Elimination of adsorptive behaviour of biomolecules at the glass-solution interface in fluorescence correlation spectroscopy

We study adsorptive behaviour of biomolecules at the glass-solution interface in fluorescence correlation spectroscopy (FCS), and propose a negatively charged coating to eliminate the adsorption of molecules. In this article, we demonstrate confocal microscopic measurements on Cy3.5-90-mer-ssDNA and Cy3.5-90-bp-dsDNA in different solutions, and use two polymers – poly (acrylic acid, sodium salt) and poly (sodium 4-styrenesulfonate) to produce the negatively charged coating on glass coverslips. This technology enables more stable FCS measurements in extremely low concentration samples and reveals that the adsorptive behaviour of biomolecules is responsible for sudden disappearance of many iomolecules in low concentration solutions

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia

The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16+, CD57+ and cytotoxic CD 8+) was studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57+, CD 16+ and cytotoxic CD 8+ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3+ lymphocytes, which were 1.7–27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4+ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57+ receptors in comparison with their peripheral blood CD 57+ lymphocytes or the CD57+ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph node

Nanomechanical properties of α-synuclein amyloid fibrils: a comparative study by nanoindentation, harmonic force microscopy, and Peakforce QNM

We report on the use of three different atomic force spectroscopy modalities to determine the nanomechanical properties of amyloid fibrils of the human α-synuclein protein. α-Synuclein forms fibrillar nanostructures of approximately 10 nm diameter and lengths ranging from 100 nm to several microns, which have been associated with Parkinson's disease. Atomic force microscopy (AFM) has been used to image the morphology of these protein fibrils deposited on a flat surface. For nanomechanical measurements, we used single-point nanoindentation, in which the AFM tip as the indenter is moved vertically to the fibril surface and back while the force is being recorded. We also used two recently developed AFM surface property mapping techniques: Harmonic force microscopy (HarmoniX) and Peakforce QNM. These modalities allow extraction of mechanical parameters of the surface with a lateral resolution and speed comparable to tapping-mode AFM imaging. Based on this phenomenological study, the elastic moduli of the α-synuclein fibrils determined using these three different modalities are within the range 1.3-2.1 GPa. We discuss the relative merits of these three methods for the determination of the elastic properties of protein fibrils, particularly considering the differences and difficulties of each method

2.19 Biophysical analysis of amyloid formation

Amyloid formation is a fascinating process with both biomedical and materials science relevance. Amyloids can be pathological, but also possess interesting potential for use as nanobiomaterials. Although amyloids have long been the focus of intense study, the amyloid formation mechanism stays unclear, including the factors that initiate and drive the aggregation process. This chapter describes the application of advanced biophysical methods, such as (single-molecule) fluorescence spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, circular dichroism (CD) spectroscopy, and high-resolution atomic force microscopy (AFM), for exploration of amyloid characteristics. Collectively, the selection of techniques provides access to all stages of amyloid aggregation: from monomers to oligomeric intermediates, and mature fibrils. Single-molecule fluorescence is shown to provide information on monomer structure and flexibility. On an ensemble level, conformational information is acquired using CD spectroscopy and EPR spectroscopy. High-resolution AFM enables detailed exploration of morphological and mechanical properties of fibrillar structures. The biophysical research approaches described are widely applicable to the broader genre of amyloid proteins to uncover the mysteries that underlie the complex biophysics of amyloid formation and their optimal utilization in bionanotechnology

Fabrication of 1d nanochannels with thin glass wafers for single molecule studies

1D nanochannels are fabricated by direct bonding between the silicon wafer containing the nanochannels, microchannels, access holes and the blank thin glass of 170 µm thickness. The thin glass wafers are used as channel covers because of their appropriate thickness for use with coverglass corrected water immersion lenses. As the nanochannel chips are used on an inverted microscope, access holes are created in the silicon wafer so that polyimide is good selection for protection during deep etching of the macro holes. Fabricated nanochannels with 50 nm depth were successfully filled with low concentration of solutions Alexa 488 could be observed through fluorescence microscopy. These nanochannels are promising for mobility studies of single molecules such as quantum dots

Studies of Interaction Between Cyanine Dye T-284 and Fibrillar Alpha-Synuclein

A key feature of Parkinson’s disease is the formation and accumulation of amyloid fibrils of the natively unfolded protein α-synuclein (ASN) inside neurons. Recently we have proposed novel sensitive monomethinecyanine dye T-284 as fluorescent probe for quantitative detection of ASN amyloid fibrils. In this study the T-284 dye complex with ASN fibril was characterized by means of fluorescence anisotropy, atomic force microscopy and time-resolved fluorescence techniques to give further insights into the mode of dye interaction with amyloid fibrils. The fluorescence anisotropy of T-284 was shown to noticeably increase upon addition of aggregated proteins indicating on stable dye/amyloid fibril complex formation. AFM imaging of fibrillar wild-type ASN revealed differences in heights between ASN fibrils alone and in presence of the T-284 dye (6.37 ± 1.0 nm and 8.0 ± 1.1 nm respectively), that is believed to be caused by embedding of T-284 dye molecules in the “binding channel” running along the fibril. Fluorescence decay analysis of the T-284 in complexes with fibrillar ASN variants revealed the fluorescence lifetime values for T-284/fibril complexes to be an order of magnitude higher as compared to the free dye. Also, the fluorescence decay of free T-284 was bi-exponential, while dye bound to protein yields tri-exponential decay. We suppose that in complexes with fibrillar ASN variants T-284 dye might exist in different “populations” due to interaction with fibrils in different conformers and ways. The exact binding mode of T-284 with ASN fibrils needs further studies. Studied parameters of dye/amyloid fibril complexes are important for the characterization and screening of newly-developed amyloid-sensitive dyes