Biosensors and Bioelectronics 28 (2011) 139–145 Contents lists available at ScienceDirect Biosensors and Bioelectronics j our na l ho me page: www.elsevier.com/locate/bios Electrochemical endotoxin sensors based on TLR4/MD-2 complexes immobilized on gold electrodes Tae Yun Yeo 1 , Ji Suk Choi 1 , Byung Kook Lee, Beob Soo Kim, Hwa In Yoon, Hyeong Yun Lee, Yong Woo Cho ∗ Department of Chemical Engineering and Department of Bionanotechnology, Hanyang University, 1271 Sa-3 dong, Sangnok-gu, Ansan, Gyeonggi-do 426-791, Republic of Korea a r t i c l e i n f o Article history: Received 29 April 2011 Received in revised form 8 July 2011 Accepted 8 July 2011 Available online 18 July 2011 Keywords: Electrochemical sensor Endotoxin TLR4/MD-2 complex Gold electrode a b s t r a c t Even low concentrations of endotoxins can be life-threatening. As such, continuous effort has been directed toward the development of sensitive and specific endotoxin detection systems. In this paper, we report the design and fabrication of a new electrochemical endotoxin sensor based on a human recom- binant toll-like receptor 4 (rhTLR4) and myeloid differentiation-2 (MD-2) complex. The rhTLR4/MD-2 complex, which specifically binds to endotoxin, was immobilized on gold electrodes through a self- assembled monolayer (SAM) technique involving the use of dithiobis(succinimidyl undecanoate) (DSU). The surface topography of the electrodes at each fabrication stage was characterized with a nanosurface profiler and atomic force microscope (AFM). The electrochemical signals generated from interactions between the rhTLR4/MD-2 complex and the endotoxin were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A linear relationship between the peak current and endo- toxin concentration was obtained in the range of 0.0005 to 5 EU/mL with a correlation coefficient (R 2 ) of 0.978. The estimated limit of detection (LOD) was fairly low, 0.0002 EU/mL. The rhTLR4/MD-2 based sensors exhibited no current responses to dipalmitoylphosphatidylcholine (DPPC) bearing two lipid chains, which is structurally similar to endotoxin, indicating the high specificity of the sensors to endotoxin. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Lipopolysaccharide (LPS), a prototypical example of an endo- toxin, is composed of a polysaccharide and a lipid known as Lipid A. LPS is a major component of the outer membrane of Gram-negative bacteria and is responsible for toxic effects (Cohen, 2000; Raetz, 1990; Raetz and Whitfield, 2002). Endotoxins are highly resistant to heat and can cause severe patient reactions when they are present in pharmaceutical drugs or medical devices. Even low concentra- tions of endotoxin can cause fever, pyrogenicity, septic shock, and sepsis in mammalian. The detection and monitoring of endotoxin in human biological products and medical devices has a signifi- cant impact on public health (Danner et al., 1991; Glauser et al., 1991). The most widely used endotoxin detection method is a limulus amebocyte lysate (LAL) assay that includes gel-clot, turbidimet- ric, and chromogenic techniques. The LAL assay focuses on the ∗ Corresponding author. Tel.: +82 31 400 5279; fax: +82 31 696 5279. E-mail address: ywcho7@hanyang.ac.kr (Y.W. Cho). 1 These authors equally contributed to this work. observation of coagulation in the hemolymph of the horseshoe crab, Limulus polyphemus (Hurley, 1995; John et al., 2010; Martinho et al., 2011). While the LAL assay has been proven to be a reli- able method for endotoxin monitoring, its use is controversial due to a few limitations such as inherent turbidity, interfer- ence by the color of a sample, and low sensitivity. Furthermore, other microbial products such as peptidoglycan and -glucan react positively in an LAL assay (Dinarello, 1983; Gutsmann et al., 2010). Recently, alternative strategies have been extensively studied to decrease reliance on horseshoe crabs. For example, research has been conducted on optical sensors that use fluorescent dyes (Voss et al., 2007), cantilever sensors based on mass change (Ooe et al., 2007), magnetoelastic sensors based on frequency-amplitude responses (Ong et al., 2006), and endotoxin recognition systems that use endotoxin-specific substances such as endotoxin neu- tralizing proteins (Hoess et al., 1993), endotoxin binding proteins (Weiss et al., 1978), cationic antibacterial proteins (Larrick et al., 1995), and peptides (Ried et al., 1996). However, despite efforts to develop new biosensors, there are still many challenges that must be overcome to improve the sensitivity and specificity of endotoxin detection. 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.07.010