Electrochemical Immunosensing Chip Using Selective Surface Modification, Capillary-Driven Microfluidic Control, and Signal Amplification by Redox Cycling Byung-Kwon Kim, a Sang-Youn Yang, a Md. Abdul Aziz, a Kyungmin Jo, a Daekyung Sung, b Sangyong Jon, b Han Young Woo, c Haesik Yang* a a Department of Chemistry, Pusan National University, Busan 609-735, Korea tel: + 82 51 510 3681; fax: + 82 51 516 7421. : b Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea c Department of Nanofusion Technology, Pusan National University, Miryang 627-706, Korea *e-mail: hyang@pusan.ac.kr Received: February 23, 2010; & Accepted: April 2, 2010 Abstract A sensitive electrochemical immunosensing chip is presented by employing (i) selective modification of protein-re- sistant surfaces; (ii) fabrication of a stable Ag/AgCl reference electrode; (iii) capillary-driven microfluidic control; (iv) signal amplification by redox cycling along with enzymatic reaction. Purely capillary-driven microfluidic control is combined with electrochemical sandwich-type immunosensing procedure. Selective modification of the surfaces is achieved by chemical reactivity-controlled patterning and electrochemical deposition. Fluidic control of the immu- nosensing chip is achieved by spontaneous capillary-driven flows and passive washing. The detection limit for mouse IgG in the immunosensing chip is 10 pg/mL. Keywords: Immunosensor, Passive washing, Microfluidic control, Nonspecific binding, Ag/AgCl reference electrode DOI: 10.1002/elan.201000148 1. Introduction Electrochemical detection is considered as one of the major sensing methods in microchip-based biosensors as it facilitates a miniature sensing system [1–6]. Although many different types of electrochemical biosensors that employ large electrodes have been developed, their prac- tical application in microchips remains challenging [1, 5– 7]. The main reason for this is that the sensitivity and re- producibility obtained with biosensors that use large elec- trodes cannot be readily achieved with microchip-based biosensors that use micropatterned electrodes. The poor sensitivity and reproducibility associated with microchip- based biosensors is largely due to (i) high nonspecific binding of biomolecules to microchamber and microchan- nel walls; (ii) low stability of microfabricated reference electrodes; (iii) complex microfluidic control required during the detection process [1, 4, 6, 8, 9] . Essentially, microchip-based electrochemical biosensors require at least four different surfaces (working, refer- ence, counter electrode, insulating surface between elec- trodes) that should be selectively modified and should share high protein-resistant properties. Therefore, a good combination of different protein-resistant surfaces and their sequential selective modification are required for microchip-based electrochemical biosensors. Generally, for selective surface modification, photolithographic pat- terning [10], electrochemical deposition [11], and chemi- cal reactivity-controlled patterning [12] have been used. Photolithographic patterning and electrochemical deposi- tion are useful in obtaining metal micropatterns, although neither are effective for selective immobilization of (bio)- molecules. However, chemical reactivity-controlled pat- terning that takes advantage of selective chemical reactiv- ity between a (bio)molecule and a solid surface is useful for selective immobilization of the (bio)molecule [12]. Se- lective chemical reactivity includes biospecific binding, such as biotin-avidin binding [13, 14], and selective chemi- cal reactions, such as selective reaction of an organo- phosphonate to a metal oxide surface over silicon oxide surface [15, 16]. For preparation of protein-resistant surfaces, covalent modification and adsorption-based modifications have been used. Surface modification using a poly(ethylene glycol) (PEG)-containing molecule or polymer is the most common approach towards covalent modification [13, 17, 18]. Blocking agents such as bovine serum albu- min, Tween 20, and skim milk are commonly used for ad- sorption-based modification [19–21]. Importantly, cova- lent modifications can be combined with chemical reac- tivity-controlled patterning. Electroanalysis 2010, 22, No. 19, 2235 – 2244  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2235 Full Paper