Superporous agarose—Reticulated vitreous carbon electrodes for electrochemical sandwich bioassays

Abstract

We report on the use of flow-through electrodes fabricated from a composite of superporous agarose (SPA) and reticulated vitreous carbon (RVC) for carrying out sandwich bioassays via a model sandwich assay scheme. The flow-through design of the SPA–RVC electrodes allows for ease in solution handling with the use of micropipettors while allowing sandwich assays to be performed on the SPA matrix inside the RVC. A sandwich bioassay was devised for detecting biotinylated bovine serum albumin (b-BSA) as a proof-of-concept scheme to demonstrate applicability of SPA–RVC electrodes to carry out sandwich assays. In this bioassay scheme, SPA–RVC electrodes with avidin molecules immobilized on the SPA matrix were incubated with low quantities of b-BSA followed by incubation with avidinylated alkaline phosphatase (av-ALP). This construct creates a sandwich bioassay whereby b-BSA is sandwiched between the two avidin complexes. Av-ALP labels captured on the bound b-BSA catalytically hydrolyze conversion of 4-aminophenylphosphate (PAPP) to electrochemically active 4-aminophenol (PAP) which is then voltammetrically detected inside the RVC. The lower concentration detection limit for b-BSA was 0.32 ± 0.1 ng mL −1 and the absolute detection limit was 32 ± 10 pg. Non-specific binding of av-ALP enzyme labels onto the avidin-activated SPA–RVC electrodes was low. Catalytic generation of PAP by non-specifically bound av-ALP occurs at a rate less than 2% of that for PAP generation by av-ALP in [(SPA-av) − (b-BSA-b) − (av-ALP)] sandwich constructs.