Projects & Collaborations 12 foundShow per page10 10 20 50 Microfluidic sample preparation for high-resolution structure determination by cryogenic electron microscopy Research Project | 1 Project MembersElectron microscopy (EM) introduced a fast and lasting change to structural and cellular biology. Direct electron detector cameras and improved image processing algorithms now allow structure determination of large biomolecules by cryogenic EM (cryo-EM) at atomic resolution using a single particle approach. For the latter, only thousands to a few million particles must be imaged, to measure high-resolution structures. This amount of protein can be delivered by microfluidic technologies. I this project we explore to the potential of combining microfluidics and electron microscopy to study protein structures for drug discovery. Microfluidic Sample Preparation for High-Resolution Electron Microscopy, Visual Proteomics and Electron Tomography Research Project | 2 Project MembersElectron microscopy (EM) introduced a fast and lasting change to structural and cellular biology. Direct electron detector cameras and improved image processing algorithms now allow structure determination of large biomolecules by cryogenic EM (cryo-EM) at atomic resolution using a single particle approach. Strategies to study the cellular ultrastructure, such as electron tomography (ET), correlative light and electron microscopy (CLEM), and the lamella milling of eukaryotic cells, opened new windows allowing biologists to study the mechanism of cellular processes at unprecedented precision. Unfortunately, sample preparation remains a bottleneck, and, surprisingly, EM is rarely used as a bioanalytical tool despite its immense potential to detect proteins on the single-molecule level. Both (i) new single-cell analysis tools and (ii) advanced methods for protein isolation and cryo-EM sample preparation are urgently needed. Here, we aim at (i) the development of a versatile system for the fast protein production, protein isolation, and cryo-EM sample preparation for the structural analysis of sensitive protein complexes, (ii) the development of a new targeted and untargeted single-cell analysis method named "single-cell visual proteomics," and (iii) the development of a new strategy for the blotting-free cryopreservation of eukaryotic cells to study cellular structures by ET. NanoWrite start-up financing Research Project | 3 Project MembersWith the Nobel prize in 2017, cryogenic electron microscopy (cryo-EM) got much attention. However, sample preparation for cryo-EM did not evolve during the last 30 years and is seen as the major methodological bottleneck. The classical preparation methods need large volumes, lose 99.99 % of the sample, are di cult to control and pose harmful conditions to the biological material during the preparation process. During the last years, we developed strategies, which overcome these shortcomings of the current state-of-the-art methods. An innovative combination of microfluidic technologies enables us to prepare EM samples from nanoliter (nL) sized volumes. The technique works virtually lossless, the preparation conditions can be precisely controlled, and harmful processing steps are eliminated. These methods now allow us to perform experiments not possible before, e.g., we can use our instrument to 'pick' an individual cell and prepare its lysate for EM analysis. Additionally, we can directly couple EM sample preparation to a novel protein purification technology, which allows protein isolation from few μLs of cell lysate, direct cryo-EM grid preparation, and subsequent high-resolution structural analysis. This technology permits the development of a novel multiplexed protein detection platform for a wide spectrum of biomedical and diagnostic applications. MiPIS - Microfluidic Protein Isolation, Stabilization and cryo-EM Preparation for High-Resolution Structural Analysis Research Project | 3 Project MembersIm Projekt MiPIS entwickeln Wissenschaftler des C-CINA (Biozentrum, Universität Basel) und der Hochschule für Life Sciences (FHNW) zusammen mit ihrem Industriepartner leadXpro (Villigen, AG) ein mikrofluidisches System für die Aufarbeitung und Probenvorbereitung von Proteinen, die mittels Kryo-Elektronenmikroskopie analysiert werden sollen. Dr. Thomas Braun vom C-CINA leitet das Projekt, das auf vorhergehenden, vom SNI unterstützen Arbeiten aufbaut. Kryo-Elektronenmikroskopie (Kryo-EM) ist heute als Standardverfahren etabliert, um die atomare Struktur komplexer Proteine aufzuklären, die beispielsweise erforderlich ist, um neue Wirkstoffe für Medikamente zu entwickeln. Im Vergleich zu anderen Verfahren benötigt die Kryo-EM deutlich geringere Proteinmengen von nur einigen Nanolitern, liefert jedoch präzise Bilder in atomarer Auflösung. Die klassischen Methoden zur Aufarbeitung von Proteine werden den Anforderungen der Kryo-EM nicht immer gerecht, da sie zeitaufwendig sind, grössere Proteinmengen benötigen und teilweise die räumliche Anordnung der Proteinkomplexe zerstören. Die Wissenschaftler im Projekt MiPIS setzen nun auf die Verwendung von Mikrofluidsystemen für die Probenaufarbeitung und erhoffen sich deutliche Vorteile gegenüber den klassischen Methoden. Am C-CINA wurde bereits ein Mikrofluid-System entwickelt, mit dem Proben direkt auf einen für die Elektronenmikroskopie benötigen Objektträger platziert werden. Innerhalb des Argovia-Projektes MiPIS soll dieses System nun weiterentwickelt werden, sodass Proteine in dem System innerhalb von zwei Stunden gereinigt, stabilisiert und unter Beibehaltung ihrer räumlichen Struktur für die Kryo-EM-Analyse vorbereitet werden können. Nanomechanical mass and viscosity measurement platform for cell imaging Research Project | 3 Project MembersDuring recent years we developed a real-time nanomechanical sensing platform to measure liquid viscosities and fluid densities based on cantilever technology, decreasing time and user interaction required for such measurements. It features (i) a droplet-generating automatic sampler for two-phase microfluidics producing microliter sample plugs, (ii) a microfluidic measurement cell containing the microcantilever sensors, (iii) dual phase-locked loop frequency tracking of a higher-mode resonance to achieve millisecond time resolution, and (iv) signal processing to extract the resonance parameters, namely the eigenfrequency and quality factor. By applying a hydrodynamic model, viscosity and density of the small liquid droplets can be measured with high temporal resolution. The platform uses an optical actuation and read-out system, which mechanically separates the transducer platform from the measurement chamber. We now will exchange the cantilevers with fully clamped Si 3 N 4 -windows. By using such a membrane-resonator at different vibrational modes, it will be possible to calculate the mass and viscosity distribution on the membrane surface. Such a platform can be used for the imaging of cell growth. Fast protein-complex isolation, sample preparation and data processing for high-resolution structural analysis and visual proteomics Research Project | 2 Project MembersMajor bottlenecks in structure determination of macromolecules by cryo-EM are: (i) the protein complexes must be produced in significant amounts for subsequent structural analysis. Unfortunately, many protein complexes of (biomedical) interest are sparsely produced in eukaryotic cells. (ii) The destabilization of complexes during isolation must be countered. Protein assemblies are significantly diluted on isolation and their inter-molecular interactions are destabilized. This leads to the dissociation of many complexes formed transiently during biological processes. (iii) The data analysis of heterogeneous samples, as they arise due to the stochastic interaction networks, is still cumbersome. Nevertheless, precisely these "interactomes" are of great interest in biological research. The above difficulties can be avoided by, first, minimizing the overall sample consumption, and, second, by reducing the time required to isolate the target complexes and prepare samples for cryo-EM. Protein isolation should be fast (one step, approx. 1h) and produce samples clean enough for imaging by cryo-EM. Ideally, the protocol should be independent of protein modifications, such as tags, as these can interfere with the biological function and assembly of the target complex. Additionally, stabilization of the complex by new cross-linking methods might be desirable directly after cell lysis. Last but not least, sample preparation for cryo-EM should be accomplished in a loss-less manner: to date, more than 99% of the protein is lost during blotting steps when classical cryo-EM grid preparation methods are used. This project aims (i) to establish a method to rapidly extract target proteins and their complexes from minimal amounts of cell lysate, and, (ii) to develop a loss-less cryo-EM grid preparation system that only consumes minute amounts of sample (5 nl) and does not involve any blotting steps. In this framework we will also explore the use of microfluidic cross-linking strategies to stabilize protein-complexes. In addition, methods for structural analysis by the single particle cryo-EM approach and de novo identification of complex subunits or interaction partners will be developed and tested on a single particle level ("visual proteomics"). Major bottlenecks in structure determination of macromolecules by cryo-EM are: (i) the protein complexes must be produced in significant amounts for subsequent structural analysis. Unfortunately, many protein complexes of (biomedical) interest are sparsely produced in eukaryotic cells. (ii) The destabilization of complexes during isolation must be countered. Protein assemblies are significantly diluted on isolation and their inter-molecular interactions are destabilized. This leads to the dissociation of many complexes formed transiently during biological processes. (iii) The data analysis of heterogeneous samples, as they arise due to the stochastic interaction networks, is still cumbersome. Nevertheless, precisely these "interactomes" are of great interest in biological research. The above difficulties can be avoided by, first, minimizing the overall sample consumption, and, second, by reducing the time required to isolate the target complexes and prepare samples for cryo-EM. Protein isolation should be fast (one step, approx. 1h) and produce samples clean enough for imaging by cryo-EM. Ideally, the protocol should be independent of protein modifications, such as tags, as these can interfere with the biological function and assembly of the target complex. Additionally, stabilization of the complex by new cross-linking methods might be desirable directly after cell lysis. Last but not least, sample preparation for cryo-EM should be accomplished in a loss-less manner: to date, more than 99% of the protein is lost during blotting steps when classical cryo-EM grid preparation methods are used. This project aims (i) to establish a method to rapidly extract target proteins and their complexes from minimal amounts of cell lysate, and, (ii) to develop a loss-less cryo-EM grid preparation system that only consumes minute amounts of sample (5 nl) and does not involve any blotting steps. In this framework we will also explore the use of microfluidic cross-linking strategies to stabilize protein-complexes. In addition, methods for structural analysis by the single particle cryo-EM approach and de novo identification of complex subunits or interaction partners will be developed and tested on a single particle level ("visual proteomics"). Single cell nanoanalytics Research Project | 4 Project MembersThe stochastic nature of biological systems inherently leads to heterogeneous cell populations. To date, most bioanalytical methods measure cell assembly averages, obscuring the cell heterogeneity and the biomolecular interaction networks. Therefore, single cell investigation is crucial for future studies. However, single cell analysis is hindered by three main obstacles: First, the tiny amount of analyte, second, the sample conditioning required for the analysis, and third, the difficulty connecting single cell cultivation and the employed detection instrumentation. The specific aim of the SCeNA project is to combine a newly developed single cell culturing and cell lysis device with various bioanalysis methods that characterize different aspects of the cell status (Fig. 1): (A) label and amplification free transcriptomics using nanomechanical viscosity sensors; (B) protein detection by enhanced reverse phase protein microarrays; (C) visual proteomics by visually analyzing the cytosolic proteins using high-speed atomic force microscopy; (D) metabolomics by mass spectrometry. SCeNA combines micro-technological, "nanoenhanced" classical and novel nanomechanical methods for cell culturing, single cell lysis and analysis. Targeted single cell proteomics using magnetic nanoparticles to study prion-like spreading of amyloid nanoparticles Research Project | 2 Project MembersStereotypic spreading of protein aggregation through the nervous system is a hallmark of many neurodegenerative diseases. This was demonstrated for Alzheimer's disease (AD, amyloid-β & tau protein) and Parkinson's disease (PD, α-synuclein, or α-syn). α-syn is a natively unfolded, presynaptic protein of unknown function and unusual conformational plasticity. Evidence accumulates that progression of synucleinopathies not only involves transmission of simple "protein misfolding" but rather specific "structural information" from one cell to the next, leading to the progressive proliferation of "structural α-syn strains". It is now suspected, that different forms of α-syn inclusions lead to different phenotypes of neurodegeneration, i.e., lead to different synucleinopathy diseases, such as PD, or Dementia with Lewy body disease. To date the patho-mechanisms are unknown, but a prion-like transmission via an intrusion of protein nanoparticles imprinting their specific folding onto native host proteins is most likely. Today's biophysical (e.g., mass spectrometry) methods can trace the presence of proteins, but do not allow detecting and monitoring the structural arrangement of the involved assemblies. In this project we will develop a novel method for single cell analyses, not only detecting proteins, but also providing structural information. This approach is geared for the study of "structural strains" of neurodegerative diseases. Project goals are: (i) Development of a targeted proteomics and electron microscopy (EM) labelling method for single cell analysis using magnetic nano particles. (ii) Study the prion-like mechanism of the spreading of α-syn nanoparticles from diseased cells to healthy cells. Identify determinants defining different structural strains of α-syn. Microfluidics to study nano-crystallization of proteins Research Project | 2 Project MembersFor high-resolution x-ray crystallography, two bottlenecks must be overcome: First, enough protein of suitable quality must be produced, and, second, three-dimensional high-resolution crystals of sufficient size must be obtained. Crystal growth is kinetically and thermodynamically controlled and exhibits two phases, i.e. nucleation and crystal extension. The latter increases the size of nuclei to macroscopic dimensions. However, the optimal conditions for nucleation and crystal extension are in general different, making it notoriously difficult to obtain crystals of suitable size. Crystal nuclei or nano-crystals are tiny and assembled from just a few hundred proteins. They can be analyzed with electron microscopy (EM) by imaging and electron nano-diffraction (ND), but are too small and decay too fast for the analysis by classical X-ray beam-lines. However, the high intensity of femto-second x-ray free-electron (FEL) pulses will allow diffraction patterns to be recorded before the sample damage occurs. Real-time viscosity sensors for label-free and functionalization-free characterization of molecular interactions Research Project | 2 Project MembersThis project targets the development of new bio-sensing platforms and methods for detection of molecular interactions. In contrast to other state-of-the-art bio-sensors, the proposed approach allows measuring bio-interactions without the need for labeling targeted bio-molecules or decorating the sensor surface with appropriate receptors. This research project has a considerable interest in both basic research (systems biology) and pharmaceutics (drug screening, diagnostics). We propose to use micro-viscometers (e.g. cantilevers operated in a dynamic oscillation mode) in combination with a micro-fluidic system to measure changes in (intrinsic) viscosity induced by the binding of molecules. This allows detecting molecular interactions in a label-free and a functionalization-free manner. The (intrinsic) viscosity of a solution depends on the shape, surface charge and molecular weight of the solved molecules. If two molecules (say A and B) bind to each other to form a new complex (having a combined molecular weight and shape), the intrinsic viscosity changes. Since the intrinsic viscosity is an additive quantity, comparing the intrinsic viscosities of A, B and A+B allows the detection of the interaction between A and B. 12 12
Microfluidic sample preparation for high-resolution structure determination by cryogenic electron microscopy Research Project | 1 Project MembersElectron microscopy (EM) introduced a fast and lasting change to structural and cellular biology. Direct electron detector cameras and improved image processing algorithms now allow structure determination of large biomolecules by cryogenic EM (cryo-EM) at atomic resolution using a single particle approach. For the latter, only thousands to a few million particles must be imaged, to measure high-resolution structures. This amount of protein can be delivered by microfluidic technologies. I this project we explore to the potential of combining microfluidics and electron microscopy to study protein structures for drug discovery.
Microfluidic Sample Preparation for High-Resolution Electron Microscopy, Visual Proteomics and Electron Tomography Research Project | 2 Project MembersElectron microscopy (EM) introduced a fast and lasting change to structural and cellular biology. Direct electron detector cameras and improved image processing algorithms now allow structure determination of large biomolecules by cryogenic EM (cryo-EM) at atomic resolution using a single particle approach. Strategies to study the cellular ultrastructure, such as electron tomography (ET), correlative light and electron microscopy (CLEM), and the lamella milling of eukaryotic cells, opened new windows allowing biologists to study the mechanism of cellular processes at unprecedented precision. Unfortunately, sample preparation remains a bottleneck, and, surprisingly, EM is rarely used as a bioanalytical tool despite its immense potential to detect proteins on the single-molecule level. Both (i) new single-cell analysis tools and (ii) advanced methods for protein isolation and cryo-EM sample preparation are urgently needed. Here, we aim at (i) the development of a versatile system for the fast protein production, protein isolation, and cryo-EM sample preparation for the structural analysis of sensitive protein complexes, (ii) the development of a new targeted and untargeted single-cell analysis method named "single-cell visual proteomics," and (iii) the development of a new strategy for the blotting-free cryopreservation of eukaryotic cells to study cellular structures by ET.
NanoWrite start-up financing Research Project | 3 Project MembersWith the Nobel prize in 2017, cryogenic electron microscopy (cryo-EM) got much attention. However, sample preparation for cryo-EM did not evolve during the last 30 years and is seen as the major methodological bottleneck. The classical preparation methods need large volumes, lose 99.99 % of the sample, are di cult to control and pose harmful conditions to the biological material during the preparation process. During the last years, we developed strategies, which overcome these shortcomings of the current state-of-the-art methods. An innovative combination of microfluidic technologies enables us to prepare EM samples from nanoliter (nL) sized volumes. The technique works virtually lossless, the preparation conditions can be precisely controlled, and harmful processing steps are eliminated. These methods now allow us to perform experiments not possible before, e.g., we can use our instrument to 'pick' an individual cell and prepare its lysate for EM analysis. Additionally, we can directly couple EM sample preparation to a novel protein purification technology, which allows protein isolation from few μLs of cell lysate, direct cryo-EM grid preparation, and subsequent high-resolution structural analysis. This technology permits the development of a novel multiplexed protein detection platform for a wide spectrum of biomedical and diagnostic applications.
MiPIS - Microfluidic Protein Isolation, Stabilization and cryo-EM Preparation for High-Resolution Structural Analysis Research Project | 3 Project MembersIm Projekt MiPIS entwickeln Wissenschaftler des C-CINA (Biozentrum, Universität Basel) und der Hochschule für Life Sciences (FHNW) zusammen mit ihrem Industriepartner leadXpro (Villigen, AG) ein mikrofluidisches System für die Aufarbeitung und Probenvorbereitung von Proteinen, die mittels Kryo-Elektronenmikroskopie analysiert werden sollen. Dr. Thomas Braun vom C-CINA leitet das Projekt, das auf vorhergehenden, vom SNI unterstützen Arbeiten aufbaut. Kryo-Elektronenmikroskopie (Kryo-EM) ist heute als Standardverfahren etabliert, um die atomare Struktur komplexer Proteine aufzuklären, die beispielsweise erforderlich ist, um neue Wirkstoffe für Medikamente zu entwickeln. Im Vergleich zu anderen Verfahren benötigt die Kryo-EM deutlich geringere Proteinmengen von nur einigen Nanolitern, liefert jedoch präzise Bilder in atomarer Auflösung. Die klassischen Methoden zur Aufarbeitung von Proteine werden den Anforderungen der Kryo-EM nicht immer gerecht, da sie zeitaufwendig sind, grössere Proteinmengen benötigen und teilweise die räumliche Anordnung der Proteinkomplexe zerstören. Die Wissenschaftler im Projekt MiPIS setzen nun auf die Verwendung von Mikrofluidsystemen für die Probenaufarbeitung und erhoffen sich deutliche Vorteile gegenüber den klassischen Methoden. Am C-CINA wurde bereits ein Mikrofluid-System entwickelt, mit dem Proben direkt auf einen für die Elektronenmikroskopie benötigen Objektträger platziert werden. Innerhalb des Argovia-Projektes MiPIS soll dieses System nun weiterentwickelt werden, sodass Proteine in dem System innerhalb von zwei Stunden gereinigt, stabilisiert und unter Beibehaltung ihrer räumlichen Struktur für die Kryo-EM-Analyse vorbereitet werden können.
Nanomechanical mass and viscosity measurement platform for cell imaging Research Project | 3 Project MembersDuring recent years we developed a real-time nanomechanical sensing platform to measure liquid viscosities and fluid densities based on cantilever technology, decreasing time and user interaction required for such measurements. It features (i) a droplet-generating automatic sampler for two-phase microfluidics producing microliter sample plugs, (ii) a microfluidic measurement cell containing the microcantilever sensors, (iii) dual phase-locked loop frequency tracking of a higher-mode resonance to achieve millisecond time resolution, and (iv) signal processing to extract the resonance parameters, namely the eigenfrequency and quality factor. By applying a hydrodynamic model, viscosity and density of the small liquid droplets can be measured with high temporal resolution. The platform uses an optical actuation and read-out system, which mechanically separates the transducer platform from the measurement chamber. We now will exchange the cantilevers with fully clamped Si 3 N 4 -windows. By using such a membrane-resonator at different vibrational modes, it will be possible to calculate the mass and viscosity distribution on the membrane surface. Such a platform can be used for the imaging of cell growth.
Fast protein-complex isolation, sample preparation and data processing for high-resolution structural analysis and visual proteomics Research Project | 2 Project MembersMajor bottlenecks in structure determination of macromolecules by cryo-EM are: (i) the protein complexes must be produced in significant amounts for subsequent structural analysis. Unfortunately, many protein complexes of (biomedical) interest are sparsely produced in eukaryotic cells. (ii) The destabilization of complexes during isolation must be countered. Protein assemblies are significantly diluted on isolation and their inter-molecular interactions are destabilized. This leads to the dissociation of many complexes formed transiently during biological processes. (iii) The data analysis of heterogeneous samples, as they arise due to the stochastic interaction networks, is still cumbersome. Nevertheless, precisely these "interactomes" are of great interest in biological research. The above difficulties can be avoided by, first, minimizing the overall sample consumption, and, second, by reducing the time required to isolate the target complexes and prepare samples for cryo-EM. Protein isolation should be fast (one step, approx. 1h) and produce samples clean enough for imaging by cryo-EM. Ideally, the protocol should be independent of protein modifications, such as tags, as these can interfere with the biological function and assembly of the target complex. Additionally, stabilization of the complex by new cross-linking methods might be desirable directly after cell lysis. Last but not least, sample preparation for cryo-EM should be accomplished in a loss-less manner: to date, more than 99% of the protein is lost during blotting steps when classical cryo-EM grid preparation methods are used. This project aims (i) to establish a method to rapidly extract target proteins and their complexes from minimal amounts of cell lysate, and, (ii) to develop a loss-less cryo-EM grid preparation system that only consumes minute amounts of sample (5 nl) and does not involve any blotting steps. In this framework we will also explore the use of microfluidic cross-linking strategies to stabilize protein-complexes. In addition, methods for structural analysis by the single particle cryo-EM approach and de novo identification of complex subunits or interaction partners will be developed and tested on a single particle level ("visual proteomics"). Major bottlenecks in structure determination of macromolecules by cryo-EM are: (i) the protein complexes must be produced in significant amounts for subsequent structural analysis. Unfortunately, many protein complexes of (biomedical) interest are sparsely produced in eukaryotic cells. (ii) The destabilization of complexes during isolation must be countered. Protein assemblies are significantly diluted on isolation and their inter-molecular interactions are destabilized. This leads to the dissociation of many complexes formed transiently during biological processes. (iii) The data analysis of heterogeneous samples, as they arise due to the stochastic interaction networks, is still cumbersome. Nevertheless, precisely these "interactomes" are of great interest in biological research. The above difficulties can be avoided by, first, minimizing the overall sample consumption, and, second, by reducing the time required to isolate the target complexes and prepare samples for cryo-EM. Protein isolation should be fast (one step, approx. 1h) and produce samples clean enough for imaging by cryo-EM. Ideally, the protocol should be independent of protein modifications, such as tags, as these can interfere with the biological function and assembly of the target complex. Additionally, stabilization of the complex by new cross-linking methods might be desirable directly after cell lysis. Last but not least, sample preparation for cryo-EM should be accomplished in a loss-less manner: to date, more than 99% of the protein is lost during blotting steps when classical cryo-EM grid preparation methods are used. This project aims (i) to establish a method to rapidly extract target proteins and their complexes from minimal amounts of cell lysate, and, (ii) to develop a loss-less cryo-EM grid preparation system that only consumes minute amounts of sample (5 nl) and does not involve any blotting steps. In this framework we will also explore the use of microfluidic cross-linking strategies to stabilize protein-complexes. In addition, methods for structural analysis by the single particle cryo-EM approach and de novo identification of complex subunits or interaction partners will be developed and tested on a single particle level ("visual proteomics").
Single cell nanoanalytics Research Project | 4 Project MembersThe stochastic nature of biological systems inherently leads to heterogeneous cell populations. To date, most bioanalytical methods measure cell assembly averages, obscuring the cell heterogeneity and the biomolecular interaction networks. Therefore, single cell investigation is crucial for future studies. However, single cell analysis is hindered by three main obstacles: First, the tiny amount of analyte, second, the sample conditioning required for the analysis, and third, the difficulty connecting single cell cultivation and the employed detection instrumentation. The specific aim of the SCeNA project is to combine a newly developed single cell culturing and cell lysis device with various bioanalysis methods that characterize different aspects of the cell status (Fig. 1): (A) label and amplification free transcriptomics using nanomechanical viscosity sensors; (B) protein detection by enhanced reverse phase protein microarrays; (C) visual proteomics by visually analyzing the cytosolic proteins using high-speed atomic force microscopy; (D) metabolomics by mass spectrometry. SCeNA combines micro-technological, "nanoenhanced" classical and novel nanomechanical methods for cell culturing, single cell lysis and analysis.
Targeted single cell proteomics using magnetic nanoparticles to study prion-like spreading of amyloid nanoparticles Research Project | 2 Project MembersStereotypic spreading of protein aggregation through the nervous system is a hallmark of many neurodegenerative diseases. This was demonstrated for Alzheimer's disease (AD, amyloid-β & tau protein) and Parkinson's disease (PD, α-synuclein, or α-syn). α-syn is a natively unfolded, presynaptic protein of unknown function and unusual conformational plasticity. Evidence accumulates that progression of synucleinopathies not only involves transmission of simple "protein misfolding" but rather specific "structural information" from one cell to the next, leading to the progressive proliferation of "structural α-syn strains". It is now suspected, that different forms of α-syn inclusions lead to different phenotypes of neurodegeneration, i.e., lead to different synucleinopathy diseases, such as PD, or Dementia with Lewy body disease. To date the patho-mechanisms are unknown, but a prion-like transmission via an intrusion of protein nanoparticles imprinting their specific folding onto native host proteins is most likely. Today's biophysical (e.g., mass spectrometry) methods can trace the presence of proteins, but do not allow detecting and monitoring the structural arrangement of the involved assemblies. In this project we will develop a novel method for single cell analyses, not only detecting proteins, but also providing structural information. This approach is geared for the study of "structural strains" of neurodegerative diseases. Project goals are: (i) Development of a targeted proteomics and electron microscopy (EM) labelling method for single cell analysis using magnetic nano particles. (ii) Study the prion-like mechanism of the spreading of α-syn nanoparticles from diseased cells to healthy cells. Identify determinants defining different structural strains of α-syn.
Microfluidics to study nano-crystallization of proteins Research Project | 2 Project MembersFor high-resolution x-ray crystallography, two bottlenecks must be overcome: First, enough protein of suitable quality must be produced, and, second, three-dimensional high-resolution crystals of sufficient size must be obtained. Crystal growth is kinetically and thermodynamically controlled and exhibits two phases, i.e. nucleation and crystal extension. The latter increases the size of nuclei to macroscopic dimensions. However, the optimal conditions for nucleation and crystal extension are in general different, making it notoriously difficult to obtain crystals of suitable size. Crystal nuclei or nano-crystals are tiny and assembled from just a few hundred proteins. They can be analyzed with electron microscopy (EM) by imaging and electron nano-diffraction (ND), but are too small and decay too fast for the analysis by classical X-ray beam-lines. However, the high intensity of femto-second x-ray free-electron (FEL) pulses will allow diffraction patterns to be recorded before the sample damage occurs.
Real-time viscosity sensors for label-free and functionalization-free characterization of molecular interactions Research Project | 2 Project MembersThis project targets the development of new bio-sensing platforms and methods for detection of molecular interactions. In contrast to other state-of-the-art bio-sensors, the proposed approach allows measuring bio-interactions without the need for labeling targeted bio-molecules or decorating the sensor surface with appropriate receptors. This research project has a considerable interest in both basic research (systems biology) and pharmaceutics (drug screening, diagnostics). We propose to use micro-viscometers (e.g. cantilevers operated in a dynamic oscillation mode) in combination with a micro-fluidic system to measure changes in (intrinsic) viscosity induced by the binding of molecules. This allows detecting molecular interactions in a label-free and a functionalization-free manner. The (intrinsic) viscosity of a solution depends on the shape, surface charge and molecular weight of the solved molecules. If two molecules (say A and B) bind to each other to form a new complex (having a combined molecular weight and shape), the intrinsic viscosity changes. Since the intrinsic viscosity is an additive quantity, comparing the intrinsic viscosities of A, B and A+B allows the detection of the interaction between A and B.