Jan Přikryl

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

Photodeposited silver nanoparticles for on-column surface-enhanced Raman spectrometry detection in capillary electrophoresis.

A new, simple photo-deposition method of silver nanoparticles induced by laser inside a fused-silica capillary is described and tested. Silver nanoparticles are immobilized using Ar-ion laser beam of a wavelength of 488 nm and power of 3.6 mW for 60 min. The photodeposited compact spot of a size of ∼10 μm is temporary and spatially stable and resistant to a hydrodynamic flow. The deposit has very good properties for surface-enhanced Raman scattering and serves well for detection in capillary electrophoresis. The advantage of this approach is that neither the silver nanoparticles nor the chemicals for their preparation are components of the background electrolyte during the electrophoretic separation. Thus, the substrate formation and separation of analytes are two independent processes and can be performed under their optimum conditions. The zone broadening due to the sorption of analytes on the immobilized nanoparticles can be significantly reduced by an addition of 20% solution of methanol. The efficiency of capillary electrophoresis and detection selectivity of surface-enhanced Raman scattering induced by He-Ne laser at 632.8 nm is demonstrated by the 3D electropherograms of rhodamines 123 and B as model samples. The limits of detection of about 49 and 150 fmol (1 and 2 μM) have been reached for rhodamine B and 123, respectively.

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.