Using 1-D Spatially Resolved Silicon-Nanowire-Field-Effect-Transistor (SiNW-FET) Platform to Characterize Cell Membrane Proteins in Their Native Bilayer Environment.
Cell membranes are the gateway for the cells to send and receive signals. More than 60% of commercial drugs interact with membrane proteins to alter cellular biochemical pathways. Since membrane proteins are primary pharmaceutical targets, information about what ligands can bind to them is useful to facilitate drug discovery and development. Most of the current methods to identify the ligand binding to membrane proteins rely on immunodetection. However, immunodetection methods may alter biomolecular interactions. Here, we develop a label-free detection method based on field effect transistor (FET) technique, which can provide rapid and sensitive responses to the ligand binding. Supported lipid bilayers (SLBs) have been widely used as platforms for studying interactions between ligands and membrane proteins in vitro. To build a FET detection platform combined with SLBs, we chose graphene as the FET semiconductive material because the two-dimensionality is compatible with the planar bilayer platform and it has high sensitivity. We fabricated graphene field effect transistor (GFET) on silica substrates and investigated the formation of SLBs on the graphene/silica substrate. We discovered that the poly(methyl methacrylate) (PMMA) residues left on graphene surface can significantly influence the formation of SLBs on graphene and applying an extra annealing step to further clean the graphene surface is required for the robust SLB formation. In addition, because the fluorescent dyes in the SLB above graphene were almost completely quenched by graphene, which hinders us from using the conventional fluorescence recovery after photobleaching (FRAP) method to verify the formation of SLBs, we developed a modified FRAP method to examine whether SLBs successfully formed on the graphene/silica substrate. Furthermore, we discovered that ions in a regular buffer can gradually adsorb to the graphene surface and cause the Dirac point to shift significantly with time during our ligand binding detection. With careful control of the history of buffer addition and flow duration in a microfluidic device, we were able to eliminate the response caused by the ion adsorption and obtained the response only from the ligand binding. We used streptavidin-biotin-lipid as a model ligand-receptor to examine the GFET response to ligand binding on the SLB. Our results show that the SLB-GFET is capable to detect the streptavidin molecules binding onto the biotin-lipids embedded in the SLB, and the consistency between the electrical response and the fluorescent quantification result shows the quantitative detection capability of our GFET device. Incorporating this platform with the technique to obtain membrane vesicles from E. coli could allow us to study the interactions between potential drug candidates and interested membrane proteins in their native-like environment.