Direct Electron Transfer Type Faradaic Enzyme EIS
Authors: Okuda-Shimazaki Junko , Ito Yuka , Loew Noya , Tsugawa Wakako , Sode Koji 
Published: February 2020

Introduction

Faradaic electrochemical impedance spectroscopy (Faradaic EIS) has been studied as the principle of biosensors. Considering that Faradaic EIS is a powerful method to investigate the surface of electrodes in details with high sensitivity, variety of Faradaic EIS based biosensors, such as immunosensors or DNA sensors have been reported. These biosensors are based on measuring the charge transfer resistance (Rct) of redox probes at the surface of electrodes. The one of the inherent limitations of currently reported Faradaic EIS based biosensors is the presence of redox probes, which is necessary to monitor the binding of target molecules to ligand which alter the flux of redox probes to the electrode surface, consequently, result the change of Rct. However, the redox probes are generally chemically unstable, and the configuration of continuous monitoring and/or in vivo monitoring type biosensors using redox probe are complicated and difficult. Therefore, the developments of alternative EIS sensor platforms, which make the robust measurements, have been expected.
Besides, enzyme-based electrochemical biosensors are categorized into three generations, based on the electron acceptors. In the 1st-generation, oxygen serves as the electron acceptor of the enzyme reaction of oxidase, and the decreasing oxygen concentration or liberating hydrogen peroxide is detected electrochemically. The 2nd-generation principle uses artificial electron acceptors (mediators) for the oxidases or dehydrogenases enzyme oxidative half reactions, instead of oxygen, and the reduced artificial electron acceptors are measured electrochemically. The 3rd-generation principle uses enzymes with the ability of direct electron transfer (DET). Although, Faradaic enzyme EIS sensors have been reported by employing redox mediators or oxygen as the external electron acceptors, there have been no reports on sensors employing DET-type redox enzymes based on Faradaic EIS principle.
In this study, we report the construction and characterization of direct electron transfer type Faradaic Enzyme EIS, as the novel biosensor employing DET-type redox enzymes without redox probe. The DET-type Faradaic Enzyme EIS employs flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase (GDH) complex (FADGDH), which is consisted of three subunits: a catalytic subunit which has FAD and 3Fe-4S-type iron-sulfur cluster, an electron transfer subunit which has 3 hemes, and a small subunit as a hitchhiker protein. Thanks to the presence of the multi heme electron transfer subunit, FADGDH is capable of DET (ref.).

Method

DET-type FADGDH- modified or non-DET-type FADGDH (fungi derived FADGDH)- modified gold disk electrodes (GDEs) were constructed to elucidate its characteristic properties. FADGDHs were modified onto GDEs via self-assembled monolayer (SAM). FADGDHs modified GDEs were evaluated by EIS to analyse faradaic impedance towards different concentrations of glucose, with Ag/AgCl and Pt wire as the reference and counter electrodes, in the absence of redox probe. EIS measurements were performed in certain frequency range by AC voltage with an amplitude of 10 mV superimposed on DC potential. Obtained EIS data were analysed with an equivalent circuit to obtain electrochemical parameters.

Result and Discussion

When EIS was performed with DET-type FADGDH-modified GDE in the presence of glucose, the obtained semicircles on the Nyquist plots became smaller with increasing depending on glucose concentrations. In contrary, EIS analyses of the non-DET-type FADGDH-modified GDEs revealed that no impedance change was observed by changing glucose concentration. These results indicated that the impedance change with DET-type FADGDH-modified GDE was observed, which results the glucose concentration dependency of Rct values. Considering the observed high sensitivity toward glucose, DET-type Faradaic Enzyme EIS sensor would provide alternative platform for future impedimetric immunosensing system, which does not use redox probe.

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Figure 1