Determination of binding curves via protein micropatterning in vitro and in living cells.

Quantification of protein interactions in living cells is of key relevance for understanding cellular signaling. With current techniques, however, it is difficult to determine binding affinities and stoichiometries of protein complexes in the plasma membrane. We introduce here protein micropatterning as a convenient and versatile method for such investigations. Cells are grown on surfaces containing micropatterns of capture antibody to a bait protein, so that the bait gets rearranged in the live cell plasma membrane. Upon interaction with the bait, the fluorescent prey follows the micropatterns, which can be readout with fluorescence microscopy. In this study, we addressed the interaction between Lck and CD4, two central proteins in early T-cell signaling. Binding curves were recorded using the natural fluctuations in the Lck expression levels. Surprisingly, the binding was not saturable up to the highest Lck expression levels: on average, a single CD4 molecule recruited more than nine Lck molecules. We discuss the data in view of protein- and lipid-mediated interactions.

Parallel Droplet Deposition via a Superhydrophobic Plate with Integrated Heater and Temperature Sensors.

A simple setup, which is suitable for parallel deposition of homogenous liquids with a precise volume (dosage), is presented. First, liquid is dispensed as an array of droplets onto a superhydrophobic dosage plate, featuring a 3 × 3 array of holes. The droplets rest on these holes and evaporate with time until they are small enough to pass through them to be used on the final target, where a precise amount of liquid is required. The system can be fabricated easily and operates in a highly parallel manner. The design of the superhydrophobic dosage plate can be adjusted, in terms of the hole positions and sizes, in order to meet different specifications. This makes the proposed system extremely flexible. The initial dispensed droplet mass is not significant, as the dosing takes place during the evaporation process, where the dosage is determined by the hole diameter. In order to speed up the evaporation process, microheaters are screen printed on the back side of the dosage plate. To characterize the temperature distribution caused by the microheaters, temperature sensors are screen printed on the top side of the dosage plate as well. Experimental data regarding the temperature sensors, the microheaters, and the performance of the setup are presented, and the improvement due to the heating of the dosage plate is assessed. A significant reduction of the total evaporation time due to the microheaters was observed. The improvement caused by the temperature increase was found to follow a power law. At a substrate temperature of 80 °C, the total evaporation time was reduced by about 79%.