Title: Fluorescence Analysis In Nanowells On Gold Surface Using Localized Surface Plasmon Field
Author: 石田昭人
Last saved by: James B. Lewis
Company: ______

Molecular Labeling of Gold Nanowells Towards Ultra-high Density “On Tip” Fluorescence Analysis  

Akito ISHIDA

PRESTO, Japan Science & Technology Corporation.
Department of Environmental Information, Kyoto Prefectural University, Shimogamo Hangi-cho 1-5, Sakyo-ku, Kyoto 606-8522, JAPAN.

Surface plasmon (SP) is the charge density wave on a gold or silver surface excited by visible and near-IR light.  The electromagnetic field is strongly localized on the surface and the propagation length is very long.  Moreover, the field intensity is remarkably enhanced compared with that of the incident light.  As an excitation source for photofunctional molecules attached on a gold or silver surface, these features offer distinct advantages over a conventional direct photoirradiation. [1]  Recently, it has been reported that the SP field distribution can be controlled by holes with a sub-micron diameter prepared on the gold surface and the SP field can be strongly localized at the rims. [2]  The regular array of such nano-sized gold holes prepared on a glass substrate may be utilized as vessels for fluorescence analysis.  The spot density will be dramatically increased compared with a conventional multi-well plate and a DNA chip.  In this study, selective molecular labeling of the “gold nanowells” and fluorescence imaging have been studied to realize the novel concept.  
The nano-holes on a 100 nm gold film have been prepared by projection method [2] using polystyrene spheres with diameters of 200, 400, 500 nm or 1000 nm.  After removal of the spheres, the gold surface and the bottom of the nanowells (i. e., exposed glass surface) were modified by a thiol derivative and an aminoalkylsilane derivative , respectively.  The amino terminated bottom of the nanowells were then modified by amine-reactive fluorescence probes such as BODIPY TMR-X SE (Molecular Probes, Inc.).  The fluorescence imaging has been carried out by a fluorescence microscope (OLYMPUS IX-70), a semiconductor laser (404, 532, or 635 nm), and a cooled-CCD camera (SPOT-JR).  Figure 1 shows fluorescence image of the BODIPY TMR modified nanowells by graze illumination of 404 nm.  The intense fluorescence demonstrates that the BODIPY molecules modified in the nanowells have been effectively excited.  Fluorescence detection of antigen-antibody interaction and hybridization of DNA in the nanowells will be discussed.  

References
1	A. Ishida, et al., Analyst, 535-540, 2000; Chem. Phys. Lett., 322, 242-246 (2000); Chem. Comm., 1299-1300, 1999; Nanotechnology, 10, 308-314 (1999) ; Chem. Commun., 57-58, 1998; Chem. Lett., 267-268, 1998.
2	C. Soennichsen, et al., App. Phys. Lett., 76, (1999) 140.