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Fast, Label-Free Tracking of Single Viruses and Weakly Scattering Nanoparticles in a Nanofluidic Optical Fiber
High-speed tracking of single particles is a gateway to understanding physical, chemical, and biological processes at the nanoscale. It is also a major experimental challenge, particularly for small, nanometer-scale particles. Although methods such as confocal or fluorescence microscopy offer both high spatial resolution and high signal-to-background ratios, the fluorescence emission lifetime limits the measurement speed, while photobleaching and thermal diffusion limit the duration of measurements. Here we present a tracking method based on elastic light scattering that enables long-duration measurements of nanoparticle dynamics at rates of thousands of frames per second. We contain the particles within a single-mode silica fiber having a subwavelength, nanofluidic channel and illuminate them using the fiber's strongly confined optical mode. The diffusing particles in this cylindrical geometry are continuously illuminated inside the collection focal plane. We show that the method can track unlabeled dielectric particles as small as 20 nm as well as individual cowpea chlorotic mottle virus (CCMV) virions-26 nm in size and 4.6 megadaltons in mass-at rates of over 3 kHz for durations of tens of seconds. Our setup is easily incorporated into common optical microscopes and extends their detection range to nanometer-scale particles and macromolecules. The ease-of-use and performance of this technique support its potential for widespread applications in medical diagnostics and micro total analysis systems.
Raman imaging to study structural and chemical features of the dentin enamel junction
The structure and chemical features of the human dentin enamel junction (DEJ) were characterized using Raman spectroscopic imaging. Slices were prepared from 10 German, and 10 Turkish teeth. Raman images were collected at 785 nm excitation and the average Raman spectra were calculated for analysis. Univariate and multivariate spectral analysis were applied for investigation. Raman images were obtained based on the intensity ratios of CH at 1450 cm-1 (matrix) to phosphate at 960 cm-1 (mineral), and carbonate to phosphate (1070/960) ratios. Different algorithms (HCA, K-means cluster and VCA) also used to study the DEJ. The obtained results showed that the width of DEJ is about 5 pm related to univariate method while it varies from 6 to 12 μm based on multivariate spectral technique. Both spectral analyses showed increasing in carbonate content inside the DEJ compared to the dentin, and the amide I (collagen) peak in dentin spectra is higher than DEJ spectra peak.
Towards on-site testing of Phytophthora species
Rapid detection and accurate identification of plant pathogens in the field is an ongoing challenge. In this study, we report for the first time on the development of a helicase-dependent isothermal amplification (HDA) in combination with on-chip hybridization for the detection of selected Phytophthora species. The HDA approach allows efficient amplification of the yeast GTP-binding protein (Ypt1) target gene region at one constant temperature in a miniaturized heating device. The assay's specificity was determined by on-chip DNA hybridization and subsequent silver nanoparticle deposition. The silver deposits serve as stable endpoint signals that enable the visual as well as the electrical readout. Our promising results point to the direction of a near future on-site application of the combined techniques for a reliable detection of Phytophthora species.
Hybrid Optical Fibers – An Innovative Platform for In‐Fiber Photonic Devices
The field of hybrid optical fibers is one of the most active research areas in current fiber optics and has the vision of integrating sophisticated materials inside fibers, which are not traditionally used in fiber optics. Novel in-fiber devices with unique properties have been developed, opening up new directions for fiber optics in fields of critical interest in modern research, such as biophotonics, environmental science, optoelectronics, metamaterials, remote sensing, medicine, or quantum optics. Here the recent progress in the field of hybrid optical fibers is reviewed from an application perspective, focusing on fiber-integrated devices enabled by including novel materials inside polymer and glass fibers. The topics discussed range from nanowire-based plasmonics and hyperlenses, to integrated semiconductor devices such as optoelectronic detectors, and intense light generation unlocked by highly nonlinear hybrid waveguides.
Rapid Colorimetric Detection of Pseudomonas aeruginosa in Clinical Isolates Using a Magnetic Nanoparticle Biosensor
A rapid, sensitive, and specific colorimetric biosensor based on the use of magnetic nanoparticles (MNPs) was designed for the detection of Pseudomonas aeruginosa in clinical samples. The biosensing platform was based on the measurement of P. aeruginosa proteolytic activity using a specific protease substrate. At the N-terminus, this substrate was covalently bound to MNPs and was linked to a gold sensor surface via cystine at the C-terminus of the substrates. The golden sensor appears black to naked eyes because of the coverage of the MNPs. However, upon proteolysis, the cleaved peptide–MNP moieties will be attracted by an external magnet, revealing the golden color of the sensor surface, which can be observed by the naked eye. In vitro, the biosensor was able to detect specifically and quantitatively the presence of P. aeruginosa with a detection limit of 102 cfu/mL in less than 1 min. The colorimetric biosensor was used to test its ability to detect in situ P. aeruginosa in clinical isolates from patients. This biochip is anticipated to be useful as a rapid point-of-care device for the diagnosis of P. aeruginosa-related infections.
Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies
The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
Characterisation of Methicillin-Resistant Staphylococcus aureus from Alexandria, Egypt
The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt’s second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.
Statistical Analysis of Scanning Fluorescence Correlation Spectroscopy Data Differentiates Free from Hindered Diffusion
Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly applicable statistical analysis pipeline for large scanning fluorescence correlation spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.
Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering
The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
Superelasticity of Plasma- and Synthetic Membranes Resulting from Coupling of Membrane Asymmetry, Curvature, and Lipid Sorting
Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the apparent initial PM area. Isolated lipids self-organize into membranes, but are prone to rupture at small (<2–4%) area strains, which limits progress for synthetic reconstitution of cellular features. Here, it is shown that by preserving PM structure and composition during isolation from cells, vesicles with cell-like elasticity can be obtained. It is found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These act as lipid reservoirs and are recruited by mechanical tension applied to the outer vesicle membrane. Both in experiment and theory, it is shown that a “superelastic” response emerges from the interplay of lipid domains and membrane curvature. This finding allows for bottom-up engineering of synthetic biomaterials that appear one magnitude softer and with threefold larger deformability than conventional lipid vesicles. These results open a path toward designing superelastic synthetic cells possessing the inherent mechanics of biological cells.