Platforms for nanopore membrane sensing

Our aim is to develop a chip-based platform for the investigation of membrane protein interaction and function. Drug screening has in recent decades suffered both a rapidly increasing cost per successfully developed drug and a decreasing success rate in number of developed drugs. Part of this problem is that drugs increasingly target complex functions manifested by membrane proteins, for which tools both for efficient research and high-throughput screening are missing. More than 50% of all drug targets are located in the cell membrane. We will make use of recent advances in the fabrication of sensors using nanotechnology that enable controlling the assembly of artificial cell membranes, including membrane proteins, so that their interactions with drug molecules can be investigated down to the single protein level. This is possible through collaboration between leading scientists at Brno University of Technology in fabricating nanoscale sensors and at University of Natural Resources and Life Sciences, Vienna, in controlling the molecular assembly of cell membrane components. By creating sensor elements that confine light to volumes corresponding to molecular and cell membrane length scale, we can study how membrane and drug targets change as they interact with drugs and other molecules. By making nanoscale pores in chips we can create stable artificial cell membranes with components from real cells, but investigate them with the most sensitive and information rich biosensors available to researchers, including transport of ions and molecules across the membrane. We aim to reach both fast measurements on many membrane components and measurements sensitive and local enough to study single proteins. A crucial advance that we will make use of to reach these goals is the ability to create brushes of polymers on nanoscale sensor elements that control where membrane molecules bind and move. Membrane proteins that are potential drug targets will be guided to areas where they behave as if in a real cell membrane but are close to a sensor element and can be investigated sensitively and in real time. Thus, the converging sciences of nanobiotechnology allow us to develop methods simultaneously for next generations devices for molecular biology research and for drug testing.

Nanostructures are increasingly used for high-tech applications in medicine, diagnostics, drug delivery, and medical research. They provide advantages such as extremely sensitive sensors capable of sensing and identifying single molecules, targeted imaging down to the molecular level in the body, and smart structures that can release drugs or capture pollutants on demand. Nanostructures in contact with biological systems require coatings that prevent biomolecules such as proteins and lipids from binding to them to achieve these functions. If biomolecules freely bind to their surfaces, the nanostructures will rapidly aggregate, lose their sensitivity and other desired properties. Providing the nanostructures with molecular coatings is the best way we know to prevent biomolecule adsorption. These coatings mimic the polymer and lipid coatings of biological surfaces, such as cell membranes known to prevent random biomolecule adsorption but allow for the specific binding of targeted biomolecules. In this project, we managed to apply methods with the highest sensitivity yet to capture the details of which type of proteins bind to polymer-functionalized nanostructures and why they do it. By measuring the heat required to displace water from the particle surfaces as proteins bound, which could find the most likely proteins to bind and the design of the polymer shell most likely to prevent them from binding. In doing so, we could demonstrate that polymers that form loops densely grafted to the nanostructure surface were the only so-called polymer brushes that could entirely prevent proteins from binding. In the future, we plan to use this finding to functionalize nanoparticles in a new sensor platform capturing light in pores smaller than cells. This sensor will allow us to detect molecules secreted by bacteria even in "dirty" environments where such sensors cannot operate today, e.g., in blood, bioreactors, or other biofluids. Our Czech partners developed these sensor chips within this project, but they were not yet functionalized and tested to detect bacteria. Further, we showed that the same platforms and methods could be used to address diverse applications such as extraction of heavy metal contaminants from solution and functionalize surfaces with membranes mimicking cell membranes but borrowing the robust properties of synthetic polymers.