Anikó Szalai

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

Effect of pH on stability and plasmonic properties of cysteine-functionalized silver nanoparticle dispersion.

Citrate-stabilized spherical silver nanoparticles (Ag NPs) with d=8.25±1.25 nm diameter were prepared and functionalized with L-cysteine (Cys) in aqueous dispersion. The nanosilver-cysteine interactions have been investigated by Raman and (1)H NMR spectroscopy. The effect of pH on stability of biofunctionalized Ag NPs was investigated. The cysteine-capped nanosilver dispersions remain stable at higher pH (pH>7), while the degree of aggregation increased as the pH decreased. Below pH ~7, the characteristic surface plasmon band of bare silver nanoparticles was back-shifted from λ(measured)(bareAgNP)=391 nm to λ(measured)(1)=387-391 nm, while the presence of a new band at λ(measured)(2)=550-600 nm was also observed depending on pH. Finite element method (FEM) was applied to numerically compute the absorption spectra of aqueous dispersions containing bare and cysteine-functionalized Ag NPs at different pH. Both the dynamic light scattering (DLS) measurements, Zeta potential values and the transmission electron microscopic (TEM) images confirmed our supposition. Namely, electrostatic interaction arose between the deprotonated carboxylate (COO(-)) and protonated amino groups (NH(3)(+)) of the amino acid resulting in cross-linking network of the Ag NPs between pH ~3 and 7. If the pH is measurable lower than ~3, parallel with the protonation of citrate and L-cysteine molecules the connection of the particles via l-cysteine is partly decomposed resulting in decrease of second plasmon band intensity.

Protection of the Blood-Brain Barrier by Pentosan Against Amyloid-β-Induced Toxicity

Endothelial cells of brain capillaries forming the blood-brain barrier play an important role in the pathogenesis and therapy of Alzheimers disease. Amyloid-β (Aβ) peptides are key pathological elements in the development of the disease. A blood-brain barrier model, based on primary rat brain endothelial cells was used in which the barrier properties were induced by glial cells. The effects of amyloid peptides have been tested on cell viability and barrier functions. Aβ showed toxic effects on primary rat brain endothelial cells measured by MTT dye conversion and the lactate dehydrogenase release. Morphologically cytoplasmic vacuolization, disruption of the structure of cytoplasmic organelles and tight junctions could be observed in brain endothelial cells. Treatment with Aβ1-42 decreased the electrical resistance, and increased the permeability of brain endothelial cell monolayers for both fluorescein and albumin. Serum amyloid P component which stabilizes Aβ fibrils in cortical amyloid plaques and cerebrovascular amyloid deposits significantly potentiated the barrier-weakening effect of Aβ1-42. Sulfated polysaccharide pentosan could decrease the toxic effects of Aβ peptides in brain endothelial cells. It could also significantly protect the barrier integrity of monolayers from damaging actions of peptides. Pentosan modified the size, and significantly decreased the number of amyloid aggregates demonstrated by atomic force microscopy. The present data further support the toxic effects of amyloid peptides on brain endothelial cells, and can contribute to the development of molecules protecting the blood-brain barrier in Alzheimers disease.

Improvement of infrared single-photon detectors absorptance by integrated plasmonic structures

Plasmonic structures open novel avenues in photodetector development. Optimized illumination configurations are reported to improve p-polarized light absorptance in superconducting-nanowire single-photon detectors (SNSPDs) comprising short- and long-periodic niobium-nitride (NbN) stripe-patterns. In OC-SNSPDs consisting of ~quarter-wavelength dielectric layer closed by a gold reflector the highest absorptance is attainable at perpendicular incidence onto NbN patterns in P-orientation due to E-field concentration at the bottom of nano-cavities. In NCAI-SNSPDs integrated with nano-cavity-arrays consisting of vertical and horizontal gold segments off-axis illumination in S-orientation results in polar-angle-independent perfect absorptance via collective resonances in short-periodic design, while in long-periodic NCAI-SNSPDs grating-coupled surface waves promote EM-field transportation to the NbN stripes and result in local absorptance maxima. In NCDAI-SNSPDs integrated with nano-cavity-deflector-array consisting of longer vertical gold segments large absorptance maxima appear in 3p-periodic designs due to E-field enhancement via grating-coupled surface waves synchronized with the NbN stripes in S-orientation, which enable to compensate fill-factor-related retrogression.

Plasmonic Structure Integrated Single-Photon Detector Configurations to Improve Absorptance and Polarization Contrast

Configurations capable of maximizing both the absorption component of system detection efficiency and the achievable polarization contrast were determined for 1550 nm polarized light illumination of different plasmonic structure integrated superconducting nanowire single-photon detectors (SNSPDs) consisting of p = 264 nm and P = 792 nm periodic niobium nitride (NbN) patterns on silica substrate. Global effective NbN absorptance maxima appear in case of p/s-polarized light illumination in S/P-orientation (γ = 90°/0° azimuthal angle) and the highest polarization contrast is attained in S-orientation of all devices. Common nanophotonical origin of absorptance enhancement is collective resonance on nanocavity gratings with different profiles, which is promoted by coupling between localized modes in quarter-wavelength metal-insulator-metal nanocavities and laterally synchronized Brewster-Zenneck-type surface waves in integrated SNSPDs possessing a three-quarter-wavelength-scaled periodicity. The spectral sensitivity and dispersion characteristics reveal that device design specific optimal configurations exist.

Impact of polar-azimuthal illumination angles on efficiency of nano-cavity-array integrated single-photon detectors

The absorptance of superconducting nanowire single-photon detectors consisting of subwavelength NbN stripes arrayed in 200 nm and 600 nm periodic patterns and integrated with nano-cavity-array and closing gold segments is maximized at the wavelength of 1550 nm via numerical computations. It is shown that the optimum azimuthal angles are γ = 90° (S-orientation) in case of p-polarized illumination, and γ = 0° (P-orientation) during s-polarized illumination. The p-polarized illumination of 200-nm-pitch design in S-orientation results in polar angle independent ~95% NbN absorptance due to collective resonances on the nano-cavity-array. In 600-nm-pitch design a local absorptance maximum (37.2%) appears as a result of near-field concentration promoted by Brewster-wave excitation during p-polarized illumination in S-orientation. For practical applications s-polarized illumination of 600-nm-pitch design in P-orientation is proposed, as ~52% absorptance larger than in case of perpendicular incidence is attainable due to total internal reflection.

Comparative Study of Plasmonic Properties of Cysteine-Functionalized Gold and Silver Nanoparticle Aggregates

The absorptance spectra of gold and silver nanoparticle (NP) aqueous dispersions were measured by UV–visible spectroscopy and computed numerically by finite element method. Both NPs were functionalized by l-cysteine amino acid (Cys) in order to develop aggregate-based localized surface plasmon resonance biosensors. Absorptance spectra measured at an analogous pH value of ∼4.9 were compared, where Au-Cys conjugates have moderately split spectra with two commensurate maxima, while Ag-Cys conjugates exhibit the most pronounced secondary peak according to the highest degree of aggregation. The purpose of our theoretical study was to determine the simplest linear chain-like and wavy aggregate geometries, which result in maxima matching the measured peaks. The aggregates were characterized by N number and d diameter of NPs, g gap between the NPs, and t thickness of the l-cysteine covering. By tuning the angle of incidence and E-field oscillation direction in p-polarized light with respect to the aggregates, the contribution of longitudinal and transversal modes was varied. The comparison of measurements and computations revealed that spectra measured on bioconjugate dispersions include effects of numerous aggregates with various geometries, illuminated from different directions and are influenced by inter-aggregate coupling. Inspecting the normalized E-field distribution surrounding the aggregates, it was shown that fundamentally different multipolar modes can be identified at primary and secondary absorptance maxima, due to coupled plasmonic resonances on NPs.

Integrated lithography to prepare arrays of rounded nano-objects

An integrated lithography method is presented to prepare rounded nano-objects with variable shape, in arrays with arbitrary symmetry and wavelength-scaled periodicity. Finite element method was applied to determine the near-field confinement under monolayers of silver and gold colloid spheres illuminated by circularly polarized beams possessing periodic intensity distribution, and to predict the shape of nano-objects, which can be fabricated on thin noble metal layers on glass substrates. It was shown that illumination by perpendicularly incident homogeneous beam results in hexagonal array of uniform nano-rings, while uniform nano-crescents appear due to single obliquely incident beam. Illumination of colloid sphere monolayers by interfering beams causes development of co-existent nano-rings and nanocrescents. It was demonstrated that the periodicity of complex patterns is determined by the wavelength and angle of incidence; the inter-object distance is controlled by the relative orientation of interference patterns with respect to colloid sphere monolayers; the nano-object size is determined by the wavelength, sphere diameter and material; while the nearfield distribution sensitively depends on the direction of illumination by circularly polarized light. We present complex patterns of various rounded nano-objects that can be uniquely fabricated via Circular Integrated Interference and Colloid sphere Lithography (CIICL), and applied as plasmonic and meta-materials.

Rotated grating coupled surface plasmon resonance on wavelength-scaled shallow rectangular gratings

Theoretical investigation of rotated grating coupling phenomenon was performed on a multilayer comprising 416-nmperiodic shallow rectangular polymer grating on bimetal film made of gold and silver layers. During the multilayer illumination by 532 nm wavelength p-polarized light the polar and azimuthal angles were varied. In presence of 0-35 nm, 0-50 nm and 15-50 nm thick polymer-layers at the valleys and hills splitting was observed on the dual-angle dependent reflectance in two regions: (i) close to 0° azimuthal angle corresponding to incidence plane parallel to the periodic pattern (P-orientation); and (ii) around ~33.5°/29°/30° azimuthal angle (C-orientation), in agreement with our previous experimental studies. The near-field study revealed that in P-orientation the E-field is enhanced at the glass side with p/2 periodicity at the first minimum appearing at 49°/50°/52° polar angles, and comprises maxima below both the valleys and hills; while E-field enhancement is observable both at the glass and polymer side with p-periodicity at the second minimum developing at 55°/63/64° tilting, comprising maxima intermittently below the valleys or above the hills. In Corientation coupled plasmonic modes are observable, involving modes propagating along the valleys at the secondary maxima appearing at ~35°/32°/32° azimuthal and ~49°/51°/56° polar angles, while modes confined along the polymer hills are observable at the primary minima, which are coupled most strongly at the ~31.5°/25°/28° azimuthal and ~55°/63°/66° polar angles. The secondary peak observable in C-orientation is proposed for biosensing applications, since the supported modes are confined along the valleys, where biomolecules prefer to attach.

Detection of biomolecules and bioconjugates by monitoring rotated grating-coupled surface plasmon resonance

Plasmonic biosensing chips were prepared by fabricating wavelength-scaled dielectric-metal interfacial gratings on thin polycarbonate films covered bimetal layers via two-beam interference laser lithography. Lysozyme (LYZ) biomolecules and gold nanoparticle (AuNP-LYZ) bioconjugates with 1:5 mass ratio were seeded onto the biochip surfaces. Surface plasmon resonance spectroscopy was performed before and after biomolecule seeding in a modified Kretschmann-arrangement by varying the azimuthal and polar angles to optimize the conditions for rotated grating-coupling. The shift of secondary and primary resonance peaks originating from rotated grating-coupling phenomenon was monitored to detect the biomolecule and bioconjugate adherence. Numerical calculations were performed to reproduce the measured reflectance spectra and the resonance peak shifts caused by different biocoverings. Comparison of measurements and calculations proved that monitoring the narrower secondary peaks under optimal rotated-grating coupling condition makes it possible to achieve enhanced sensitivity in biodetection. The sensitivity is further increased in case of bioconjugates due to coupled localized resonances on Au NPs. The enlarged resonance peak shift is resulted by the two-fold antisymmetric long-range plamonic modes propagating at the edge of the valleys and hills, which originate from Bragg scattered surface plasmon polaritons. Optimal configuration of ideal chips supporting rotated grating-coupled long-range plasmonic modes are proposed for biosensing.