Multiple Label-Free Detection of Antigen-Antibody Reaction Using Localized Surface Plasmon Resonance-Based Core-Shell Structured Nanoparticle Layer Nanochip Tatsuro Endo, † Kagan Kerman, ‡ Naoki Nagatani, ⊥ Ha Minh Hiepa, ‡ Do-Kyun Kim, ‡ Yuji Yonezawa, § Koichi Nakano, § and Eiichi Tamiya* ,‡ Department of Mechano-Micro Engineering, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8502, Japan, School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST) 1-1, Asahidai, Nomi City, Ishikawa, 923-1292, Japan, Industrial Research Institute of Ishikawa, 2-1 Kuratsuki, Kanazawa City, Ishikawa 920-8203, Japan, and Department of Biotechnology and Applied Chemistry, Faculty of Engineering, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan In this research, a localized surface plasmon resonance (LSPR)-based bioanalysis method for developing multi- array optical nanochip suitable for screening bimolecular interactions is described. LSPR-based label-free monitor- ing enables to solve the problems of conventional methods that require large sample volumes and time-consuming labeling procedures. We developed a multiarray LSPR- based nanochip for the label-free detection of proteins. The multiarray format was constructed by a core-shell- structured nanoparticle layer, which provided 300 nano- spots on the sensing surface. Antibodies were immobi- lized onto the nanospots using their interaction with Protein A. The concentrations of antigens were deter- mined from the peak absorption intensity of the LSPR spectra. We demonstrated the capability of the array measurement using immunoglobulins (IgA, IgD, IgG, IgM), C-reactive protein, and fibrinogen. The detection limit of our label-free method was 100 pg/mL. Our nanochip is readily transferable to monitor the interac- tions of other biomolecules, such as whole cells or receptors, with a massively parallel detection capability in a highly miniaturized package. We anticipate that the direct label-free optical immunoassay of proteins reported here will revolutionize clinical diagnosis and accelerate the development of hand-held and user-friendly point-of- care devices. The post-genome era of life sciences is rapidly moving beyond functional genomics to proteomics. 1 Since genome sequencing has evolved to become a routine technology, many efforts have been in progress to improve the proteomics technologies. 2 As an emerging field in life sciences, proteomics is not only based on, but also being developed beyond, genomics. However, a large- scale, rapid, and ultrasensitive assay system is highly desired. The large-scale and partly high-throughput characterization of the human proteome has become possible with the sophisticated biochemical techniques. Although enzyme-linked immunosorbent assay and two-dimensional gel electrophoresis are currently the most widely used bioanalysis tools for monitoring protein-protein interactions, they bring along disadvantages with regard to throughput, reproducibility, and sensitivity. 3 Surface plasmon resonance (SPR) is a strong alternative to the existing technologies for monitoring the biomolecular interactions. Unfortunately, conventional SPR reflectometry requires sophisticated optical instrumentation associated with the detection system. This latter limitation is significant, because biochips are urgently in demand for high-throughput and cost-effective monitoring. To overcome these disadvantages of conventional detection systems, biochip assay systems employing micro and nano electromechanical systems (MEMS and NEMS) have been developed. 4 A highly developing trend in the application of MEMS and NEMS tech- nologies in life sciences and biotechnology is directed toward miniaturization and multidetection. These chip technologies have several advantages over the conventional bioanalysis systems. First, the chip-based assays enable rapid analysis of a large number of samples in a single experiment. Second, the amount of material required is significantly small. Reaction volumes are lower than the amount that is typically used in conventional microtiter plates. Third, the signal-to-noise ratio exhibited by micro or nano- fabricated biochips is much better than that observed for conventional microtiter plate assay systems. 5,6 In addition to the advantages, such as the reduced reagent consumption and the laboratory space conservation, lab-on-a-chip technology offers new prospects for laboratory innovation and automation. Biochips * Corresponding author. E-mail: tamiya@jaist.ac.jp. † Tokyo Institute of Technology. ‡ JAIST. § Industrial Research Institute of Ishikawa. ⊥ Okayama University of Science. (1) Drewes, G.; Bouwmeester, T. Curr. Opin. Cell Biol. 2003, 15, 199-205. (2) Timperman, A. T.; Aebersold, R. Anal. Chem. 2000, 72, 4115-4121. (3) Petach, H.; Gold, L. Curr. Opin. Biotechnol. 2002, 13, 309-314. (4) Talapatra, A.; Rouse, R.; Hardiman, G. Pharmacogenomics 2002, 3, 527- 536. (5) Workman, J., Jr.; Koch, M.; Veltkamp, D. Anal. Chem. 2005, 77, 3789- 3806. (6) Gardeniers, H.; Van Den Berg, A. Int. J. Environ. Anal. Chem. 2004, 84, 809-819. Anal. Chem. 2006, 78, 6465-6475 10.1021/ac0608321 CCC: $33.50 © 2006 American Chemical Society Analytical Chemistry, Vol. 78, No. 18, September 15, 2006 6465 Published on Web 07/22/2006