27 1.96 ( 0.21
161.7 ( 34.10 33.6 ( 1.20
71.2 17.1 ( 1.22
1.76 ( 0.10
36.6 ( 2.90
20.8 ( 0.52
The catalytic activities of the HRP immobilized on GO with the lower and higher enzyme loadings were further characterized by turnover number (Kcat) and enzyme efficiency (Kcat/Km). Km and Kcat values were obtained according to the Lineweaver-Burk equation as described in the Supporting Information (Figure S2). The values of the kinetic parameters Km and Kcat are summarized in Table 1. The similar Km values for the GO immobilized HRP with the lower and higher enzyme loadings, and free HRP indicated that they all have a similar affinity to the reducing substrate. However, Kcat/Km values of the immobilized HRP are lower than those of free HRP. Noticeably, the comparable Kcat/ Km values for the HRP immobilized on GO with the higher and lower enzyme loadings confirmed that increasing enzyme loading does not affect the enzyme efficiency. The catalytic reactions of the immobilized HRP (with the higher and lower enzyme loadings) with a bulky reducing substrate, 2,4,6-trimethylphenol, exhibited similar activity, further supporting this result. Thus, combined with the AFM imaging results, we believe that the observed lower enzymatic activity for the immobilized HRP is mainly due to the HRP conformational changes induced by its binding to GO. According to the number of oxygen containing groups on the GO surface and the transverse area of one HRP molecule, there should be at least an average of two oxygen containing groups of the GO surface interacting with one HRP molecule (Figure 3c). Multiple interactions between the substrate and the enzyme molecule could change the enzyme conformation.11 Thus, to maintain the conformation and catalytic capability of the immobilized enzyme, the distribution, number, and property of the functional groups on the substrate surface must be optimized to match the surface of the enzyme being immobilized.
Conclusion In summary, we have demonstrated that individual GO sheets could be used as substrates to study enzyme immobilization. Pronouncedly, the rich surface functional groups of GO make the immobilization of the enzymes happen quickly through electrostatic interaction without using any cross-linking reagents; the unique flat surface of GO made it possible to observe the native immobilized enzyme in situ using AFM. We found that the catalytic performance of the immobilized enzymes is determined by the interaction of enzyme molecules with the surface functional groups of the substrate, but the enzyme specific activity is not influenced by the enzyme loading as far as the substrate surface was not fully covered by the enzyme. Based on the AFM images and enzyme activity assay, we conclude that full retention of the conformation of immobilized enzyme should be the key to improve its catalytic performance. Acknowledgment. This work was supported by the National “973 Program” (Nos. 2007CB936000 and 2010CB933900) and the NSFC of China (Nos. 20774029 and 20671034). Supporting Information Available: FT-IR spectrum of GO and catalytic data of the immobilized HRP. This material is available free of charge via the Internet at http://pubs.acs.org. DOI: 10.1021/la904014z
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