5:15 PM - 6:45 PM
[AOS13-P14] Development of electrochemical real-time Megabalanus roseus gene measurement system
1 Introduction
Global warming caused by greenhouse gases, i.e., the temperature rise with increasing atmospheric CO2 concentration, has become a serious problem. Since approximately 70% of the Earth's surface consists of oceans, exploring changes in the marine environment caused by greenhouse gases will lead to global environmental conservation. Genetic analysis, which requires complicated operations and large equipment, is considered difficult to conduct in real time in the ocean. In order to understand the marine ecological environment more accurately, this study aimed to conduct in-situ gene analysis using an electrochemical biosensor that can be miniaturized and used for continuous measurement. The sensor electrode was developed by targeting a polluted barnacle, Megabalanus roseus, and immobilizing its gene and complementary sequence as a probe on the electrode surface (Fig. 1). In this presentation, we report on the performance of the fabricated DNA sensor.
2 Experiment
2.1 Target DNA and probe design
We designed a target DNA containing a sequence unique to Megabalanus roseus (ACTAGT) and a probe that has a complementary sequence to this DNA and can form a loop structure. A thiol group was introduced at the 5' end and an amino group was introduced at the 3' end (Table 1).
2.2 Fabrication of probe-modified electrode
A screen-printed electrode (SPE) was used as the electrode. A washed gold electrode was modified by dropping 5 micro l of the loop-forming probe onto the electrode and allowing it to stand overnight. Loop formation of the probe was performed by annealing at 95 degrees Celsius for 5 min, followed by 15 min at room temperature and 5 min on ice. The probe was then washed with PBS, and 10 micro l of 5 mM Amine-reactive PES was added dropwise and allowed to stand for 1.5 hours, followed by washing with PBS to modify the mediator at the end of the probe. Then 10 micro l drops of 10 mM 6-Mercapto-1-hexanol as blocking agent were added and washed with PBS after 1 hour to make probe-modified electrode.
3. Results and discussion
3.1 Evaluation of electrode surface
The impedance of an untreated gold electrode, a probe without a loop structure, and a gold electrode modified with a probe having a loop structure were each measured to confirm whether the loop structure was properly formed (Fig. 2). The diameter of the semicircle in the graph showing charge transfer resistance increased significantly for the probe-modified electrode without the refolding treatment compared to the untreated electrode, suggesting that the probe was immobilized on the electrode. In addition, the diameter of the semicircle of the probe-modified electrode with refolding treatment increased further, suggesting that the probe formed a loop structure, which increased the charge transfer resistance on the electrode surface, suggesting that the probe was modified with an appropriate loop structure.
3.2 Detection of target DNA by CV measurement
The results of CV measurement without and with target DNA are shown in Fig. 3. A decrease in the oxidation peak was observed when the target DNA was added. It is thought that the mediator, which had formed a loop structure and approached the electrode surface, was moved away from the electrode when the probe complemented the target, thereby decreasing the electron transfer efficiency and decreasing the current.
3.3 Specificity and selectivity evaluation of probe-modified electrode
Megabalanus roseus DNA and DNA of a different sequence were added as probe targets (Fig. 4 (a) and (b)). The decrease in the oxidation peak was observed only when Megabalanus roseus DNA was added, confirming the specificity of the newly designed probe. Furthermore, when two types of DNA were added to the same electrode, a large decrease in the oxidation peak was observed only when Megabalanus roseus DNA was added (Fig. 5), confirming the selectivity of the newly designed probe.
Global warming caused by greenhouse gases, i.e., the temperature rise with increasing atmospheric CO2 concentration, has become a serious problem. Since approximately 70% of the Earth's surface consists of oceans, exploring changes in the marine environment caused by greenhouse gases will lead to global environmental conservation. Genetic analysis, which requires complicated operations and large equipment, is considered difficult to conduct in real time in the ocean. In order to understand the marine ecological environment more accurately, this study aimed to conduct in-situ gene analysis using an electrochemical biosensor that can be miniaturized and used for continuous measurement. The sensor electrode was developed by targeting a polluted barnacle, Megabalanus roseus, and immobilizing its gene and complementary sequence as a probe on the electrode surface (Fig. 1). In this presentation, we report on the performance of the fabricated DNA sensor.
2 Experiment
2.1 Target DNA and probe design
We designed a target DNA containing a sequence unique to Megabalanus roseus (ACTAGT) and a probe that has a complementary sequence to this DNA and can form a loop structure. A thiol group was introduced at the 5' end and an amino group was introduced at the 3' end (Table 1).
2.2 Fabrication of probe-modified electrode
A screen-printed electrode (SPE) was used as the electrode. A washed gold electrode was modified by dropping 5 micro l of the loop-forming probe onto the electrode and allowing it to stand overnight. Loop formation of the probe was performed by annealing at 95 degrees Celsius for 5 min, followed by 15 min at room temperature and 5 min on ice. The probe was then washed with PBS, and 10 micro l of 5 mM Amine-reactive PES was added dropwise and allowed to stand for 1.5 hours, followed by washing with PBS to modify the mediator at the end of the probe. Then 10 micro l drops of 10 mM 6-Mercapto-1-hexanol as blocking agent were added and washed with PBS after 1 hour to make probe-modified electrode.
3. Results and discussion
3.1 Evaluation of electrode surface
The impedance of an untreated gold electrode, a probe without a loop structure, and a gold electrode modified with a probe having a loop structure were each measured to confirm whether the loop structure was properly formed (Fig. 2). The diameter of the semicircle in the graph showing charge transfer resistance increased significantly for the probe-modified electrode without the refolding treatment compared to the untreated electrode, suggesting that the probe was immobilized on the electrode. In addition, the diameter of the semicircle of the probe-modified electrode with refolding treatment increased further, suggesting that the probe formed a loop structure, which increased the charge transfer resistance on the electrode surface, suggesting that the probe was modified with an appropriate loop structure.
3.2 Detection of target DNA by CV measurement
The results of CV measurement without and with target DNA are shown in Fig. 3. A decrease in the oxidation peak was observed when the target DNA was added. It is thought that the mediator, which had formed a loop structure and approached the electrode surface, was moved away from the electrode when the probe complemented the target, thereby decreasing the electron transfer efficiency and decreasing the current.
3.3 Specificity and selectivity evaluation of probe-modified electrode
Megabalanus roseus DNA and DNA of a different sequence were added as probe targets (Fig. 4 (a) and (b)). The decrease in the oxidation peak was observed only when Megabalanus roseus DNA was added, confirming the specificity of the newly designed probe. Furthermore, when two types of DNA were added to the same electrode, a large decrease in the oxidation peak was observed only when Megabalanus roseus DNA was added (Fig. 5), confirming the selectivity of the newly designed probe.