Abstract
Cytokines play a vital role in immune system signaling, making their detection crucial for continuous health monitoring. Among the various cytokines, tumor necrosis factor-alpha (TNF-α) stands out as a key regulator of the immune response. Notably, TNF-α can be detected in sweat at concentrations as low as pg/mL, with levels strongly correlated with those in blood. Despite its importance, sensitive, wearable, and continuous monitoring of TNF-α in sweat remains limited. To address this gap, this study presents a flexible electrochemical sensor integrated into a microfluidic system for the sensitive and selective detection of TNF-α under continuous sweat flow. First, we present the fabrication of two distinct, miniaturized designs of flexible screen-printed carbon three-electrode platforms, which are subsequently biofunctionalized with gold nanoparticles (AuNPs) coated with TNF-α-specific thiolated aptamers. Next, we compare the two geometrically distinct AuNP-aptamer-functionalized sensors, utilizing experimental and novel simulation-based characterization techniques. Finally, the sensors are integrated into a custom-built microfluidic system enabling the detection of TNF-α ranging from 0.2 to 1000 pg/mL under constant artificial sweat flow conditions, exhibiting high selectivity with negligible responses to non-specific analytes. These findings highlight the feasibility of integrating wearable cytokine sensors for detecting TNF-α under continuous sweat flow conditions, achieving clinically relevant sensitivity within the pg/mL range.