Preparation and characterization of superporous agarose–reticulated vitreous carbon electrodes as platforms for electrochemical bioassays

Three-dimensional flow-through electrodes were fabricated using superporous agarose (SPA) and reticulated vitreous carbon (RVC) composite materials that were suitable as a platform for sandwich assays. These SPA–RVC composite electrodes were fabricated by fitting a SPA–RVC composite cylinder inside a graphite tube and subsequently fixing the graphite tube onto a polypropylene micropipette tip. The electrode design allows for ease in reagent/washing steps involved in sandwich assay protocols and could easily be made portable. The electrode materials were characterized with respect to pore-size distribution, total free volume, ligament and bulk densities of the RVC, and physical structural characteristics. Coulometric detection of redox molecules such as K 3Fe(CN) 6 and 4-aminophenol was possible using SPA–RVC electrodes by the trapping of these redox molecules inside the SPA–RVC electrodes. Avidin affinity molecules were covalently immobilized onto the SPA matrix inside the RVC electrodes by periodate-activation followed by reductive amination. The amount of avidin immobilized inside the SPA–RVC electrodes was (5 ± 0.06) × 10 −11 mol, which was determined by saturating the avidin sites with biotinylated fluorescein (b-fluo) and subsequently determining the amount of immobilized b-fluo via a standard addition method using fluorescence spectroscopy. Non-specific binding of labeled enzymes such as biotinylated alkaline phosphatase (b-ALP) onto the SPA–RVC electrodes without avidin capture sites was determined to be less than 1% compared to the specific binding of b-ALP on avidinylated SPA–RVC electrodes.