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Walther Von Der Vegelweide

mendel

Výzkum

Identification of quantum dots labeled metallothionein by fast scanning laser-induced breakdown spectroscopy

Determination of metallothionein (MT) has become important due to its potential role as a tumor disease marker. Numerous studies have shown an increased expression of MT in tumors of breast, colon, kidney, liver, lung, nasopharynx, ovary, prostate, salivary gland, testes, thyroid and urinary bladder [1-3]. From the point of view of its structure, which could be one of the keys to the answering of the questions what is key feature of these proteins to be related to cancer, structure of MT is based on 61 amino acids that can bind essential and/or toxic metals in two distinct cluster structures in the molecule. One cluster closer to the N-terminal can bind three metal atoms to nine cysteines using three bridging sulfur atoms per a metal ions, while the second cluster, closer to the C-terminal, binds four metal atoms using 11 cysteines with five bridging sulfur atoms. The most of biological functions suggested for MT is related to its metal-binding function. Therefore MT protects cells against metal toxicity and due to high cysteine content protects against oxidative stress, too [2]. For MT determination enzyme-linked immunosorbent assay (ELISA) with polyclonal or monoclonal antibodies can be advantageously used [4, 5]. Immunoassays are widely used in bioanalysis. The conventional heterogeneous immunoassays have some advantages such as high specificity, low background and high throughput. The most frequently used labels are enzymes or fluorescent molecules or nanoparticles [6-8]. The concentration of analyte is then proportional to absorbance and/or fluorescence. However, the results of optical measurements can be affected by optical properties of the samples or solutions, or by the fluorescence or matrix interference. Another issue is the linear range of the assay and the low stability of the signal. Better results can be obtained by non-optical methods including electrochemistry, where electrochemical activity of the product of enzymatic activity is measured [9-13].

All above mentioned assays highly demand on robust laboratory equipment, which is hardly to be used in situ. Different strategies leading to miniaturization, automation, microfluidics and portable devices development are, therefore, investigated [14, 15]. From this reason a non-optical dry and automated method of immunoassay evaluation is beneficial. An example of such application is a postponed evaluation of immunoassay results after long-term remote samples collecting. One possibility of the sample storage is injection of the liquid sample (i.e. blood) on the specific surface, especially filtration paper in the form of dried spots. According to the particular applications the dried samples are stored at dry place and/or at -20 °C. The spot is then analyzed by ELISA, polymerase chain reaction (PCR) or mass spectrometry (MS) [16-18]. Quantitative or semiquantitative analysis can be provided by dipsticks or by lateral flow tests [19, 20]. The wide range of various applications and types of samples has been already analyzed by laser-induced breakdown spectroscopy (LIBS) [21-23]. One of the fastest evolving fields of LIBS application is the analysis of bio-samples as it was described by Kaiser et al. [24], where selected biological LIBS applications including cells, microorganisms, animal tissues, plant samples or biominerals are overviewed. The main advantage of LIBS method is the minimum need of sample preparation, what is extremely important for bio-samples and the possibility of investigation of the elemental spatial distribution. Kidneys slices from a mouse were analyzed by LIBS after injection of a solution of gadolinium-based nanoparticles [25]. The presented procedure of quantifying Gd in the tissue by LIBS was in good agreement with measurement performed by ICP-OES. Determination of nanoparticles by LIBS was described on the example of two metaloxide nanoparticles, titanium dioxide (TiO2) and a rare earth sesquioxide nanoparticle, holmium oxide (Ho2O3) [26]. Nanoparticles in aqueous solution are identified and quantified by complementary LIBS and Raman spectroscopy. This approach can be applied to fully characterize the nanoparticle elemental composition in aqueous environments and to determine their size with a detection limit of a few nanometers. In addition, gold on ferromagnetic nanoparticles was analyzed and quantified by LIBS method after the fixation of powdered samples in the form of UV hardened gel [27]. The new approach in nanoparticle utilization is their deposition on the surface of the metallic sample which results in Nanoparticle-Enhanced LIBS. In the presence of silver nanoparticles an increase of 1-2 orders of magnitude in LIBS signals was obtained [28].

In this study the combination of LIBS with immunoanalysis procedures is described. The detection of biomolecules is based on the labeling with Cd-containing quantum dots. Cadmium spatial distribution is determined using its emission line at 508.58 nm. In the first part quantum dots or their conjugates with metallothionein are injected on the polystyrene surface of microtitration plate and the basic analytical characteristics of LIBS measurements are defined. The distribution of QDs on the surface is also observed by fluorescence measurement as a comparative technique. The total amount of Cd in prepared solutions is determined by atomic absorption spectrometry. In the second part of our work the antibody sandwich immunoassay is prepared and LIBS map of Cd spatial distribution is measured.

Práce je spojená s projektem CEITEC CZ.1.05/1.1.00/02.0068.

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