Bioelectrochemistry of non-covalent immobilized alcohol dehydrogenase on oxidized diamond nanoparticles Eduardo Nicolau, Jessica Méndez, José J. Fonseca, Kai Griebenow, Carlos R. Cabrera ⁎ Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, PO Box 23346, San Juan, Puerto Rico NASA Center for Advanced Nanoscale Materials, University of Puerto Rico, Rio Piedras Campus, PO Box 23346, San Juan, Puerto Rico abstract article info Article history: Received 12 July 2011 Received in revised form 7 October 2011 Accepted 11 November 2011 Available online 20 November 2011 Keywords: Adsorption Alcohol dehydrogenase Nanodiamonds Bioelectrochemistry Diamond nanoparticles are considered a biocompatible material mainly due to their non-cytotoxicity and re- markable cellular uptake. Model proteins such as cytochrome c and lysozyme have been physically adsorbed onto diamond nanoparticles, proving it to be a suitable surface for high protein loading. Herein, we explore the non-covalent immobilization of the redox enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (E.C.1.1.1.1) onto oxidized diamond nanoparticles for bioelectrochemical applications. Diamond nanoparticles were first oxidized and physically characterized by X-ray diffraction (XRD), FT-IR and TEM. Langmuir isotherms were constructed to investigate the ADH adsorption onto the diamond nanoparticles as a function of pH. It was found that a higher packing density is achieved at the isoelectric point of the en- zyme. Moreover, the relative activity of the immobilized enzyme on diamond nanoparticles was addressed under optimum pH conditions able to retain up to 70% of its initial activity. Thereafter, an ethanol bioelectro- chemical cell was constructed by employing the immobilized alcohol dehydrogenase onto diamond nanopar- ticles, this being able to provide a current increment of 72% when compared to the blank solution. The results of this investigation suggest that this technology may be useful for the construction of alcohol biosensors or biofuel cells in the near future. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Protein interaction with solid surfaces is believed to play a key role in the development of future nanotechnologies, biomaterials, and biotechnological processes [1]. The immobilization of proteins onto nanomaterials widely opens the door for fundamental and applied re- search [2]. The use of nanodiamonds holds an interesting and bright future for the development of a variety of biological and analytical plat- forms. This material has been investigated during the last years due to its properties: hardness, dopability, and biocompatibility [3,4]. In fact, the immobilization of different biomacromolecules (e.g. DNA [5], proteins [6], and cells [7]) onto diamond nanoparticles has shown the feasibility of using these platforms for biological and bio- medical applications [5,7–12]. For instance, it has been demonstrated that diamond nanoparticles are more compatible than multi-walled and single-walled carbon nanotubes for different cell types [13]. However, since it is particularly important to understand the im- mobilization behavior of these biomacromolecules, research has been conducted to assess the adsorption capacities of certain proteins on nanodiamonds. In 2004, Chang et al. reported on the noncovalent immobilization of cytochrome c (12,000 g/mol) onto nanodiamonds of different sizes [14]. This investigation revealed the nanodiamond size effect on the adsorption of the protein, revealing a packing den- sity of 8.0 × 10 12 molecules/cm 2 and 3.0 × 10 12 molecules/cm 2 for 100 nm and 5 nm diamond particles (mostly agglomerates). In addi- tion to the nanodiamond size affecting the adsorption behavior of proteins on nanodiamonds, the solution pH has been also considered in another investigation conducted in 2007. In this research, the hen egg white lysozyme (14,000 g/mol) was noncovalently immobilized on 100 nm diamond particles with pH variations [9,15]. The results demonstrated that besides hydrogen bonding, electrostatic interac- tions play a key role in the adsorption behavior of the selected protein onto the nanodiamonds. In fact, the packing density of the protein was dependent on the solution pH. Also, insulin (5,800 g/mol) has been physically immobilized onto nanodiamonds as a strategy for protein delivery, by using pH changes as the trigger to release the protein [16]. The latter is another example of the outstanding adsorp- tion capacity of nanodiamonds and their applicability in the nanome- dicine field. Accordingly, it has been well established the tremendous capability of nanodiamonds to be used for biological and biomedical applications [17]. For instance, these nanodiamond platforms are suitable to be used as protein supports for bioanalytical applications. Due to this, and to further extend the current knowledge in the area of nanodiamonds for analytical applications here we present Bioelectrochemistry 85 (2012) 1–6 ⁎ Corresponding author. Tel.: + 1 787 764 0000 1 4807; fax: + 1 787 756 8242. E-mail address: carlos.cabrera2@upr.edu (C.R. Cabrera). 1567-5394/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2011.11.002 Contents lists available at SciVerse ScienceDirect Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem