Microfluidic devices are widely used for the sensing of small quantities of analytes. In these applications, the measurement can be easily perturbed by loss of analyte due to binding of the analyte outside the sensing area. We studied the binding of small molecules and nanoparticles up to 400 nm in a state-of-the-art sensing platform – receptor gradients on supported lipid bilayers (SLBs) – in a microfluidic device over time. Biotin-streptavidin was used as the model interaction motif for specific binding and a biotin-modified dye, which can bind to the streptavidin on the SLB, as a small-molecule model analyte. We used finite element simulations to show that the time-dependent binding of analytes in the sensing area depends strongly on the extent of the nonspecific binding of the vesicles, used in a preceding step to make the SLB platform, outside of the sensing area (e. g., in the tubing). At sufficiently high flow rates, proteins and nanoparticles were only partially depleted by nonspecifically adsorbed lipids, and no delayed onset of binding was observed, because of their lower diffusion coefficients. As a practical solution, a flow cell with two inlets was used to avoid the presence of nonspecifically adsorbed receptors in the sample inlet, which allowed us to decouple the formation of the sensor layer on the surface from the subsequent sensing event. We found that in the absence of lipids adsorbed to the tubing, the nonspecific binding of dye molecules was negligible.