Electrochemical Aptasensor Employing Enzyme-Aptamer One-to-One Complex Showing Quasi-Direct Electron Transfer Ability

Abstract

1. Introduction In this study, we report an innovative electrochemical aptasensor based on the labeling aptamer with quasi-direct electron transfer (quasi-DET)-type redox-enzyme, by developing a breakthrough technological platform to prepare one-to-one complex of enzyme and aptamer.The chronoamperometric-based electrochemical aptasensor is an ideal sensor principle using aptamers for biomolecular recognition considering signal amplification by the labelled redox-enzyme. However, conventional methods to label aptamers with enzymes are chemical modification using cross-linking reagents, which often result in uncontrollable multimer-complexes. Therefore, the method to prepare enzyme-aptamer one-to-one complexes which yields in reproducible and designed enzyme-aptamer complex is technologically challenging. We here report the development of a novel technological platform to prepare enzyme-aptamer one-to-one complex, based on biomolecular engineering concept. 2. Methods Enzyme-aptamer one-to-one complex was constructed utilizing monomeric streptavidin (mSA) and biotin complex formation [1] [2]. We used FAD-dependent glucose dehydrogenase (GDH) from Aspergillus flavus as an aptamer labelling enzyme, and anti-vascular endothelial growth factor (VEGF) aptamer, as to proof of this conceptional study. VEGF is a biomarker for cancer diagnosis. We created the recombinant fusion protein of GDH and mSA (GDH-mSA). The combination of GDH-mSA with biotinylated anti-VEGF aptamer results in their one-to-one complex. To evaluate the formation of the complex and detect VEGF using GDH activity, we performed enzyme-linked aptamer assay (ELAA). Next, GDH-mSA was evaluated as a label enzyme for electrochemical aptasensor, by chronoamperometry analysis using one-to-one complex of GDH-mSA and thiolated and biotinylated aptamer on the gold electrode. Before the analysis, we modified GDH-mSA covalently with phenazine ethosulfate (PES) which was a redox probe enabling redox-enzymes to possess quasi-DET ability [3]. The formation of the enzyme-aptamer one-to-one complex on the electrode and quasi-DET ability of PES-modified GDH-mSA was evaluated. 3. Results GDH-mSA showed dye-mediated GDH activity, indicating the fusion of mSA did not negatively affect the catalytic activity. As a result of ELAA, GDH-mSA showed remarkable GDH activity in the presence of VEGF and biotinylated anti-VEGF aptamer. This indicated one-to-one complex of GDH-mSA and biotinylated anti-VEGF aptamer was formed as we designed, and showed specific recognition and binding with VEGF. Chronoamperometry analysis was carried out, and the signal was increased strikingly upon the addition of glucose only with the gold electrode immobilizing PES-modified GDH-mSA with biotinylated aptamer. Unmodified GDH-mSA did not show quasi-DET. These results indicated our success in the construction of a novel technological platform to prepare enzyme-aptamer one-to-one complex, based on biomolecular engineering concept. 4. Conclusions In this study, to circumvent the formation of uncontrollable multimer-complexes of enzymes and aptamers, we created enzyme-aptamer one-to-one complex based on the mSA-biotin interaction. Thus designed enzyme-aptamer one-to-one complex was prepared without losing both characteristics and acknowledging both benefits. These results suggested that GDH-mSA and biotinylated aptamer complex can be applied for the development of quasi-DET-type aptasensor by simply modifying GDH-mSA with PES. These benefits of utilizing GDH-mSA combined with aptamer will lead to the further application of aptamer-based biosensing of various targets. 5.