171 research outputs found
Design of Surface Modifications for Nanoscale Sensor Applications
Nanoscale biosensors provide the possibility to miniaturize optic, acoustic and electric sensors to the dimensions of biomolecules. This enables approaching single-molecule detection and new sensing modalities that probe molecular conformation. Nanoscale sensors are predominantly surface-based and label-free to exploit inherent advantages of physical phenomena allowing high sensitivity without distortive labeling. There are three main criteria to be optimized in the design of surface-based and label-free biosensors: (i) the biomolecules of interest must bind with high affinity and selectively to the sensitive area; (ii) the biomolecules must be efficiently transported from the bulk solution to the sensor; and (iii) the transducer concept must be sufficiently sensitive to detect low coverage of captured biomolecules within reasonable time scales. The majority of literature on nanoscale biosensors deals with the third criterion while implicitly assuming that solutions developed for macroscale biosensors to the first two, equally important, criteria are applicable also to nanoscale sensors. We focus on providing an introduction to and perspectives on the advanced concepts for surface functionalization of biosensors with nanosized sensor elements that have been developed over the past decades (criterion (iii)). We review in detail how patterning of molecular films designed to control interactions of biomolecules with nanoscale biosensor surfaces creates new possibilities as well as new challenges
Nucleation in mesoscopic systems under transient conditions: Peptide-induced pore formation in vesicles
Attachment of lytic peptides to the lipid membrane of virions or bacteria is often accompanied by their aggregation and pore formation, resulting eventually in membrane rupture and pathogen neutralization. The membrane rupture may occur gradually via formation of many pores or abruptly after the formation of the first pore. In academic studies, this process is observed during interaction of peptides with lipid vesicles. We present an analytical model and the corresponding Monte Carlo simulations focused on the pore formation in such situations. Specifically, we calculate the time of the first nucleation-limited pore-formation event and show the distribution of this time in the regime when the fluctuations of the number of peptides attached to a vesicle are appreciable. The results obtained are used to clarify the mechanism of the pore formation and membrane destabilization observed recently during interaction of highly active alpha-helical peptide with sub-100-nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature. The model proposed and the analysis presented are generic and may be applicable to other meso- and nanosystems
Two-Dimensional Flow Nanometry of Biological Nanoparticles for Accurate Determination of Their Size and Emission Intensity
Biological nanoparticles (BNPs) are of high interest due to their key role in
various biological processes and use as biomarkers. BNP size and molecular
composition are decisive for their functions, but simultaneous determination of
both properties with high accuracy remains challenging, which is a severe
limitation. Surface-sensitive microscopy allows one to precisely determine
fluorescence or 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. We here show that attaching BNPs (specifically, vesicles and
functionalized gold NPs) to a supported lipid bilayer, subjecting them to a
hydrodynamic flow, and tracking their motion via surface-sensitive imaging
enable to determine their diffusion coefficients and flow-induced drift
velocities and to accurately quantify both BNP size and emission intensity. For
vesicles, the high accuracy is demonstrated by resolving the expected
radius-squared dependence of their fluorescence intensity.Comment: 28 pages, 5 figure
Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment
Lipid vesicles immobilized via molecular linkers at a solid support represent a convenient platform for basic and applied studies of biological processes occurring at lipid membranes. Using total internal reflection fluorescence microscopy (TIRFM), one can track such processes at the level of individual vesicles provided that they contain dyes. In such experiments, it is desirable to determine the size of each vesicle, which may be in the range from 50 to 1000 nm. Fortunately, TIRFM in combination with nanoparticle tracking analysis makes it possible to solve this problem as well. Herein, we present the formalism allowing one to interpret the TIRFM measurements of the latter category. The analysis is focused primarily on the case of unpolarized light. The specifics of the use of polarized light are also discussed. In addition, we show the expected difference in size distribution of suspended and immobilized vesicles under the assumption that the latter ones are deposited under diffusion-controlled conditions. In the experimental part of our work, we provide representative results, showing explicit advantages and some shortcomings of the use of TIRFM in the context under consideration, as well as how our refined formalism improves previously suggested approaches
Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions
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
RNA and DNA interactions with zwitterionic and charged lipid membranes — A DSC and QCM-D study
AbstractThe aim of the present study is to establish under which conditions tRNA associates with phospholipid bilayers, and to explore how this interaction influences the lipid bilayer. For this purpose we have studied the association of tRNA or DNA of different sizes and degrees of base pairing with a set of model membrane systems with varying charge densities, composed of zwitterionic phosphatidylcholines (PC) in mixtures with anionic phosphatidylserine (PS) or cationic dioctadecyl-dimethyl-ammoniumbromide (DODAB), and with fluid or solid acyl-chains (oleoyl, myristoyl and palmitoyl). To prove and quantify the attractive interaction between tRNA and model-lipid membrane we used quartz crystal microbalance with dissipation (QCM-D) monitoring to study the tRNA adsorption to deposit phospholipid bilayers from solutions containing monovalent (Na+) or divalent (Ca2+) cations. The influence of the adsorbed polynucleic acids on the lipid phase transitions and lipid segregation was studied by means of differential scanning calorimetry (DSC). The basic findings are: i) tRNA adsorbs to zwitterionic liquid-crystalline and gel-phase phospholipid bilayers. The interaction is weak and reversible, and cannot be explained only on the basis of electrostatic attraction. ii) The adsorbed amount of tRNA is higher for liquid-crystalline bilayers compared to gel-phase bilayers, while the presence of divalent cations show no significant effect on the tRNA adsorption. iii) The adsorption of tRNA can lead to segregation in the mixed 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC)-1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and DMPC–DODAB bilayers, where tRNA is likely excluded from the anionic DMPS-rich domains in the first system, and associated with the cationic DODAB-rich domains in the second system. iv) The addition of shorter polynucleic acids influence the chain melting transition and induce segregation in a mixed DMPC–DMPS system, while larger polynucleic acids do not influence the melting transition in these system. The results in this study on tRNA–phospholipid interactions can have implications for understanding its biological function in, e.g., the cell nuclei, as well as in applications in biotechnology and medicine
Dual-angle interferometric scattering microscopy for optical multiparametric particle characterization
Traditional single-nanoparticle sizing using optical microscopy techniques
assesses size via the diffusion constant, which requires suspended particles in
a medium of known viscosity. However, these assumptions are typically not
fulfilled in complex natural sample environments. Here, we introduce dual-angle
interferometric scattering microscopy (DAISY), enabling optical quantification
of both size and polarizability of individual nanoparticles without requiring a
priori information regarding the surrounding media or super-resolution imaging.
DAISY achieves this by combining the information contained in concurrently
measured forward and backward scattering images through twilight off-axis
holography and interferometric scattering (iSCAT). Going beyond particle size
and polarizability, single-particle morphology can be deduced from the fact
that hydrodynamic radius relates to the outer particle radius while the
scattering-based size estimate depends on the internal mass distribution of the
particles. We demonstrate this by optically differentiating biomolecular
fractal aggregates from spherical particles in fetal bovine serum at the single
particle level
A virus biosensor with single virus-particle sensitivity based on fluorescent vesicle labels and equilibrium fluctuation analysis
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
Determination of Nano-sized Adsorbate Mass in Solution using Mechanical Resonators: Elimination of the so far Inseparable Liquid Contribution
Assumption-free mass quantification of nanofilms, nanoparticles, and
(supra)molecular adsorbates in liquid environment remains a key challenge in
many branches of science. Mechanical resonators can uniquely determine the mass
of essentially any adsorbate; yet, when operating in liquid environment, the
liquid dynamically coupled to the adsorbate contributes significantly to the
measured response, which complicates data interpretation and impairs
quantitative adsorbate mass determination. Employing the Navier-Stokes equation
for liquid velocity in contact with an oscillating surface, we show that the
liquid contribution can be eliminated by measuring the response in solutions
with identical kinematic viscosity but different densities. Guided by this
insight, we used quartz crystal microbalance (QCM), one of the most
widely-employed mechanical resonator, to demonstrate that kinematic-viscosity
matching can be utilized to accurately quantify the dry mass of systems such as
adsorbed rigid nanoparticles, tethered biological nanoparticles (lipid
vesicles), as well as highly hydrated polymeric films. The same approach
applied to the simultaneously measured energy dissipation made it possible to
quantify the mechanical properties of the adsorbate and its attachment to the
surface, as demonstrated by, for example, probing the hydrodynamic stablization
induced by nanoparticles crowding. Finally, we envision that the possibility to
simultaneously determine the dry mass and mechanical properties of adsorbates
as well as the liquid contributions will provide the experimental tools to use
mechanical resonators for applications beyond mass determination, as for
example to directly interrogate the orientation, spatial distribution, and
binding strength of adsorbates without the need for complementary techniques.Comment: 22 pages, 7 figure
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