Square-wave voltammetry assays for glycoproteins on nanoporous gold

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• 作者：Binod Pandey^a ; ^b ; Jay K. Bhattarai^a ; ^b ; Papapida Pornsuriyasak^a ; Kohki Fujikawa^a ; Rosa Catania^a ; Alexei V. Demchenko^a ; Keith J. Stine^a ; ^b ; ^{kstine@umsl.edu" class="auth_mail}
• 关键词：Enzyme-linked lectin sorbent assay ; Nanoporous gold ; Square wave voltammetry ; Glycoprotein ; Lectin ; Self-assembled monolayer
• 刊名：Journal of Electroanalytical Chemistry
• 出版年：15 March, 2014
• 年：2014
• 卷：717-718
• 期：Complete
• 页码：47-60
• 全文大小：1611 K

文摘

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^鈭? BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl 伪-d-mannopyranoside for the binding of TSF and IgG to Con A-ALP; IC₅₀ values were found to be 90 渭M and 286 渭M, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay.