The main objective of the present study was to investigate the use of in situ 2D fluorometry for monitoring key bioprocess variables in mammalian cell cultures, namely the concentration of viable cells and the concentration of recombinant proteins. All studies were conducted using a recombinant Baby Hamster Kidney (BHK) cell line expressing a fusion glycoprotein IgG1-IL2 cultured in batch and fed-batch modes. It was observed that the intensity of fluorescence signals in the excitation/emission wavelength range of amino acids, vitamins and NAD(P)H changed along culture time, although the dynamics of single fluorophors could not be correlated with the dynamics of the target state variables. Therefore, multivariate chemometric modeling was adopted as a calibration methodology. 2D fluorometry produced large volumes of redundant spectral data, which were first filtered by principal components analysis (PCA). Then, a partial least squares (PLS) regression was applied to correlate the reduced fluorescence maps with the target state variables. Two validation strategies were used to evaluate the predictive capacity of the developed PLS models. Accurate estimations of viable cells density (r(2) = 0.95; 99.2% of variance captured in the training set; r(2) = 0.91; 97.7% of variance captured in the validation set) and of glycoprotein concentration (r(2) = 0.99 and 99.7% of variance captured in the training set; r(2) = 0.99 and 99.3% of variance captured in the validation set) were obtained over a wide range of reactor operation conditions. The results presented herein confirm that 2D fluorometry constitutes a reliable methodology for on-line monitoring of viable cells and recombinant protein concentrations in mammalian cell cultures.

A study is presented of the molecular dynamics and of the viscosity in pure [Aliquat][Cl] ionic liquid and in a mixture of [Aliquat][Cl] with 1% (v/v) of [Aliquat][FeCl4]. The (1)H spin-lattice relaxation rate, R1, was measured by NMR relaxometry between 8 and 300 MHz. In addition, the translation self-diffusion, D, was measured by pulse field gradient NMR. The ILs' viscosity was measured as a function of an applied magnetic field, B, and it was found that the IL mixture's viscosity decreased with increasing B, whereas the [Aliquat][Cl] viscosity is independent of B. All experimental results were analyzed taking into account the viscosity's magnetic field dependence, assuming a modified Stokes-Einstein diffusion/viscosity relation. The main difference between the relaxation mechanisms responsible for R1 in the two IL systems is related to the additional paramagnetic relaxation contribution associated with the (1)H spins-[FeCl4] paramagnetic moments' interactions. Cross-relaxation cusps in the R1 dispersion, associated with (35)Cl and (1)H nuclear spins in the IL systems, were detected. The R1 model considered was successfully fitted to the experimental results, and it was possible to estimate the value of D at zero field in the case of the IL mixture which was consistent with the values of D measured at 7 and 14.1 T and with the magnetic field dependence estimated from the viscosity measurements. It was observed that a small concentration of [Aliquat][FeCl4] in the [Aliquat][Cl] was enough to produce a "superparamagnetic"-like effect and to change the IL mixture's molecular dynamics and viscosity and to allow for their control with an external magnetic field.

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A proton nuclear magnetic relaxation dispersion (1)H NMRD study of the molecular dynamics in mixtures of magnetic ionic liquid [P66614][FeCl4] with [P66614][Cl] ionic liquid and mixtures of [P66614][FeCl4] with dimethyl sulfoxide (DMSO) is presented. The proton spin-lattice relaxation rate, R1, was measured in the frequency range of 8 kHz-300 MHz. The viscosity of the binary mixtures was measured as a function of an applied magnetic field, B, in the range of 0-2 T. In the case of DMSO/[P66614][FeCl4] the viscosity was found to be independent from the magnetic field, while in the case of the [P66614][Cl]/[P66614][FeCl4] system viscosity decreased with the increase of the magnetic field strength. The spin-lattice relaxation results were analyzed for all systems taking into account the relaxation mechanisms associated with the molecular motions with correlation times in a range between 10(-11) and 10(-7)s, usually observed by NMRD, and the paramagnetic relaxation contributions associated with the presence of the magnetic ions in the systems. In the case of the DMSO/[P66614][FeCl4] system the R1 dispersion shows the relaxation enhancement due to the presence of the magnetic ions, similar to that reported for contrast agents. For the [P66614][Cl]/[P66614][FeCl4] system, the R1 dispersion presents a much larger paramagnetic relaxation contribution, in comparison with that observed for the DMSO/[P66614][FeCl4] mixtures but different from that reported for other magnetic ionic liquid system. In the [P66614][Cl]/[P66614][FeCl4] system the relaxation enhancement associated with the paramagnetic ions is clearly not proportional to the concentration of magnetic ions, in contrast with what is observed for the DMSO/[P66614][FeCl4] system.

This work aims to present a comprehensive study about the macroscopic characteristics of globular vegetable proteins, in terms of their gelling ability, by understanding their molecular behaviour, when submitted to a thermal gelling process. The gels of soy, pea and lupin proteins were characterized by rheological techniques. Gelation kinetics, mechanical spectra, as well as the texture of these gels were analyzed and compared. Additionally, capillary viscometry, steady-state fluorescence and fluorescence anisotropy were used to monitor the structural changes induced by the thermal denaturation, which constitutes the main condition for the formation of a gel structure. Based on these techniques it was possible to establish a relationship between the gelling ability of each protein isolate and their structural resistance to thermal unfolding, enabling us to explain the weakest and the strongest gelling ability observed for lupin and soy proteins isolates, respectively.

This work shows the ability to reversibly modulate the hydrophilicity of the hydrogels doped with iron oxide nanoparticles (MNPs) in a noninvasive way when exposed to a cyclic variation of the intensity (ON/OFF) of an external magnetic field. A reversible switching of surface contact angles was observed for magnetic PVA hydrogels when exposed to consecutive variation of the magnetic field intensity between 0 and 0.08 T. Motivated by the magnetic dependence of the hydrophilicity of these hybrid hydrogels, the impact of the magnetic field on protein sorption was also evaluated. The noninvasive regulation of protein sorption-released mechanisms was achieved by ON/OFF magnetic field switches, suggesting the possible influence of magnetic-induced hydrogel shrinking effect and changes of surface wettability on protein sorption. The capacity to magnetically modulate surface wettability and protein sorption make these magnetic hydrogels promising candidates for development of functional devices for tissue engineering, drug release applications, or biosensor systems, where the control of protein sorption and mobility are essential steps to improve the efficiency of these processes.

Preparation of porous membranes from PMAA-b-PMMA copolymers and magnetic iron oxide nanoparticles and their performance under magnetic fields.

The objective of this work is to develop an appropriate technology for environmentally sound membrane-based purification of a tannery effluent assuring, simultaneously, the recovery of chromium, considered as the most hazardous inorganic water pollutant extensively used in leather tanning. A comparison between the permeate fluxes obtained during treatment of a synthetic tannery effluent through nanofiltration (NF270 and NF90 membranes) and reverse osmosis (BW30 and SW30) membranes was first performed. Then, a dedicated polymeric membrane was prepared by coating chitosan (cs) on a polyethersulfone (PES) microfiltration membrane (cs-PES MFO22) support. The resulting membrane was characterized by Fourier Transforms Infrared Spectroscopy Attenuated Total Reflectance (FTIR-ATR), Emission Scanning Electronic Microscopy (SEM) to confirm the process of surface modification and cross-linking of chitosan with glutaraldehyde. This membrane was found to be highly effective for chromium removal (>99%), which was more than eight times higher in reference to monovalent cations (e.g., Na+ and K+) and more than six times higher in reference to the divalent cations (Mg2+ and Ca2+) studied. The reverse osmosis permeate conforms to local Algerian regulations regarding being discharged directly into the natural environment (in this case, Reghaia Lake) or into urban sewers linked to wastewater biological treatment stations. While the SW30 membrane proved to be the most effective for purification of the tannery effluent, the chitosan modified membrane proved to be appropriate for recovery of chromium from the reverse osmosis concentrate.