Sensitive and selective detection of superoxide secreted from neutrophils based on one-electrode redox reactions

Abstract

Subclinical mastitis is an infectious disease of dairy cows. To develop an on-site diagnostic device for mastitis, we used the number of neutrophils in raw milk samples as an indicator. The number was measured by detecting superoxide anion (O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-</sup> ) secreted from neutrophils. To achieve highly sensitive detection of O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-</sup> , we used one-electrode redox reactions. In this method, O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-</sup> secreted from neutrophils was converted into copper deposited on an electrode via superoxide dismutase (SOD). The amount of the deposited copper was measured by coulometry. In addition, hydrogen peroxide, which is produced from O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-</sup> , was not oxidized by copper ions because of the relation between the redox potentials of these reactions. A clear liner relationship was obtained between the output charge and the number of neutrophils in the sensing region. Also, a significant improvement in sensitivity was observed compared with our previous method.