Antibody microarrays: the crucial impact of mass transport on assay kinetics and sensitivity

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.