title: Square-wave voltammetry assays for glycoproteins on nanoporous gold.
abstract: Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A - ALP (or soybean agglutinin - ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A-ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL-1BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay.
authors.affiliation: Department of Chemistry and Biochemistry University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121 ; Center for Nanoscience University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121.
authors.forename: Binod
authors.initials: B
authors.lastname: Pandey
authors.affiliation: Department of Chemistry and Biochemistry University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121 ; Center for Nanoscience University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121.
authors.forename: Jay K
authors.initials: JK
authors.lastname: Bhattarai
authors.affiliation: Department of Chemistry and Biochemistry University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121.
authors.forename: Papapida
authors.initials: P
authors.lastname: Pornsuriyasak
authors.affiliation: Department of Chemistry and Biochemistry University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121.
authors.forename: Kohki
authors.initials: K
authors.lastname: Fujikawa
authors.affiliation: Department of Chemistry and Biochemistry University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121.
authors.forename: Rosa
authors.initials: R
authors.lastname: Catania
authors.affiliation: Department of Chemistry and Biochemistry University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121.
authors.forename: Alexei V
authors.initials: AV
authors.lastname: Demchenko
authors.affiliation: Department of Chemistry and Biochemistry University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121 ; Center for Nanoscience University of Missouri-St. Louis One University Boulevard Saint Louis, MO 63121.
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Stine
keywords: Enzyme-linked lectin sorbent assay; glycoprotein; lectin; nanoporous gold; self-assembled monolayer; square wave voltammetry