Detection of Light Scattering for Lab-On-A-Chip Immunoassays Using Optical Fibers

Abstract

This dissertation develops technology for microfluidic point-of-care immunoassay devices. This research (2004–2007) improved microfluidic immunoassay performance by reducing reagent consumption, decreasing analysis time, increasing sensitivity, and integrating processes using a lab-on-a-chip. Estimates show that typical hospital laboratories can save $1.0 million per year by using microfluidic chips. Our first objective was to enhance mixing in a microfluidic channel, which had been one of the main barriers to using these devices. Another goal of our studies was to simplify immunoassays by eliminating surfactants. Manufacturers of latex immunoassays add surfactants to prevent non-specific aggregation of microspheres. However, these same surfactants can cause false positives (and negatives) during diagnostic testing. This work, published in Appendix A (© 2006 Elsevier) shows that highly carboxylated polystyrene (HCPS) microspheres can replace surfactants and induce rapid mixing via diffusion in microfluidic devices. Our second objective was to develop a microfluidic device using fiber optics to detect static light scattering (SLS) of microspheres in Appendix B (© 2007 Elsevier). Fiber optics were used to deliver light emitting diode (LED) or laser light. A miniature spectrometer was used to measure 45° forward light scattering collected by optical fiber. Latex microspheres coated with PR3 proteins were used to test for the vasculitis marker, anti-PR3. No false negatives or positives were observed. A limit of detection (LOD) of 50 ng mL⁻¹ was demonstrated. This optical detection system works without fluorescence or chemiluminescence markers. It is cost effective, small, and re-usable with simple rinsing. The final objective in this dissertation, published in Appendix C (© 2007 Elsevier), developed a multiplex immunoassay. A lab-on-a-chip was used to detect multiple antibodies using microsphere light scattering and quantum dot (QD) emission. We conjugated QDs onto microspheres and named this configuration “nano-on-micro” or “NOM”. Upon radiation with UV light, strong light scattering is observed. Since QDs also provide fluorescent emission, we are able to use increased light scattering for detecting antigen-antibody reactions, and decreased QD emission to identify which antibody is present.