Biosensors and Bioelectronics 21 (2006) 1077–1085 Application of europium(III) chelate-dyed nanoparticle labels in a competitive atrazine fluoroimmunoassay on an ITO waveguide C.M. Cummins a , M.E. Koivunen b , A. Stephanian a , S.J. Gee b , B.D. Hammock b , I.M. Kennedy c,∗ a Department of Biomedical Engineering, University of California Davis, Davis, CA 95616, USA b Department of Entomology and the UCDavis Cancer Center, University of California Davis, Davis, CA 95616, USA c Department of Mechanical and Aeronautical Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA Received 22 January 2005; received in revised form 7 April 2005; accepted 8 April 2005 Available online 28 April 2005 Abstract We have demonstrated the use of an optical indium tin oxide (ITO) (quartz) waveguide as a new platform for immunosensors with fluorescent europium(III) chelate nanoparticle labels (Seradyn) in a competitive atrazine immunoassay. ITO as a solid surface facilitated the successful use of particulate labels in a competitive assay format. The limit of detection in the new nanoparticle assay was similar to a conventional ELISA. The effect of particle size on bioconjugate binding kinetics was studied using three sizes of bioconjugated particle labels (107, 304, and 396 nm) and a rabbit IgG/anti-IgG system in a 96-well plate. A decrease in particle size resulted in faster binding but did not increase the assay sensitivity. Flux calculations based on the particle diffusivity prove that faster binding of the small particles in this study was primarily due to diffusion kinetics and not necessarily to a higher density of antibodies on the particle surface. The results suggest that ITO could make a good platform for an optical immunosensor using fluorescent nanoparticle labels in a competitive assay format for small molecule detection. However, when used in combination with fluorescent particulate labels, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size. Different regeneration methods tested in this study showed that repeated washings with 0.1 M glycine–HCl facilitated the reuse of the ITO waveguide. © 2005 Elsevier B.V. All rights reserved. Keywords: ITO; Immunoassay; Nanoparticles; Lanthanide; Fluorescence; Atrazine 1. Introduction Immunoassays using antibody/antigen recognition for the detection and quantification of target analytes are applied in both clinical and environmental monitoring, in which her- bicides and their metabolites are important target analytes. Compounds such as atrazine or atrazine mercapturate can be detected using an enzyme-linked immunosorbent assay (ELISA) or other immunochemical methods (Karu et al., 1991; Wortberg et al., 1995; Jaeger et al., 1998; Reimer et al., 1998). Atrazine as a member of the triazine herbicide family is of interest because it is one of the most widely used ∗ Corresponding author. Tel.: +1 530 752 2796; fax: +1 530 210 8220. E-mail address: imkennedy@ucdavis.edu (I.M. Kennedy). herbicides in the US, and it has been shown to be present in groundwater (Cohen et al., 1986). More importantly, atrazine is classified as a possible human carcinogen by the US En- vironmental Protection Agency, and it is a known endocrinic disruptor posing a potential health risk to humans (Yu, 2005) as well as to wildlife (Renner, 2002). Current immunochem- ical methods have been able to detect atrazine at a concen- tration of 0.1–0.2 ng mL -1 (Wortberg et al., 1995; Ciumasu et al., 2005). Recently, ELISAs have been replaced by more sensitive and faster fluoroimmunoassays (Hall et al., 1999; Schobel et al., 2000). Quantum dots (Chan et al., 2002; Goldman et al., 2002), lanthanide oxide (Feng et al., 2003), and sil- ica nanoparticles doped with lanthanide chelates (Hai et al., 2004; Ye et al., 2004, 2005) or organic dyes (Yang et al., 0956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2005.04.003