Biodistribution and stability of CdSe core quantum dots in mouse digestive tract following per os administration: advantages of double polymer/silica coated nanocrystals.

CdSe-core, ZnS-capped semiconductor quantum dots (QDs) are of great potential for biomedical applications. However, applications in the gastrointestinal tract for in vivo imaging and therapeutic purposes are hampered by their sensitivity to acidic environments and potential toxicity. Here we report the use of coatings with a combination of polythiol ligands and silica shell (QDs PolyT-APS) to stabilize QDs fluorescence under acidic conditions. We demonstrated the stability of water-soluble QDs PolyT-APS both in vitro, in strong acidic solutions, and in vivo. The biodistribution, stability and photoluminescence properties of QDs in the gastrointestinal tract of mice after per os administration were assessed. We demonstrated that QDs coated with current traditional materials - mercapto compounds (QDs MPA) and pendant thiol group (QDs PolyT) - are not capable of protecting QDs from chemically induced degradation and surface modification. Polythiol ligands and silica shell quantum dots (QDs PolyT-APS) are suitable for biological and biomedical applications in the gastrointestinal tract.

Using lanthanide-based resonance energy transfer for in vitro and in vivo studies of biological processes.

This review describes key directions in the development of different probes based on complex compounds of lanthanides for in vitro and in vivo researches. The role of microsecond fluorescence of lanthanides for overcoming problems of background fluorescence is considered. The basic classes of synthetic and genetically encoded complex compounds of lanthanides are summarized. Main principles of designing lanthanide-based molecular sensors, including FRET sensors based on lanthanides and colored fluorescent proteins are described. Their applications in bioanalytical research and cellular bioimaging are described. The main principles of cellular bioimaging using lanthanides are formulated, questions of their delivery into cells are considered, and the problem of their potential toxicity for living organisms is discussed. A technique using multiphoton excitation of lanthanides is described.

[Induction-resonance energy transfer between the terbium-binding peptide and the red fluorescent proteins Dsred2 and TagRFP].

Two novel FRET-pairs: Tb3+ -binding peptide-DsRed2 and Tb3+ -binding peptide-TagRFP have been produced based on the terbium-binding peptide and the red fluorescent proteins DsRed2 and TagRFP. Two induction-resonance energy transfer processes in both FRET-pairs have been registered, from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to the acceptor, the chromophore, DsRed2 or TagRFP. The lifetimes of terbium in the presence and absence of the acceptor have been determined. It has been shown that the lifetime of Tb3+ in the presence of 150 mM NaCl decreases in both FRET-pairs. The efficiency of fluorescent resonance transfer from Tb3+ to the acceptor proteins is 28 and 35% for Tb3+ -binding peptide-DsRed2 and Tb3+ -binding peptide-TagRFP, respectively.

[Genetically encoded FRET-pair on the basis of terbium-binding peptide and red fluorescent protein].

The genetically encoded FRET-pair was developed on the basis of terbium-binding peptide and red fluorescent protein DsRed2. To study fluorescence resonance energy transfer within FRET-pair, the gene-engineered construction was obtained, where sequences of terbium-binding peptide and red fluorescent protein DsRed2 were fused in single reading frame. The expression of this construction in strain E. coli BL21 (DE3) was studied and conditions of synthesis, isolation and purification of recombinant protein were optimized. The hydrodynamic radius of hybrid protein was determined by the method of dynamic light scattering. Energy transfer between sensitized terbium and red fluorescent protein was confirmed by the methods of phosporescent spectroscopy. The obtained FRET-pair can be used both for studies in vitro and as a reporter in living cells.

[Lanthanide fluorescent immune analysis for determination of hormone thyroxine in dry blood spots]. / Ispol'zovanie metoda lantanidnogo fluorestsentnogo immunoanaliza dlia opredeleniia gormona tiroksina v sukhikh piatnakh krovi.

The determination of thyroxin (T4) is a basic confirmative test for congenital hypothyroidism. 70% cases of this period inborn disease are taking asymptomatic course. We developed immunofluorescent T4-assay in dried blood spots with anti-T4 monoclonal antibody and europium chelate (dianhydride of diethylentriaminipentaacetic acid (DA-DTPA)). This method requires DELFIA Plate Fluorometer 1232 (Wallac, Finland) or its sub modifications. Panel of monoclonal antibodies for T4 has been obtained. Type/subtype (IgG2b, IgG1), affinity constants (10(7)-10(8) M-1), cross reactivity to homologous structures (0-2%) were determined. Stable clones with high affinity and viability were selected for the development of the assay. Conjugates of thyroxin and europium chelate were synthesized. Inclusion of sodium salicylate (2 mg/ml) and EDTA (2 mM) into the buffer reduced the nonspecific signal. Limit for T4 detection (T4 standarts) was 10 nM with not more than 15% variation coefficient. Accuracy was estimated by Bio-Rad Lipochek Immunoassay Plus Control Kit. Obtained results were within control confidence interval.