Tuning the Activities and Structures of Enzymes Bound to Graphene Oxide with a Protein Glue Ajith Pattammattel, † Megan Puglia, † Subhrakanti Chakraborty, ‡ Inoka K. Deshapriya, † Prabir K. Dutta, ‡ and Challa V. Kumar* ,† † Department of Chemistry, University of Connecticut, Department of Molecular and Cell Biology, and the Institute of Material Science, 55 North Eagleville Road, Unit 3060, Storrs, Connecticut 06269-3060, United States ‡ Department of Chemistry, Biochemistry, Ohio State University, Newman & Wolfrom Lab, 100 West 18th Avenue, Columbus, Ohio 43210-1340, United States * S Supporting Information ABSTRACT: Graphene oxide (GO) is being investigated extensively for enzyme and protein binding, but many enzymes bound to GO denature considerably and lose most of their activities. A simple, novel, and efficient approach is described here for improving the structures and activities of enzymes bound to GO such that bound enzymes are nearly as active as those of the corresponding unbound enzymes. Our strategy is to preadsorb highly cationized bovine serum albumin (cBSA) to passivate GO, and cBSA/GO (bGO) served as an excellent platform for enzyme binding. The binding of met-hemoglobin, glucose oxidase, horseradish peroxidase, BSA, catalase, lysozyme, and cytochrome c indicated improved binding, structure retention, and activities. Nearly 100% of native-like structures of all the seven proteins/ enzymes were noted at near monolayer formation of cBSA on GO (400% w/ w), and all bound enzymes indicated 100% retention of their activities. A facile, benign, simple, and general method has been developed for the biofunctionalization of GO, and this approach of coating with suitable protein glues expands the utility of GO as an advanced biophilic nanomaterial for applications in catalysis, sensing, and biomedicine. ■ INTRODUCTION Graphene and graphene oxide (GO) are emerging as excellent materials for various practical applications due to their remarkable mechanical, electrical, and optoelectronic proper- ties. 1 A general, facile, simple approach for biophilization of GO (bGO) is reported here, and enzymes bound to bGO indicated structures/activities that are nearly the same as those of unbound enzymes. Graphene is considered as a new generation nanoelectronic material because of its high carrier mobility, room temperature quantum hall effect, and others. 2 These properties ensure graphene as a promising candidate for the fabrication of advanced devices such as field effect transistors (FETs), supercapacitors, solar cells, etc. 1,3,4 Application of GO in biocatalysis, 5-7 biosensors, 8,9 bioelectronics, 8 and biomedi- cine 10-12 is highly promising. The nanosheets of GO (surface area = 7.05 × 10 22 Å 2 /g) 5 are decorated with COOH, OH, epoxide groups, and hydrophobic patches that enabled adsorption of biomolecules. 13-15 So far, functionalization of GO with amines, 16 proteins, 17 chemical reduction, 18 and PEGylation 10 were used to improve the nature or affinity of GO for biomolecules. Despite these efforts, enzyme binding to GO often results in significant loss of structure and/or function, which is not desirable. Horseradish peroxidase (HRP) and esterase bound to GO, for example, lost ∼70% of their activities. 5,18 However, in the case of lipase and oxalate oxidase, the activities were improved due to extensive distortion of bound protein. 18 The biomolecule-GO interactions often result in significant loss of biomolecular structure cited as a fundamental problem. 18,19 These interactions are not well-understood, 18 and reports suggest that they are primarily hydrophobic and/or electrostatic in nature. 18-20 Systematic manipulation of these interactions under chemical or biochemical control will be central for the rapid development of biological applications of GO, as well as in assessing, predicting, and controlling the biological toxicity of GO. We have previously investigated the molecular details of enzyme binding to inorganic layered solids and developed specific strategies to control these interactions. 21 Highly hydrated and strongly polar or ionic surfaces provide a benign environment for favorable binding of biomolecules, 22,23 and the hydration layer on the host surface promotes the retention of bound enzyme structure and activity to a significant extent. These strategies are now being extended to control enzyme- GO interactions in a predictable manner. Our hypothesis is that the conversion of hydrophobic regions of GO into highly Received: October 21, 2013 Revised: November 22, 2013 Published: November 25, 2013 Article pubs.acs.org/Langmuir © 2013 American Chemical Society 15643 dx.doi.org/10.1021/la404051c | Langmuir 2013, 29, 15643-15654