Multiplexed Enzymatic Biosensors with On-Chip pH Control for Lactate and pH Monitoring in Calf Saliva Operating Under an IoT Enabled Electronics Interface

Non-invasive biomarker assessment in saliva offers a promising route for early detection of bovine respiratory disease (BRD) and scour in calves. However, accurate electrochemical sensing in this complex matrix remains challenging due to variable pH, viscosity, and interfering species. Here, we report an integrated dual-function interdigitated microelectrode (IDE) platform capable of simultaneous detection of lactate and pH, incorporating a novel in-situ electrochemical pH control strategy that enhances sensor performance in saliva by producing molecular oxygen. Microband Au IDEs were fabricated using multilayer photolithography and metal deposition, followed by sequential surface modification with Pt-black, o-phenylenediamine/β-cyclodextrin (o-PD/ β-CD), and lactate oxidase (LOx) to enable selective and sensitive lactate detection. Finite element simulations confirmed rapid local acidification and oxygen generation at the protonator electrodes, enabling precise pH tuning at the sensing interface. The pH sensor exhibited a linear Nernst-like response from pH 3-9 (-65.9 mV.pH-1, R2 = 0.999), while the lactate biosensor demonstrated a linear range of 0.02-7 mM in artificial saliva, achieving a limit of detection of 0.4 µM with pH control. A portable, battery-powered electronics interface with wireless data transmission was developed and benchmarked against laboratory instrumentation, achieving comparable sensitivities for both lactate (-0.47 nA.mM-1) and pH (-117 mV.pH-1). In calf saliva, the platform accurately distinguished healthy vs. sick animals, consistent with clinical health scoring. This multiplexed, pH-regulated sensing system offers a robust pathway toward field-deployable, non-invasive diagnostic tools for continuous monitoring of animal health and welfare.