ABSTRACT
Integration of optical components into microfluidic devices can enhance particle manipulations, separations, and analyses. We present a method to fabricate microscale diffractive lenses composed of aperiodically spaced concentric rings milled into a thin metal film to precisely position optical tweezers within microfluidic channels. Integrated thin-film microlenses perform the laser focusing required to generate sufficient optical forces to trap particles without significant off-device beam manipulation. Moreover, the ability to trap particles with unfocused laser light allows multiple optical traps to be powered simultaneously by a single input laser. We have optically trapped polystyrene particles with diameters of 0.5, 1, 2, and 4 µm over microlenses fabricated in chromium and gold films. Optical forces generated by these microlenses captured particles traveling at fluid velocities up to 64 µm/s. Quantitative trapping experiments with particles in microfluidic flow demonstrate size-based differential trapping of neutrally buoyant particles where larger particles required a stronger trapping force. The optical forces on these particles are identical to traditional optical traps, but the addition of a continuous viscous drag force from the microfluidic flow introduces tunable size selectivity across a range of laser powers and fluid velocities.
ABSTRACT
Orbital dynamics of a dielectric microparticle in air using a lensed counter-propagating dual-beam trap was studied experimentally and by numerical simulations. Relationships between the dynamic parameters, trap geometry, and optical power were examined both experimentally and computationally. We found that this scheme can provide narrow bandwidth (δν/ν≈10-3) detection that is at least two orders of magnitude below typical values attainable with previously studied geometries. We predict that this characteristic makes the approach suitable for ultrasensitive in-situ detection of particle mass changes. In our experimental conditions, silica microspheres orbited on trajectories spanning tens of µm, at frequencies of up to â¼2kHz, at atmospheric pressure. With the help of simulations, we briefly discuss how the dual-beam lensed orbital trap approach can be further enhanced to gain unmatched capabilities to measure changes in the physical parameters associated with a particle interacting with its surrounding medium.
ABSTRACT
While extensive studies of virus capsid assembly in environments mimicking in vivo conditions have led to an understanding of the thermodynamic driving forces at work, applying this knowledge to virus assembly in other solvents than aqueous buffers has not been attempted yet. In this study, Brome mosaic virus (BMV) capsid proteins were shown to preserve their self-assembly abilities in an aprotic polar solvent, dimethyl sulfoxide (DMSO). This facilitated protein cage encapsulation of nanoparticles and dye molecules that favor organic solvents, such as ß-NaYF4-based upconversion nanoparticles and BODIPY dye. Assembly was found to be robust relative to a surprisingly broad range of DMSO concentrations. Cargos with poor initial stability in aqueous solutions were readily encapsulated at high DMSO concentrations and then transferred to aqueous solvents, where they remained stable and preserved their function for months.
Subject(s)
BromovirusABSTRACT
Non-enveloped RNA viruses pervade all domains of life. In a cell, they co-assemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native one. However, they contain large native structure fragments that can be recycled to form BMV virions, should a viral genome become available. The existence of such structures suggest the possibility of a previously unknown regulatory pathway for the packaging process inside cells.
Subject(s)
Bromovirus , Bromovirus/genetics , Capsid , Capsid Proteins , Cryoelectron Microscopy , RNA, Viral , Virion , Virus AssemblyABSTRACT
Here, we investigate scanning photothermal microspectroscopic imaging of metal nanoparticles with reflective objectives. We show that correction-less collection of spectra from single spherical nanoparticles embedded in a polymer is possible over a wide spectral band, with large depth of focus, long working distance, and high lateral spatial resolution. We posit that these beneficial characteristics are inherent of the Bessel-Gauss character of the focused beam. When compared with other types of optical microscopy, the combination of these characteristics give photothermal imaging with reflective objectives unique appeal for material characterization applications.
ABSTRACT
The precise control of assembly and packing of proteins and colloids on curved surfaces has fundamental implications in nanotechnology. In this paper, we describe dynamical simulations of the self-assembly of conical subunits around a spherocylindrical template, and a continuum theory for the bending energy of a triangular lattice with spontaneous curvature on a surface with arbitrary curvature. We find that assembly depends sensitively on mismatches between subunit spontaneous curvature and the mean curvature of the template, as well as anisotropic curvature of the template (mismatch between the two principal curvatures). Our simulations predict assembly morphologies that closely resemble those observed in experiments in which virus capsid proteins self-assemble around metal nanorods. Below a threshold curvature mismatch, our simulations identify a regime of optimal assembly leading to complete, symmetrical particles. Outside of this regime we observe defective particles, whose morphologies depend on the degree of curvature mismatch. To learn how assembly is affected by the nonuniform curvature of a spherocylinder, we also study the simpler cases of assembly around spherical and cylindrical cores. Our results show that both the intrinsic (Gaussian) and extrinsic (mean) curvatures of a template play significant roles in guiding the assembly of anisotropic subunits, providing a rich design space for the formation of nanoscale materials.
ABSTRACT
A virus binding to a surface causes stress of the virus cage near the contact area. Here, we investigate the potential role of substrate-induced structural perturbation in the mechanical response of virus particles to adsorption. This is particularly relevant to the broad category of viruses stabilized by weak noncovalent interactions. We utilize atomic force microscopy to measure height distributions of the brome mosaic virus upon adsorption from solution on atomically flat substrates and present a continuum model that captures our observations and provides estimates of elastic properties and of the interfacial energy of the virus, without recourse to indentation.
Subject(s)
Bromovirus , Microscopy, Atomic Force , Virion , AdsorptionABSTRACT
Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.
ABSTRACT
UNLABELLED: The three subsets of virions that comprise the Brome mosaic virus (BMV) were previously thought to be indistinguishable. This work tested the hypothesis that distinct capsid-RNA interactions in the BMV virions allow different rates of viral RNA release. Several results support distinct interactions between the capsid and the BMV genomic RNAs. First, the deletion of the first eight residues of the BMV coat protein (CP) resulted in the RNA1-containing particles having altered morphologies, while those containing RNA2 were unaffected. Second, subsets of the BMV particles separated by density gradients into a pool enriched for RNA1 (B1) and for RNA2 and RNA3/4 (B2.3/4) were found to have different physiochemical properties. Compared to the B2.3/4 particles, the B1 particles were more sensitive to protease digestion and had greater resistivity to nanoindentation by atomic force microscopy and increased susceptibility to nuclease digestion. Mapping studies showed that portions of the arginine-rich N-terminal tail of the CP could interact with RNA1. Mutational analysis in the putative RNA1-contacting residues severely reduced encapsidation of BMV RNA1 without affecting the encapsidation of RNA2. Finally, during infection of plants, the more easily released RNA1 accumulated to higher levels early in the infection. IMPORTANCE: Viruses with genomes packaged in distinct virions could theoretically release the genomes at different times to regulate the timing of gene expression. Using an RNA virus composed of three particles, we demonstrated that the RNA in one of the virions is released more easily than the other two in vitro. The differential RNA release is due to distinct interactions between the viral capsid protein and the RNAs. The ease of RNA release is also correlated with the more rapid accumulation of that RNA in infected plants. Our study identified a novel role for capsid-RNA interactions in the regulation of a viral infection.
Subject(s)
Bromovirus/genetics , Capsid/metabolism , Gene Expression Regulation, Viral/physiology , RNA, Viral/metabolism , Virion/genetics , Virus Release/physiology , Agrobacterium , Blotting, Northern , Capsid Proteins/genetics , DNA Mutational Analysis , Gene Expression Regulation, Viral/genetics , Gene Transfer Techniques , Microscopy, Atomic Force , Peptide Mapping , Time Factors , Virion/chemistry , Virion/metabolism , Virus Release/geneticsABSTRACT
UNLABELLED: Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behaviors. We report a cryo-electron microscopy (cryo-EM) structure of the WHV capsid at nanometer resolution and characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions than hCp149: at 23 °C and at 100 mM ionic strength, the pseudocritical concentrations of assembly of wCp149 and hCp149 are 1.8 µM and 43.3 µM, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, nonicosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the â¼ 5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 °C to 37 °C; van't Hoff analyses relate the differences in temperature dependence to the high positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host. IMPORTANCE: In this paper, we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that the assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a nonhomeostatic environment. These studies yield insight into the interplay between core protein self-assembly and the host environment, which may be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.
Subject(s)
Hepadnaviridae/physiology , Hepatitis B Virus, Woodchuck/physiology , Viral Core Proteins/metabolism , Virion/metabolism , Virus Assembly/radiation effects , Amino Acid Sequence , Animals , Cryoelectron Microscopy , Hepadnaviridae/radiation effects , Hepadnaviridae/ultrastructure , Hepatitis B Virus, Woodchuck/radiation effects , Hepatitis B Virus, Woodchuck/ultrastructure , Humans , Mass Spectrometry , Microscopy, Electron, Transmission , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Alignment , Temperature , Virion/ultrastructureABSTRACT
Viral nanoparticles used for biomedical applications must be able to discriminate between tumor or virus-infected host cells and healthy host cells. In addition, viral nanoparticles must have the flexibility to incorporate a wide range of cargo, from inorganic metals to mRNAs to small molecules. Alphaviruses are a family of enveloped viruses for which some species are intrinsically capable of systemic tumor targeting. Alphavirus virus-like particles, or viral nanoparticles, can be generated from in vitro self-assembled core-like particles using nonviral nucleic acid. In this work, we expand on the types of cargo that can be incorporated into alphavirus core-like particles and the molecular requirements for packaging this cargo. We demonstrate that different core-like particle templates can be further enveloped to form viral nanoparticles that are capable of cell entry. We propose that alphaviruses can be selectively modified to create viral nanoparticles for biomedical applications and basic research.
Subject(s)
Alphavirus/physiology , Nanoparticles/administration & dosage , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Virus Assembly , Alphavirus/chemistry , Alphavirus/genetics , Alphavirus/metabolism , Glycoproteins/metabolism , Luminescent Proteins/metabolismABSTRACT
The photothermally induced nanoscale dynamics of rapid melting and resolidification of a thin layer of molecular material surrounding a nanoparticle is examined in real time by an all-optical approach. The method employs pulsed periodic modulation of the medium's dielectric constant through absorption of a low-duty-cycle laser pulse train by a single nanoparticle that acts as a localized heating source. Interpretation of experimental data, including inference of a phase change and of the liquid/solid interface dynamics, is obtained by comparing experimental data with results from coupled optical-thermal numerical simulations. The combined experimental/computational workflow presented in this proof-of-principle study will enable future explorations of material parameters at nanoscale, which are often different from their bulk values and in many cases difficult to infer from macroscopic measurements.
ABSTRACT
Solving Maxwell's equations numerically to map electromagnetic fields in the vicinity of nanostructured metal surfaces can be a daunting task when studying non-periodic, extended patterns. However, for many nanophotonic applications such as sensing or photovoltaics it is often important to have an accurate description of the actual, experimental spatial field distributions near device surfaces. In this article, we show that the complex light intensity patterns formed by closely-spaced multiple apertures in a metal film can be faithfully mapped with sub-wavelength resolution, from near-field to far-field, in the form of a 3D solid replica of isointensity surfaces. The permittivity of the metal film plays a role in shaping of the isointensity surfaces, over the entire examined spatial range, which is captured by simulations and confirmed experimentally.
ABSTRACT
Reducing hot-carrier relaxation rates is of great significance in overcoming energy loss that fundamentally limits the efficiency of solar energy utilization. Semiconductor quantum dots are expected to have much slower carrier cooling because the spacing between their discrete electronic levels is much larger than phonon energy. However, the slower carrier cooling is difficult to observe due to the existence of many competing relaxation pathways. Here we show that carrier cooling in colloidal graphene quantum dots can be 2 orders of magnitude slower than in bulk materials, which could enable harvesting of hot charge carriers to improve the efficiency of solar energy conversion.
ABSTRACT
Simple RNA viruses self-assemble spontaneously and encapsulate their genome into a shell called the capsid. This process is mainly driven by the attractive electrostatics interaction between the positive charges on capsid proteins and the negative charges on the genome. Despite its importance and many decades of intense research, how the virus selects and packages its native RNA inside the crowded environment of a host cell cytoplasm in the presence of an abundance of nonviral RNA and other anionic polymers has remained a mystery. In this paper, we perform a series of simulations to monitor the growth of viral shells and find the mechanism by which cargo-coat protein interactions can impact the structure and stability of the viral shells. We show that coat protein subunits can assemble around a globular nucleic acid core by forming nonicosahedral cages, which have been recently observed in assembly experiments involving small pieces of RNA. We find that the resulting cages are strained and can easily be split into fragments along stress lines. This suggests that such metastable nonicosahedral intermediates could be easily reassembled into the stable native icosahedral shells if the larger wild-type genome becomes available, despite the presence of a myriad of nonviral RNAs.
Subject(s)
Virus Assembly , Viruses , Capsid/chemistry , Capsid Proteins/genetics , Capsid Proteins/chemistry , RNA/analysisABSTRACT
Radiation brightening was recently observed in a multifluorophore-conjugated brome mosaic virus (BMV) particle at room temperature under pulsed excitation. On the basis of its nonlinear dependence on the number of chromophores, the origins of the phenomenon were attributed to a collective relaxation. However, the mechanism remains unknown. We present ultrafast transient absorption and fluorescence spectroscopic studies which shed new light on the collective nature of the relaxation dynamics in such radiation-brightened, multifluorophore particles. Our findings indicate that the emission dynamics is consistent with a superradiance mechanism. The ratio between the rates of competing radiative and nonradiative relaxation pathways depends on the number of chromophores per virus. The findings suggest that small icosahedral virus shells provide a unique biological scaffold for developing nonclassical, deep subwavelength light sources and may open new avenues for the development of photonic probes for medical imaging applications.
Subject(s)
Bromovirus , Viruses , Fluorescent Dyes , Spectrometry, FluorescenceABSTRACT
Biological systems offer more than an inspiration for the spontaneous hierarchical organization of matter at length scales between 1 and 1000 nm. They also provide useful principles and molecular building blocks that have recently emerged with the proven ability to generate extended three-dimensional structures of hybrid biotic/abiotic components arranged with molecular precision. These principles and tools draw from the methods of molecular biology and modern biochemistry and are expected to provide unmatched flexibility in building supramolecular architectures, notably structures made of artificial atoms whose coupled responses to electromagnetic or elastic excitations have been predicted to yield astonishing properties unparalleled by any conventional materials. To illustrate the potential of merging bio-enabled organization with metamaterials synthesis, we provide here a succinct overview of the architectural constraints leading to metamaterial behavior together with examples of biological material assembly that are particularly promising to comply with these constraints.
Subject(s)
Biocompatible Materials/chemistry , Nanoparticles/chemistry , Physics/instrumentation , NanotechnologyABSTRACT
We recently reported the design and synthesis of a series of conformationally dynamic chromophores that are built on the C(3)-symmetric tris(N-salicylideneaniline) platform. This system utilizes cooperative structural folding-unfolding motions for fluorescence switching, which is driven by the assembly and disassembly of hydrogen bonds between the rigid core and rotatable peripheral part of the molecule. Here, we report detailed time-resolved spectroscopic studies to investigate the structure-property relationships of a series of functionalized tris(N-salicylideneaniline)s. Time-resolved fluorescence decay spectroscopy was applied to determine the main relaxation mechanisms of these π-extended fluorophores, and to address the effects of hydrogen bonding, steric constraints, and extension of the π-conjugation on their relaxation dynamics. Our results agree well with the conformational switching model that was previously suggested from steady-state experiments. Notably, extension of the π-conjugation from peripheral aryl groups resulted in the stabilization of the excited states, as evidenced by longer lifetimes and lower nonradiative decay constants. As a consequence, an increase in the fluorescence quantum yields was observed, which could be explained by the suppression of the torsional motions about the C-N bonds from an overall increase in the quinoid character of the excited states. A combination of time-resolved and steady-state techniques also revealed intermolecular interactions through π-π stacking at higher concentrations, which provide additional de-excitation pathways that become more pronounced in solid samples.
Subject(s)
Aniline Compounds/chemistry , Fluorescence , Schiff Bases/chemistry , Thermodynamics , Hydrogen Bonding , Molecular Conformation , Molecular StructureABSTRACT
Viruses avoid exposure of the viral genome to harmful agents with the help of a protective protein shell known as the capsid. A secondary effect of this protective barrier is that macromolecules that may be in high concentration on the outside cannot freely diffuse across it. Therefore, inside the cell and possibly even outside, the intact virus is generally under a state of osmotic stress. Viruses deal with this type of stress in various ways. In some cases, they might harness it for infection. However, the magnitude and influence of osmotic stress on virus physical properties remains virtually unexplored for single-stranded RNA viruses-the most abundant class of viruses. Here, we report on how a model system for the positive-sense RNA icosahedral viruses, brome mosaic virus (BMV), responds to osmotic pressure. Specifically, we study the mechanical properties and structural stability of BMV under controlled molecular crowding conditions. We show that BMV is mechanically reinforced under a small external osmotic pressure but starts to yield after a threshold pressure is reached. We explain this mechanochemical behavior as an effect of the molecular crowding on the entropy of the "breathing" fluctuation modes of the virus shell. The experimental results are consistent with the viral RNA imposing a small negative internal osmotic pressure that prestresses the capsid. Our findings add a new line of inquiry to be considered when addressing the mechanisms of viral disassembly inside the crowded environment of the cell.