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

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 (
$\text{C}_{{1}}{)}$
module of a hydroxylase from the soil microbe
$\textit {Acinetobacter baumannii}$
catalyzes the oxidation of
$\beta $
-nicotinamide adenine dinucleotide (NADH) by oxygen with hydrogen peroxide (
$\text{H}_{{2}}\text{O}_{{2}}{)}$
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 that produces a functioning analytical tool for
$\text{C}_{{1}}$
-based 4-HPA biosensing with interference-free
$\text{H}_{{2}}\text{O}_{{2}}$
amperometric readout. In phosphate buffer with
$\text{C}_{{1}}$
, the Cu disk is operated at
$\text{H}_{{2}}\text{O}_{{2}}$
reduction potential. Sample addition and the start of electrochemical cell current acquisition are followed by NADH injection, with the resultant
$\text{H}_{{2}}\text{O}_{{2}}$
formation monitored as an increase in current. Samples containing 4-HPA produce allosteric activation of
$\text{C}_{{1}}$
, while 4-HPA-free samples have no effect. The activation of 4-HPA, resulting in a steeper
$\text{H}_{{2}}\text{O}_{{2}}$
current trace, is dependent on the 4-HPA concentration: calibration plots showed a sensitivity of about 20-
$\mu \text{M} \text{H}_{{2}}\text{O}_{{2}} \text{s}^{-{1}}/\mu \text{M}$
4-HPA and were linear up to
$20 ~\mu \text{M}$
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. The development of the proposed methodology into a diagnostic assay for clinical analysis and personal healthcare is feasible by transferring to screen-printed Cu electrode platforms in combination with handheld potentiostats.