PENTEL actin-G immunoelectrode: Immunoassay at the tip of a pencil

Here we present design and assay methodology of an actin-G immunoelectrode for the quantitation of muscle protein, actin. The immunoelectrode was assembled using a proprietary pencil material composed of a polymer-resin and graphite composite as an antibody support and electronic conductor. An effective immobilisation procedure for the capture antibody and surface blocking protein is cited based on a two-step physisorption chemistry. BSA coverage was demonstrated to reduce non-specific interactions to below 8% of the base polymer-graphite binding capacity. Immunoassays performed with the actin immunoelectrode utilise an o-benzoquinone-diimine electrochemistry which is formed in-situ by interfacial peroxidase oxidation. Cyclic voltammetry data revealed the immunoelectrode surface to be porous to the diimine signalling molecule, while remaining inaccessible to macromolecules of the ELISA. The molecular structure and poly-functional nature of the immunoelectrode was further investigated using an anti-antibody peroxidase conjugate. The findings suggest that capture antibody is randomly dispersed over the surface, exposing both Fc and actin binding sites. A saturation current density - concentration curve was analysed by Langmuir adsorption theory and an estimate for the antibody binding affinity derived (K_a≈1×10⁵M^-1). The actin immunoelectrode was applied as a peroxidase detector in a double-antibody immunoassay. The electrode shows a dose-response characteristic which is most sensitive to actin over the concentration range 1-100ngmL^-1. Immunoelectrode sensitivity for actin is of the order of 5nAmm^-2/ngmL^-1 (10-100ngmL^-1) with a limit of detection estimated at 10ngmL^-1 and measurement precision RSD 7.1% (100ngmL^-1 actin). At higher concentrations of actin, >100ngmL^-1 the immunoelectrode response is more complex. A high-dose hook characteristic becomes evident which arises from macromolecular steric interactions and other binding site limitations at the electrode surface.