Volume Determination of Human Metaphase Chromosomes by Scanning Force Microscopy

The scanning force microscope (SFM) yields the topography of the investigated surface. A procedure was developed which starts from this three-dimensional information to estimate the volume of a biological specimen. The volume of spread human metaphase chromosomes was determined in air and rehydrated in aqueous buffer. A difference of the determined volume of a air-dried metaphase chromosome set was found compared to values from electron microscopic investigations, and could be correlated with differences in the hydration state of the chromosomes. SFM-based relative volumes of air-dried chromosomes resembles literature data regarding volume range and distribution. Possible application of SFM-based relative volume measurements for chromosome classification purposes is discussed

Scanning Force Microscopy of Chromatin

Scanning force microscopy (SFM) is a new method to obtain the topography of surfaces with nanometer-resolution. The ability to image under liquids makes the technique attractive for biological applications, especially for the determination of the ultrastructure of biomolecules under native conditions. One growing field of interest is the investigation of chromatin and chromatin-related structures. Different levels of chromatin condensation were the subject of several previous SFM investigations, from the nucleosomal chain, to the 30-nm fiber, ending with the metaphase chromosome. The SFM yielded new information on such fundamental problems as the core spacing of the nucleosomal chain, the internal structure of the 30-nm fiber and the banding mechanism of metaphase chromosomes. Other investigations dealt with the SFM characterization of polytene chromosomes. This paper reviews the state-of-the-art in SFM chromatin research and discusses future developments in this field

Norrin stimulates cell proliferation in the superficial retinal vascular plexus and is pivotal for the recruitment of mural cells

Mutations in Norrin, the ligand of a receptor complex consisting of FZD4, LRP5 and TSPAN12, cause severe developmental blood vessel defects in the retina and progressive loss of the vascular system in the inner ear, which lead to congenital blindness and progressive hearing loss, respectively. We now examined molecular pathways involved in developmental retinal angiogenesis in a mouse model for Norrie disease. Comparison of morphometric parameters of the superficial retinal vascular plexus (SRVP), including the number of filopodia, vascular density and number of branch points together with inhibition of Notch signaling by using DAPT, suggest no direct link between Norrin and Notch signaling during formation of the SRVP. We noticed extensive vessel crossing within the SRVP, which might be a loss of Wnt- and MAP kinase-characteristic feature. In addition, endomucin was identified as a marker for central filopodia, which were aligned in a thorn-like fashion at P9 in Norrin knockout (Ndpy/−) mice. We also observed elevated mural cell coverage in the SRVP of Ndpy/− mice and explain it by an altered expression of PDGFβ and its receptor (PDGFRβ). In vivo cell proliferation assays revealed a reduced proliferation rate of isolectin B4-positive cells in the SRVP from Ndpy/− mice at postnatal day 6 and a decreased mitogenic activity of mutant compared with the wild-type Norrin. Our results suggest that the delayed outgrowth of the SRVP and decreased angiogenic sprouting in Ndpy/− mice are direct effects of the reduced proliferation of endothelial cells from the SRV

Propagating and localized surface plasmon resonance sensing — A critical comparison based on measurements and theory

With its potential for ultrasensitive, label-free detection of molecular interactions, sensing methods based on the surface plasmon resonance (SPR) effect fully meet the requirements for modern analytical techniques. Already established by using propagating SPR in thin gold layers, the last years witnessed the emergence of another related technique utilizing extremely miniaturized noble metal sensor structures, based on a localized SPR. This paper provides a critical comparison of these kinds of SPR sensing, reviews the foundation of both general approaches, presents experimental data on exactly the same molecular model system using both techniques, as well as theoretical considerations in order to allow reasonable comparison. It highlights the specific features and effects, in order to provide guidance in choosing the right technique for given bioanalytical tasks. The study demonstrated the capabilities of LSPR for sensing of molecular layers even in the lower nanometer dimension. For the detection of small (bio)molecules, smaller particle diameters are favored regarding highest sensitivity. It also presents an approach to obtain refractive index and the thickness of a molecular film by analyzing the signal response of plasmonic sensors with metal nanoparticles. Moreover, an additional method for the improvement of the parameters' determination is introduced

Modification of Surface Bond Au Nanospheres by Chemically and Plasmonically Induced Pd Deposition

In this work we investigated methods of modifying gold nanospheres bound to a silicon surface by depositing palladium onto the surfaces of single nanoparticles. Bimetallic Au-Pd nanoparticles can thus be gained for use in catalysis or sensor technology. For Pd deposition, two methods were chosen. The first method was the reduction of palladium acetate by ascorbic acid, in which the amounts of palladium acetate and ascorbic acid were varied. In the second method we utilized light-induced metal deposition by making use of the plasmonic effect. Through this method, the surface bond nanoparticles were irradiated with light of wavelengths capable of inducing plasmon resonance. The generation of hot electrons on the particle surface then reduced the palladium acetate in the vicinity of the gold nanoparticle, resulting in palladium-covered gold nanospheres. In our studies we demonstrated the effect of both enhancement methods by monitoring the particle heights over enhancement time by atomic force microscopy (AFM), and investigated the influence of ascorbic acid/Pd acetate concentration as well as the impact of the irradiated wavelengths on the enhancement effect. It could thus be proven that both methods were valid for obtaining a deposition of Pd on the surface of the gold nanoparticles. Deposition of Pd on the gold particles using the light-assisted method could be observed, indicating the impact of the plasmonic effect and hot electron for Pd acetate reduction on the gold particle surface. In the case of the reduction method with ascorbic acid, in addition to Pd deposition on the gold nanoparticle surface, larger pure Pd particles and extended clusters were also generated. The reduction with ascorbic acid however led to a considerably thicker Pd layer of up to 54 nm in comparison to up to 11 nm for the light-induced metal deposition with light resonant to the particle absorption wavelength. Likewise, it could be demonstrated that light of non-resonant wavelengths was not capable of initiating Pd deposition, since a growth of only 1.6 nm (maximum) was observed for the Pd layer

2-LED-μspectrophotometer for rapid on-site detection of pathogens using noble-metal nanoparticle-based colorimetric assays

Novel point-of-care compatible methods such as colorimetric assays have become increasingly important in the field of early pathogen detection. A simple and hand-held prototype device for carrying out DNA-amplification assay based on plasmonic nanoparticles in the colorimetric detection is presented. The low-cost device with two channels (sample and reference) consists of two spectrally different light emitting diodes (LEDs) for detection of the plasmon shift. The color change of the gold-nanoparticle-DNA conjugates caused by a salt-induced aggregation test is examined in particular. A specific and sensitive detection of the waterborne human pathogen Legionella pneumophila is demonstrated. This colorimetric assay, with a simple assay design and simple readout device requirements, can be monitored in real-time on-site. © 2020 by the authors

A new strategy for silver deposition on Au nanoparticles with the use of peroxidase-mimicking DNAzyme monitored by Localized Surface Plasmon Resonance technique

Peroxidase-mimicking DNAzyme was applied as a catalyst of silver deposition on gold nanoparticles. This DNAzyme is formed when hemin binds to the G-quadruplex-forming DNA sequence. Such a system is able to catalyze a redox reaction with a one- or two-electron transfer. The process of silver deposition was monitored via a localized surface plasmon resonance technique (LSPR), which allows one to record scattering spectrum of a single nanoparticle. Our study showed that DNAzyme is able to catalyze silver deposition. The AFM experiments proved that DNAzyme induced the deposition of silver shells of approximately 20 nm thickness on Au nanoparticles (AuNPs). Such an effect is not observed when hemin is absent in the system. However, we noticed non-specific binding of hemin to the capture oligonucleotides on a gold NP probe that also induced some silver deposition, even though the capture probe was unable to form G-quadruplex. Analysis of SEM images indicated that the surface morphology of the silver layer deposited by DNAzyme is different from that obtained for hemin alone. The proposed strategy of silver layer synthesis on gold nanoparticles catalyzed by DNAzyme is an innovative approach and can be applied in bioanalysis (LSPR, electrochemistry) as well as in material sciences

Ultrastructural characterization of colloidal metal films for bioanalytical applications by scanning force microscopy

Colloidal metal films (CMFs) are prepared by the attachment of silver, gold, or platinum (or other metal) particles to a glass slide modified by silanization with 3‐mercaptopropyl silane. The covalent attachment of the metal particles occurs through the metal–sulfur bond. In these samples the local electromagnetic field is enhanced near the surface of the CMF due to excitation of plasmon resonances. This phenomenon can be used for a variety of analytical applications. Because the optical properties are strongly dependent on the morphology of the film, its structural characterization becomes of great importance. To further characterize CMFs we have used scanning force microscopy (SFM). Initial studies revealed lateral dimensions of the particles as well as the particle density. Height measurements were made using the three‐dimensional topographic image of the surface yielded by SFM, and were used to evaluate the selective deposition of a silica spacer layer onto the metal particles. Comparative SFM measurements in water and air using different imaging modes were performed to investigate the mechanical stability of CMFs against the forces exerted by the scanning tip