The light-scattering properties of submicroscopic metal particles ranging from 40 to 120 nm in diameter have recently been investigated. These particles scatter incident white light to generate monochromatic light, which can be seen either by the naked eye or by dark-field microscopy. The nanoparticles are well suited for detection in microchannel-based immunoassays. The goal of the present study was to detect Helicobacter pyloriand Escherichia coli O157:H7-specific antigens with biotinylated polyclonal antibodies. Gold particles (diameter, 80 nm) functionalized with a secondary antibiotin antibody were then used as the readout. A dark-field stereomicroscope was used for particle visualization in poly(dimethylsiloxane) microchannels. A colorimetric quantification scheme was developed for the detection of the visual color changes resulting from immune reactions in the microchannels. The microchannel immunoassays reliably detected H. pylori and E. coli O157:H7 antigens in quantities on the order of 10 ng, which provides a sensitivity of detection comparable to those of conventional dot blot assays. In addition, the nanoparticles within the microchannels can be stored for at least 8 months without a loss of signal intensity. This strategy provides a means for the detection of nanoparticles in microchannels without the use of sophisticated equipment. In addition, the approach has the potential for use for further miniaturization of immunoassays and can be used for long-term archiving of immunoassays.Immunoassays are based on specific antibody-antigen reactions (26). Quantification of immunoassays is generally achieved by measuring the specific activity of a label, for example, radioactivity, fluorescence, chemiluminescence, bioluminescence, or electrical conductivity (20,21,26). However, these labels share a common drawback, which is that they are not suitable for long-term preservation (26). While different isotopes have various half-lives, the use of radioactivity is more difficult because of issues of disposition and potential harmful health effects. Furthermore, fluorescence suffers from the issue of photobleaching (15).Recently, nanoparticles, especially gold and silver particles, have been successfully applied for labeling because of their easily controlled size distribution, long-term stability, and compatibility with biological macromolecules, including proteins and nucleic acids (4, 11). Multiple nanoparticle detection methods have been developed, including scanning and transmission electron microscopy (12, 25), Raman spectroscopy (6, 9, 27), and the naked eye (16,24). Detection by the naked eye may be preferable, as other techniques are expensive and require specialized equipment and additional preparations. Both a DNA microarray and a heterogeneous immunoassay have been developed with 10-nm gold particles amplified with silver, which can be detected by the naked eye (16,24).Most recently, the light-scattering properties of submicroscopic metal particles, such as gold nanoparticles, have been investigated ...