Electrochemical biosensors represent an attractive analytical platform for the detection
of biologically relevant analytes due to their high sensitivity, low cost, and compatibility
with miniaturized devices. Lactate is an important metabolic biomarker widely monitored
in medical diagnostics, sports medicine, physiological research, and biotechnology. The
performance of enzymatic lactate biosensors strongly depends on the properties of the
electrode material and the efficiency of enzyme immobilization.
This bachelor’s thesis investigates the applicability of graphene acid-based materials as
electrode modifiers for screen-printed carbon electrodes (SPCEs) intended for amperometric lactate biosensing. Graphene acid (GA), nitrogen-doped graphene acid (NGA),
and single copper atom anchored nitrogen-doped graphene acid (NGA-Cu) were evaluated as sensing platforms. Two enzyme immobilization strategies, namely physical
adsorption and covalent immobilization via EDC/NHS coupling chemistry, were implemented and compared using lactate oxidase (LOx) as the biorecognition element.
Electrochemical impedance spectroscopy, cyclic voltammetry, and amperometric measurements were employed to characterize the modified electrodes and evaluate their
suitability for lactate detection. EIS measurements demonstrated that graphene acid
based modifications significantly altered the electrochemical properties of the electrodes,
with GA exhibiting the lowest charge-transfer resistance. Cyclic voltammetry identified
hexaammineruthenium(II/III) chloride as the most suitable redox mediator among the
investigated systems. However, despite favorable electrochemical characteristics, neither
adsorption-based nor covalent enzyme immobilization resulted in a measurable amperometric response toward lactate.
To identify the origin of this limitation, the modified electrodes were subsequently evaluated through direct hydrogen peroxide detection. The results confirmed that all investigated electrode materials remained electrochemically active and capable of generating
stable concentration-dependent signals. NGA-Cu exhibited the highest sensitivity toward
hydrogen peroxide, indicating that the absence of lactate response was primarily associated with enzyme immobilization rather than insufficient electrochemical performance
of the electrode materials.
A measurable lactate response was achieved only after employing an alternative enzyme
immobilization strategy based on a glutaraldehyde-cross-linked gelatin matrix protected
by a cellulose membrane. Under these conditions, the NGA-Cu biosensor exhibited a
concentration-dependent response over a narrower concentration range than that investigated, however demonstrated approximately fourfold higher sensitivity than a graphene
oxide-based reference biosensor. The obtained results highlight the critical role of enzyme
stabilization in biosensor construction.