Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF.

Ellie D Wilson,Koji Sode,Jinhee Lee,Kaori Tsukakoshi,Atsuro Tatsumi,Kazunori Ikebukuro,Koichi Abe

doi:10.3390/s20143878

Abstract

Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.

Highlights

Aptamers are DNA or RNA ligands that recognize a specific target molecule with comparable affinity and specificity to antibodies [1,2]
Inspired by our previous study [18], we have focused on the zinc finger protein (ZF), a monomeric double-stranded DNA-binding protein, to create a glucose dehydrogenase (GDH)-labelled aptamer
We present a novel GDH labeling method using ZF-fused GDH for aptamers

Summary

Introduction

Aptamers are DNA or RNA ligands that recognize a specific target molecule with comparable affinity and specificity to antibodies [1,2]. The utilization of aptamers in biosensing has several advantages over antibodies owing to their small size, high thermal stability, and ease of chemical modification. Sensors 2020, 20, 3878 times, ease of miniaturization, and cost-effectiveness owing to their established platform over other electrochemical detection principles. To construct such electrochemical sensors, enzymes are attractive and often used as labeling molecules for high-sensitivity detection owing to their signal amplification ability. In reported electrochemical aptamer sensors, alkaline phosphatase and horse radish peroxidase are the most common enzymatic labels [4]. The procedure to chemically conjugate and purify avidin and enzymes of interest before their use is laborious, but it can result in heterogenous molecules due to random conjugation [5] as well as the loss of protein function [6]

Methods

Results

Discussion

Conclusion