What has been done so far?
We have designed, fabricated and tested the performance of different microfluidic devices. The device consists of a functionalized glass slide, which serves for the attachment of the DNA origami nanostructures, and a fluidic chamber made of PDMS with inlet and outlet. Once optimized the performance of the fluidic device, the system for the study of the EGFR clustering on breast cancer cells was set up within the environmental chamber of a TIRF fluorescence microscope. The complete set up includes a valve selector, that allows to select the sample to flow, the fluidic device, the pumps, and the syringes that enable fluidic operation.
“For the first time a fluidic platform to study the effect of receptor clustering in early cell signaling has been developed"
Rectangular origami structures of approximately 60 nm x 100 nm were prepared as molecular pegboards for the assembly of nanoscaled EGF (epidermal growth factor) patterns through streptavidin-biotin bridges. These origami structures possess ssDNA strands protruding from the rectangular construct to enable their binding to complementary, surface-bound capture oligonucleotides on the glass surface. They also contain fluorescent dye labels to allow for their detection by fluorescence microscopy imaging. The ligand-origami constructs were anchored on the glass surface by hybridization with complementary ssDNA capture strands, covalently tethered to the solid surface. This process was then established inside the microfluidic reaction chamber. For implementation of origami assembly into the microfluidic system, the fluidic parameters were optimized to enable the routine preparation of high quality origami-surfaces for cell culture experiments. Once the OMICS platform was established, the activation of the EGFR was studied in breast cancer cells.
For the first time a fluidic platform to study the effect of receptor clustering in early cell signaling has been developed. Further refinement of experimental conditions is currently under way to quantitatively characterize the activation of EGFR and to allow for the detailed benchmarking. Once benchmarked, we envisage that this device will be applied to the analysis and comparison of different cell lines and under the effect of different anticancer drugs. Owing to its robustness, we anticipate that the here developed system can be implemented in other laboratories working in this research field. This project has provided a unique concept to develop a fully integrated and robust assay for addressing fundamental aspects of early stages of cell signaling in living cells. In the future, this platform may be used to study new ways to target cancer cell stimulation by interfering with the receptor clustering process.