Development of Nanozyme and Nanozyme-Enhanced Electrode for Electrochemical Lactate Sensing

Abstract

Nickel oxide (NiO) nanostructures deposited by glancing angle deposition (GLAD) are fabricated to achieve highly specific catalytic electrooxidation of lactate, replacing the natural enzyme lactate oxidase for electrochemical detection of lactate in sweat. GLAD NiO electrodes exhibit high sensitivity (412 μA mM-1 cm-2), wide linear detection range (1–45 mM), low detection limit (3 μM), and excellent specificity in artificial sweat samples. The unique microporous structure of the GLAD NiO electrodes, combined with their high surface area, high catalytic activity, and excellent conductivity, enhance the performance of the sensor and demonstrate their exceptional effectiveness in the sensitive detection of lactate. In-house fabricated gold counter, and stable solid-state Ag/AgCl reference electrodes, all fabricated on a flexible PET substrate along with the GLAD NiO working electrode, demonstrate performance comparable to commercial Pt auxiliary and Ag/AgCl (1M KCl) reference electrodes in lactate detection, along with outstanding flexibility, tested at various radii of curvature (15 mm, 7.5 mm, and 5 mm). The durable and long-lasting GLAD NiO electrode chips overcome numerous challenges in transport, storage, and operation, paving the way for the development of wearable lactate sensors that can detect lactate levels in sweat. A wearable integrated nanozyme-based electrochemical sensor is developed for sweat lactate monitoring. The sensor comprises cobalt oxide (Co3O4) and cobalt phosphate (Co3(PO4)2) nanoflakes on a screen-printed carbon electrode (SPCE), and the complete device includes a microfluidic unit for sweat collection and transport to the electrode surface. Cobalt oxide nanoflakes are directly electrodeposited onto a SPCE and electrochemically activated to form Co3O4/Co3(PO4)2/SPCE which is used to detect lactate. The structural morphologies, surface composition and chemical states, and crystalline information of the modified electrodes are analyzed using various analytical techniques, such as scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and transmission electron microscopy. The Co3O4/Co3(PO4)2/SPCE exhibits robust electrocatalytic activity for lactate detection at neutral pH, yielding a dynamic range of 1 - 80 mM with a sensitivity of 8.3 uA mM-1 cm-2 and a limit of detection of 0.3 mM. The developed sensor offers good selectivity against common electroactive sweat components (ascorbic acid, uric acid, and glucose). The microfluidic integrated sensor is tested dynamically with different concentrations of lactate, yielding a sensitivity of 5.2 uA mM-1 cm-2, with a sensor response time of 35 s. The sensing platform is conveniently attached to the upper limb of an athlete, enabling the collection of eccrine sweat samples both at rest and during exercise. The integrated electrochemical sensor unit facilitates real-time measurement of sweat lactate concentrations in real sweat samples. The developed platform offers significant potential for non-invasive physiological analysis and personalized health tracking. Copper–cobalt nanocomposites supported on carboxylated graphitic carbon nitride (GCN) were developed as a versatile platform for nonenzymatic lactate sensing under neutral conditions. Structural analysis using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy revealed the formation of multiple oxide phases, including CuO, Co3O4, and CuCo2O4, depending on the Cu/Co ratio. Differential pulse voltammetry (DPV) and chronoamperometry (CA) measurements of lactate using the prepared nanocomposite electrode in neutral pH media demonstrated linear detection ranges of 1 to 30 mM with sensitivities of 10.3 nA mM-1 (R2 = 0.978) and 53 nA mM-1 (R2 = 0.98) for DPV and CA, respectively. Mechanistic studies revealed that the DPV signal arises from H2O2 reduction by Cu1+/Cu2+ species during lactate oxidation while the CA response is associated with the reduction of H2O2, generated during lactate oxidation, mediated by bimetal redox couples (Cu1+/Cu2+, Co2+/Co3+). Dissolved oxygen monitoring, using an oxygen meter in CA experiments, confirmed oxygen evolution upon lactate addition, supporting the proposed catalytic reaction pathway. Selectivity experiments against known interfering compounds such as glucose, ascorbic acid, uric acid and urea did not produce measurable currents. The synergistic Cu–Co–GCN heterojunction material thus provides a robust and selective nanozyme platform for lactate detection under physiologically relevant conditions, demonstrating promise for wearable lactate sensors and biomedical diagnostics.