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- ItempH sensing and imaging in living cells based on fluorescence lifetime of carbon dot nanosensors(2026-01-01) Kalytchuk, Sergii; Malina, Tomaš; Mravec, Filip; Poláková, Kateřina; Zdražil, Lukáš; Kment, Štěpan; Rogach, Andrey L.; Otyepka, Michal; Zbořil, RadekThe pH value is one of the most frequently measured chemical parameters, yet developing nanometric sensors capable of accurately mapping pH distribution and dynamics with high spatial and temporal resolution remains a significant challenge. Such sensors are vital for advancing our understanding of numerous physiological and pathological processes. Nanoparticle-based sensors, commonly referred to as nanosensors, represent a promising class of optical sensors, with fluorescence lifetime-based probes offering superior sensitivity and quantitative reliability. However, existing pH nanosensors relying on fluorescence lifetime are challenging to synthesize and often suffer from poor biocompatibility, narrow pH response ranges, low stability, and calibration-dependent performance. Here, we overcome these limitations by introducing a water-dispersible pH nanosensor based on fluorescence lifetime of colloidal carbon dots (CDs) derived via a one-step reaction from a single precursor Rhodamine B. These CDs are biocompatible, non-toxic, and stable in highly acidic/basic conditions, which makes them well-suited for intracellular applications. The intrinsic fluorescence lifetime of these CDs exhibits a pseudo-linear, self-referencing response across exceptionally broad pH range (1–11), driven by pH-induced transformations of their electronic structure occurring during protonation and deprotonation of CD surface. By applying micrometer-resolution, quantitative pH imaging via fluorescence lifetime imaging microscopy, we demonstrate how CDs are preferentially sequestered in lysosomes of human skin fibroblasts, enabling precise quantification of inhibitor-induced pH changes within these organelles. Our findings highlight a significant potential of the CD nanosensors for precise monitoring of lysosomal pH in living cells, offering broad utility in biomedical research and potential studies of pH-associated cellular dysfunction.
- ItemPilot-scale production of poly--hydroxybutyrate with the cyanobacterium Synechocytis sp. CCALA192 in a non-sterile tubular photobioreactor(Elsevier, 2018-07-20) Troschl, Clemens; Meixner, Katharina; Fritz, Ines; Leitner, Klaus; Palacios Romero, Patricia Alejandra; Kovalčík, Adriána; Sedláček, Petr; Drosg, BernhardThe biopolymer poly--hydroxybutyrate (PHB) can be used as a promising bioplastic. It has a broad range of applications and is degraded relatively rapidly by soil organisms. Like many prokaryotes, the cyanobacterium Synechocystis sp. CCALA192 produces this biopolymer as a storage compound, especially under nutrient limitation. In a 200-L tubular photobioreactor, we cultivated Synechocystis sp. CCALA192 semi-continuously over a period of 75days with CO2 as sole carbon source. A two-stage cultivation strategy was performed, where after 5–7days nitrogen was depleted and the culture started to produce PHB and gradually turned from blue-green to yellow. After 16–20days, 90% of the culture were harvested and the residual 10% were used as inoculum for the following cycle. The harvested culture had an average biomass concentration of 1.0g/L with an average PHB content of 12.5% of cell dry weight. After restarting with fresh nutrients, the yellow culture turned blue-green again and degraded the PHB within 24–48h. When nitrogen of the medium was consumed, PHB was produced again and the cycle continued. In the late stage of each production cycle, a ripening process was observed, where no CO2 was consumed but the PHB concentration was still rising at the expense of the existing glycogen rich biomass. Establishing a stable Synechocystis sp. CCALA192 culture under non-sterile conditions turned out to be difficult, as this small unicellular organism is very sensitive and easily grazed by protozoa. Therefore, a special cultivation strategy with partially anoxic conditions was necessary.
- ItemNon-Equilibrium Thermodynamics View on Kinetics of Autocatalytic Reactions—Two Illustrative Examples(MDPI, 2021-01-22) Pekař, MiloslavAutocatalytic reactions are in certain contrast with the linear algebra of reaction stoichiometry, on which rate equations respecting the permanence of atoms are constructed. These mathematical models of chemical reactions are called conservative. Using a non-equilibrium thermodynamicsbased theory of chemical kinetics, it is shown how to introduce autocatalytic step into such (conservative) rate equation properly. Further, rate equations based on chemical potentials or affinities are derived, and conditions for the consistency of rate equations with the entropic inequality (the second law of thermodynamics) are illustrated. The theory illustrated here can be viewed as a tool for verifying and generalizing traditional mass-action kinetics by means of modern non-equilibrium thermodynamics, which is able to deal also with such rather problematic cases.
- ItemTransesterification of Lactic Acid Oligomers with Ethanol, a Way to Anhydrous Ethyl Lactate: A Kinetic Study(MDPI, 2018-08-15) Figalla, Silvestr; Petrůj, Jaroslav; Tulková, TerezaA new method for the preparation of anhydrous ethyl ester of lactic acid was studied. The selected method is based on catalytic transesterification of lactic acid oligomers, which were prepared for this purpose by autocatalytic oligomerization of lactic acid. In this work, a kinetic model for the case of catalytic alcoholysis of oligoesters was derived assuming a first-order reaction and equimolar content of reactants in the reaction mixture. The model makes it possible to obtain the values of the reaction rate and equilibrium constants and the equilibrium alcohol concentration by regression analysis at one time. The model was verified by measuring the rate of consumption of ethanol over the time at various reaction temperatures with anhydrous FeCl3 as the catalyst. The reaction was studied at overpressure under autogenous conditions in the temperature range of 100–180 °C. For the catalyst concentration of 1 mol %, the activation energy value was 64.35 kJ·mol1. The dependence of equilibrium composition and rate constant on the temperature was obtained. The derived model is generally applicable to all first-order equilibrium reactions. The presumption is that the forward and reverse reactions are of the same order and have the same stoichiometry and equivalent amounts of reactants at the beginning of the reaction.
- ItemStructural Changes of Sodium Warfarin in Tablets Affecting the Dissolution Profiles and Potential Safety of Generic Substitution(MDPI, 2021-08-30) Muselík, Jan; Urbanová, Martina; Bartoníčková, Eva; Palovčík, Jakub; Vetchý, David; Czernek, Jiří; Janišová, Larisa; Velychkivska, Nadiia; Franc, Aleš; Brus, JiříAt present, the risk of generic substitutions in warfarin tablets is still being discussed. The aim of this study was to assess whether API interactions with commonly used excipients may affect the safety of generic replacement of warfarin sodium tablets. These interactions were observed during an accelerated stability study, and the effect of the warfarin solid phase (crystalline/amorphous form) as well as the API particle size distribution was studied. Commercial tablets and prepared tablets containing crystalline warfarin or amorphous warfarin were used. In addition, binary mixtures of warfarin with various excipients were prepared. The structural changes before and after the stability study were monitored by dissolution test in different media, solid-state NMR spectroscopy and Raman microscopy. During the stability study, the conversion of the sodium in warfarin to its acid form was demonstrated by some excipients (e.g., calcium phosphate). This change in the solid phase of warfarin leads to significant changes in dissolution, especially with the different particle sizes of the APIs in the tablet. Thus, the choice of suitable excipients and particle sizes are critical factors influencing the safety of generic warfarin sodium tablets.