Development of a Nanoparticle-Labeled Microfluidic Immunoassay for Detection of Pathogenic Microorganisms

摘要

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 pylori- and 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.