Development of a Direct Electron Transfer-Type Enzymatic Sensor for the Spermine in Saliva, a Marker of Pancreatic Cancer
Authors: Yaegashi Yuki , Sheng Tong , Yanase Takumi , Okuda-Shimazaki Junko , Asano Ryutaro , Ikebukuro Kazunori , Sode Koji , Tsugawa Wakako 
Published: December 2024

Introduction

Pancreatic cancer (PC) has one of the worst prognoses among cancers because of the scarcity of early symptoms and the delay of early diagnosis. Current most available and advanced diagnosis methods of PC are based on imaging technologies such as computed tomography, positron emission tomography, magnetic resonance imaging, and endoscopic ultrasonography. However, such imaging methods cannot diagnose PC at an early stage. Patients diagnosed with PC often have metastasized, and only less than 30% of PC patients can undergo surgical resection. Therefore, there is an urgent need for more accurate, simple, and innovative methods for PC early diagnosis. Recently, salivary spermine has attracted attention as a new biomarker for PC <Asai et.al.,
Cancers, 2018>, and studies aimed at detecting spermine are attempted. Thus, we focused on the electrochemical measurements of spermine using enzyme.

Electrochemical enzymatic biosensors are categorized into three generation principles based on the electron acceptors used for the oxidative half reaction. The 1st generation principle uses oxygen as the electron acceptor. However, the signal is affected by the oxygen concentration and a high applied potential is required to detect hydrogen peroxide. The 2nd generation principle requires the use of synthetic electron acceptors or mediators. The 3rd generation principle utilizes enzymes which are capable of direct electron transfer (DET) to the electrode, does not require any additional electron acceptors. Notably, the 3rd generation principle-based sensor can monitor target by applying relatively lower potentials, therefore will not be influenced by redox-active interferents. Considering the salivary sample contains variety of ingredient potentially interferants for enzymatic spermine monitoring, the DET-type enzymatic sensors are ideal. However, those have never been reported. In this work, we present the development of the 3rd generation principle based enzymatic sensor for spermine by employing a unique

Methods

SpdH was recombinantly produced using E. coli and purified by Ni2+ affinity chromatography. An electrochemical enzymatic sensor was constructed by immobilizing SpdH on gold electrodes. The DET properties of the enzyme-immobilized electrode were evaluated by cyclic voltammetry with an Ag/AgCl reference electrode and a Pt wire counter electrode, respectively. Chronoamperometry measurement was performed with SpdH immobilized gold electrodes using artificial saliva or phosphate buffer, where spermine was spiked with different concentrations.

Results and Discussions

For spermine measurement, we selected SpdH which was expected to be capable of DET with electrode, which was predicted from its unique protein structure. The crystal structure of SpdH shows the presence of heme b and FAD as the co-factors of the enzyme with a distinctive character. The heme b locates on the surface of the enzyme and is expected to accept electron from FAD and can transfer electrons to the electrode. The cyclic voltammetry observation showed the spermine-concentration dependent peak current increase in the absence of any additional electron acceptor, revealing that the enzyme harbors DET ability with electrode. The spermine concentration dependent currents increase was also observed by chronoamperometry measurement. The limit of detection of the enzymatic electrode was sub-micromolar level, which covers the physiologically relevant ranges of spermine. Furthermore, the electrode did not respond to electrochemically active interferants due to its characteristic of low redox potentials of heme being measurable at low potentials.

This is the first report to construct a spermine sensor with a DET-type enzyme. DET-type enzymes allow simple electrode configurations operating at lower potentials. These features are less susceptible to redox-active interferents. The current achievements promise the future realization of a simple diagnosis method for PC using a non-invasive specimen, saliva.

Figure 1