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In this study, an ultra-sensitive hydrogen peroxide sensor was fabricated 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 interaction would proceed until all hydrogen peroxide are reacted. It leads to the high sensitivity of the sensor due to the signal contributed from all reacted hydrogen peroxide. The detection limit of the hydrogen peroxide sensor is 0.7 nM. The detecting range of concentration of H 2 O 2 is from 0.7 nM to 1 µM. The simple fabrication for the sensor allows the sensor to be costeffective and disposable. This ultra-sensitive hydrogen peroxide sensor is promising in applications for low concentration hydrogen peroxide detections, such as the reactive oxygen species (ROS) in oxidative stress studies.

In this study, a cost-effective and highly sensitive cholesterol microsensor, which is consisted of cholesterol oxidase (ChOx), horseradish peroxidase (HRP), and polyaniline (PANI), was developed based on the enzyme-induced conductivity change of PANI with fast response. Hydrogen peroxide is produced via the reaction between cholesterol and ChOx, which was immobilized in a dialysis membrane. The produced hydrogen peroxide can oxidize HRP, which can be reduced by oxidizing PANI, thus resulting in decreased conductivity of the polyaniline thin film. The reduced HRP can be oxidized again by hydrogen peroxide and the cycle of the oxidation/reduction continues until all hydrogen peroxide are reacted, leading to the high sensitivity of the sensor due to the signal contributed from all hydrogen peroxide molecules. Cholesterol was detected near the physiological concentrations ranging from 100 mg/dl to 400 mg/dl with the cholesterol microsensors. The results show linear relation between cholesterol concentration and the conductivity change of the PANI. The microsensor showed no response to cholesterol when the PANI was standalone without cholesterol oxidase immobilized, indicating that the enzymatic reaction is required for cholesterol detection. The simple process of the sensor fabrication allows the sensor to be cost-effective and disposable usage. This electronic cholesterol microsensor is promising for point-of-care health monitoring in cholesterol level with low cost and fast response.

Hydrogen peroxide is a metabolic by-product and a kind of stable reactive oxygen species (ROS). When the ROS levels increase, it may causes several harmful effects of ROS on the cell structure, such as damage of DNA and protein oxidation, and this is known as oxidative stress. The oxidative stress is also the important clinical indicators of cause of aging, Alzheimer disease, and kidney diseases. Now the common test method of oxidative stress is Enzyme-linked immunosorbent assay (ELISA). ELISA needs color giving dyes, complicated preparation of sample, and optical system, so it is very expensive and inconvenient. In our study, we present a high sensitivity hydrogen peroxide sensor with ultra-low detection limit. The sensor can directly test the sample and only need very small amount of sample. And because of the simple structural design and fabrication, the sensor can be used as a cheap, efficient, and portable sensor system. In the future, the novel hydrogen peroxide sensor has various applications in studying oxidative stress and detecting reactive oxygen species for cells. Furthermore, we can combine the PANI sensor with different enzymes to fabricate other highly sensitive sensors to detect diverse materials, such as glucose, lactic acid and cholesterin. In our study, the PANI layer and gold electrodes were deposited on silicon nitride substrate, and the PANI was applied to fabricate a thin film between two electrodes. Then the PANI layer was sultonated by propane sultone and modified with HRP. During the measurement, the sensor was operated at 100mV and different concentrations of hydrogen peroxide citrate buffer solutions (pH=5.4) were dropped on it. The current change was measured when the hydrogen peroxide reacted with the HRP Immobilized PANI thin film. We tested the hydrogen peroxide solution from 0.1 nm to 1mM. The HRP-modified resistive sensors based on n-alkylated polyaniline(PANI) detect hydrogen peroxide in solution with very high sensitivity, ultra-low limit, and short response time. The sensitivity is higher than that of other sensing methods, such as electrochemical sensors or transistor sensors. The detection limit of PANI sensor is 0.7 nM (Figure 1. a, b). It is three orders smaller than that of other common methods with detection limit around 1 μM. To the best of our knowledge, it is the lowest detection limit that has ever been reported. And We combine the hydrogen peroxide sensor with glucose oxidase to build up the glucose sensor. In summary, the hydrogen peroxide sensor can provide a more exact hydrogen peroxide concentration and quick detection. The simple process for the sensor fabrication also allows the sensor to be cheap, disposable and combinable with other sensors to build up sensing system. This work was partially supported by National Science Council grant (No.99B20495A & 101-2221-E-007-102-MY3) and by the research grant (100N2049E1) at National Tsing Hua University.