Biosensors and Bioelectronics 22 (2007) 3021–3027 Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization Zi-Xia Zhao, Ming-Qiang Qiao, Feng Yin, Bin Shao, Bao-Yan Wu, Yan-Yan Wang, Xin-Sheng Wang, Xia Qin, Sha Li, Lei Yu, Qiang Chen ∗ The Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China Received 5 September 2006; received in revised form 10 November 2006; accepted 8 January 2007 Available online 20 January 2007 Abstract Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H 2 O 2 permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10 -3 AM -1 cm -2 , also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 A responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing. © 2007 Elsevier B.V. All rights reserved. Keywords: Biosensor; Self-assembly; Hydrophobin; Glucose oxidase 1. Introduction Enzyme immobilization has become a widely concerned issue in biosensing and surface functionalizing for years. As for amperometric biosensors, the generally applied strategy to get stronger enzymatic responses is to increase the quantity of immobilized enzyme, such as deposition of enzyme multilayers (Chen et al., 1998; Hoshi et al., 2001; Ferreira et al., 2004; Davis and Higson, 2005), or bringing in nanomaterials with large spe- cific surface area to immobilize more enzyme (Zhou et al., 2005; Guan et al., 2005; Yang and Zhu, 2006; Xian et al., 2006). While we focus on another promising strategy: to improve the efficiency of enzyme utilization. That means making best use of the catalytic activity for per unit of enzyme and produc- ing electrochemical signal as much as possible under limited ∗ Corresponding author. Tel.: +86 22 23506173; fax: +86 22 23506122. E-mail address: qiangchen@nankai.edu.cn (Q. Chen). enzyme quantity. To realize high efficiency of enzyme utiliza- tion, the crucial first step should be searching for a highly biocompatible immobilization matrix which well conserves the catalytic activity of enzymes and provides a mild and stable environment for enzymatic reactions. Hydrophobins, small nat- ural self-assembling proteins, provide new choices with great potential. Hydrophobins consisting approximately 100 amino acid residues are the most powerful surface-active proteins known (Hakanp¨ a¨ a et al., 2004). They are ubiquitously produced by filamentous fungi, playing various roles in fungal physiology related to surface phenomena, such as adhesion, formation of surface layers, and lowering of surface tension (Linder et al., 2002). As a remarkable property, hydrophobins have both hydrophobic and hydrophilic parts and self-assemble into strong, highly ordered, amphiphilic films at almost any inter- faces, helping fungi adapt to a wide variety of environmental conditions and to attach to various surfaces. Depending on the type of surface, various molecular interactions, such as van 0956-5663/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2007.01.007