Introduction We propose a new biosensor based on the bipolar phenomenon. The bipolar phenomenon polarizes a conductor (bipolar electrode, BPE) inserted into an electric field formed between driving electrodes (DEs) connected to an external power source. Electrochemical biosensing using the bipolar phenomenon for detecting biomolecules has recently been reported. In biosensing, BPE separates the molecular recognition site from the detection site. This feature is scalable and enables the exploration of new detection reactions. Therefore, the bipolar phenomenon shows potential as a new tool for biosensors. Here, we demonstrate the electrochemical biosensor with a simple design using an open BPE. The biosensor detects C-reactive protein (CRP) by electrochemical impedance measurement under non-faradic conditions. Experimental Gold electrodes (10 mm × 10 mm) were immersed in 10 mM 3-mercaptopropionic acid (MPA) solution for 1 h to form an MPA self-assembled monolayer. The carboxyl groups of the MPA monolayer were treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (0.2 M/0.1 M). Subsequently, the electrodes were incubated with 10 µg/mL of anti-CRP antibody in 0.1 M phosphate buffer (PB, pH 7.0) to immobilize the antibody. Following the blocking treatment, the electrodes were exposed to 10 μg/mL CRP in 0.1 M PB at 37°C for 0-2 h to react with CRP. For impedance measurements, Pt-black-coated gold wires were used as the DEs and connected to an impedance analyzer. The BPE was placed at the bottom of an electrochemical cell filled with 0.1 M PB (pH 7.0), and the DE was positioned directly above the BPE (refer to Fig. 1A). The measurements were performed in a frequency range from 1 Hz to 8 MHz, with an amplitude of 0.1 V and a DC bias voltage of 0 V. Results and Discussion Figure 1B shows the relationship between CRP immobilization time and impedance value at 1 kHz. A linear decrease in impedance was observed during the immobilization time of 0-1 h, which caused the increased in capacitance of the BPE surface resulting from the increased amount of immobilized CRP. Over 1 h of immobilization, the impedance change slowed down, indicating the saturation of CRP immobilization. These results suggest that the sensor developed in this study detects the antigen-antibody reaction on the BPE through impedance change. Figure 1
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