RESUMO
The emergence of antibiotic and antifungal resistant microorganisms represents nowadays a major public health issue that might push humanity into a post-antibiotic/antifungal era. One of the approaches to avoid such a catastrophe is to advance rapid antibiotic and antifungal susceptibility tests. In this study, we present a compact, optical fiber-based nanomotion sensor to achieve this goal by monitoring the dynamic nanoscale oscillation of a cantilever related to microorganism viability. High detection sensitivity was achieved that was attributed to the flexible two-photon polymerized cantilever with a spring constant of 0.3 N/m. This nanomotion device showed an excellent performance in the susceptibility tests of Escherichia coli and Candida albicans with a fast response in a time frame of minutes. As a proof-of-concept, with the simplicity of use and the potential of parallelization, our innovative sensor is anticipated to be an interesting candidate for future rapid antibiotic and antifungal susceptibility tests and other biomedical applications.
Assuntos
Antibacterianos , Antifúngicos , Fibras Ópticas , Testes de Sensibilidade Microbiana , Candida albicans , Escherichia coliRESUMO
The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson's disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments. We show that protofilaments form from the direct molecular assembly of unfolded monomeric α-synuclein polypeptide chains. To unravel protofilaments' internal structure and elastic properties, we manipulated nanomechanically these species by atomic force spectroscopy. The single-molecule scale identification and characterization of the fundamental unit of amyloid assemblies provide insights into early events underlying their formation and shed light on opportunities for therapeutic intervention at the early stages of aberrant protein self-assembly.
Assuntos
Amiloide/química , Desdobramento de Proteína , alfa-Sinucleína/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Humanos , Microscopia de Força Atômica , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , alfa-Sinucleína/metabolismoRESUMO
We propose an all-dielectric magneto-photonic crystal with a hybrid magneto-optical response that allows for the simultaneous measurements of the surface and bulk refractive index of the analyzed substance. The approach is based on two different spectral features of the magneto-optical response corresponding to the resonances in p- and s-polarizations of the incident light. Angular spectra of p-polarized light have a step-like behavior near the total internal reflection angle which position is sensitive to the bulk refractive index. S-polarized light excites the TE-polarized optical Tamm surface mode localized in a submicron region near the photonic crystal surface and is sensitive to the refractive index of the near-surface analyte. We propose to measure a hybrid magneto-optical intensity modulation of p-polarized light obtained by switching the magnetic field between the transverse and polar configurations. The transversal component of the external magnetic field is responsible for the magneto-optical resonance near total internal reflection conditions, and the polar component reveals the resonance of the Tamm surface mode. Therefore, both surface- and bulk-associated features are present in the magneto-optical spectra of the p-polarized light.
RESUMO
Chemical characterization at the nanoscale is of significant importance for many applications in physics, analytical chemistry, material science, and biology. Despite the intensive studies in the infrared range, high-spatial-resolution and high-sensitivity imaging for compositional identification in the visible range is rarely exploited. In this work, we present a gap-plasmon-enhanced imaging approach based on photothermal-induced resonance (PTIR) for nanoscale chemical identification. With this approach, we experimentally obtained a high spatial resolution of â¼5 nm for rhodamine nanohill characterization and achieved monolayer sensitivity for mapping the single-layer chlorophyll-a islands with the thickness of only 1.9 nm. We also successfully characterized amyloid fibrils stained with methylene blue dye, indicating that this methodology can be also utilized for identification of the radiation-insensitive macromolecules. We believe that our proposed high-performance visible PTIR system can be used to broaden the applications of nanoscale chemical identification ranging from nanomaterial to life science areas.
RESUMO
We performed dynamic force spectroscopy of single dextran and titin I27 molecules using small-amplitude and low-frequency (40-240 Hz) dithering of an atomic force microscope tip excited by a sine wave voltage fed onto the tip-carrying piezo. We show that for such low-frequency dithering experiments, recorded phase information can be unambiguously interpreted within the framework of a transparent theoretical model that starts from a well-known partial differential equation to describe the dithering of an atomic force microscope cantilever and a single molecule attached to its end system, uses an appropriate set of initial and boundary conditions, and does not exploit any implicit suggestions. We conclude that the observed phase (dissipation) signal is due completely to the dissipation related to the dithering of the cantilever itself (i.e., to the change of boundary conditions in the course of stretching). For both cases, only the upper bound of the dissipation of a single molecule has been established as not exceeding 3â 10(-7)kg/s. We compare our results with previously reported measurements of the viscoelastic properties of single molecules, and we emphasize that extreme caution must be taken in distinguishing between the dissipation related to the stretched molecule and the dissipation that originates from the viscous damping of the dithered cantilever. We also present the results of an amplitude channel data analysis, which reveal that the typical values of the spring constant of a I27 molecule at the moment of module unfolding are equal to 4±1.5mN/m, and the typical values of the spring constant of dextran at the moment of chair-boat transition are equal to 30-50mN/m.
Assuntos
Dextranos/química , Microscopia de Força Atômica , Proteínas/química , Elasticidade , Desenho de Equipamento , Escherichia coli , Humanos , Modelos Teóricos , Solventes/química , Viscosidade , Água/químicaRESUMO
The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVß3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Laminina/metabolismo , Fusão de Membrana , Estresse Mecânico , Internalização do Vírus , Humanos , Concentração de Íons de Hidrogênio , Integrina alfaVbeta3/metabolismo , Cinética , Ligantes , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismo , Análise Espectral , Termodinâmica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
Amyloid polymorphism is a hallmark of almost all amyloid species, yet the mechanisms underlying the formation of amyloid polymorphs and their complex architectures remain elusive. Commonly, two main mesoscopic topologies are found in amyloid polymorphs characterized by non-zero Gaussian and mean curvatures: twisted ribbons and helical fibrils, respectively. Here, a rich heterogeneity of configurations is demonstrated on insulin amyloid fibrils, where protofilament packing can occur, besides the common polymorphs, also in a combined mode forming mixed-curvature polymorphs. Through AFM statistical analysis, an extended array of heterogeneous architectures that are rationalized by mesoscopic theoretical arguments are identified. Notably, an unusual fibrillization pathway is also unraveled toward mixed-curvature polymorphs via the widespread recruitment and intertwining of protofilaments and protofibrils. The results present an original view of amyloid polymorphism and advance the fundamental understanding of the fibrillization mechanism from single protofilaments into mature amyloid fibrils.
RESUMO
A label-free biosensor device based on registration of photonic crystal surface waves is described. Angular interrogation of the optical surface wave resonance is used to detect changes in the thickness of an adsorbed layer, while an additional simultaneous detection of the critical angle of total internal reflection provides independent data of the liquid refractive index. The abilities of the device are demonstrated by measuring of biotin molecule binding to a streptavidin monolayer, and by measuring association and dissociation kinetics of immunoglobulin G proteins. Additionally, deposition of PSS / PAH polyelectrolytes is recorded in situ resulting calculation of PSS and PAH monolayer thicknesses separately.
Assuntos
Técnicas Biossensoriais/instrumentação , Óptica e Fotônica/instrumentação , Animais , Biotina/metabolismo , Biotinilação , Cristalização , Eletrólitos/química , Proteínas Imobilizadas/metabolismo , Imunoglobulina G/metabolismo , Ligantes , Camundongos , Coelhos , Processamento de Sinais Assistido por Computador , Estreptavidina/metabolismo , Propriedades de SuperfícieRESUMO
We propose and demonstrate dendrimer-based coatings for a sensitive biochip surface that enhance the high-performance sorption of small molecules (i.e., biomolecules with low molecular weights) and the sensitivity of a label-free, real-time photonic crystal surface mode (PC SM) biosensor. Biomolecule sorption is detected by measuring changes in the parameters of optical modes on the surface of a photonic crystal (PC). We describe the step-by-step biochip fabrication process. Using oligonucleotides as small molecules and PC SM visualization in a microfluidic mode, we show that the PAMAM (poly-amidoamine)-modified chip's sorption efficiency is almost 14 times higher than that of the planar aminosilane layer and 5 times higher than the 3D epoxy-dextran matrix. The results obtained demonstrate a promising direction for further development of the dendrimer-based PC SM sensor method as an advanced label-free microfluidic tool for detecting biomolecule interactions. Current label-free methods for small biomolecule detection, such as surface plasmon resonance (SPR), have a detection limit down to pM. In this work, we achieved for a PC SM biosensor a Limit of Quantitation of up to 70 fM, which is comparable with the best label-using methods without their inherent disadvantages, such as changes in molecular activity caused by labeling.
RESUMO
The phenomenon of amyloid polymorphism is a key feature of protein aggregation. Unravelling this phenomenon is of great significance for understanding the underlying molecular mechanisms associated with neurodegenerative diseases and for the development of amyloid-based functional biomaterials. However, the understanding of the molecular origins and the physicochemical factors modulating amyloid polymorphs remains challenging. Herein, we demonstrate an association between amyloid polymorphism and environmental stress in solution, induced by an air/water interface in motion. Our results reveal that low-stress environments produce heterogeneous amyloid polymorphs, including twisted, helical, and rod-like fibrils, whereas high-stress conditions generate only homogeneous rod-like fibrils. Moreover, high environmental stress converts twisted fibrils into rod-like fibrils both in-pathway and after the completion of mature amyloid formation. These results enrich our understanding of the environmental origin of polymorphism of pathological amyloids and shed light on the potential of environmentally controlled fabrication of homogeneous amyloid biomaterials for biotechnological applications.
Assuntos
Amiloide , Hidrodinâmica , Proteínas Amiloidogênicas , ÁguaRESUMO
A current-driven source of long-range surface plasmons (LRSPs) on a duplex metal nanolayer is reported. Electrical excitation of LRSPs was experimentally observed in a planar structure, where an organic light-emitting film was sandwiched between two metal nanolayers that served as electrodes. To achieve the LRSP propagation in these metal nanolayers at the interface with air, the light-emitting structure was bordered by a one-dimensional photonic crystal (PC) on the other side. The dispersion of the light emitted by such a hybrid PC/organic-light-emitting-diode structure (PC/OLED) comprising two thin metal electrodes was obtained, with a clearly identified LRSP resonance peak.
RESUMO
The xanthophyll cycle is a regulatory mechanism operating in the photosynthetic apparatus of plants. It consists of the conversion of the xanthophyll pigment violaxanthin to zeaxanthin, and vice versa, in response to light intensity. According to the current understanding, one of the modes of regulatory activity of the cycle is associated with the influence on a molecular organization of pigment-protein complexes. In the present work, we analyzed the effect of violaxanthin and zeaxanthin on the molecular organization of the LHCII complex, in the environment of membranes formed with chloroplast lipids. Nanoscale imaging based on atomic force microscopy (AFM) showed that the presence of exogenous xanthophylls promotes the formation of the protein supramolecular structures. Nanoscale infrared (IR) absorption analysis based on AFM-IR nanospectroscopy suggests that zeaxanthin promotes the formation of LHCII supramolecular structures by forming inter-molecular ß-structures. Meanwhile, the molecules of violaxanthin act as "molecular spacers" preventing self-aggregation of the protein, potentially leading to uncontrolled dissipation of excitation energy in the complex. This latter mechanism was demonstrated with the application of fluorescence lifetime imaging microscopy. The intensity-averaged chlorophyll a fluorescence lifetime determined in the LHCII samples without exogenous xanthophylls at the level of 0.72 ns was longer in the samples containing exogenous violaxanthin (2.14 ns), but shorter under the presence of zeaxanthin (0.49 ns) thus suggesting a role of this xanthophyll in promotion of the formation of structures characterized by effective excitation quenching. This mechanism can be considered as a representation of the overall photoprotective activity of the xanthophyll cycle.
Assuntos
Membranas Intracelulares/enzimologia , Complexos de Proteínas Captadores de Luz/química , Narcissus/química , Spinacia oleracea/enzimologia , Zeaxantinas/química , Clorofila A/química , Estrutura Secundária de Proteína , Xantofilas/químicaRESUMO
The formation of amyloid fibrils is a characterizing feature of a range of protein misfolding diseases, including Parkinson's disease. The propensity of native proteins to form such amyloid fibril, both in vitro and in vivo, is highly sensitive to the surrounding environment, which can alter the aggregation kinetics and fibrillization mechanisms. Here, we investigate systematically the influence of several representative environmental stimuli on α-synuclein aggregation, including hydrodynamic mixing, the presence of an air-water interface and sedimentation. Our results show that hydrodynamic mixing and interfacial effects are critical in promoting several microscopic steps of α-synuclein aggregation and amyloid fibril formation. The presence of an air-water interface under agitation significantly promoted primary nucleation. Secondary processes were facilitated by hydrodynamic mixing, produced by 3D rotation and shaking either in the presence or in the absence of an air-water interface. Effects of sedimentation, as investigated in a microgravity incubator, of α-synuclein lead only to minor changes on the aggregation kinetics rates in comparison to static conditions. These results forward the understanding of α-synuclein fibrillization, paving the way for the development of high-throughput assays for the screening of pharmacological approaches targeting Parkinson's disease.
RESUMO
Atomic force microscopy was used to image single-stranded DNA (ssDNA) adsorbed on mica modified by Mg(2+), by 3-aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing, thus this method could be important for studying certain processes involving ssDNA in more details.
Assuntos
Silicatos de Alumínio , DNA de Cadeia Simples/ultraestrutura , Grafite , Microscopia de Força AtômicaRESUMO
Optical biosensors based on photonic crystal surface waves (PC SWs) offer a possibility to study binding interactions with living cells, overcoming the limitation of rather small evanescent field penetration depth into a sample medium that is characteristic for typical optical biosensors. Besides this, simultaneous excitation of s- and p-polarized surface waves with different penetration depths is realized here, permitting unambiguous separation of surface and volume contributions to the measured signal. PC-based biosensors do not require a bulk signal correction, compared to widely used surface plasmon resonance-based devices. We developed a chitosan-based protocol of PC chip functionalization for bacterial attachment and performed experiments on antibody binding to living bacteria measured in real time by the PCSW-based biosensor. Data analysis reveals specific binding and gives the value of the dissociation constant for monoclonal antibodies (IgG2b) against bacterial lipopolysaccharides equal to KD = 6.2 ± 3.4 nM. To our knowledge, this is a first demonstration of antibody-binding kinetics to living bacteria by a label-free optical biosensor.
Assuntos
Anticorpos , Bactérias , Técnicas Biossensoriais , Membrana Celular , Fótons , Cinética , Ligação ProteicaRESUMO
Currently, sensors invade into our everyday life to bring higher life standards, excellent medical diagnostic and efficient security. Plasmonic biosensors demonstrate an outstanding performance ranking themselves among best candidates for different applications. However, their sensitivity is still limited that prevents further expansion. Here we present a novel concept of magnetoplasmonic sensor with ultranarrow resonances and high sensitivity. Our approach is based on the combination of a specially designed one-dimensional photonic crystal and a ferromagnetic layer to realize ultralong-range propagating magnetoplasmons and to detect alteration of the environment refractive index via observation of the modifications in the Transversal Magnetooptical Kerr Effect spectrum. The fabrication of such a structure is relatively easy in comparison with e.g. nanopatterned samples. The fabricated heterostructure shows extremely sharp (angular width of 0.06°) surface plasmon resonance and even sharper magnetoplasmonic resonance (angular width is 0.02°). It corresponds to the propagation length as large as 106 µm which is record for magnetoplasmons and promising for magneto-optical interferometry and plasmonic circuitry as well as magnetic field sensing. The magnitude of the Kerr effect of 11% is achieved which allows for detection limit of 1â10(-6). The prospects of further increase of the sensitivity of this approach are discussed.
RESUMO
ELISA and Western blot immunochemical data attest an effective and highly specific interaction of the surface glycoprotein E domain II (DII) of the tick born encephalitis and Dengue viruses with the laminin binding protein (LBP). Based on a highly conservative structure of the DII in different flaviviruses we propose a similarly effective interaction between the LBP and the DII of the surface glycoprotein E of the West Nile virus. We report the results of studies of this interaction by immunochemical and single molecule force spectroscopy methods. The specific binding between these species is confirmed by both methods.