Realization of an ultra-sensitive hydrogen peroxide sensor with conductance change of horseradish peroxidase-immobilized polyaniline and investigation of the sensing mechanism

• An electronic, disposable, miniaturized, and ultra-sensitive hydrogen peroxide sensor is developed with a detection limit of 0.7 nM.
• The detectable H2O2 concentration of the hydrogen peroxide sensor is from 0.7 nM–1 μM.
• Fabrication process, characterizations, and sensing mechanism of the hydrogen peroxide sensor based on enzyme immobilized N-alkylated polyaniline are revealed.
• The hydrogen peroxide sensor has potential applications in detecting reactive oxygen species (ROS) in oxidative stress studies.

In this study, we fabricate an ultra-sensitive hydrogen peroxide sensor by using horseradish peroxidase (HRP)-immobilized conducting polymer, polyaniline (PANI). With the proposed detection mechanism, hydrogen peroxide first oxidizes HRP, which then oxidizes polyaniline, thus resulting in decreased conductivity of the polyaniline thin film. The reduced HRP can be further oxidized by hydrogen peroxide and the cycle of the oxidation/reduction would continue until all hydrogen peroxide are reacted, leading to the high sensitivity of the sensor due to the signal contributed from all hydrogen peroxide molecule. The detection limit of this sensor is only 0.7 nM. The detectable concentration of H2O2 is from 0.7 nM to 1 μM. Beyond 1 μM, the sensor gradually saturates and some H2O2 remains, indicating the inhibition of HRP activity at high concentration of H2O2. There is no response to hydrogen peroxide once the PANI is standalone without HRP immobilized, showing the enzymatic reaction is required in the process of hydrogen peroxide detection. The simple process for the sensor fabrication allows the sensor to be cost-effective and disposable. This electronic hydrogen peroxide sensor is promising in applications for low concentration hydrogen peroxide detections, such as the reactive oxygen species (ROS) in oxidative stress studies.