Projects / Programmes
Novel nanopores for sensing applications
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| P310 |
Natural sciences and mathematics |
Proteins, enzymology |
| Code |
Science |
Field |
| 3.04 |
Medical and Health Sciences |
Medical biotechnology |
Nanopores, proteins, model membrane systems, sensorics
Researchers (26)
Organisations (2)
Abstract
Nanopore sensing has become an important high-throughput technology for direct sensing of diverse analytes such as small molecules or larger molecules such as DNA, RNA and proteins. Biological nanopores represent an important group of molecules that enabled sensing and attracted a lot of attention in recent years due to successful application in DNA sequencing. In this project we will identify novel pores and membrane systems that could be used for sensing of small molecules or large polymeric structures such as DNA, RNA or proteins. We will focus on families of natural pores that have superior properties for nanopore sensing applications. We will engineer them to be suitable for sensing applications aimed at characterising peptides and proteins and we will explore novel membrane systems suitable for employment in sensing applications. Our main goal is to develop novel nanopores based on existing pore forming proteins (PFPs) families and not yet explored membrane systems. The use of novel materials in the case of lipid membranes will enable extension of sensing capabilities and development of sensors that could offer supreme functioning even at less optimal conditions found in complex samples (such as serum or environmental samples). The research directions of this project will thus follow these specific objectives: i) Exploring structural and functional properties of nanopores derived from aerolysin-like PFPs and actinoporin protein families; ii) Development of model membrane systems in order to make them useful for sensing applications and formation of model membrane systems (liposomes, nanodiscs); iii) Sensing of analytes by some of the newly developed nanopores and membrane systems. Data on novel nanopore properties in different membrane supports, together with sensing data for various analytes, will provide a solid foundation of optimal membrane environment for future research directions on nanopore sensing with many implications in biomedicine and environmental monitoring.
Significance for science
This project proposal aims to identify novel biological pores that may be used for sensing of small molecules or large polymeric structures such as DNA or proteins. Such sensing can be employed in various applications, from medicine, biotechnology to material science or engineering. In this study, we will focus on two families of natural pores that have superior properties for nanopore sensing applications already in their native, wild-type form. Moreover, we will engineer these pores further at different levels to tune them to be highly suitable for sensing of peptides, proteins and nucleic acids. Furthermore, we will also explore novel membrane systems suitable for employment of these pores in sensing applications. We will explore pores from both classes or pore forming proteins (PFPs), those that form stable beta-barrels in the lipid bilayers (i.e. beta-PFPs), as well as those of a more flexible nature, containing alpha-helical pores (i.e. alpha-PFPs). The use of novel nanopores as well as membrane materials will enable extension of sensing capabilities and development of sensors that could offer supreme functioning even at less optimal conditions found in complex samples (such as serum or environmental samples).
We expect that our research will provide original results at the level of basic science as well as in application. Novel natural pores, their biochemical and biophysical properties as well as structures at the atomic level will give us insight into mechanism of pore formations and thus biological roles of these proteins, many of which are actually toxins. This knowledge will be a crucial stepping-stone for further research into biological as well as medicinal direction. On the other hand, knowing the capabilities, capacities as well as selectivity of these pores to either translocate or bind particular molecules can be used for the purposes of application as stated as several points throughout this proposal. Miniaturisation of sensing devices and point-of-care diagnostic approaches require novel molecular systems. aPFPS and actinoporins, which will be studied within this proposal, represent a novel alternative to existing biological nanopores. Furthermore, biological nanopores have many advantages over solid-state nanopores. They have well-defined properties of the conductive pathway, which can be easily modified by site-directed mutagenesis or by introducing small organic molecules that can serve as adaptors. We believe that their usage, together with more stable lipid membrane mimics, will extend the spectrum of molecules that could be targeted by nanopore sensing applications in the future.
Significance for the country
The project proposal is responsive and aligned with Slovenian and EU scientific development goals and is highly intersectorial. The proposed project follows the research priorities of the Horizon 2020 regarding Future Emerging Technologies, Industrial Technologies and Health. The project is also aligned with the activities within ARRS programme group Molecular Interactions (P10391), where the majority of the activities will be performed.
The project will provide crucial information on important families of pore forming proteins (PFPs), since many members of these families are toxins, which targets are usually eukaryotic organisms, including animals, plants and humans. For example, members of aerolysin-like PFPs are present in many different pathogenic bacteria, such as Aeromonas, Clostridium or Bacillus, where they serve as crucial virulence factors in infection and food poisoning. In the same time, important information arising from this project will open doors for further exploitation of these proteins in medical and sensing applications. We already collaborate with Oxford Nanopore Technologies, UK, which already use biological nanopores in their successful DNA sequencing platforms, and thus can greatly benefit form the knowledge coming out of our studies. Moreover, since such nanopores can be used also in other fields of sensing as well as potentially in development of other systems that are based on conductance or transduction of ions and molecules (i.e. batteries), new technological platforms could arise from our results.
This project will also allow further development and employment of methods and protocols for assessing molecular interactions by using state-of-the-art biophysical equipment. This will further strengthen the role of our research department in the region and Europe in general and the developed approaches will be available for other researchers in Slovenia and abroad. In particular, the local pharmaceutical industry, with which the principal investigator has already established a strong collaboration, will be interested in using them. It will also provide good training opportunities for young scientist, which will be included in all stages of the project.
Most important scientific results
Final report
Most important socioeconomically and culturally relevant results
Final report
