The In Vivo Fate of Polycatecholamine Coated Nanoparticles Is Determined by a Fibrinogen Enriched Protein Corona.

Polycatecholamine coatings have attracted significant attention in the past 10 years owing to their ability to functionalize a wide range of materials. Here we apply the use of such coatings to drug nanocrystals, made from a poorly soluble drug compound, to postfunctionalize the nanocrystal surface with the aim of providing steric stabilization and extending their circulation time after intravenous injection. We show that both polydopamine and polynorepinephrine can be used to successfully modify drug nanocrystals and subsequently incorporate end-functionalized PEG to the surface. Even though high grafting densities of PEG were achieved, we observed rapid clearance and increased liver uptake for polycatecholamine functionalized drug nanocrystals. Using both surface sensitive model systems and protein corona profiling, we determine that the rapid clearance was correlated with an increase in adsorption of proteins involved in coagulation to the polycatecholamine surface, with fibrinogen being the most abundant. Further analysis of the most abundant proteins revealed a significant increase in thiol-rich proteins on polycatecholamine coated surfaces. The observed interaction with coagulation proteins highlights one of the current challenges using polycatecholamines for drug delivery but might also provide insights to the growing use of these materials in hemostatic applications.

Stable trapping of multiple proteins at physiological conditions using nanoscale chambers with macromolecular gates.

The possibility to detect and analyze single or few biological molecules is very important for understanding interactions and reaction mechanisms. Ideally, the molecules should be confined to a nanoscale volume so that the observation time by optical methods can be extended. However, it has proven difficult to develop reliable, non-invasive trapping techniques for biomolecules under physiological conditions. Here we present a platform for long-term tether-free (solution phase) trapping of proteins without exposing them to any field gradient forces. We show that a responsive polymer brush can make solid state nanopores switch between a fully open and a fully closed state with respect to proteins, while always allowing the passage of solvent, ions and small molecules. This makes it possible to trap a very high number of proteins (500-1000) inside nanoscale chambers as small as one attoliter, reaching concentrations up to 60 gL-1. Our method is fully compatible with parallelization by imaging arrays of nanochambers. Additionally, we show that enzymatic cascade reactions can be performed with multiple native enzymes under full nanoscale confinement and steady supply of reactants. This platform will greatly extend the possibilities to optically analyze interactions involving multiple proteins, such as the dynamics of oligomerization events.

Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion.

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.

Nanoplasmonic Sensor Detects Preferential Binding of IRSp53 to Negative Membrane Curvature.

Biosensors based on plasmonic nanostructures are widely used in various applications and benefit from numerous operational advantages. One type of application where nanostructured sensors provide unique value in comparison with, for instance, conventional surface plasmon resonance, is investigations of the influence of nanoscale geometry on biomolecular binding events. In this study, we show that plasmonic "nanowells" conformally coated with a continuous lipid bilayer can be used to detect the preferential binding of the insulin receptor tyrosine kinase substrate protein (IRSp53) I-BAR domain to regions of negative surface curvature, i.e., the interior of the nanowells. Two different sensor architectures with and without an additional niobium oxide layer are compared for this purpose. In both cases, curvature preferential binding of IRSp53 (at around 0.025 nm-1 and higher) can be detected qualitatively. The high refractive index niobium oxide influences the near field distribution and makes the signature for bilayer formation less clear, but the contrast for accumulation at regions of negative curvature is slightly higher. This work shows the first example of analyzing preferential binding of an average-sized and biologically important protein to negative membrane curvature in a label-free manner and in real-time, illustrating a unique application for nanoplasmonic sensors.

Optical properties of plasmonic nanopore arrays prepared by electron beam and colloidal lithography.

Solid state nanopores are central structures for many applications. To date, much effort has been spent on controlled fabrication of single nanopores, while relatively little work has focused on large scale fabrication of arrays of nanopores. In this work we show wafer-scale fabrication of plasmonic nanopores in 50 nm thick silicon nitride membranes with one or two 30 nm gold films, using electron beam lithography with a negative resist or a new version of colloidal lithography. Both approaches offer good control of pore diameter (even below 100 nm) and with high yield (>90%) of intact membranes. Colloidal lithography has the advantage of parallel patterning without expensive equipment. Despite its serial nature, electron beam lithography provides high throughput and can make arbitrary array patterns. Importantly, both methods prevent metal from ending up on the membrane pore sidewalls. The new fabrication methods make it possible to compare the optical properties of structurally identical plasmonic nanopore arrays with either long-range order (e-beam) or short-range order (colloidal). The resonance features in the extinction spectrum are very similar for both structures when the pitch is the same as the characteristic spacing in the self-assembled colloidal pattern. Long-range ordering slightly enhances the magnitude of the extinction maximum and blueshift the transmission maximum by tens of nm. Upon reducing the diameter in long-range ordered arrays, the resonance is reduced in magnitude and the transmission maximum is further blue shifted, just like for short-range ordered arrays. These effects are well explained by interpreting the spectra as Fano interference between the grating-type excitation of propagating surface plasmons and the broad transmission via individual pores in the metal film. Furthermore, we find that only the short-range ordered arrays scatter light, which we attribute to the highly limited effective period in the short-range ordered system and the corresponding lack of coherent suppression of scattering by interference effects.

Gating Protein Transport in Solid State Nanopores by Single Molecule Recognition.

Control of molecular translocation through nanoscale apertures is of great interest for DNA sequencing, biomolecular filters, and new platforms for single molecule analysis. However, methods for controlling the permeability of nanopores are very limited. Here, we show how nanopores functionalized with poly(ethylene glycol) brushes, which fully prevent protein translocation, can be reversibly gated to an "open" state by binding of single IgG antibodies that disrupt the macromolecular barrier. On the basis of surface plasmon resonance data we propose a two-state model describing the antibody-polymer interaction kinetics. Reversibly (weakly) bound antibodies decrease the protein exclusion height while irreversibly (strongly) bound antibodies do not. Our results are further supported by fluorescence readout from pore arrays and high-speed atomic force microscopy on single pores. This type of dynamic barrier control on the nanoscale provides new possibilities for biomolecular separation and analysis.

Antibody-Antigen Interaction Dynamics Revealed by Analysis of Single-Molecule Equilibrium Fluctuations on Individual Plasmonic Nanoparticle Biosensors.

Antibody-antigen interactions are complex events central to immune response, in vivo and in vitro diagnostics, and development of therapeutic substances. We developed an ultrastable single-molecule localized surface plasmon resonance (LSPR) sensing platform optimized for studying antibody-antigen interaction kinetics over very long time scales. The setup allowed us to perform equilibrium fluctuations analysis of the PEG/anti-PEG interaction. By time and frequency domain analysis, we demonstrate that reversible adsorption of monovalently bound anti-PEG antibodies is the dominant factor affecting the LSPR fluctuations. The results suggest that equilibrium fluctuation analysis can be an alternative to established methods for determination of interaction rates. In particular, the methodology is suited to analyze molecular systems whose properties change during the initial interaction phases, for example, due to mass transport limitations or, as demonstrated here, because the effective association rate constant varies with surface concentration of adsorbed molecules.

Nanoplasmonic Sensing Architectures for Decoding Membrane Curvature-Dependent Biomacromolecular Interactions.

Nanoplasmonic sensors have emerged as a promising measurement approach to track biomacromolecular interactions involving lipid membrane interfaces. By taking advantage of nanoscale fabrication capabilities, it is possible to design sensing platforms with various architectural configurations. Such capabilities open the door to fabricating lipid membrane-coated nanoplasmonic sensors with varying degrees of membrane curvature in order to understand how biomacromolecular interaction processes are influenced by membrane curvature. Herein, we employed an indirect nanoplasmonic sensing approach to characterize the fabrication of supported lipid bilayers (SLBs) on silica-coated nanowell and nanodisk sensing platforms and to investigate how membrane curvature influences membrane-peptide interactions by evaluating the corresponding measurement responses from different spectral signatures that are sensitive to specific regions of the sensor geometries. SLBs were prepared by the vesicle fusion method, as monitored in real-time by nanoplasmonic sensing measurements and further characterized by fluorescence recovery after photobleaching (FRAP) experiments. By resolving different spectral signatures in the nanoplasmonic sensing measurements, it was determined that peptide binding induces membrane disruption at positively curved membrane regions, while peptide binding without subsequent disruption was observed at planar and negatively curved regions. These findings are consistent with the peptide's known preference to selectively form pores in positively curved membranes, providing validation to the nanoplasmonic sensing approach and highlighting how the integration of nanoplasmonic sensors with different nanoscale architectures can be utilized to study the influence of membrane curvature on biomacromolecular interaction processes.

Polymer brushes in solid-state nanopores form an impenetrable entropic barrier for proteins.

Polymer brushes are widely used to prevent the adsorption of proteins, but the mechanisms by which they operate have remained heavily debated for many decades. We show conclusive evidence that a polymer brush can be a remarkably strong kinetic barrier towards proteins by using poly(ethylene glycol) grafted to the sidewalls of pores in 30 nm thin gold films. Despite consisting of about 90% water, the free coils seal apertures up to 100 nm entirely with respect to serum protein translocation, as monitored label-free through the plasmonic activity of the nanopores. The conclusions are further supported by atomic force microscopy and fluorescence microscopy. A theoretical model indicates that the brush undergoes a morphology transition to a sealing state when the ratio between the extension and the radius of curvature is approximately 0.8. The brush-sealed pores represent a new type of ultrathin filter with potential applications in bioanalytical systems.

Detecting Selective Protein Binding Inside Plasmonic Nanopores: Toward a Mimic of the Nuclear Pore Complex.

Biosensors based on plasmonic nanostructures offer label-free and real-time monitoring of biomolecular interactions. However, so do many other surface sensitive techniques with equal or better resolution in terms of surface coverage. Yet, plasmonic nanostructures offer unique possibilities to study effects associated with nanoscale geometry. In this work we use plasmonic nanopores with double gold films and detect binding of proteins inside them. By thiol and trietoxysilane chemistry, receptors are selectively positioned on the silicon nitride interior walls. Larger (~150 nm) nanopores are used detect binding of averaged sized proteins (~60 kg/mol) with high signal to noise (>100). Further, we fabricate pores that approach the size of the nuclear pore complex (diameter down to 50 nm) and graft disordered phenylalanine-glycine nucleoporin domains to the walls, followed by titration of karyopherinß1 transport receptors. The interactions are shown to occur with similar affinity as determined by conventional surface plasmon resonance on planar surfaces. Our work illustrates another unique application of plasmonic nanostructures, namely the possibility to mimic the geometry of a biological nanomachine with integrated optical sensing capabilities.

Corrigendum: Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules.

This corrects the article DOI: 10.1038/srep30410.

Switchable Plasmonic Metasurfaces with High Chromaticity Containing Only Abundant Metals.

Plasmonic color generation offers several advantages but is also limited by the cost and availability of noble metals like gold. In this work, we present color-tunable metasurfaces with high chromaticity and reflectivity consisting of an aluminum mirror, a dielectric spacer, and a plasmonic nanohole array in copper. Copper is shown to be an excellent alternative to gold when properly protected from oxidation and makes it possible to generate a wide RGB gamut covering 27% of the standard RGB. By patterning the metasurfaces into microscale pixel triplets, color photos can be well reproduced with high resolution over wafer-sized areas. Further, we demonstrate active modulation of the reflected intensity using an electrochromic conductive polymer deposited on top of the nanostructures by screen printing. This technology opens up for ultrathin and flexible reflective displays in full color, that is, plasmonic electronic paper, compatible with large-scale sustainable production.

Fabrication and Characterization of Plasmonic Nanopores with Cavities in the Solid Support.

Plasmonic nanostructures are widely used for various sensing applications by monitoring changes in refractive index through optical spectroscopy or as substrates for surface enhanced Raman spectroscopy. However, in most practical situations conventional surface plasmon resonance is preferred for biomolecular interaction analysis because of its high resolution in surface coverage and the simple single-material planar interface. Still, plasmonic nanostructures may find unique sensing applications, for instance when the nanoscale geometry itself is of interest. This calls for new methods to prepare nanoscale particles and cavities with controllable dimensions and curvature. In this work, we present two types of plasmonic nanopores where the solid support underneath a nanohole array has been etched, thereby creating cavities denoted as 'nanowells' or 'nanocaves' depending on the degree of anisotropy (dry or wet etch). The refractometric sensitivity is shown to be enhanced upon removing the solid support because of an increased probing volume and a shift of the asymmetric plasmonic field towards the liquid side of the finite gold film. Furthermore, the structures exhibit different spectral changes upon binding inside the cavities compared to the gold surface, which means that the structures can be used for location-specific detection. Other sensing applications are also suggested.

Superior LSPR substrates based on electromagnetic decoupling for on-a-chip high-throughput label-free biosensing.

Localized surface plasmon resonance (LSPR) biosensing based on supported metal nanoparticles offers unparalleled possibilities for high-end miniaturization, multiplexing and high-throughput label-free molecular interaction analysis in real time when integrated within an opto-fluidic environment. However, such LSPR-sensing devices typically contain extremely large regions of dielectric materials that are open to molecular adsorption, which must be carefully blocked to avoid compromising the device readings. To address this issue, we made the support essentially invisible to the LSPR by carefully removing the dielectric material overlapping with the localized plasmonic fields through optimized wet-etching. The resulting LSPR substrate, which consists of gold nanodisks centered on narrow SiO2 pillars, exhibits markedly reduced vulnerability to nonspecific substrate adsorption, thus allowing, in an ideal case, the implementation of thicker and more efficient passivation layers. We demonstrate that this approach is effective and fully compatible with state-of-the-art multiplexed real-time biosensing technology and thus represents the ideal substrate design for high-throughput label-free biosensing systems with minimal sample consumption.

Plasmonic Metasurfaces with Conjugated Polymers for Flexible Electronic Paper in Color.

A flexible electronic paper in full color is realized by plasmonic metasurfaces with conjugated polymers. An ultrathin large-area electrochromic material is presented which provides high polarization-independent reflection, strong contrast, fast response time, and long-term stability. This technology opens up for new electronic readers and posters with ultralow power consumption.

Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles.

Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.

Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules.

The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.

Biosensing using plasmonic nanohole arrays with small, homogenous and tunable aperture diameters.

Plasmonic nanohole arrays are widely used for optical label-free molecular detection. An important factor for many applications is the diameter of the apertures. So far nanohole arrays with controllable diameters below 100 nm have not been demonstrated and it has not been systematically investigated how the diameter influences the optical properties. In this work we fine-tune the diameter in short range ordered nanohole arrays down to 50 nm. The experimental far field spectra show how the wavelength of maximum extinction remains unaffected while the transmission maximum blue shifts with smaller diameters. The near field is visualized by numerical simulations, showing a homogenous enhancement throughout the cylindrical void at the transmission maximum for diameters between 50 and 100 nm. For diameters below 50 nm plasmon excitation is no longer possible experimentally or by simulations. Further, we investigate the refractive index sensing capabilities of the smaller holes. As the diameter was reduced, the sensitivity in terms of resonance shift with bulk liquid refractive index was found to be unaltered. However, for the transmission maximum the sensitivity becomes more strongly localized to the hole interior. By directing molecular binding to the bottom of the holes we demonstrate how smaller holes enhance the sensitivity in terms of signal per molecule. A real-time detection limit well below one protein per nanohole is demonstrated. The smaller plasmonic nanoholes should be suitable for studies of molecules confined in small volumes and as mimics of biological nanopores.

Location-specific nanoplasmonic sensing of biomolecular binding to lipid membranes with negative curvature.

The biochemical processes of cell membranes are sensitive to the geometry of the lipid bilayer. We show how plasmonic "nanowells" provide label-free real-time analysis of molecules on membranes with detection of preferential binding at negative curvature. It is demonstrated that norovirus accumulate in invaginations due to multivalent interactions with glycosphingolipids.

Strongly stretched protein resistant poly(ethylene glycol) brushes prepared by grafting-to.

We present a new grafting-to method for resistant "non-fouling" poly(ethylene glycol) brushes, which is based on grafting of polymers with reactive end groups in 0.9 M Na2SO4 at room temperature. The grafting process, the resulting brushes, and the resistance toward biomolecular adsorption are investigated by surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy. We determine both grafting density and thickness independently and use narrow molecular weight distributions which result in well-defined brushes. High density (e.g., 0.4 coils per nm(2) for 10 kDa) and thick (40 nm for 20 kDa) brushes are readily achieved that suppress adsorption from complete serum (10× dilution, exposure for 50 min) by up to 99% on gold (down to 4 ng/cm(2) protein coverage). The brushes outperform oligo(ethylene glycol) monolayers prepared on the same surfaces and analyzed in the same manner. The brush heights are in agreement with calculations based on a simple model similar to the de Gennes "strongly stretched" brush, where the height is proportional to molecular weight. This result has so far generally been considered to be possible only for brushes prepared by grafting-from. Our results are consistent with the theory that the brushes act as kinetic barriers rather than efficient prevention of adsorption at equilibrium. We suggest that the free energy barrier for passing the brush depends on both monomer concentration and thickness. The extraordinary simplicity of the method and good inert properties of the brushes should make our results widely applicable in biointerface science.