Time‐Dependent Binding of Molecules and Nanoparticles at Receptor‐Modified Supported Lipid Bilayer Gradients in a Microfluidic Device

Abstract

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.

Multivalent Affinity Profiling: Direct Visualization of the Superselective Binding of Influenza Viruses

“…The sensing area in the MAP chip is divided into three positions with a total of 21–48 corrals, depending on the chip layout and the alignment of the microchannel. Two inlets are used to decouple the SLB formation from the binding of receptors and viruses . Using a previously described method, we formed SLBs from vesicles of zwitterionic MPPC (1-myristoyl-2-palmitoyl- sn -glycero-3-phosphocholine) with 0.3–0.8% biotin-DOPE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(cap biotinyl)), which has a negative charge.…”

“…Two inlets are used to decouple the SLB formation from the binding of receptors and viruses . Using a previously described method, we formed SLBs from vesicles of zwitterionic MPPC (1-myristoyl-2-palmitoyl- sn -glycero-3-phosphocholine) with 0.3–0.8% biotin-DOPE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(cap biotinyl)), which has a negative charge. Electrophoretic gradients were formed by applying a potential over the SLBs.…”

“…Flow cells were fabricated according to the procedure described earlier. 40 A bilayer lift-off recipe was used for fabricating Au electrodes on Mempax glass wafers (Schott). First, LOR 5A (MicroChem) was spin-coated, after which normal lithography was performed on top with Olin OiR 907−17 photoresist (FujiFilm) to create a bilayer resist stack.…”

“…39 The resulting gradients were then post-functionalized and used as a receptor density gradient. 33,40 Recently, we introduced a method, called "multivalent affinity profiling" (MAP), to measure the avidity of influenza viruses using receptor density gradients to obtain binding profiles as a function of receptor density. 15 The equilibrium constant of the multivalent virus−surface interaction is a receptor densitydependent quantity reflected in the shape of the binding profile obtained by MAP.…”

“…By using lipids that are in the gel state at room temperature, gradients have been formed at elevated temperatures and locked-in by cooling . The resulting gradients were then post-functionalized and used as a receptor density gradient. , …”