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

Abstract

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 ...

Diagnostic microarray for 14 water and foodborne pathogens using a flatbed scanner

“…The array was washed with distilled water and dried. Signal preservation was achieved by quickly dipping the slide in archive solution (QIAGEN, Valencia, CA) as suggested by previous researchers (Lin et al, 2005; Reichert et al, 2000) and stored for scanning.…”

Rapid detection of pathogens using antibody-coated microbeads with bioluminescence in microfluidic chips

“…Other investigators have reported that the KPL goat anti-E. coli O157:H7 polyclonal antibody is highly specific for E. coli O157:H7. Its cross-reactivity to other bacteria genus such as S. enteritidis 4931, S. typhimirium 13311 (Moore and Griffith 2002;Su and Li 2004;Lee et al 2006), staphylococcal enterotoxin B (Yacoub-George et al 2007), L. monocytogenes (Shriver-Lake et al 2007) and other E. coli stains such as E. coli B, CDC, Crook, O111:B4, O26:B6, W (Yu and Bruno 1996), O55 (Subramanian et al 2006), K-12 (Yu and Bruno 1996;Shriver-Lake et al 2007), HB101, F18 and C25 (Lin et al 2005) are minimal.…”

“…In order to determine the relation between changes in output RLUs and the number of E. coli O157:H7 bacteria cells (Subramanian et al 2006;Lin et al 2005;Su and Li 2004), the bioluminescence was measured by photodiode and the results are presented in Fig. 5(a).…”

Fine Particles in Medicine and Pharmacy

“…Some of the recently published examples of therapies include applications of gold nanoparticles to detection and control of microorganisms, enhanced activity of antibiotics, and so on [415][416][417][418][419]. Dykman et al studied the capacity of colloidal gold for enhancing immune response in laboratory animals [420].…”