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- Block, Stephan, 1978, et al.
(author)
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Two-dimensional flow nanometry of biological nanoparticles for accurate determination of their size and emission intensity
- 2016
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 7, s. art no 12956 -
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Journal article (peer-reviewed)abstract
- Biological nanoparticles (BNPs) are of high interest due to their key role in various biological processes and use as biomarkers. BNP size and composition are decisive for their functions, but simultaneous determination of both properties with high accuracy remains challenging. Optical microscopy allows precise determination of fluorescence/scattering intensity, but not the size of individual BNPs. The latter is better determined by tracking their random motion in bulk, but the limited illumination volume for tracking this motion impedes reliable intensity determination. Here, we show that by attaching BNPs to a supported lipid bilayer, subjecting them to hydrodynamic flows and tracking their motion via surface-sensitive optical imaging enable determination of their diffusion coefficients and flow-induced drifts, from which accurate quantification of both BNP size and emission intensity can be made. For vesicles, the accuracy of this approach is demonstrated by resolving the expected radius-squared dependence of their fluorescence intensity for radii down to 15 nm.
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| 2. |
- Carlred, Louise M, 1985, et al.
(author)
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Imaging of Amyloid-β in Alzheimer’s disease transgenic mouse brains with Time-of-Flight Secondary Ion Mass Spectrometry using Immunoliposomes
- 2016
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In: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 11:2, s. 1-11
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Journal article (peer-reviewed)abstract
- Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-β (Aβ), a biologically relevant peptide in Alzheimer's disease (AD), in transgenic mouse braintissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aβ. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aβ deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid–protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.
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| 3. |
- Johansson Fast, Björn, 1986
(author)
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Hydrodynamic flow for deterministic sorting of cell-membrane components
- 2016
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Doctoral thesis (other academic/artistic)abstract
- The biological membrane, an amphiphilic structure that is the barrier between the cell interior and exterior, is one of the vital building blocks of all cells. Not only does it define the outer boundaries of the cell, it also carries important biological function by virtue of the proteins and other molecules that constitute the membrane, the function of which is in turn intimately coupled to their association with the lipid-based membrane.That the function of the protein is linked to its amphiphilicity necessitates preservation of the amphiphilic environment when probing the function of membrane associated proteins. The low expression levels of, even overexpressed, membrane proteins in a cell membrane crowded with many different types of proteins presents a barrier for direct studies of this class of proteins in their native membrane environment.In the work leading up to this thesis, the aim has been to overcome some of these hurdles and enable membrane protein accumulation and purification in a near native cell membrane. In the articles appended to the thesis, steps have been taken towards being able to move, concentrate, purify and in the end visualize single membrane proteins, using a combination of surface-sensitive imaging, microfluidics and hydrodynamic flow. The last development led to the insight that using this approach we were able to determine the exact size of nanometer-sized objects bound to the two-dimensional interface that is the supported membrane by measuring both the nanoparticles' deterministic and stochastic movement.Looking forward, this thesis work has provided a solid foundation for deterministic sorting of membrane proteins, without the need for detergent solubilization. While this in itself is rather enticing, the possibility to simultaneously determine both the size and the biomolecular content of biological nanoparticles, as demonstrated in the final paper, might help telling whether it is the size, the amount of a specific molecule, or a precise combination of the two, that is decisive for their biological function, such as; infectivity, gene transfer or drug delivery in the context of virions, exosomes and nanoscale drug carriers, respectively.
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