No abstract

In this report we examine the limitations of existing microarray immunoassays and investigate how best to optimize them using theoretical and experimental approaches. Derived from DNA technology, microarray immunoassays present a major technological challenge with much greater physicochemical complexity. A key physicochemical limitation of the current generation of microarray immunoassays is a strong dependence of antibody microspot kinetics on the mass flux to the spot as was reported by us previously. In this report we analyze, theoretically and experimentally, the effects of microarray design parameters (incubation vessel geometry, incubation time, stirring, spot size, antibody-binding site density, etc.) on microspot reaction kinetics and sensitivity. Using a two-compartment model, the quantitative descriptors of the microspot reaction were determined for different incubation and microarray design conditions. This analysis revealed profound mass transport limitations in the observed kinetics, which may be slowed down as much as hundreds of times compared with the solution kinetics. The data obtained were considered with relevance to microspot assay diffusional and adsorptive processes, enabling us to validate some of the underlying principles of the antibody microspot reaction mechanism and provide guidelines for optimal microspot immunoassay design. For an assay optimized to maximize the reaction velocity on a spot, we demonstrate sensitivities in the aM and low fM ranges for a system containing a representative sample of antigen-antibody pairs. In addition, a separate panel of low abundance cytokines in blood plasma was detected with remarkably high signal-to-noise ratios. Molecular

Although they are superficially similar to DNA microarrays, immunoassay microarrays represent a daunting technological challenge owing to the much wider diversity of proteins. Yet, as the leading edge of bioscience migrates from genomics to proteomics, the complexity and enormous dynamic range of proteins in a cell necessitate an analytic tool with exceptional specificity and sensitivity. In theory, microspot immunoassays could fulfill this need. However, antibody microarrays have had limited success to date, and have often required a highly sensitive detection system and/or sophisticated immobilization approach to be of any use for the profiling of complex specimens. There is a solid body of work on the theory of microspot reaction kinetics, yet much of the published experimental work on protein microarray development pays insufficient attention to the kinetic aspects of this interaction. This review explains that one of the main limitations for the sensitivity of current generation microspot immunoassays is the strong dependence of antibody microspot kinetics upon mass flux to the spot. This not only involves migration of analyte in solution, but also across the surface of the solid phase. Understanding of this effect will be discussed, along with several related effects and their significance to improving existing microarray designs. It is concluded that current efforts may be too focused on areas that cannot improve performance significantly, and that other critical areas of design should receive more attention. Finally, the review addresses the question of whether ambient analyte immunoassay is truly a separate category of microspot assay, with the conclusion that this may be a flawed concept.

No abstract

No abstract