Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor-acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Förster radius R(0) could directly be determined from the distance dependence of energy transfer yielding R(0)=5.3±0.3 nm for the Cy3-Cy5 pair.

Anionic and cationic lipids are key molecules involved in many cellular processes; their distribution in biomembranes is highly asymmetric, and their concentration is well-controlled. Graphene solution-gated field-effect transistors (SGFETs) exhibit high sensitivity toward the presence of surface charges. Here, we establish conditions that allow the observation of the formation of charged lipid layers on solution-gated field-effect transistors in real time. We quantify the electrostatic screening of electrolyte ions and derive a model that explains the influence of charged lipids on the ion sensitivity of graphene SGFETs. The electrostatic model is validated using structural information from X-ray reflectometry measurements, which show that the lipid monolayer forms on graphene. We demonstrate that SGFETs can be used to detect cationic lipids by self-exchange of lipids. Furthermore, SGFETs allow measuring the kinetics of layer formation induced by vesicle fusion or spreading from a reservoir. Because of the high transconductance and low noise of the electrical readout, we can observe characteristic conductance spikes that we attribute to bouncing-off events of lipid aggregates from the SGFET surface, suggesting a great potential of graphene SGFETs to measure the on-off kinetics of small aggregates interacting with supported layers.

Cellular adhesion is a central element in tissue mechanics, biological cell−cell signaling, and cell motility. In this context, the cell−substrate distance has been investigated in the past by studying natural cells and biomimetic cell models adhering on solid substrates. The amount of water in the membrane substrate gap, however, is difficult to determine. Here, we present a neutron reflectivity (NR) structural study of confluent epithelial cell monolayers on silicon substrates. In order to ensure valid in vitro conditions, we developed a cell culture sample chamber allowing us to grow and cultivate cells under proper cell culture conditions while performing in vitro neutron reflectivity measurements. The cell chamber also enabled perfusion with cell medium and hence allowed for contrast variation in situ by sterile exchange of buffer with different H 2 O-to-D 2 O ratio. Contrast variation reduces the ambiguity of data modeling for determining the thickness and degree of hydration of the interfacial cleft between the adherent cells and the substrate. Our data suggest a three-layer interfacial organization. The first layer bound to the silicon surface interface is in agreement with a very dense protein film with a thickness of 9 ± 2 nm, followed by a highly hydrated 24 ± 4 nm thick layer, and a several tens of nanometers thick layer attributed to the composite membrane. Hence, the results provide clear evidence of a highly hydrated intermediate region between the composite cell membrane and the substrate, reminiscent of the basal lamina.