The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application
in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and
can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The
needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes
are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or
In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of
phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of
contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions
of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells). We have
shown, that carbon nanoparticles do not have any hemolytic effects, if judged by the dynamics of acidic hemolysis,
although they are capable of permeabilizating the cells and facilitating the internalization of propidium iodide into the nuclei.
Here we describe a novel approach to sialidase activity estimation. Sialidases (EC 18.104.22.168, exo-α-sialidases), also
known as neuraminidases, are the group of enzymes, which hydrolyze the glycoside bound between terminal sialic acid
and subsequent carbohydrate residue in glycoproteins and glycolipids. Sialic acids are the group of monosaccharides
with acidic properties, since they are acetylated or glycolylated derivates of neuraminic acid. Flu and some other viruses
use neuraminidase activity to infect host cells. The level of sialylation was shown to be tightly connected with tumor cell
invasiveness and metastatic potential, sialylation level also determines the clearance of aged or virus-infected cells. Thus,
detection of sialidase activity is of primary importance for clinical diagnostics as well as life science research.
The authors developed the assay for both visualization and estimation of sialidase activity in living cells. Previously
known methods for sialidase activity detection required destruction of cellular material, or were low-sensitive, or
provided no information on the activity localization in certain intracellular compartment. To overcome these problems, a
fluorogenic neuraminidase substrate, 4-MUNA was utilized, and the method for detection of neuraminidase activity
using fluorescent microscopy was proposed, it provided a high signal level and information on cellular localization of the
studied enzyme. By using this approach the increase of sialidase activity on apoptotic cells was demonstrated in
comparison to viable and primary necrotic cells.
Method of rapid detection of bacterial cells by light scattering is described. Determination of quantitative changes of
bacteria is the given method based on the changes of their size distributing in the process of cultivation. Liquid medial
are diluted and analyzed by the proposed technology to determine presence of bacteria. A method includes sounding of
flow suspended bacterial cells by monochromatic coherent light, registration of signals of co-operation of sounding
radiation with the explored microbiological objects by detects amplitudes and durations of scattered light impulses.
Distribution of particles by sizes is determined from the measured functional dependence of number of registered
particles from amplitude and duration of the proper electric impulses on the output photoreceiver. Detection is done for a
range of particle size from 0.1 to 10 mkm, and thus particle's size distribution is determined. The results of studying of
rapid detection of Escherichia coli by light scattering are described.
Monitoring of bacterial cell numbers is of great importance not only in microbiological industry but also for control of
liquids contamination in the food and pharmaceutical industries. Here we describe a novel low-cost and highly efficient
technology for bacterial cell monitoring during cultivation process. The technology incorporates previously developed
monitoring device and algorithm of its action. The devise analyses light scattered by suspended bacterial cells. Current
stage utilizes monochromatic coherent light and detects amplitudes and durations of scattered light impulses, it does not
require any labeling of bacterial cell. The system is calibrated using highly purificated bacteria-free water as standard.
Liquid medial are diluted and analyzed by the proposed technology to determine presence of bacteria. Detection is done
for a range of particle size from 0.1 to 10 μm, and thus particles size distribution is determined. We analyzed a set of
different bacterial suspensions and also their changes in quantity and size distribution during cultivation. Based on the
obtained results we conclude that proposed technology can be very effective for bacteria monitoring during cultivation
process, providing benefits of low simplicity and low cost of analysis with simultaneous high detection precision.
The determination algorithm of aerosol microparticles' size distribution by iteration process is described. In the described method the particle, registered by optoelectronic devices is characterized by parameters of amplitude and duration of impulse. Distribution of particles' size is determined from the measured functional dependence of number of registered particles from amplitude and duration of the proper electric impulses on the output photo-detector. Given dependence, within limits of statistical errors, is repeated when performing measurement series in the medium with identical optical parameters. It is linked by functional dependence to relative particles' fraction of different sizes, which is expressed by integral first kind Fredholm equation.
Apoptosis, or programmed cell death, is a form of cell death occurring during normal physiological processes and is used by the multicellular organism for elimination of "old" and impaired cells. Apoptosis is characterized by specific morphological changes such as plasma membrane blebbing, nucleus condensation, and cell wrinkling with further destruction into apoptotic bodies. Apoptosis detection is in focus of instrumental methods used in modern biomedical sciences. The available methods for such purpose are either very expensive, or require time-consuming operations. Their specificity and sensitivity are frequently not sufficient for biomedical diagnostics. We propose to use light scattering analysis for evaluation of apoptosis in cell population, especially for the detection of physical changes of cells, such as cell condensation and degradation into apoptotic bodies. The method proved to be very effective, providing quantitative estimation and high precision, simplicity and low costs of analysis (UA Patent No.64090). Another approach for the detection of apoptosis is based on the recently discovered fact (Bilyy, Stoika 2003; Bilyy et al, 2004; 2005) that apoptotic cells are characterized by increased expression levels of specific glycoproteins in the plasma membrane, which were proved to be selective and specific markers of apoptotic cells. Specific carbohydrate-binding proteins - lectins - were used for identification of mentioned glycoproteins; fluorescent conjugates of lectins were proved to be another novel tool for apoptosis identification using approaches of biophotonics.
In report a new method of quantitative determination of apoptosis parameters in cellular suspensions using correlation optic methods is considered. The principle of the new optical verification method is described. This method includes sounding of liquid flow with monochromatic coherent light, registration of impulse signals resulting from interaction of sounding radiation with cells, and measuring impulses' amplitude and duration and construction on the basis of obtained measurement results functions of two-dimensional pulses' amplitudes and duration distribution that express statistical intensity characteristics of light scattering by cells. On the basis of obtained functions the size distribution of cells is determined and apoptosis parameters are estimated. Results of apoptotic cells of K562 line (human leukemia), induced by cisplatin action, are described.
Estimation of cell proliferation and apoptosis are in focus of instrumental methods used in modern biomedical sciences. Present study concerns monitoring of functional state of cells, specifically the development of their programmed death or apoptosis. The available methods for such purpose are either very expensive, or require time-consuming operations. Their specificity and sensitivity are frequently not sufficient for making conclusions which could be used in diagnostics or treatment monitoring. We propose a novel method for apoptosis measurement based on quantitative determination of cellular functional state taking into account their physical characteristics. This method uses the patented device -- laser microparticle analyser PRM-6 -- for analyzing light scattering by the microparticles, including cells. The method gives an opportunity for quick, quantitative, simple (without complicated preliminary cell processing) and relatively cheap measurement of apoptosis in cellular population. The elaborated method was used for studying apoptosis expression in murine leukemia cells of L1210 line and human lymphoblastic leukemia cells of K562 line. The results obtained by the proposed method permitted measuring cell number in tested sample, detecting and quantitative characterization of functional state of cells, particularly measuring the ratio of the apoptotic cells in suspension.