Marcela Lišková

Conjugation reactions in the preparations of quantum dot-based immunoluminescent probes for analysis of proteins by capillary electrophoresis

AbstractA number of biologically important molecules, such as DNA, proteins, and antibodies, are routinely conjugated with fluorescent tags for high-sensitivity analyses. Here, the application of quantum dots in the place of bright and size-tunable luminophores is studied. Several selected bioconjugation reactions via zero-length cross-linkers, long-chain linkers, and oriented methods for linking of quantum dots with proteins were tested. Anti-ovalbumin, anti-proliferating cell nuclear antigen, anti-hemagglutinin, and anti-CD3 membrane protein as model antibodies and annexin V were used as high-specificity selectors. The reaction yield and efficiency of the prepared immunoluminescent probes were tested by capillary zone electrophoresis with laser-induced fluorescence detection. FigureScheme of antibody-quantum dot conjugate

Determination of ζ-potential, charge, and number of organic ligands on the surface of water soluble quantum dots by capillary electrophoresis.

The number of charges and/or organic ligands covalently attached to the surface of CdTe quantum dot nanoparticles has been determined from their electrophoretic mobilities measured in capillaries filled with free electrolyte buffers. Three sizes of water soluble CdTe quantum dots with 3‐mercaptopropionic and thioglycolic acids as surface ligands were prepared. Their electrophoretic mobilities in different pH and ionic strength values of separation buffers were measured by capillary electrophoresis with laser induced fluorescence detection. The ζ‐potentials determined from electrophoretic mobilities using analytical solution of Henry function proposed by Ohshima were in the range from −30 to −100 mV. Charges of QDs were calculated from ζ‐potentials. As a result, numbers of organic ligands bonded to QDs surface were determined to be 13, 14, and 15 for the sizes of 3.1, 3.5, and 3.9 nm, respectively. The dissociation constants of organic ligands bonded on QDs surfaces estimated from the dependence of QDs charge on pH of the separation buffer were 7.8 and 7.9 for 3‐mercaptopropionic acid and 6.9 for thioglycolic acid.

Capillary electrophoresis immunoassays with conjugated quantum dots.

Water‐soluble CdTe quantum dots (QDs) and their conjugates with antibodies and antigenes were prepared by optimized procedures for applications in CE immunoassays. The QD size of 3.5 nm, excitation spectrum in the range of 300–500 nm, the maximum wavelength of the emission spectrum at 610 nm, quantum yield of 0.25 and luminescence lifetimes in the range of 3.6–43 ns were determined. The 0.1 M solution of TRIS/TAPS (pH 8.3) was found to be the optimum buffer for the separation of the antiovalbumin–ovalbumin immunocomplex from the free conjugates of QDs.

Bioluminescence determination of active caspase-3 in single apoptotic cells

Caspase‐3 is an executive caspase, in the central position within apoptotic machinery. Apoptosis as a way of programmed cell death is a physiological process that plays an essential role in the development and homeostasis maintenance; moreover, its deregulations are linked to tumor progression or various autoimmune disorders. Therefore, an investigation of apoptosis pathways on the level of individual cells is not only of biological but also medical importance. In this work we report on the development of a high‐sensitivity instrumentation and protocol for detection of active caspase‐3 in individual mammalian apoptotic cells. The technology is based on the specific cleavage of modified luciferin by caspase‐3, an immediate bioluminescence reaction of free luciferin with luciferase followed by emissions of photons and their detection by photomultiplier tube working in the photon counting regime. Three different instrumental arrangements are compared for the determination of caspase‐3 in free cells or tissue samples. Thus, in our best miniaturized system the mean amount as low as about 6.5 fg corresponding to 122 000 molecules of caspase‐3 can be detected in individual apoptotic mouse leg cells.

Analysis of quantum dots and their conjugates by capillary electrophoresis with detection of laser-induced luminescence.

In many bioanalytical applications, important molecules such as DNA, proteins, and antibodies are routinely conjugated with fluorescent tags to reach an extraordinary sensitivity of analyses. Semiconductor nanoparticles, quantum dots, have already proved to be suitable components of highly luminescent tags, probes, and sensors with a broad applicability in analytical chemistry. Quantum dots provide high extinction coefficients together with a wide range of excitation wavelengths, size- and composition-tunable emissions, narrow and symmetric emission spectra, good quantum yields, relatively long size-dependent luminescence lifetime, and practically no photobleaching. Most of these properties are superior when compared with conventional organic fluorescent dyes. In this chapter, optimized procedures for the preparation of water-dispersed cadmium telluride (CdTe) quantum dots, conjugating reactions with antibodies, DNA, and macrocycles as well as their analyses by capillary electrophoresis are described. The potential of capillary electrophoresis for fast analyses of nanoparticles, their conjugates with antibodies, and immunocomplexes with targeted antigens is demonstrated on examples.

Detection of active apoptotic molecules with femtogram sensitivity in single cells: a pilot study

T epidermis is a dynamic epithelium with constant renewal throughout life. Epidermal homeostasis depends on two types of proliferative cells, keratinocyte stem cells (KSCs), and transit amplifying (TA) cells. In the case of chronologic aging, levels of KSCs tend to decrease and change functionally. However, little is known about the effect of photoaging on epidermal proliferative subtype populations. The aim of this study was to validate involucrin/beta1-integrin ratio as a molecular marker of epidermal photoaging, and to investigate the effects of photoaging caused by chronic UV exposure on the proliferative subtype populations. A total of 15 male volunteers (age range 20-24 and 77-85 years) provided sun-exposed and sun-protected skin samples. The expression of beta1-integrin was found to be significantly reduced in photoaged skin and ratios of the expressions of involucrin to beta1-integrin were increased 2.6-fold only in elderly subjects. Interestingly, immunostaining of the sun-exposed skins of elderly subjects showed aberrant beta1-integrin expression over the basal layer and greater numbers of Ki-67-positive cells than in sun-protected buttock skin. Flow cytometric analysis revealed that the proportion of KSCs to TA cells was reversed in sun-exposed and sun-protected skins of elderly subjects. Our results suggest that KSC numbers may be lower in photoaged skin than in chronologically aged skin and could be applied to hyperplastic pattern of photoaging. These findings suggest that the epidermis of photoaged skin is impaired in terms of its proliferative potential by attempting to repair chronic UV exposure and that photoaging may be associated with alteration in the two proliferative cell fractions.N exhibit modified chemical and physical properties that give them the ability to interact with the biological systems at the cellular and molecular levels. These interactions enhance the biomedical applications of nanotechnology in the field of tissue regeneration. A wide range of nanomaterials made of organic and inorganic composites could be self assembled in nanoscale size that simulates more accurately the dimensions of the natural human tissues such as nanoparticles, nanosurfaces and nanoscaffolds. These novel nanomaterials significantly influence the behavior and development of stem cells. The applications of nanotechnology in specific tissue regeneration, such as bone, cartilage, cardiovascular and neural tissues were investigated by several researchers. Nanostructures have been used to promote stem cell viability, proliferation and differentiation. Nanotechnology provides biodegradable and biocompatible biomimetic fabricates that restore and improve the tissue functions. Nanocomposition and nanotopography of a tissue engineered material determine the implant fate providing 3 dimentional tissue culture systems that promote normal cell growth and differentiation without adverse tissue reaction. Recent progress in the synthesis allows the cultured cells to react to the internal and external stimuli and to exchange the signaling factors between those cells and the external environment. However, further understanding to the interactions of nanomaterials with the biological system and more investigations of the safety of these nanostructures are still required before their full application in human tissue repair