Enhanced Sensitivity of a Surface-plasmon-resonance (SPR) Sensor for 2,4-D by Controlled Functionalization of Self-assembled Monolayer-based Immunosensor Chip Sook Jin Kim, 1 K. Vengatajalabathy Gobi, 2 Hiroyuki Tanaka, 3 Yukihiro Shoyama, 3 and Norio Miura Ã1;2 1 Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Kasuga, Fukuoka 816-8580 2 Art, Science and Technology Center for Cooperative Research, Kyushu University, Kasuga, Fukuoka 816-8580 3 Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8582 (Received July 18, 2006; CL-060803; E-mail: miura@astec.kyushu-u.ac.jp) A highly sensitive SPR immunosensor has been fabricated by covalent binding of a protein conjugate of 2,4-D (2,4-D- BSA) on SAM of 3-mercaptopropionic acid. With indirect inhib- ition immunoassay, it demonstrated a low detection limit of 0.1 ppb 2,4-D. Taking advantage of the combination of the SAM technique and immobilization of 2,4-D-BSA at controlled surface coverage, a remarkable enhancement of the low detec- tion limit by 10-fold to 10 ppt with no use of any labels was dem- onstrated, while keeping the response time constant at 4 min. Public concern on protection of the environment and the quality of food products has been ever more increasing, because of widespread use of various toxic chemicals and of the increase in the number of environmental pollutants. 2,4-Dichlorophen- oxyacetic acid (2,4-D) is a systemic herbicide and has been as- sociated with potential endocrine-disrupting activities. 1 Primary screening methods for 2,4-D detection should be inexpensive and usable for automated analysis of a large number of food and potable water samples. Immunosensors based on specific antigen–antibody interaction are immensely explored for direct determination from complex sample matrices. SPR immunosensor systems have received wide acknow- ledgement for applications into various fields, because of their inherent advantages over other type of biosensors in its versatil- ity and compatibility with miniaturization, high throughput analysis and label-free detection. 2,3 Integration of biological receptors with physicochemical transducers plays a very signifi- cant role in the fabrication of biosensors, where the reactivity of bioreceptor should not be affected while maintaining high resistivity to irrelevant sample matrices. Various methods em- ployed in the fabrication of functional sensing surfaces include physisorption, self-assembly method, binding of complementary ssDNA units, etc. Physisorption of proteins had been primarily investigated because of its advantages such as simplicity, applicable to various biomolecules, etc. We had fabricated SPR immunosensors with the use of simple physical adsorption of a protein–analyte conjugate, and reliable highly sensitive immunosensor systems had been fabricated for various low- molecular-weight analytes (MW < 500 Da) of biological and environmental interest. 4–6 Another well-investigated approach is the binding of target molecule directly on SAM surfaces, where interference due to nonspecific adsorption was detrimen- tal to sensor performance. 7 In this communication, we demonstrate the development of a novel SAM based SPR immunosensor chip for 2,4-D detection, where a combination of stable SAM structure using low-cost commercially available materials and 2,4-D-BSA conjugate used for antibody production has been applied. An on-line in- direct competitive immunoreaction principle has been investiga- ted, in which the immunoreactivity between 2,4-D and its anti- body is modulated by controlled surface modification procedures to enhance the sensitivity of the SPR immunosensor. SPR experiments were performed using SPR-670 analyzer from NLE, Japan. Au-sputtered glass plates with an inner Cr la- yer (5-nm Cr layer and 50-nm-Au layer) performed as SPR ac- tive sensing platforms. Initially, the Au surface of the SPR sen- sor chip was exposed to the flow of ethanol to cleanse the sensor surface. After a stable baseline with PBS (pH 7.2), the thin-film Au chip was exposed to the flow of 10 mM MPA in PBS for 20 min. A stable increase in SPR angle was observed indicating irreversible binding of MPA. Carboxyl end groups of the resul- tant MPA layer were activated for amide bonding by the flow of a mixture of EDC and NHS and were covalently bound to 2,4-D-BSA conjugate. The sensor response observed for the covalent binding process (Figure 1) showed a gradual increase in SPR angle indicating the uniform binding of the conjugate on MPA monolayer. After the immobilization of 2,4-D-BSA, a high concentration of BSA (1000 ppm) was let to flow to block any free active sites available on the sensor chip. The fabricated sensor chip exhibits a highly specific and sensitive immunoreactivity towards the monoclonal anti-2,4-D antibody (2,4-D-mAb). Flow of 2,4-D-mAb at 15 ppm (mg mL À1 ) showed a smooth and stable increase in SPR angle by ca. 0.14 deg. (Figure 2a), indicating the binding of 2,4-D- mAb by specific immunoaffinity reaction. Dissociation of the antigen–antibody complex without affecting the beneath 2,4- D-BSA conjugate had been investigated. The antibody bound sensor surface was treated with an acidic glycine–HCl buffer (pH 2.0) for 30 s, and the resonance angle of the sensor chip was found to decrease to the original level, indicating complete removal of the antibody from the sensor surface and the stability 15 min 0.1 deg. EDC + NHS 25 μg mL -1 2,4-D-BSA 1000 μg mL -1 BSA SAM of MPA PBS PBS PBS PBS 0.18 deg. Figure 1. Activation of the SAM of MPA by a mixture of EDC and NHS (2 mg mL À1 each) and followed covalent binding of 2,4-D-BSA on sensor surface. 1132 Chemistry Letters Vol.35, No.10 (2006) Copyright Ó 2006 The Chemical Society of Japan