| 1. |
- Bally, Marta, 1981, et al.
(författare)
-
A virus biosensor with single virus-particle sensitivity based on fluorescent vesicle labels and equilibrium fluctuation analysis
- 2013
-
Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Biosensors allowing for the rapid and sensitive detection of viral pathogens in environmental or clinical samples are urgently needed to prevent disease outbreaks and spreading. We present a bioanalytical assay for the detection of whole viral particles with single virus sensitivity. Specifically, we focus on the detection of human norovirus, a highly infectious virus causing gastroenteritis. In our assay configuration, virus-like particles are captured onto a supported lipid bilayer containing a virus-specific glycolipid and detected after recognition by a glycolipid-containing fluorescent vesicle. Read-out is performed after illumination of the vesicle labels by total internal reflection fluorescence microscopy. This allows for visualization of individual vesicles and for recording of their binding kinetics under equilibrium conditions (equilibrium fluctuation analysis), as demonstrated previously. In this work we extend the concept and demonstrate that this simple assay setup can be used as a bioanalytical assay for the detection of virus particles at a limit of detection of 16 fM. Furthermore, we demonstrate how the analysis of the single vesicle-virus-like particle interaction dynamics can contribute to increase the accuracy and sensitivity of the assay by discriminating specific from non-specific binding events. This method is suggested to be generally applicable, provided that these events display different interaction kinetics.
|
|
| 2. |
- Bally, Marta, 1981, et al.
(författare)
-
Fluorescent vesicles for signal amplification in reverse phase protein microarray assays
- 2011
-
Ingår i: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 416:2, s. 145-151
-
Tidskriftsartikel (refereegranskat)abstract
- Developments in microarray technology promise to lead to great advancements in the biomedical and biological field. However, implementation of these analytical tools often relies on signal amplification strategies that are essential to reach the sensitivity levels required for a variety of biological applications. This is true especially for reverse phase arrays where a complex biological sample is directly immobilized on the chip. We present a simple and generic method for signal amplification based on the use of antibody-tagged fluorescent vesicles as labels for signal generation. To assess the gain in assay sensitivity, we performed a model assay for the detection of rabbit immunoglobulin G (IgG) and compared the limit of detection (LOD) of the vesicle assay with the LOD of a conventional assay performed with fluorescent reporter molecules. We evaluated the improvements for two fluorescence-based transduction setups: a high-sensitivity microarray reader (ZeptoREADER) and a conventional confocal scanner. In all cases, our strategy led to an increase in sensitivity. However, gain in sensitivity widely depended on the type of illumination; whereas an approximately 2-fold increase in sensitivity was observed for readout based on evanescent field illumination, the contribution was as high as more than 200-fold for confocal scanning. (C) 2011 Elsevier Inc. All rights reserved.
|
|
| 3. |
- Bally, Marta, 1981, et al.
(författare)
-
Interaction of Single Viruslike Particles with Vesicles Containing Glycosphingolipids
- 2011
-
Ingår i: Physical Review Letters. - : American Physical Society. - 0031-9007 .- 1079-7114. ; 107:18
-
Tidskriftsartikel (refereegranskat)abstract
- Glycosphingolipids are involved in the first steps of virus-cell interaction, where they mediate specific recognition of the host cell membrane. We have employed total-internal-reflection fluorescence microscopy to explore the interaction kinetics between individual unlabeled noroviruslike particles, which are attached to a glycosphingolipid-containing lipid bilayer, and fluorescent vesicles containing different types and concentrations of glycosphingolipids. Under association equilibrium, the vesicle-binding rate is found to be kinetically limited, yielding information on the corresponding activation energy. The dissociation kinetics are logarithmic over a wide range of time. The latter is explained by the vesicle-size-related distribution of the dissociation activation energy. The biological, pharmaceutical, and diagnostic relevance of the study is briefly discussed.
|
|
| 4. |
- Bally, Marta, 1981, et al.
(författare)
-
Interaction of virions with membrane glycolipids
- 2012
-
Ingår i: Physical Biology. - : IOP Publishing. - 1478-3967 .- 1478-3975. ; 9:2
-
Tidskriftsartikel (refereegranskat)abstract
- Cellular membranes contain various lipids including glycolipids (GLs). The hydrophilic head groups of GLs extend from the membrane into the aqueous environment outside the cell where they act as recognition sites for specific interactions. The first steps of interaction of virions with cells often include contacts with GLs. To clarify the details of such contacts, we have used the total internal reflection fluorescence microscopy to explore the interaction of individual unlabelled virus-like particles (or, more specifically, norovirus protein capsids), which are firmly bound to a lipid bilayer, and fluorescent vesicles containing glycosphingolipids (these lipids form a subclass of GLs). The corresponding binding kinetics were earlier found to be kinetically limited, while the detachment kinetics were logarithmic over a wide range of time. Here, the detachment rate is observed to dramatically decrease with increasing concentration of glycosphingolipids from 1% to 8%. This effect has been analytically explained by using a generic model describing the statistics of bonds in the contact area between a virion and a lipid membrane. Among other factors, the model takes the formation of GL domains into account. Our analysis indicates that in the system under consideration, such domains, if present, have a characteristic size smaller than the contact area between the vesicle and the virus-like particle.
|
|
| 5. |
- Bally, Marta, 1981, et al.
(författare)
-
Liposome and lipid bilayer arrays towards biosensing applications
- 2010
-
Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 6:22, s. 2481-2497
-
Tidskriftsartikel (refereegranskat)abstract
- Sensitive and selective biosensors for high-throughput screening are having an increasing impact in modern medical care. The establishment of robust protein biosensing platforms however remains challenging, especially when membrane proteins are involved. Although this type of proteins is of enormous relevance since they are considered in >60% of the pharmaceutical drug targets, their fragile nature (i.e., the requirement to preserve their natural lipid environment to avoid denaturation and loss of function) puts strong additional prerequisites onto a successful biochip. In this review, the leading approaches to create lipid membrane-based arrays towards the creation of membrane protein biosensing platforms are described. Liposomes assembled in micro- and nanoarrays and the successful set-ups containing functional membrane proteins, as well as the use of liposomes in networks, are discussed in the first part. Then, the complementary approaches to create cell-mimicking supported membrane patches on a substrate in an array format will be addressed. Finally, the progress in assembling free-standing (functional) lipid bilayers over nanopore arrays for ion channel sensing will be reported. This review illustrates the rapid pace by which advances are being made towards the creation of a heterogeneous biochip for the high-throughput screening of membrane proteins for diagnostics, drug screening, or drug discovery purposes.
|
|
| 6. |
- Bally, Marta, 1981, et al.
(författare)
-
Norovirus GII.4 Virus-like Particles Recognize Galactosylceramides in Domains of Planar Supported Lipid Bilayers.
- 2012
-
Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 51:48, s. 12020-4
-
Tidskriftsartikel (refereegranskat)abstract
- A sticky situation: Domain-dependent recognition of the glycosphingolipid galactosylceramide by norovirus-like particles (see picture; red/yellow) is shown using supported lipid bilayers (purple) as model membranes. Optimal ligand presentation is found to promote strong binding to GalCer. This presentation can be found at the edges of the glycosphingolipid-enriched domains (green) and binding is repressed in the absence of these domains.
|
|
| 7. |
- de Lange, V., et al.
(författare)
-
Microarrays Made Easy: Biofunctionalized Hydrogel Channels for Rapid Protein Microarray Production
- 2011
-
Ingår i: ACS Applied Materials & Interfaces. - : American Chemical Society (ACS). - 1944-8252 .- 1944-8244. ; 3:1, s. 50-57
-
Tidskriftsartikel (refereegranskat)abstract
- We present a simple, inexpensive, and sensitive technique for producing multiple copies of a hydrogel-based protein microarray. An agarose block containing 25 biofunctionalized channels is sliced perpendicularly to produce many identical biochips. Each microarray consists of 500 mu m spots, which contain protein-coated microparticles physically trapped in porous SeaPrep agarose. Proteins diffuse readily through SeaPrep agarose while the larger microparticles are immobilized in the hydrogel matrix Without major assay optimization the limit of detection is 12 pM for a sandwich assay detecting human IgG. These highly flexible, multiplexed arrays can produced rapidly without any special instrumentation and are compatible with standard fluorescence-based read-out.
|
|
| 8. |
- Gunnarsson, Anders, 1981, et al.
(författare)
-
Time-resolved surface-enhanced ellipsometric contrast imaging for label-free analysis of biomolecular recognition reactions on glycolipid domains
- 2012
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:15, s. 6538-6545
-
Tidskriftsartikel (refereegranskat)abstract
- We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ∼1 Hz with a sensitivity in terms of surface coverage of ∼1 ng/cm2. Despite the significant improvement in spatial resolution compared to alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry. The potential of the technique to discriminate local differences in protein binding kinetics was demonstrated by time-resolved imaging of anti-GalCer antibodies binding to phase-separated lipid bilayers consisting of phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher antibody binding capacity was observed on the GalCer-diluted fluid region in comparison to the GalCer-rich gel phase domains. This observation is tentatively attributed to differences in the presentation of the GalCer epitope in the two phases, resulting in differences in availability of the ligand for antibody binding. The complementary information obtained by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes was used to support the data interpretation. The simplicity and generic applicability of the concept is discussed in terms of microfluidic applications.
|
|
| 9. |
- Kunze, Angelika, 1978, et al.
(författare)
-
Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions
- 2013
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 3
-
Tidskriftsartikel (refereegranskat)abstract
- Carbohydrate-carbohydrate interactions (CCIs) are of central importance for several biological processes. However, the ultra-weak nature of CCIs generates difficulties in studying this interaction, thus only little is known about CCIs. Here we present a highly sensitive equilibrium-fluctuation-analysis of single liposome binding events to supported lipid bilayers (SLBs) based on total internal reflection fluorescence (TIRF) microscopy that allows us to determine apparent kinetic rate constants of CCIs. The liposomes and SLBs both contained natural Le(x) glycosphingolipids (Gal beta 4( Fuc alpha 3) GlcNAc beta 3Gal beta 4Glc beta 1Cer), which were employed to mimic cell-cell contacts. The kinetic parameters of the self-interaction between Le(x)-containing liposomes and SLBs were measured and found to be modulated by bivalent cations. Even more interestingly, upon addition of cholesterol, the strength of the CCIs increases, suggesting that this interaction is strongly influenced by a cholesterol-dependent presentation and/or spatial organization of glycosphingolipids in cell membranes.
|
|
| 10. |
- Movérare-Skrtic, Sofia, et al.
(författare)
-
Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures.
- 2014
-
Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 20:11, s. 1279-88
-
Tidskriftsartikel (refereegranskat)abstract
- The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need.
|
|
| 11. |
- Nasir, Waqas, et al.
(författare)
-
Parvovirus B19 VLP recognizes globoside in supported lipid bilayers
- 2014
-
Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 456, s. 364-369
-
Tidskriftsartikel (refereegranskat)abstract
- Studies have suggested that the glycosphingolipid globoside (Gb4Cer) is a receptor for human parvovirus B19. Virus-like particles bind to Gb4Cer on thin-layer chromatograms, but a direct interaction between the virus and lipid membrane-associated Gb4Cer has been debated. Here, we characterized the binding of parvovirus B19 VP1/VP2 virus-like particles to glycosphingolipids (i) on thin-layer chromatograms (TLCs) and (ii) incorporated into supported lipid bilayers (SLBs) acting as cell-membrane mimics. The binding specificities of parvovirus B19 determined in the two systems were in good agreement; the VLP recognized both Gb4Cer and the Forssman glycosphingolipid on TLCs and in SLBs compatible with the role of Gb4Cer as a receptor for this virus.
|
|
| 12. |
- Rupert, Deborah, 1986, et al.
(författare)
-
Determination of exosome concentration in solution using surface plasmon resonance spectroscopy.
- 2014
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 86:12, s. 5929-5936
-
Tidskriftsartikel (refereegranskat)abstract
- Exosomes are cell-secreted nanometer-sized extracellular vesicles that have been reported to play an important role in intercellular communication. They are also considered potential diagnostic markers for various health disorders, and intense investigations are presently directed towards their use as carriers in drug-delivery and gene-therapy applications. This has generated a growing need for sensitive methods capable of accurately and specifically determining the concentration of exosomes in complex biological fluids. Here, we explore the use of label-free surface-based sensing with surface plasmon resonance (SPR) read-out to determine the concentration of exosomes in solution. Human mast cell secreted exosomes carrying the tetraspanin membrane protein CD63 were analyzed by measuring their diffusion-limited binding rate to an SPR sensor surface functionalized with anti-CD63 antibodies. The concentration of suspended exosomes was determined by first converting the SPR response into surface-bound mass. The increase in mass uptake over time was then related to the exosome concentration in solution using a formalism describing diffusion-limited binding under controlled flow conditions. The proposed quantification method is based on a calibration and control measurements performed with proteins and synthetic lipid vesicles and takes into account i) the influence of the broad size distribution of the exosomes on the surface coverage, ii) the fact that their size is comparable to the ~150 nm probing depth of SPR, and iii) possible deformation of exosomes upon adsorption. Under those considerations, the accuracy of the concentration determination was estimated to be better than ±50% and significantly better if exosome deformation is negligible.
|
|
