Sub-Nanomolar Detection of Oligonucleotides Using Molecular Beacons Immobilized on Lightguiding Nanowires.

The detection of oligonucleotides is a central step in many biomedical investigations. The most commonly used methods for detecting oligonucleotides often require concentration and amplification before detection. Therefore, developing detection methods with a direct read-out would be beneficial. Although commonly used for the detection of amplified oligonucleotides, fluorescent molecular beacons have been proposed for such direct detection. However, the reported limits of detection using molecular beacons are relatively high, ranging from 100 nM to a few µM, primarily limited by the beacon fluorescence background. In this study, we enhanced the relative signal contrast between hybridized and non-hybridized states of the beacons by immobilizing them on lightguiding nanowires. Upon hybridization to a complementary oligonucleotide, the fluorescence from the surface-bound beacon becomes coupled in the lightguiding nanowire core and is re-emitted at the nanowire tip in a narrower cone of light compared with the standard 4π emission. Prior knowledge of the nanowire positions allows for the continuous monitoring of fluorescence signals from each nanowire, which effectively facilitates the discrimination of signals arising from hybridization events against background signals. This resulted in improved signal-to-background and signal-to-noise ratios, which allowed for the direct detection of oligonucleotides at a concentration as low as 0.1 nM.

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 to be 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 (radius <170 nm) 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 the 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 differentiating biomolecular fractal aggregates from spherical particles in fetal bovine serum at the single-particle level.

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.

Label-free quantification of protein binding to lipid vesicles using transparent waveguide evanescent-field scattering microscopy with liquid control.

Recent innovations in microscopy techniques are paving the way for label-free studies of single nanoscopic biological entities such as viruses, lipid-nanoparticle drug carriers, and even proteins. One such technique is waveguide evanescent-field microscopy, which offers a relatively simple, yet sensitive, way of achieving label-free light scattering-based imaging of nanoparticles on surfaces. Herein, we extend the application of this technique by incorporating microfluidic liquid control and adapting the design for use with inverted microscopes by fabricating a waveguide on a transparent substrate. We furthermore formulate analytical models describing scattering and fluorescence intensities from single spherical and shell-like objects interacting with evanescent fields. The models are then applied to analyze scattering and fluorescence intensities from adsorbed polystyrene beads and to temporally resolve cholera-toxin B (CTB) binding to individual surface-immobilized glycosphingolipid GM1 containing vesicles. We also propose a self-consistent means to quantify the thickness of the CTB layer, revealing that protein-binding to individual vesicles can be characterized with sub-nm precision in a time-resolved manner.

Fluorescence excitation enhancement by waveguiding nanowires.

The optical properties of vertical semiconductor nanowires can allow an enhancement of fluorescence from surface-bound fluorophores, a feature proven useful in biosensing. One of the contributing factors to the fluorescence enhancement is thought to be the local increase of the incident excitation light intensity in the vicinity of the nanowire surface, where fluorophores are located. However, this effect has not been experimentally studied in detail to date. Here, we quantify the excitation enhancement of fluorophores bound to a semiconductor nanowire surface by combining modelling with measurements of fluorescence photobleaching rate, indicative of the excitation light intensity, using epitaxially grown GaP nanowires. We study the excitation enhancement for nanowires with a diameter of 50-250 nm and show that excitation enhancement reaches a maximum for certain diameters, depending on the excitation wavelength. Furthermore, we find that the excitation enhancement decreases rapidly within tens of nanometers from the nanowire sidewall. The results can be used to design nanowire-based optical systems with exceptional sensitivities for bioanalytical applications.

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.

Single-shot self-supervised object detection in microscopy.

Object detection is a fundamental task in digital microscopy, where machine learning has made great strides in overcoming the limitations of classical approaches. The training of state-of-the-art machine-learning methods almost universally relies on vast amounts of labeled experimental data or the ability to numerically simulate realistic datasets. However, experimental data are often challenging to label and cannot be easily reproduced numerically. Here, we propose a deep-learning method, named LodeSTAR (Localization and detection from Symmetries, Translations And Rotations), that learns to detect microscopic objects with sub-pixel accuracy from a single unlabeled experimental image by exploiting the inherent roto-translational symmetries of this task. We demonstrate that LodeSTAR outperforms traditional methods in terms of accuracy, also when analyzing challenging experimental data containing densely packed cells or noisy backgrounds. Furthermore, by exploiting additional symmetries we show that LodeSTAR can measure other properties, e.g., vertical position and polarizability in holographic microscopy.

Probing the Separation Distance between Biological Nanoparticles and Cell Membrane Mimics Using Neutron Reflectometry with Sub-Nanometer Accuracy.

Nanoparticle interactions with cellular membranes are controlled by molecular recognition reactions and regulate a multitude of biological processes, including virus infections, biological nanoparticle-mediated cellular communication, and drug delivery applications. Aided by the design of various supported cell membrane mimics, multiple methods have been employed to investigate these types of interactions, revealing information on nanoparticle coverage, interaction kinetics, as well as binding strength; however, precise quantification of the separation distance across which these delicate interactions occur remains elusive. Here, we demonstrate that carefully designed neutron reflectometry (NR) experiments followed by an attentive selection and application of suitable theoretical models offer a means to quantify the distance separating biological nanoparticles from a supported lipid bilayer (SLB) with sub-nanometer precision. The distance between the nanoparticles and SLBs was tuned by exploiting either direct adsorption or specific binding using DNA tethers with different conformations, revealing separation distances of around 1, 3, and 7 nm with nanometric accuracy. We also show that NR provides precise information on nanoparticle coverage, size distribution, material composition, and potential structural changes in the underlying planar SLB induced upon nanoparticle binding. The precision with which these parameters could be quantified should pave an attractive path for investigations of the interactions between nanoparticles and interfaces at length scales and resolutions that were previously inaccessible. This thus makes it possible to, for example, gain an in-depth understanding of the molecular recognition reactions of inorganic and biological nanoparticles with cellular membranes.

Time-Resolved Thickness and Shape-Change Quantification using a Dual-Band Nanoplasmonic Ruler with Sub-Nanometer Resolution.

Time-resolved measurements of changes in the size and shape of nanobiological objects and layers are crucial to understand their properties and optimize their performance. Optical sensing is particularly attractive with high throughput and sensitivity, and label-free operation. However, most state-of-the-art solutions require intricate modeling or multiparameter measurements to disentangle conformational or thickness changes of biomolecular layers from complex interfacial refractive index variations. Here, we present a dual-band nanoplasmonic ruler comprising mixed arrays of plasmonic nanoparticles with spectrally separated resonance peaks. As electrodynamic simulations and model experiments show, the ruler enables real-time simultaneous measurements of thickness and refractive index variations in uniform and heterogeneous layers with sub-nanometer resolution. Additionally, nanostructure shape changes can be tracked, as demonstrated by quantifying the degree of lipid vesicle deformation at the critical coverage prior to rupture and supported lipid bilayer formation. In a broader context, the presented nanofabrication approach constitutes a generic route for multimodal nanoplasmonic optical sensing.

Enhanced Optical Biosensing by Aerotaxy Ga(As)P Nanowire Platforms Suitable for Scalable Production.

Sensitive detection of low-abundance biomolecules is central for diagnostic applications. Semiconductor nanowires can be designed to enhance the fluorescence signal from surface-bound molecules, prospectively improving the limit of optical detection. However, to achieve the desired control of physical dimensions and material properties, one currently uses relatively expensive substrates and slow epitaxy techniques. An alternative approach is aerotaxy, a high-throughput and substrate-free production technique for high-quality semiconductor nanowires. Here, we compare the optical sensing performance of custom-grown aerotaxy-produced Ga(As)P nanowires vertically aligned on a polymer substrate to GaP nanowires batch-produced by epitaxy on GaP substrates. We find that signal enhancement by individual aerotaxy nanowires is comparable to that from epitaxy nanowires and present evidence of single-molecule detection. Platforms based on both types of nanowires show substantially higher normalized-to-blank signal intensity than planar glass surfaces, with the epitaxy platforms performing somewhat better, owing to a higher density of nanowires. With further optimization, aerotaxy nanowires thus offer a pathway to scalable, low-cost production of highly sensitive nanowire-based platforms for optical biosensing applications.

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles.

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Dissimilar Deformation of Fluid- and Gel-Phase Liposomes upon Multivalent Interaction with Cell Membrane Mimics Revealed Using Dual-Wavelength Surface Plasmon Resonance.

The mechanical properties of biological nanoparticles play a crucial role in their interaction with the cellular membrane, in particular for cellular uptake. This has significant implications for the design of pharmaceutical carrier particles. In this context, liposomes have become increasingly popular, among other reasons due to their customizability and easily varied physicochemical properties. With currently available methods, it is, however, not trivial to characterize the mechanical properties of nanoscopic liposomes especially with respect to the level of deformation induced upon their ligand-receptor-mediated interaction with laterally fluid cellular membranes. Here, we utilize the sensitivity of dual-wavelength surface plasmon resonance to probe the size and shape of bound liposomes (∼100 nm in diameter) as a means to quantify receptor-induced deformation during their interaction with a supported cell membrane mimic. By comparing biotinylated liposomes in gel and fluid phases, we demonstrate that fluid-phase liposomes are more prone to deformation than their gel-phase counterparts upon binding to the cell membrane mimic and that, as expected, the degree of deformation depends on the number of ligand-receptor pairs that are engaged in the multivalent binding.

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles.

Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.

Diffusion of Lipid Nanovesicles Bound to a Lipid Membrane Is Associated with the Partial-Slip Boundary Condition.

During diffusion of nanoparticles bound to a cellular membrane by ligand-receptor pairs, the distance to the laterally mobile interface is sufficiently short for their motion to depend not only on the membrane-mediated diffusivity of the tethers but also in a not yet fully understood manner on nanoparticle size and interfacial hydrodynamics. By quantifying diffusivity, velocity, and size of individual membrane-bound liposomes subjected to a hydrodynamic shear flow, we have successfully separated the diffusivity contributions from particle size and number of tethers. The obtained diffusion-size relations for synthetic and extracellular lipid vesicles are not well-described by the conventional no-slip boundary condition, suggesting partial slip as well as a significant diffusivity dependence on the distance to the lipid bilayer. These insights, extending the understanding of diffusion of biological nanoparticles at lipid bilayers, are of relevance for processes such as cellular uptake of viruses and lipid nanoparticles or labeling of cell-membrane-residing molecules.

Investigation of Self-Emulsifying Drug-Delivery System Interaction with a Biomimetic Membrane under Conditions Relevant to the Small Intestine.

Self-emulsifying drug-delivery systems (SEDDS) have been extensively shown to increase oral absorption of solvation-limited compounds. However, there has been little clinical and commercial use of these formulations, in large part because the demonstrated advantages of SEDDS have been outweighed by our inability to precisely predict drug absorption from SEDDS using current in vitro assays. To overcome this limitation and increase the biological relevancy of in vitro assays, an absorption function can be incorporated using biomimetic membranes. However, the effects that SEDDS have on the integrity of a biomimetic membrane are not known. In this study, a quartz crystal microbalance with dissipation monitoring and total internal reflection fluorescence microscopy were employed as complementary methods to in vitro lipolysis-permeation assays to characterize the interaction of various actively digested SEDDS with a liquescent artificial membrane comprising lecithin in dodecane (LiDo). Observations from surface analysis showed that interactions between the digesting SEDDS and LiDo membrane coincided with inflection points in the digestion profiles. Importantly, no indications of membrane damage could be observed, which was supported by flux profiles of the lipophilic model drug felodipine (FEL) and impermeable marker Lucifer yellow on the basal side of the membrane. There was a correlation between the digestion kinetics of the SEDDS and the flux of FEL, but no clear correlation between solubilization and absorption profiles. Membrane interactions were dependent on the composition of lipids within each SEDDS, with the more digestible lipids leading to more pronounced interactions, but in all cases, the integrity of the membrane was maintained. These insights demonstrate that LiDo membranes are compatible with in vitro lipolysis assays for improving predictions of drug absorption from lipid-based formulations.

Time-Resolved and Label-Free Evanescent Light-Scattering Microscopy for Mass Quantification of Protein Binding to Single Lipid Vesicles.

In-depth understanding of the intricate interactions between biomolecules and nanoparticles is hampered by a lack of analytical methods providing quantitative information about binding kinetics. Herein, we demonstrate how label-free evanescent light-scattering microscopy can be used to temporally resolve specific protein binding to individual surface-bound (∼100 nm) lipid vesicles. A theoretical model is proposed that translates protein-induced changes in light-scattering intensity into bound mass. Since the analysis is centered on individual lipid vesicles, the signal from nonspecific protein binding to the surrounding surface is completely avoided, offering a key advantage over conventional surface-based techniques. Further, by averaging the intensities from less than 2000 lipid vesicles, the sensitivity is shown to increase by orders of magnitude. Taken together, these features provide a new avenue in studies of protein-nanoparticle interaction, in general, and specifically in the context of nanoparticles in medical diagnostics and drug delivery.

Formation of Supported Lipid Bilayers Derived from Vesicles of Various Compositional Complexity on Conducting Polymer/Silica Substrates.

Supported lipid bilayers (SLBs) serve important roles as minimalistic models of cellular membranes in multiple diagnostic and pharmaceutical applications as well as in the strive to gain fundamental insights about their complex biological function. To further expand the utility of SLBs, there is a need to go beyond simple lipid compositions to thereby better mimic the complexity of native cell membranes, while simultaneously retaining their compatibility with a versatile range of analytical platforms. To meet this demand, we have in this work explored SLB formation on PEDOT:PSS/silica nanoparticle composite films and mesoporous silica films, both capable of transporting ions to an underlying conducting PEDOT:PSS film. The SLB formation process was evaluated by using the quartz crystal microbalance with dissipation (QCM-D) monitoring, total internal reflection fluorescence (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) for membranes made of pure synthetic lipids with or without the reconstituted membrane protein ß-secretase 1 (BACE1) as well as cell-derived native lipid vesicles containing overexpressed BACE1. The mesoporous silica thin film was superior to the PEDOT:PSS/silica nanoparticle composite, providing successful formation of bilayers with high lateral mobility and low defect density even for the most complex native cell membranes.

Control of Polymer Brush Morphology, Rheology, and Protein Repulsion by Hydrogen Bond Complexation.

Polymer brushes are widely used to alter the properties of interfaces. In particular, poly(ethylene glycol) (PEG) and similar polymers can make surfaces inert toward biomolecular adsorption. Neutral hydrophilic brushes are normally considered to have static properties at a given temperature. As an example, PEG is not responsive to pH or ionic strength. Here we show that, by simply introducing a polymeric acid such as poly(methacrylic acid) (PMAA), the highly hydrated brush barrier can change its properties entirely. This is caused by multivalent hydrogen bonds in an extremely pH-sensitive process. Remarkably, it is sufficient to reduce the pH to 5 for complexation to occur at the interface, which is two units higher than in the corresponding bulk systems. Below this critical pH, PMAA starts to bind to PEG in large amounts (comparable to the PEG amount), causing the brush to gradually compact and dehydrate. The brush also undergoes major rheology changes, from viscoelastic to rigid. Furthermore, the protein repelling ability of PEG is lost after reaching a threshold in the amount of PMAA bound. The changes in brush properties are tunable and become more pronounced when more PMAA is bound. The initial brush state is fully recovered when releasing PMAA by returning to physiological pH. Our findings are relevant for many applications involving functional interfaces, such as capture-release of biomolecules.