Development of in-Situ Electrochemical Megabalanus Roseusgene Measurement System
Authors: Uejima Rino , Sugimoto Mai , Arima Masakazu , Saiki Tsunemasa , Nagai Satoshi , Takamura Eiichiro , Sakamoto Hiroaki 
Published: December 2024

In recent years, global warming due to greenhouse gases has become a serious problem, causing problems such as atmospheric CO2 concentration and temperature rise. On the other hand, since about 70% of the earth is ocean, it is expected that exploring changes in the ocean environment not only due to atmospheric gases but also due to greenhouse gases will lead to conservation of the global environment.

In order to accurately understand and predict the marine ecological environment, we need to acquire chemical information such as CO2 concentration, salinity, and pH, physical information such as temperature and pressure, and biological information such as genetic analysis from multiple angles. Chemical and physical information can be obtained in real time by installing high-precision sensors on underwater robots. However, since biological information requires complicated operations and large equipment, it is generally obtained by conducting experiments on land after sampling water. Here, there is a concern that genetic measurements in the ocean may involve losses such as decomposition of biological samples and changes in traits between the time the seawater sample is collected and the sample is transported to the laboratory. Therefore, in order to more accurately understand the marine ecological environment, we aimed for in-situ gene measurement using an electrochemical biosensor that is compact and capable of continuous measurement.

The purpose of this research is to detect the DNA of the larvae of Megabalanus roseus, which are fouling barnacles, using an electrochemical sensor. Megabalanus roseus attach to the ships, cooling waterway systems of thermal power plants, and nuclear power plants, causing problems such as increased fuel consumption and decreased flow of cooling water. We developed a sensor electrode by designing a probe with a complementary sequence to ACTAGT, a sequence unique to Megabalanus roseus (Table 1), and immobilizing it on the electrode surface. The probe has a thiol group at the 5' end and a mediator at the 3' end, forming a loop structure. When the probe captures the target, the loop structure is loosened, and the distance between the electrode and the mediator changes, reducing the electron transfer efficiency and making it possible to quantify the target (Fig. 1).

A 25×11 mm screen-printed electrode (SPE : working electrode - carbon, counter electrode - carbon, reference electrode - Ag/AgCl) was used as the electrode. The working electrode was electrodeposited with gold, and after washing, 5 µl of the probe with a loop structure was dropped onto the gold electrode and allowed to stand overnight for modification. Loop formation of the probe was performed by heat treatment at 95° C. for 5 minutes, followed by standing at room temperature for 15 minutes and on ice for 5 minutes. After that, the probe was washed with PBS, and 10 µl of 5 mM Amine-reactive PES was added dropwise, left to stand for 1.5 hours, and then washed with PBS to modify the end of the probe with a mediator. Next, 10 µl of 10 mM 6-Mercapto-1-hexanol was added dropwise as a blocking agent, and after 1 hour, the electrode was washed with PBS and used as a probe-modified electrode.

A mediator peak was confirmed by cyclic voltammetry (CV), confirming the modification of the probe. When target DNA was added thereto, the oxidation peak current decreased. This is thought to be due to the loop unraveling when the probe, which had a loop structure, captured the target DNA. When the probe has a loop structure, the mediator and electrode are close to each other, but when the loop is unraveled, the mediator moves away from the electrode, reducing electron transfer efficiency and reducing the oxidation peak.

Figure 1