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(3)Fe(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)x10(-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.
Rao
Ashwin K
AK
Advanced Material Research Laboratories, 91 Technology Drive, Anderson, SC 29625, United States.
Creager
Stephen E
SE
D001360:Avidin / Q000737:chemistry; D001665:Binding Sites; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D002244:Carbon / Q000737:chemistry*; D004563:Electrochemistry; D004566:Electrodes; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D016062:Porosity; D012685:Sepharose / Q000737:chemistry*
D001360:Avidin; D001710:Biotin; D002244:Carbon; D012685:Sepharose