Characterization of the Aeration and Hydrodynamics in Vertical-Wheel™ Bioreactors.

In this work, the oxygen transport and hydrodynamic flow of the PBS Vertical-Wheel MINI™ 0.1 bioreactor were characterized using experimental data and computational fluid dynamics simulations. Data acquired from spectroscopy-based oxygenation measurements was compared with data obtained from 3D simulations with a rigid-lid approximation and LES-WALE turbulence modeling, using the open-source software OpenFOAM-8. The mass transfer coefficients were determined for a range of stirring speeds between 10 and 100 rpm and for working volumes between 60 and 100 mL. Additionally, boundary condition, mesh refinement, and temperature variation studies were performed. Lastly, cell size, energy dissipation rate, and shear stress fields were calculated to determine optimal hydrodynamic conditions for culture. The experimental results demonstrate that the kL can be predicted using Sh=1.68Re0.551Sc13G1.18, with a mean absolute error of 2.08%. Using the simulations and a correction factor of 0.473, the expression can be correlated to provide equally valid results. To directly obtain them from simulations, a partial slip boundary condition can be tuned, ensuring better near-surface velocity profiles or, alternatively, by deeply refining the mesh. Temperature variation studies support the use of this correlation for temperatures up to 37 °C by using a Schmidt exponent of 1/3. Finally, the flow was characterized as transitional with diverse mixing mechanisms that ensure homogeneity and suspension quality, and the results obtained are in agreement with previous studies that employed RANS models. Overall, this work provides new data regarding oxygen mass transfer and hydrodynamics in the Vertical-Wheel bioreactor, as well as new insights for air-water mass transfer modeling in systems with low interface deformation, and a computational model that can be used for further studies.

Molecular crowding effects on the distribution of amphiphiles in biological media.

Biological systems are the result of the interactions established among their many distinct molecules and molecular assemblies. The high concentration of small molecules dissolved in the aqueous media alter the water properties with important consequences in the interactions established. In this work, the effects of high concentrations of the disaccharide trehalose on the solubility of a homologous series of fluorescent amphiphiles (NBD-Cn, n=4-16) and on their interaction with a lipid bilayer and a serum protein are quantitatively characterized. Both kinetic and equilibrium aspects are reported for a better understanding of the effects observed. The aqueous solubility of the most hydrophobic amphiphiles (n ≥ 8) is strongly increased by 1 M trehalose, while no signifcant effect is observed for the most polar amphiphile (n = 4). This results from a decrease in the magnitude of the hydrophobic effect at molecular crowding conditions. A small decrease is observed on the equilibrium association with serum albumin. This is most significant for amphiphiles with longer alkyl chains, in agreement with their increased solubility in the aqueous media containing trehalose. The effects on the association of the amphiphiles with lipid bilayers are influenced by both equilibrium and kinetic aspects. On the one hand, the decreased magnitude of the hydrophobic effect leads to a decrease in the affinity of the amphiphiles towards the membrane. However, this tendency may be overbalanced by the effects on the kinetics of the interaction (insertion/desorption) due to the increase in the viscosity of the aqueous media. It is shown that the distribution of amphiphilic drugs in the crowded biological media is significantly different from that predicted from studies in dilute solutions and that the effects are dependent on the solute's hydrophobicity.

Calixarenes as High Temperature Matrices for Thermally Activated Delayed Fluorescence: C70 in Dihomooxacalix[4]arene.

Thermally activated delayed fluorescence (TADF) of 12C70 and 13C70 was observed up to 140 °C in a p-tert-butyldihomooxacalix[4]arene solid matrix, a temperature range significantly higher than that of previous TADF quantitative studies. An effective singlet-triplet energy gap of 29 kJ/mol and triplet formation quantum yields of 0.97 and 0.99 were measured for 12C70 and 13C70, respectively. The photophysical properties of the two fullerenes in this new matrix are comparable to those obtained in polystyrene at a lower temperature range. Calixarenes are proposed to be suitable matrices for high temperature TADF studies and applications.

The Role of Local Triplet Excited States and D-A Relative Orientation in Thermally Activated Delayed Fluorescence: Photophysics and Devices.

Here, a comprehensive photophysical investigation of a the emitter molecule DPTZ-DBTO2, showing thermally activated delayed fluorescence (TADF), with near-orthogonal electron donor (D) and acceptor (A) units is reported. It is shown that DPTZ-DBTO2 has minimal singlet-triplet energy splitting due to its near-rigid molecular geometry. However, the electronic coupling between the local triplet (3LE) and the charge transfer states, singlet and triplet, (1CT, 3CT), and the effect of dynamic rocking of the D-A units about the orthogonal geometry are crucial for efficient TADF to be achieved. In solvents with low polarity, the guest emissive singlet 1CT state couples directly to the near-degenerate 3LE, efficiently harvesting the triplet states by a spin orbit coupling charge transfer mechanism (SOCT). However, in solvents with higher polarity the emissive CT state in DPTZ-DBTO2 shifts below (the static) 3LE, leading to decreased TADF efficiencies. The relatively large energy difference between the 1CT and 3LE states and the extremely low efficiency of the 1CT to 3CT hyperfine coupling is responsible for the reduction in TADF efficiency. Both the electronic coupling between 1CT and 3LE, and the (dynamic) orientation of the D-A units are thus critical elements that dictate reverse intersystem crossing processes and thus high efficiency in TADF.

Dinuclear Zinc(II) Macrocyclic Complex as Receptor for Selective Fluorescence Sensing of Pyrophosphate.

A new diethylenetriamine-derived macrocycle known as L, bearing 2-methylquinoline arms and containing m-xylyl spacers, was prepared in good yield by a one-pot [2 + 2] Schiff base condensation procedure, followed by reduction with sodium borohydride. Up to now this is the first hexaazamacrocycle with appended fluorophore units. Single-crystal X-ray diffraction determination of the dinuclear zinc(II) complex of L showed that metal centers are located at about 7.20(2) Å from one another. This complex exhibits only weak fluorescence in aqueous solution, but the addition of 1 equiv of pyrophosphate (PPi) caused a 21-fold enhancement of the fluorescence intensity. The sensor response is linear up to a value of 10 µM HPPi(3-) and has a detection limit of 300 nM. The receptor behaves as a highly selective sensor for pyrophosphate as other anions, including phosphate, phenylphosphate (PhP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP), failed to induce any fluorescence change and practically do not affect the fluorescence intensity of the sensor in the presence of HPPi(3-). Competition titrations carried out in aqueous solution at pH 7.4 [in 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer] by spectrofluorometry revealed a high association constant value of 6.22 log units for binding of PPi by the dinuclear zinc(II) receptor, one of the highest reported values for colorimetric/fluorometric sensors able to work under real aqueous physiological conditions, while association constant values for binding of the other phosphorylated substrates are in the 5.51-4.03 log unit range.

Interaction of NBD-labelled fatty amines with liquid-ordered membranes: a combined molecular dynamics simulation and fluorescence spectroscopy study.

A complete homologous series of fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl-(NBD) labelled fatty amines of varying alkyl chain lengths, NBD-Cn, inserted in 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) or N-palmitoyl sphingomyelin (SpM) bilayers, with 50 mol% and 40 mol% cholesterol (Chol), respectively, was studied using atomistic molecular dynamics simulations. For all amphiphiles in both bilayers, the NBD fluorophore locates at the interface, in a more external position than that previously observed for pure POPC bilayers. This shallower location of the NBD group agrees with the lower fluorescent quantum yield, shorter fluorescence lifetime, and higher ionisation constants (smaller pKa) determined experimentally. The more external location is also consistent with the changes measured in steady-state fluorescence anisotropy from POPC to POPC/Chol (1 : 1) vesicles. Accordingly, the equilibrium location of the NBD group within the various bilayers is mainly dictated by bilayer compositions, and is mostly unaffected by the length of the attached alkyl chain. Similarly to the behaviour observed in POPC bilayers, the longer-chained NBD-Cn amphiphiles show significant mass density near the mixed bilayers' midplanes, and the alkyl chains of the longer derivatives, mainly NBD-C16, penetrate the opposite bilayer leaflet to some extent. However, this effect is quantitatively less pronounced in these ordered bilayers than in POPC. Similarly to POPC bilayers, the effects of these amphiphiles on the structure and dynamics of the host lipid were found to be relatively mild, in comparison with acyl-chain phospholipid analogues.

Influence of excited-state absorption on time-resolved luminescence: general formalism and application to the phosphorescence of polycyclic aromatic hydrocarbons.

The luminescence decay of a species in an absorbing medium whose optical thickness changes with time, as occurs with triplet-triplet absorption following excitation cut-off, is studied theoretically and experimentally. A general luminescence decay function based on a distribution of optical thicknesses is presented. A simple decay function previously used empirically is shown to result from an exponential distribution of optical thicknesses. The general approach introduced allows the adequate description of the phosphorescence decays of two polycyclic aromatic hydrocarbons, coronene and triphenylene (normal and perdeuterated forms for both molecules), in polymer films in the presence of excited-state absorption.

Temperature dependence of the phosphorescence and of the thermally activated delayed fluorescence of 12C70 and 13C70 in amorphous polymer matrices. Is a second triplet involved?

The phosphorescence and thermally activated delayed fluorescence (TADF) lifetimes of 12C70 and 13C70 in two different glassy hydrocarbon polymers, one aliphatic (cyclic polyolefin) and one aromatic (polystyrene), were measured between -200 and 100 ºC. The temperature dependence of the lifetimes is equally well described by a three-state mechanism (ground state, S0, and two excited states in thermal equilibrium, T1 and S1, the lifetime of T1 being temperature dependent) and by a four-state mechanism (ground state, S0, and three excited states in thermal equilibrium, T1, T2 and S1, all with temperature-independent lifetimes). The estimated S1-T1 and T2-T1 energy gaps (four-state mechanism) are in good agreement with spectroscopic measurements. These and the determined quantum yield of triplet formation, 0.997 ± 0.001, are found to be essentially independent of the polymer matrix and of the isotopic composition of the fullerene. On the other hand, the lifetimes of both T1 and T2 (four-state mechanism) are weakly dependent on the polymer matrix but strongly vary with the fullerene isotopic composition, nearly doubling when going from 12C70 to 13C70. A parameter useful for the characterization of TADF, the on-set temperature T0, is also introduced.