3 research outputs found
Gold Nanorods as Plasmonic Sensors for Particle Diffusion
Plasmonic
gold nanoparticles are normally used as sensor to detect
analytes permanently bound to their surface. If the interaction between
the analyte and the nanosensor surface is negligible, it only diffuses
through the sensorās sensing volume, causing a small temporal
shift of the plasmon resonance position. By using a very sensitive
and fast detection scheme, we are able to detect these small fluctuations
in the plasmon resonance. With the help of a theoretical model consistent
with our detection geometry, we determine the analyteās diffusion
coefficient. The method is verified by observing the trends upon changing
diffusor size and medium viscosity, and the diffusion coefficients
obtained were found to reflect reduced diffusion close to a solid
interface. Our method, which we refer to as NanoPCS (for nanoscale
plasmon correlation spectroscopy), is of practical importance for
any application involving the diffusion of analytes close to nanoparticles
Application of DNA Machineries for the Barcode Patterned Detection of Genes or Proteins
The study introduces an analytical
platform for the detection of
genes or aptamer-ligand complexes by nucleic acid barcode patterns
generated by DNA machineries. The DNA machineries consist of nucleic
acid scaffolds that include specific recognition sites for the different
genes or aptamer-ligand analytes. The binding of the analytes to
the scaffolds initiate, in the presence of the nucleotide mixture,
a cyclic polymerization/nicking machinery that yields displaced strands
of variable lengths. The electrophoretic separation of the resulting
strands provides barcode patterns for the specific detection of the
different analytes. Mixtures of DNA machineries that yield, upon sensing
of different genes (or aptamer ligands), one-, two-, or three-band
barcode patterns are described. The combination of nucleic acid scaffolds
acting, in the presence of polymerase/nicking enzyme and nucleotide
mixture, as DNA machineries, that generate multiband barcode patterns
provide an analytical platform for the detection of an individual
gene out of many possible genes. The diversity of genes (or other
analytes) that can be analyzed by the DNA machineries and the barcode
patterned imaging is given by the Pascalās triangle. As a proof-of-concept,
the detection of one of six genes, that is, TP53, Werner syndrome,
Tay-Sachs normal gene, BRCA1, Tay-Sachs mutant gene, and cystic fibrosis
disorder gene by six two-band barcode patterns is demonstrated. The
advantages and limitations of the detection of analytes by polymerase/nicking
DNA machineries that yield barcode patterns as imaging readout signals
are discussed
Multiple Internalization Pathways of Polyelectrolyte Multilayer Capsules into Mammalian Cells
Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (<i>e</i>.<i>g</i>., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching-off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phagolysosomes, but not with other endocytic vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to heterophagolysosomes