The miniaturization of laboratory processes offers substantial advantages over traditional techniques in terms of cost, speed, and potential for multistage automation. To date, only a few studies have reported successful microfluidics-based immunoassays, most of which rely on fluorescence detection technologies. The goal of this study was to develop a poly(dimethylsiloxane) microfluidics-based immunoassay methodology and a versatile colorimetric quantification scheme for the detection of visual colour changes resulting from immune reactions in microchannels. The novel immunoassay technique was applied towards the detection of Helicobacter pylori infection using 20 human serum samples of known infection status, and results compared with conventional nitrocellulose membrane-based dot-ELISA. The microchannel immunoassay reliably detected H. pylori antigens in quantities on the order of 10 ng, which provides a sensitivity of detection comparable to conventional dot-blot assays. Sensitivity was 100%, specificity was 90%, positive predictive value 91%, and negative predictive value 100%, with an overall accuracy of 95%. The software developed generated results that were consistent with visual observations and by automatically taking into account background intensity changes, the software minimized subjectivity. Volumes of solutions used were 100-fold less compared with conventional immunoassays. Miniaturization of the ELISA using this technique provides a means for the accurate diagnosis of microbial infection while minimizing waste production.

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 ...