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New technique on a chip for rapid detection of biological materials
Pejović Simeunović, Jelena ; Foret,, František (referee) ; Táborský,, Petr (referee) ; Hubálek, Jaromír (advisor)
Tato práce navrhuje techniku separace a detekce na čipu pro kvantové tečky (QD, „quantum dots“) konjugované s různými proteiny, za účelem sledování vlivu vazebného činidla na potlačení intenzity uorescence QD způsobené konjugací s proteinem a za účelem provedení multianalytické imunoanalýzy k identifikaci malých množství daného proteinu. Za optimálních podmínek byly biokonjugované QD úspěšně odděleny od těch nezkonjugovaných během 10 minut. Částice a cílové roztoky byly smíchány a detekce na čipu byla provedena za pomoci zařízení vyvinutého v naší laboratoři. Byl použit pouze jeden zdroj excitačního světla v kombinaci s několika filtry pro různé emisní vlnové délky. Fluorescence emitovaná dvěma typy konjugovaných QD mohla být poté zaznamenána současně, protože QD emitovaly světlo na různých vlnových délkách, ačkoliv byly excitovány při stejné vlnové délce. Smícháním dvou typů QD biokonjugovaných se dvěma druhy proteinů a protilátek jsme dokázali detekovat imunokomplexní píky s různými plochami. Plocha pod píkem závisela na koncentraci QD a antigenů, na postupu reakcí protilátka–antigen a ukázalo se, že je lineárně korelována s koncentrací antigenu. Ukázali jsme, že kapilární elektroforéza QD na čipu může být použita jako citlivá technika pro detekci biologických molekul. Hlavními výhodami této metody jsou jednoduchost, malé požadavky na objem vzorku i činidla a také vysoká účinnost separace.
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Synthesis of self-immolative linkers suitable for bioconjugation
Taraj, Lukáš ; Baszczyňski, Ondřej (advisor) ; Jindřich, Jindřich (referee)
Self-immolative (SI) linkers are chemical constructs that undergo controlled self- fragmentation based on an appropriate stimulus, e.g., activation by light or a chemical agent. SI linkers are used in the targeted delivery of drugs, in the construction of probes for biochemistry, or in various polymers. The aim of the thesis will be the synthesis of new phosphorus-based SI linkers, which will contain a reactive chemical function for conjugation with thiols. Such a function will be, for example, a vinyl phosphonate or alkynyl phosphonate group. The aim of the work will be to examine the synthesis, self-immolation, stability, and the possibility of conjugability of such linkers with thiols, e.g., cysteine, glutathione, etc. The content of the student's work will be the synthesis of SI linkers, analysis of the obtained data, planning and monitoring of chemical experiments, and writing the diploma thesis. Keywords: self-immolative, bioconjugation, phosphorus, drug delivery
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Modification of nucleic acids by reactive groups for bioconjugations and cross-linking with lysine-containing peptides and proteins
Ivancová, Ivana
In the first part of this thesis, I developed reactive DNA probe for selective cross-linking with lysine residues of DNA-binding proteins. I synthesized 2'-deoxycytidine 5'-O-mono- and triphosphate bearing squaramate moiety tethered to the position 5 via propargylamine linker. The monophosphate was used as a model compound to test the reactivity of this mixed squaramate in cross-linking reactions with lysine and short lysine containing peptides. Squaramate modified 2'-deoxycytidine 5'-O-triphosphate was found to be suitable substrate for KOD XL polymerase in both PEX and PCR synthesis of modified DNA. Squaramate modified DNA forms stable diamide linkage with primary amines. I tested the reactivity of this DNA probe in bioconjugation reactions with sulfo-Cy5-amine and lysine containing peptides. Afterwards, squaramate-linked DNA was successfully cross-linked with lysine rich histone proteins. This reactive squaramate modified nucleotide showed potential for following bioconjugation reactions of nucleic acids with amines or lysine containing peptides and proteins without the need of external reagent. Based on positive results of experiments with squaramate modified DNA, in the second part of the thesis I developed and synthesized squaramate modified ribonucleotide to study cross- linking with RNA...
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Modification of nucleic acids by reactive groups for bioconjugations and cross-linking with lysine-containing peptides and proteins
Ivancová, Ivana ; Hocek, Michal (advisor) ; Míšek, Jiří (referee) ; Urban, Milan (referee)
In the first part of this thesis, I developed reactive DNA probe for selective cross-linking with lysine residues of DNA-binding proteins. I synthesized 2'-deoxycytidine 5'-O-mono- and triphosphate bearing squaramate moiety tethered to the position 5 via propargylamine linker. The monophosphate was used as a model compound to test the reactivity of this mixed squaramate in cross-linking reactions with lysine and short lysine containing peptides. Squaramate modified 2'-deoxycytidine 5'-O-triphosphate was found to be suitable substrate for KOD XL polymerase in both PEX and PCR synthesis of modified DNA. Squaramate modified DNA forms stable diamide linkage with primary amines. I tested the reactivity of this DNA probe in bioconjugation reactions with sulfo-Cy5-amine and lysine containing peptides. Afterwards, squaramate-linked DNA was successfully cross-linked with lysine rich histone proteins. This reactive squaramate modified nucleotide showed potential for following bioconjugation reactions of nucleic acids with amines or lysine containing peptides and proteins without the need of external reagent. Based on positive results of experiments with squaramate modified DNA, in the second part of the thesis I developed and synthesized squaramate modified ribonucleotide to study cross- linking with RNA...
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Synthesis of chelators for use in diagnostic imaging
Kretschmer, Jan ; Polášek, Miloslav (advisor) ; Kubíček, Vojtěch (referee) ; Hrubý, Martin (referee)
Metals play a crucial role in medicine as a part of therapeutic or diagnostic preparations. However, in the majority of cases, their properties cannot be utilized entirely in free ionic form. Organic molecules capable of chelation are used to open the full potential of the metal. The molecules are called chelators and are the core theme of this thesis. The most important function of these molecules is the chelation and coordination of the metal, but chelators can provide other important functionalities. This work, therefore, focuses on the design, synthesis, and application of such polyfunctional chelators and is divided into two parts: DO3A-Hyp This part of the thesis deals with chelators that can be used as amino acids to incorporate lanthanides into peptides. The developed chelators provide a short and rigid connection of the metal to the peptide chain. Tripeptides containing two units of such chelators with a central amino acid bearing a CF3 group were synthesized to demonstrate the capability of DO3A-Hyp building blocks. Two paramagnetic metals were combined within this tripeptide, and it was shown that such a rigid and locked system could be used for combining their magnetic susceptibility tensors. These magnetic susceptibility tensors were used for manipulation of the 19 F NMR shift of the CF3...
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Application of fluoroalkyl hypervalent iodine reagents in C-H functionalization of small molecules and aromatic amino acid residues
Rahimidashaghoul, Kheironnesae ; Beier, Petr (advisor) ; Kvíčala, Jaroslav (referee) ; Urban, Milan (referee)
The chemistry of fluorolkyl hypervalent iodine reagents has witnessed a great boast in recent years. These compounds are highly attractive as drug candidates, advanced materials and agrochemicals as described in detail in the Introduction. Despite this fact, applications of these reagents in biological studies are rather rare and under developed. The goal of this thesis is therefore the development of mild and metal-free methods in order to fill this gap. Two ways of application of fluoroalkyl hypervalent iodine reagents in labeling of biologically relevant compounds was explored. First, the applicability of previously reported parent Togni CF3 and their analogous tetrafluoroethyl reagents in radical fluoroalkylation of electron-rich substrates such as indole and pyrrole derivatives using sodium ascorbate as reductant was described. This afforded trifluoromethyl or 1,1,2,2-tetrafluoroethyl containing products in moderate to high yields. Next, same reagents were applied for labeling of several peptides and proteins bearing aromatic amino acids in their structure. This way, peptides and proteins containing electron-rich aromatics such as Trp, Phe, Tyr and His were reacted with fluoroalkyl groups with high selectivity toward Trp. In the second part of the work, a different approach of radical...
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Photon-upconversion nanoparticles for single-molecule immunosensing of cancer biomarkers and bacteria
Farka, Z. ; Mickert, M. J. ; Hlaváček, Antonín ; Poláchová, V. ; Kostiv, Uliana ; Pastucha, M. ; Horák, Daniel ; Gorris, H. H. ; Skládal, P.
The recent progress in the field of immunoassays has been driven by introduction of various kinds of nanomaterials. In particular, photon-upconversion nanoparticles (UCNPs) proved to be excellent immunoassay labels due to their ability to emit light of shorter wavelengths under near-infrared excitation (anti-Stokes emission), which prevents autofluorescence, minimizes light scattering, and thus reduces the optical background interference. These unique photoluminescent properties allow counting of individual biomolecules labeled with UCNPs by conventional wide-field epiluminescence microscopy and enable the development of single-molecule (digital) immunoassays. We have introduced a novel label based on UCNPs conjugated with streptavidin via poly(ethylene glycol) and applied it in a digital upconversion-linked immunosorbent assay (ULISA) for the detection of a cancer biomarker prostate specific antigen (PSA). The digital readout based on counting of individual immunocomplexes improved the sensitivity 16× compared to conventional analog readout and allowed to reach a limit of detection (LOD) of 23 fg·mL−1 (800 aM). Human serum samples were successfully analyzed achieving an excellent correlation with electrochemiluminescence reference method. The conjugates of UCNPs with streptavidin are also suitable for the detection of pathogenic bacterium Melissococcus plutonius, the causative agent of honeybee disease European foulbrood. The ULISA assay provided an LOD of 340 CFU·mL−1 and successfully analyzed real samples of bees, larvae and bottom hive debris. Due to the high reliability and relatively simple detection scheme, the digital ULISA can pave the way for a new generation of digital immunoassays with a strong potential for commercialization.
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New technique on a chip for rapid detection of biological materials
Pejović Simeunović, Jelena ; Foret,, František (referee) ; Táborský,, Petr (referee) ; Hubálek, Jaromír (advisor)
Tato práce navrhuje techniku separace a detekce na čipu pro kvantové tečky (QD, „quantum dots“) konjugované s různými proteiny, za účelem sledování vlivu vazebného činidla na potlačení intenzity uorescence QD způsobené konjugací s proteinem a za účelem provedení multianalytické imunoanalýzy k identifikaci malých množství daného proteinu. Za optimálních podmínek byly biokonjugované QD úspěšně odděleny od těch nezkonjugovaných během 10 minut. Částice a cílové roztoky byly smíchány a detekce na čipu byla provedena za pomoci zařízení vyvinutého v naší laboratoři. Byl použit pouze jeden zdroj excitačního světla v kombinaci s několika filtry pro různé emisní vlnové délky. Fluorescence emitovaná dvěma typy konjugovaných QD mohla být poté zaznamenána současně, protože QD emitovaly světlo na různých vlnových délkách, ačkoliv byly excitovány při stejné vlnové délce. Smícháním dvou typů QD biokonjugovaných se dvěma druhy proteinů a protilátek jsme dokázali detekovat imunokomplexní píky s různými plochami. Plocha pod píkem závisela na koncentraci QD a antigenů, na postupu reakcí protilátka–antigen a ukázalo se, že je lineárně korelována s koncentrací antigenu. Ukázali jsme, že kapilární elektroforéza QD na čipu může být použita jako citlivá technika pro detekci biologických molekul. Hlavními výhodami této metody jsou jednoduchost, malé požadavky na objem vzorku i činidla a také vysoká účinnost separace.
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Photon-upconverting nanoparticles as a novel background-free label in immunoassays
Farka, Z. ; Hlaváček, Antonín ; Mickert, M. J. ; Skládal, P. ; Gorris, H. H.
Photon-upconverting nanoparticles (UCNPs) have become an attractive label in immunoassays because their anti-Stokes luminescence can be excited by the NIR laser and detected in the VIS region without optical background interference. Further advantages of UCNPs include good photostability, large anti-Stokes shifts, and multiple narrow emission bands that can be used for multiplexed detection. We have developed a competitive upconversion-linked immunosorbent assay (ULISA) for detection of the pharmaceutical diclofenac (DCF) in surface waters. Silica-coated UCNPs (50 nm in diameter) with carboxyl groups on the surface were synthesized and conjugated with the secondary anti-IgG antibody. The structure and monodispersity of the nanoconjugates was studied by TEM and agarose gel electrophoresis. Using a highly affine anti-DCF primary antibody, the optimized ULISA provided a detection limit of 50 pg·mL−1.
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Separation and Detection of Bioconjugated Quantum Dots Using on a Chip Electrophoresis
Pejović, Jelena
Semiconductor nanocrystals, quantum dots (QDs), are nanoscale particles that have been attract a lot of attention due to their unique optic and electronic properties. Due to very diverse and numerous applications, it is really important to make a tool for precise and controlled detection of QDs. In this study, we investigate on a chip detection and separation of QDs bioconjugated with BSA (bovine serum albumin) using homemade equipment based on principle of capillary electrophoresis (CE) and optical detection. Quenching effect of different concentration of BSA on fluorescence intensity of QDs was monitored. It was found that with increasing concentration of BSA fluorescence intensity of QDs is decreasing. This research can lead to a better understanding of interaction between different size QDs and biomolecules.
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