Label-Free Detection of Transport Kinetics and Inhibitor Binding of Membrane Transport Proteins with a Two-Mode Plasmonic Sensor

Membrane transporters translocate molecules across cell membranes and are involved in a variety of cellular processes. However, there is still no label-free technique for measuring the transport kinetics of non-charged species through membrane transporters. Here, we develop a new method based on surface plasmon resonance (SPR) and plasmon-waveguide resonance (PWR) to measure transporter function and ligand binding simultaneously. The PWR/SPR-combined sensor chip comprises a silica layer with micron-sized pores on a thin gold film, and the geometry allows the appearance of two independent plasmonic modes. The electromagnetic enhancement regions of the SPR-associated mode and the PWR-associated mode are localized inside the pore directly above the gold film and outside the pore above the silica layer, respectively, and can be used to detect refractive index or environmental changes in the corresponding regions. We deposited cell membrane vesicles onto the sensor chip to form pore-spanning cell membrane patches containing transporters of interest. Only species transported across the membrane can enter into the pore region and be detected by the SPR-associated mode, while ligand binding above the membrane can be detected by the PWR-associated mode. We used glucose transporters in HeLa cell membranes to demonstrate that the platform can be used to study how inhibitor binding can influence transport behaviors. More importantly, we successfully measured the glucose transport kinetics across the membrane. The obtained kinetic parameters of glucose transporters were comparable to those reported in studies using radiolabeled glucose, suggesting that this new label-free method can accurately characterize the membrane transport kinetics.