# Construction and Characterization of a Novel Direct Electron Transfer Type Enzymatic Sensor Using Spermidine Dehydrogenase

## TL;DR

This paper describes a new sensor using an enzyme called spermidine dehydrogenase to detect spermine in saliva with high accuracy.

## Contribution

The study demonstrates the direct electron transfer capability of spermidine dehydrogenase for the first time in a sensor application.

## Key findings

- SpDH exhibits direct electron transfer from FAD to heme b, confirmed by a reduction peak at 560 nm.
- The sensor detected spermine in artificial saliva with a detection limit of 0.084 µM.
- Structure analysis shows conserved histidine residues in SpDH homologs, suggesting potential for DET with electrodes.

## Abstract

This study reports on the direct electron transfer (DET) ability of the enzyme spermidine dehydrogenase (SpDH) and its use in a DET-type enzymatic sensor for detecting spermine. SpDH was found to exhibit internal electron transfer from its cofactor, flavin adenine dinucleotide (FAD), to heme b. This was confirmed by observing the heme b-derived reduction peak at 560 nm in the presence of spermine, the substrate. SpDH was immobilized on a gold electrode via a dithiobis (succinimidyl hexanoate) self-assembled monolayer. The cyclic voltammetry analysis of the SpDH-immobilized gold electrode revealed an increased oxidation current in the presence of 0.1 mM spermine with an onset potential of −0.14 V vs. Ag/AgCl in the absence of an additional external electron acceptor. This result confirmed that SpDH is capable of DET. Chronoamperometric analyses were conducted using an SpDH-immobilized gold electrode with spermine as the substrate under a 0 V oxidation potential vs. Ag/AgCl using an artificial saliva matrix containing 10 µM ascorbic acid and 100 µM uric acid. The sensor exhibited good linear correlation between the current increase and spermine concentration from 0.2 to 2.0 µM, with a limit of detection of 0.084 µM, which encompasses the physiologically relevant spermine concentration found in the saliva. Primary structure alignments and 3D structure predictions revealed that all SpDH homologs possess two conserved histidine residues in the same location on the surface as the heme b ligand of SpDH. This indicates their potential for DET-ability with an electrode.