Development of a Direct Electron Transfer-Type Enzymatic Sensor for the Spermine in Saliva, a Marker of Pancreatic Cancer

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 1 st 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 2 nd generation principle requires the use of synthetic electron acceptors or mediators. The 3 rd generation principle utilizes enzymes which are capable of direct electron transfer (DET) to the electrode, does not require any additional electron acceptors. Notably, the 3 rd 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 3 rd generation principle based enzymatic sensor for spermine by employing a unique Methods SpdH was recombinantly produced using E. coli and purified by Ni 2+ 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 a