A Copper Disk Electrode Biosensor Assay for Urinary 4-Hydroxyphenyl Acetate Testing

Abstract

An increase in the urinary concentration of 4-hydroxyphenyl acetate (4-HPA) accompanies several tumors and brain defects or may be a sign of intestinal microbial imbalance, also known as gut dysbiosis. The reductase (C <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> ) module of a hydroxylase from the soil microbe <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Acinetobacter baumannii</i> catalyzes the oxidation of NADH by oxygen with hydrogen peroxide (H <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> ) as a by-product, in a reaction that is allosterically activated by concentration-dependent binding of the disease biomarker 4-HPA. Here, we show an economical, facile fabrication of a disk electrode based on an electrical copper (Cu) cable produces a functioning analytical tool for C <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> -based 4-HPA biosensing with interference-free H <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> amperometric readout. In phosphate buffer with C <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> , the Cu disk is operated at H <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> reduction potential. Sample addition and the start of electrochemical cell current acquisition is followed by NADH injection, with the resultant H <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> formation monitored as an increase in current. Samples containing 4-HPA produce allosteric activation of C <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> , while 4-HPA-free samples have no effect. The activation of 4-HPA, resulting in a steeper H <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> current trace, is dependent on the 4-HPA concentration: calibration plots showed a sensitivity of about 20 μM H <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> 0 <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> s <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-1</sup> /μM 4-HPA and were linear up to 20 μM before approaching a signal plateau on saturation of the effector binding sites. The methodology was able to differentiate between healthy and pathological 4-HPA concentrations in urine samples. Development of the proposed methodology into a diagnostic assay for clinical analysis and personal healthcare is feasible by transfer to screen-printed copper electrode platforms in combination with hand-held potentiostats.