Development of the Electrochemical Detection System Using the Combination of Aptamer and Enzyme

Abstract

Introduction Aptamers are DNA or RNA molecules that bind to targets (proteins, small compounds, cells etc.) with high affinity and specificity. In particular, DNA aptamers have attractive properties for construction of biosensors; they are chemically-synthesized and easy to be modified by functional group with low production cost. Aptamer-introduced materials have been utilized to develop numerous biosensors, and indeed, proteomic microarray-based platform using modified aptamers is commercially available. Due to its several useful properties, we have employed DNA aptamers for development of electrochemical biosensors. We focused on glucose dehydrogenases (GDHs), enzymes have been already adopted by industries and utilized in commercially available glucose monitoring systems, as signal transducers for electrochemical detection of targets by aptamers. In addition, it is well-known that thrombin cleaves substrate peptide conjugated electroactive compound, and aptameric enzyme subunit (AES), a homogeneous detection method based on enzymatic activity, was developed utilizing thrombin in our laboratory. Thus, we are developing electrochemical measurement platform for AES based on thrombin activity. In this presentation, we describe electrochemical target detection systems based on GDH-labeled DNA aptamers and AES utilizing thrombin. Methods 1. Electrochemical detection of targeted molecules using GDH-labeled aptamers We developed GDH-employed electrochemical aptasensors with three method of GDH-labeling to DNA aptamer; (i) avidin-biotin interaction1,2, (ii) GDH-binding aptamers3,4 and (iii) GDH-fused zinc finger protein (unpublished result, but the enzyme-labeling method using enzyme-fused zinc finger protein has been already reported5.) DNA aptamers were labeled with recombinant PQQGDH from Acinetobacter calcoaceticus or FADGDH from Aspergillus flavus. GDH-fused zinc finger protein was expressed in Escherichia coli BL21(DE3) and purified by affinity chromatography. Thiolated aptamers or biotinylated antibody were immobilized on gold electrode. After incubation with target molecules, the gold electrode was immersed into GDH-labeled aptamer solution. We measured the response current to addition of glucose and m-PMS. 2. Electrochemical measurement of thrombin activity via interdigitated array electrodes We aim at measurement of target molecules by using interdigitated array (IDA) electrodes. IDA electrodes are suitable for redox cycling, which lead to achieve highly sensitive detection with a combination of a redox agent. Therefore, we measured thrombin activity using substrate peptide conjugated p-aminophenol. p-aminophenol is characterized as a redox agent. First, thrombin activity without inhibition for p-aminophenol-conjugating peptide was measured by IDA electrodes and the increased sensitivity via redox cycling was confirmed. Next, the activity with inhibition was investigated. Results and discussion 1. We found significant decreasing enzymatic activity of PQQGDH conjugated with streptavidin, but measurement of thrombin was attained and the detection threshold was indicated as 40 nM due to high catalytic efficiency of original PQQGDH. GDH fusing zinc finger protein showed 50% of enzymatic activity compared to the wild type GDH. DNA aptamer binds to GDHs did not affect the enzymatic activity. Sandwich-type sensing platform was utilized for protein detection, and target measurement based on the structural change was utilized for detection of adenosine. We successfully detected 500 pM-15 nM of VEGF and 0.2-5 mM of adenosine, respectively. 2. We observed changes in the electric current on addition of the electroactive substrate in the thrombin concentration dependent way. Under the redox cycling condition, level of the current was twice as high as the usual, non-redox cycling condition. Inhibition of thrombin was also found by addition of DNA aptamers for thrombin. These results suggested that a combination of electrochemical measurement of thrombin activity with IDA electrodes and thrombin inhibition system utilizing AES has the potential to achieve simplified diagnostic method in the future. Acknowledgement A part of this work was supported by AMED-SENTAN program.