Anal. Chem.

The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240 000 molecules) of analyte