Colloids and Surfaces B: Biointerfaces 71 (2009) 102–106 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing Zi-Xia Zhao a,b , Hui-Cai Wang a , Xia Qin a , Xin-Sheng Wang a , Ming-Qiang Qiao a , Jun-ichi Anzai b , Qiang Chen a,∗ a The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, PR China b Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan article info Article history: Received 15 October 2008 Received in revised form 9 January 2009 Accepted 12 January 2009 Available online 20 January 2009 Keywords: Hydrophobin Self-assemble Surface wettability Choline oxidase (ChOx) Biosensor abstract Hydrophobins are small fungal proteins which self-assemble on interfaces and significantly change the surface wettability. The self-assembled film of hydrophobin HFBI on a gold surface improved the surface hydrophilicity with water contact angle changing from 73.8 ± 1.8 ◦ to 45.3 ± 1.4 ◦ . A quartz crystal microbal- ance (QCM) analysis indicated that the HFBI coverage density on a gold surface was 588 ng cm -2 , and the self-assembled film remained stable under different pH values ranging from 1 to 13. A hydrophilic protein such as choline oxidase (ChOx) was then successfully immobilized on the HFBI modified gold surface. To evaluate the bioactivity of immobilized enzyme, an amperometric choline biosensor was constructed based on the Gold/HFBI/ChOx electrode, which produced as large as 4578.27nA response current by 0.238 g immobilized ChOx, when saturated by choline substrate. Comparing with our choline biosen- sors previously reported, the HFBI self-assembled film exhibited excellent capability to preserve the bioactivity of ChOx, hence a great potential in electrochemical biosensing is suggested. © 2009 Elsevier B.V. All rights reserved. 1. Introduction In the study of fungal biology, interesting phenomena have been observed that cell wall surfaces of the hypha growing in a moist substrate are hydrophilic, while the surfaces of aerial hyphae and airborne spores are hydrophobic. Genetic investigations revealed that the adjusting of fungal cell wall surface wettability is real- ized through secretion of hydrophobins, which are a family of small proteins produced exclusively by filamentous fungi [1,2]. Hydrophobins help fungi fulfill a series of interface related bio- logical functions, such as mediating adhesion of fungal structures, lowering of water surface tension, and formation of coatings [3,4]. Firstly named by Wessels and co-workers in 1991 [1], hydrophobins are found easy to self-assemble at various interfaces to form robust and orderly membranes, which reverse the wettabil- ity of surfaces [5,6]. Structural analysis indicates that a hydrophobin consists of approximately 100 amino acid residues with a character- istic pattern of eight cysteine residues forming four disulfide bonds [7]. Their structural fold allows the display of a large flat hydropho- bic patch by utilizing aliphatic side chains located near the loop regions of two adjoining hairpins [2]. Therefore one side of the pro- ∗ Corresponding author. Tel.: +86 22 23506173; fax: +86 22 23506122. E-mail address: qiangchen@nankai.edu.cn (Q. Chen). tein consists solely of hydrophobic aliphatic side chains that form a planar hydrophobic patch on the otherwise mostly hydrophilic pro- tein surface. With this unique amphiphatic structure, hydrophobins are able to change a hydrophobic surface into hydrophilic and vice versa [4]. Recently, hydrophobin modification on various kinds of sur- faces such as Teflon, Nafion, Lycra [6], glass [8], mica, PDMS [9], and glassy carbon electrode [10–14] has been reported, which has greatly changed the surface wettability. This remarkable surface activity of hydrophobins has implied great prospects in biofunc- tionalization of many hydrophobic surfaces. Since biomolecules are mostly hydrophilic, highly hydrophilic surfaces are required in their immobilization. The amphiphilic structure helps hydrophobins to easily self-assemble on relatively hydrophobic surfaces and improve the surface hydrophilicity, which would facilitate sub- sequent biomolecular immobilization. Furthermore, with high biocompatibility [15,16], hydrophobin matrix could well preserve the bioactivities of immobilized biomolecules and may serve as a suitable biomoleculer immobilizing biomaterial. We are interested in immobilizing redox enzymes on gold sur- faces via hydrophobin self-assembled film. Redox enzymes catalyze the electron transfer from electron donors to electron acceptors, which can be quantitatively recorded by electrochemical measure- ments. A 7.5-kDa hydrophobin, HFBI from Trichoderma reesei, has been used in this work to immobilize choline oxidase (ChOx). An amperometric choline biosensor has been constructed based on 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.01.011