Studies in Developing Electrochemical Immunoassay Microarrays
by Fritsch, Ingrid; Williams, Caitlin R.
The current project addresses different steps necessary to miniaturize an immunoassay in an array format, where primary antibodies are patterned on a single substrate with a spotting arrayer. The sandwich immunoassay is an anal. method that employs specific antibody-antigen interactions to detect a target analyte, creating a versatile system that is well-suited for small-vols. We use a heterogeneous, sandwich-type, mouse IgG immunoassay as a model system. The primary antibody is immobilized on a u-functionalized alkylthiol self-assembled monolayer (SAM) on gold. It is this step that is most affected by the conditions used during arraying, which forms small spots of primary antibodies that covalently couple to the SAM. The assay is completed with the sample contg. antigen (mouse IgG), followed by a secondary antibody-alk. phosphatase (AP) conjugate. The AP converts p-aminophenylphosphate to the electrochem.-detectable p-aminophenol (PAP). We will present recent results involving a variety of methods that follow the assay assembly and det. the effects of the spotting conditions (e.g. exposure to air and drying) on assay performance. Characterization methods include electrochem. detection of PAP from complete and active assays, scanning electrochem. microscopy, electrochem. capacitance and permeation studies, mass spectrometry, and surface plasmon resonance. One issue we are focused on is that although SAMs are well-ordered and easy to form as a basis for attachment, organothiols on gold can air-oxidize. Another is that during the spotting process, the biochem. components of the assay dry and must be rehydrated, which could also change binding and enzyme activity.