Abstract
Continuous biomarker monitoring with affinity biosensors remains limited by probe saturation, slow dissociation, signal carryover, and regeneration methods that require chemical washing, harsh buffers, or external reset modules. Here, we report a flow–programmed, self–regenerative electrochemical aptamer–based biosensor for continuous 17β–estradiol monitoring. The platform restores the sensing interface by tuning buffer flow through the device, enabling repeated sensing–regeneration cycles without chemical stripping or active-reset hardware. The biosensor achieves a 1.46 pM limit of detection, and a log-linear dynamic range from 100 pM to 1 μM in real human sweat, covering physiologically relevant estradiol concentrations. It exhibits high selectivity, with responses to potential molecularly similar interferents remaining below 10% of the 17β–estradiol signal. Under dynamic flow operation, the device shows reversible signal recovery and stable electrochemical performance, with higher buffer–wash flow reducing baseline carryover between measurements. A coupled binding–transport simulation captures the flow-dependent regeneration trends and maps how wash–flow rate, wash duration, and apparent aptamer affinity affect regeneration efficiency. Ex vivo testing in real human sweat, including samples from pregnant women, supports translational relevance. This work establishes buffer-flow programming as a reagent–free route to regenerable electrochemical aptamer sensing for automated, wearable–compatible estradiol monitoring.