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SDS-PAGE as a Tool for Size-Dependent Separation of Quantum Dots

Quantum dots (QDs) are semiconductor nanocrystals with diameters in the range of 1-10 nm. They have unique spectral properties featured mainly by the size and/or composition-tuneable emissions due to quantum size effects [1-4]. The range of their use varies from chemical to physical, computing and biological sciences [5]. In biological applications, QDs are employed in imaging, labelling and sensing. The unique optical properties enable to use them as in vivo and in vitro fluorophores, as well as tools for the labelling of cells and tissues; they are also efficient donors in fluorescence resonance energy transfer (FRET) [6-8]. QDs can form bioconjugates with proteins, small molecules and oligonucleotides by a direct link to defined binding sites. These bioconjugates have a wide field of applications, from immunoassays to cells and organelles imaging or mutation detection. Other important applications of QDs appear to be traceable drug delivery, intracellular sensors [9], vectors for gene therapy [10], quantum computers, [11] and photodetectors [12].

Several analytical techniques, namely laser dynamic light scattering (DLS), laser Doppler electrophoresis (LDE), capillary electrophoresis and gel electrophoresis are suitable to characterize the nanocolloids [13-17]. Optical characterization of QDs is usually provided by UV-VIS and photoluminescence spectroscopy, which offers fast, non-destructive and contactless option. The size of QDs is generally calculated using conventional techniques such as scanning electron microscopy (SEM) and/or transmission electron microscopy (TEM). Also a number of different techniques have been employed to characterize QDs surface chemistry: particularly X-ray photoelectron spectroscopy, nuclear magnetic resonance spectroscopy, and Rutherford backscattering may be utilized [1]. Gel electrophoresis is a widely applied technique used for biomolecules analysis. As well as for biomolecules, gel electrophoresis has been used to analyse nanoparticles. Agarose gel electrophoresis (AGE) using TAE or TBE buffer, commonly used for nucleic acids analysis is most frequently employed. Main purposes are to purify QDs conjugates from unconjugated ligands [18, 19], to check the interaction of QDs with ligand [20, 21] and/or to prove the stability of the conjugates.

Polyacrylamide electrophoresis (PAGE) generally allows more effective separation of both proteins and nucleic acids. Unlike in native DNA, where all molecules have similar charge and shape and the only resolution factor is the size of the molecule, proteins have different size, charge, shape, and surface modifications [22]. As well as proteins, QDs generally may differ in all abovementioned parameters [23, 24]. Tris-Glycine (T-G) SDS-PAGE has been commonly used for protein analysis for more than four decades. This method enables separation of proteins mixture according to their molecular weight. This is achieved by reducing of proteins charge and shape influence by SDS. Effectiveness of proteins separation is increased by use of stacking and resolving gels, which differ in polyacrylamide concentration, ionic strength and pH. This system allows effective separation of proteins in the range approximately from 300 kDa to 15 kDa. Tris-Tricine (T-T) SDS-PAGE is based on the same separation principle, but replacement of glycine by tricine allows more effective separation of proteins and peptides in the range between 100 and 2 kDa [25].

Práce je spojená s projektem NanoBioTECell GA CR P102/11/1068.

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