Presentation Information
[AOS25-P12]Development of a highly sensitive and rapid electrochemical sensing technology for marine genetic monitoring
*Hiromu Oki1, Rino Uejima1, Tsunemasa Saiki2, Masakazu Arima3, Hirofumi Sumoto4, Sakamoto Hiroaki1 (1.University of Fukui, 2.University of Fukutiyama, 3.Osaka Metropolitan University, 4.Kagoshima University)
Keywords:
Electrochemical biosensor,Environmental sensing,Real-time monitoring,Chaetoceros,Probe,Marine gene
1 Introduction
Atmospheric CO2 levels have increased and caused ocean acidification, leading to deterioration of corals and calcium-carbonate shell organisms and impacting marine ecosystems. Accurate monitoring of marine environments requires rapid and precise analysis; however, biological information collection remains difficult due to cost, time, and complicated procedures. Therefore, this study focused on an electrochemical biosensor.
The biosensor detects structural changes caused by hybridization between immobilized probe DNA and target microbial DNA as an electrical signal. Because conventional systems rely on one-to-one hybridization, sensitivity depends on target concentration. To overcome this limitation, an electrode interface utilizing the specific cleavage reaction of Exonuclease III (Exo III) was designed to enable target DNA recycling and signal amplification (Fig. 1). This improved detection sensitivity from the n–µM level to the n–fM level, enabling simpler and faster marine gene detection.
A gene of phytoplankton in the genus Chaetoceros, abundant in marine environments and sensitive to CO2, was selected as the detection target.
2 Experimental
2-1 Probe design
The sequences of the signal probe (SP), assistant probe (AP), and target DNA used in this study are shown in Table 1. The base lengths were 23 bp for SP and 25 bp for AP, and they were prepared by HPLC purification. Thiolation of the 3′ end of SP enabled immobilization of the probe on the Au electrode via Au–S bonding. In addition, amination of the 5′ end enabled modification with a mediator via amide bonding. The base length of the target DNA was 14 bp.
2-2 Preparation of the SP-AP-MB probe
3 µL of 2 µM SP solution modified at the 5′ end with MB and 3 µL of 2 µM AP solution were mixed in 54 µL of 1×PBS (−), kept at 85°C for 10 min, and then left at room temperature for 1 h to form the SP-AP-MB probe, which is dsDNA of SP-MB and AP. To reduce disulfide bonds between probes, 2 µL of 10 mM TCEP was added and the mixture was left for 1 h.
2-3 Sensor evaluation by square wave voltammetry (SWV)
15 µL of the probe solution generated in section 2-2 was dropped onto the Au electrode to immobilize the SP-AP-MB probe on the electrode surface, thereby obtaining a probe-modified electrode. Measurements were performed by square wave voltammetry (SWV) using a three-electrode system with this electrode as the working electrode. After that, 100 nM target DNA solution was dropped onto the electrode and measured by SWV. Finally, the electrode surface was immersed in a solution containing 5 U of Exo III, incubated at 37°C for 2 h, and measured by SWV.
3 Results of detection of the Chaetoceros genus gene by SWV
Evaluation by SWV was performed for sensor application. The SWV results for detection using Exo III with 100 nM target DNA are shown in Fig. 2. When only the target DNA was present, no significant change in the oxidation peak current value was observed. A large increase in the oxidation peak current value was observed in the presence of both target DNA and Exo III. This is considered to be because Exo III reacted with the complex of the SP-AP-MB probe and the target DNA, converting the SP-AP-MB probe into a single strand of SP-MB and changing the structure of SP from an extended state to a folded structure. As a result, the mediator became more accessible to the electrode surface and the electron transfer rate increased. Furthermore, it was suggested that the target DNA released by the enzymatic degradation reaction of Exo III was reused. Consequently, the reused target DNA repeatedly bound to new SP-AP-MB probes, and the amount of SP with a stem–loop structure on the electrode increased.
Atmospheric CO2 levels have increased and caused ocean acidification, leading to deterioration of corals and calcium-carbonate shell organisms and impacting marine ecosystems. Accurate monitoring of marine environments requires rapid and precise analysis; however, biological information collection remains difficult due to cost, time, and complicated procedures. Therefore, this study focused on an electrochemical biosensor.
The biosensor detects structural changes caused by hybridization between immobilized probe DNA and target microbial DNA as an electrical signal. Because conventional systems rely on one-to-one hybridization, sensitivity depends on target concentration. To overcome this limitation, an electrode interface utilizing the specific cleavage reaction of Exonuclease III (Exo III) was designed to enable target DNA recycling and signal amplification (Fig. 1). This improved detection sensitivity from the n–µM level to the n–fM level, enabling simpler and faster marine gene detection.
A gene of phytoplankton in the genus Chaetoceros, abundant in marine environments and sensitive to CO2, was selected as the detection target.
2 Experimental
2-1 Probe design
The sequences of the signal probe (SP), assistant probe (AP), and target DNA used in this study are shown in Table 1. The base lengths were 23 bp for SP and 25 bp for AP, and they were prepared by HPLC purification. Thiolation of the 3′ end of SP enabled immobilization of the probe on the Au electrode via Au–S bonding. In addition, amination of the 5′ end enabled modification with a mediator via amide bonding. The base length of the target DNA was 14 bp.
2-2 Preparation of the SP-AP-MB probe
3 µL of 2 µM SP solution modified at the 5′ end with MB and 3 µL of 2 µM AP solution were mixed in 54 µL of 1×PBS (−), kept at 85°C for 10 min, and then left at room temperature for 1 h to form the SP-AP-MB probe, which is dsDNA of SP-MB and AP. To reduce disulfide bonds between probes, 2 µL of 10 mM TCEP was added and the mixture was left for 1 h.
2-3 Sensor evaluation by square wave voltammetry (SWV)
15 µL of the probe solution generated in section 2-2 was dropped onto the Au electrode to immobilize the SP-AP-MB probe on the electrode surface, thereby obtaining a probe-modified electrode. Measurements were performed by square wave voltammetry (SWV) using a three-electrode system with this electrode as the working electrode. After that, 100 nM target DNA solution was dropped onto the electrode and measured by SWV. Finally, the electrode surface was immersed in a solution containing 5 U of Exo III, incubated at 37°C for 2 h, and measured by SWV.
3 Results of detection of the Chaetoceros genus gene by SWV
Evaluation by SWV was performed for sensor application. The SWV results for detection using Exo III with 100 nM target DNA are shown in Fig. 2. When only the target DNA was present, no significant change in the oxidation peak current value was observed. A large increase in the oxidation peak current value was observed in the presence of both target DNA and Exo III. This is considered to be because Exo III reacted with the complex of the SP-AP-MB probe and the target DNA, converting the SP-AP-MB probe into a single strand of SP-MB and changing the structure of SP from an extended state to a folded structure. As a result, the mediator became more accessible to the electrode surface and the electron transfer rate increased. Furthermore, it was suggested that the target DNA released by the enzymatic degradation reaction of Exo III was reused. Consequently, the reused target DNA repeatedly bound to new SP-AP-MB probes, and the amount of SP with a stem–loop structure on the electrode increased.