Sample volume as a key design parameter in affinity-based biosensors

Affinity-based biosensors have become indispensable in modern diagnostics and health monitoring. While considerable research has focused on optimizing analyte transport and binding kinetics, a fundamental parameter – sample volume – remains largely underexplored in biosensor design. This is critical because biosensor performance depends on the absolute number of target molecules present, not solely their concentration, making volume a key consideration where sample availability is limited. To address this gap, we developed a tractable two-compartment mathematical model integrating simplified mass transport, Langmuir binding kinetics, and mass conservation under finite volume constraints. Validated against experimental measurements and numerical simulations, the model accurately predicts critical performance metrics including assay time and minimum required sample volume while achieving more than a 10,000-fold reduction in computational time compared to commercial simulation packages. Through systematic analysis, we derived quantitative design rules for biosensor optimization that explicitly account for measurement time and sample volume as primary decision variables. We validated this framework experimentally by optimizing flow rate parameters for a quartz crystal microbalance (QCM) biosensor and retrospectively applied it to enhance sensitivity of published biosensor designs. Released as open-source software, our model enables researchers to gain mechanistic insights, optimize device performance, and make informed design decisions tailored to specific healthcare contexts, including point-of-care testing and resource-constrained environments.