| For medical diagnostic applications, immunoassays provide a relatively simple, quick, and reliable method to measure analytes. Immunoassays identify and quantify substances by harnessing the sensitivity and specificity of nature's antibody-antigen detection system. Since clinically relevant analytes tend to have lower concentrations in physiological fluids other than blood, sensitive immunoassays with low detection limits would facilitate safer and painless noninvasive measurements of these analytes. Practical limitations to immunoassays, such as autofluorescence and nonspecific binding, often confine detection limits to higher levels, and functional immunoassays with extremely low detection limits are rare, particularly ones with rapid turnaround times.; To address these issues, an engineering approach to immunoassay development, as opposed to the typical empirical one, was used to characterize and optimize an immunoassay that utilized up-converting phosphor (UCP) technology. With this immunoassay development platform, which featured 2-D imaging and quantification of UCP labels in immunoassays, as well as temperature control, a microfluidic immunoassay for interferon-gamma (IFN-gamma) was developed to demonstrate a UCP-based immunoassay for cytokines (regulatory proteins involved in the body's immune response) for potential use as an early indicator of infection and stress. Image processing techniques were employed to extract the signals from the 2-D images, which were proportional to the IFN-gamma concentrations present in the samples. Concentrations as low as 3 pM (50 pg/mL) and as high as 600 pM (10 ng/mL) were detected from 100-muL samples with a total assay time of under an hour, including the 8-minute readout time.; To enhance immunoassay sensitivity, a novel mathematical model for immunoassays that incorporates the effects of both temperature and nonspecific binding was used to predict the kinetic behavior of the immunoassay based on reagent and substrate properties. Temperature-controlled immunoassays experimentally verified that temperature modulation could be employed to significantly reduce nonspecific binding levels by over 50%. This model used in conjunction with the immunoassay development platform provides a valuable tool to speed development, reduce errors, and potentially improve the accuracy and sensitivity of immunoassays, whether they are applied to biological research or medical diagnostics. |
