Nanoscale fluoro-immuno assays with lanthanide oxide nanoparticles I.M. Kennedy *a , M. Koivunen b , S. Gee b , C. Cummins a , R. Perron a , D. Dosev a , B.D. Hammock b a Department of Mechanical and Aeronautical Engineering, b Department of Entomology University of California Davis, Davis CA USA 95616 ABSTRACT The use of polystyrene nanoparticles with europium chelate has been demonstrated as fluorescent reporters in an immunoassay for atrazine. The limit of detection with the nanoparticles was similar to that achieved with a conventional ELISA. It was shown that as the particle size decreased the time required for binding decreased and the sensitivity of the assay increased. This suggests that the use of smaller particles would greatly speed up the reaction and simultaneously increase sensitivity. However, the detection system used sets limits to the particle size as well. There is clearly a point where our detection system would not be sensitive enough to detect the emission from small particles. Therefore, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size. Keywords: immunoassay, nanoparticles, lanthanide, fluorescence, atrazine 1. INTRODUCTION Immunoassays are biological assays, which use antibody/antigen recognition to detect and quantify target analytes. Immunoassays are used for both clinical and environmental monitoring. Enzyme-linked immunosorbent assay (ELISA) are competitive heterogeneous assays, which can be performed immobilizing one of the reagents on a variety of solid supports (tubes, microtiter plates, plastic-baked nitrocellulose membranes, magnetic particles, etc.). Measurements are based on light absorption proportional to color developed during the immuno reaction. Even though ELISAs are widely used, applications with fluorescent markers can increase sensitivity. Microchannels, or lab-on-a-chip, have become a major thrust of biosensor research over the past decade owing to the use of smaller samples volumes and shorter assay times. Biological assays in microchannels have shown an increase in the assay sensitivity and a decrease in the assay time 1-8 . The two major ways to construct microchannels are in a fully pre- sealed or detachable format. A pre-sealed channel is constructed using a photoepoxy such as Su8 9 and all of the immunoassay steps are performed after the channel is constructed. An immunoassay using this type of channel has not been shown to be effective until recently 10 . The detachable channel uses poly(dimethylsiloxane) (PDMS) and is placed on top of a glass surface to create a sealable channel. This creates a channel where surface treatments to attach antibodies or antigens can be done prior to channel formation 11 . More research needs to be done to optimize these biosensors for more specific applications. However, as science progresses future biological detection devices will use more microchannels to move the lab to a more mobile environment. New labels can be useful in improving the sensitivity of fluoroimmunoassays. Polystyrene particles dyed with europium (III) chelates and europium (III) oxide nanoparticles have been shown to be effective fluorophores for time resolved fluorescence measurements in biological assays 12-21 . Europium(III) chelates – dyed polystyrene particles have major excitation peak at around 333 nm and fluorescence peak at 613 nm with a half life of 0.5 ms (Figure 1). Similarly, europium(III)oxide has excitation peaks at 280 nm, 394 nm and 466 nm with emission at 615 nm (Figure 2) and similar fluorescence lifetime. In contrast, most of the widely used organic fluophores have fluorescence lifetime in the pico- to nanosecond range. Thus, europium compounds are excellent markers for biological assays in which a clear differentiation between the signal and background is needed for increased sensitivity and detection. Recently, * imkennedy@ucdavis.edu ; Phone 530 752 2796; FAX 503 210 8220 Invited Paper Nanosensing: Materials and Devices, edited by M. Saif Islam, Achyut K. Dutta, Proceedings of SPIE Vol. 5593 (SPIE, Bellingham, WA, 2004) 0277-786X/04/$15 · doi: 10.1117/12.571483 329