7 research outputs found
A microfluidic device with fluorimetric detection for intracellular components analysis
An integrated microfluidic system that coupled lysis of two cell lines: L929 fibroblasts and A549 epithelial cells, with fluorescence-based enzyme assay was developed to determine β-glucocerebrosidase activity. The microdevice fabricated in poly(dimethylsiloxane) consists of three main parts: a chemical cell lysis zone based on the sheath flow geometry, a micromeander and an optical fibers detection zone. Unlike many methods described in literature that are designed to analyse intracellular components, the presented system enables to perform enzyme assays just after cell lysis process. It reduces the effect of proteases released in lysis process on determined enzymes. Glucocerebrosidase activity, the diagnostic marker for Gaucher’s disease, is the most commonly measured in leukocytes and fibroblasts using 4-methylumbelliferyl-β-D-glucopyranoside as synthetic β-glucoside. The enzyme cleavage releases the fluorescent product, i.e. 4-methylumbelliferone, and its fluorescence is measured as a function of time. The method of enzyme activity determination described in this paper was adapted for flow measurements in the microdevice. The curve of the enzymatic reaction advancement was prepared for three reaction times obtained from application of different flow rates of solutions introduced to the microsystem. Afterwards, determined β-glucocerebrosidase activity was recalculated with regard to 105 cells present in samples used for the tests. The obtained results were compared with a cuvette-based measurements. The lysosomal β-glucosidase activities determined in the microsystem were in good correlation with the values determined during macro-scale measurements
Utilization of oriented crystal growth for screening of aromatic carboxylic acids cocrystallization with urea
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Propensity of salicylamide and ethenzamide cocrystallization with aromatic carboxylic acids.
The cocrystallization of salicylamide (2-hydroxybenzamide, SMD) and
ethenzamide (2-ethoxybenzamide, EMD) with aromatic carboxylic acids was
examined both experimentally and theoretically. The supramolecular
synthesis taking advantage of the droplet evaporative crystallization
(DEC) technique was combined with powder diffraction and vibrational
spectroscopy as the analytical tools. This led to identification of
eleven new cocrystals including pharmaceutically relevant coformers such
as mono- and dihydroxybenzoic acids. The cocrystallization abilities of
SMD and EMD with aromatic carboxylic acids were found to be
unexpectedly divers despite high formal similarities of these two
benzamides and ability of the R2,2(8) heterosynthon formation. The
source of diversities of the cocrystallization landscapes is the
difference in the stabilization of possible conformers by adopting
alternative intramolecular hydrogen boding patterns. The stronger
intramolecular hydrogen bonding the weaker affinity toward
intermolecular complexation potential. The substituent effects on
R2,2(8) heterosynthon properties are also discussed
Exploring the cocrystallization potential of urea and benzamide
The cocrystallization landscape of benzamide and urea interacting with
aliphatic and aromatic carboxylic acids was studied both experimentally
and theoretically. Ten new cocrystals of benzamide were synthesized
using an oriented samples approach via a fast dropped evaporation
technique. Information about types of known bi-component cocrystals
augmented with knowledge of simple binary eutectic mixtures was used for
the analysis of virtual screening efficiency among 514 potential pairs
involving aromatic carboxylic acids interacting with urea or benzamide.
Quantification of intermolecular interaction was achieved by estimating
the excess thermodynamic functions of binary liquid mixtures under
supercooled conditions within a COSMO-RS framework. The smoothed
histograms suggest that slightly more potential pairs of benzamide are
characterized in the attractive region compared to urea. Finally, it is
emphasized that prediction of cocrystals of urea is fairly direct, while
it remains ambiguous for benzamide paired with carboxylic acids. The
two known simple eutectics of urea are found within the first two
quartiles defined by excess thermodynamic functions, and all known
cocrystals are outside of this range belonging to the third or fourth
quartile. On the contrary, such a simple separation of positive and
negative cases of benzamide miscibility in the solid state is not
observed. The difference in properties between urea and benzamide
R2,2(8) heterosynthons is also documented by alterations of substituent
effects. Intermolecular interactions of urea with para substituted
benzoic acid analogues are stronger compared to those of benzamide.
Also, the amount of charge transfer from amide to aromatic carboxylic
acid and vice versa is more pronounced for urea. However, in both cases,
the greater the electron withdrawing character of the substituent, the
higher the binding energy, and the stronger the supermolecule
polarization via the charge transfer mechanism
Applicability of Phenolic Acids as Effective Enhancers of Cocrystal Solubility of Methylxanthines
Applicability of phenolic acids as potential cocrystal formers for
methylxanthine derivatives was analyzed both in terms of
cocrystallization probabilities and solubility advantage. The cocrystal
formation abilities were evaluated using mixing enthalpy estimated
within the conductor like screening model for real solvents (COSMO-RS)
framework. The solubility improvement of potential cocrystals was
estimated by formulation of the model relating experimental values to
predicted solubilities. This enabled for ranking of potential cocrystals
formers according to their solubility enhancement potential. According
to the calculation results, a highly linear relationship (R2 = 0.989)
was found between estimated theophylline and caffeine cocrystal
solubility values. It has been found that many phenolic acids,
especially ones with several hydroxyl groups attached to phenyl ring,
are the most promising candidates for cocrystallization with caffeine or
theophylline. Experimental verification of the proposed protocol for
caffeine and theophylline resulted in eight new molecular complexes,
which were synthesized via a mechanochemical approach. All new solids
were characterized using powder X-ray diffractometry and Fourier
transform infrared spectroscopy combined with a attenuated total
reflection technique
Longitudinal Evaluation of Biomarkers in Wound Fluids from Venous Leg Ulcers and Split-thickness Skin Graft Donor Site Wounds Treated with a Protease-modulating Wound Dressing
Venous leg ulcers represent a clinical challenge and impair the quality of life of patients. This study examines impaired wound healing in venous leg ulcers at the molecular level. Protein expression patterns for biomarkers were analysed in venous leg ulcer wound fluids from 57 patients treated with a protease-modulating polyacrylate wound dressing for 12 weeks, and compared with exudates from 10 acute split-thickness wounds. Wound healing improved in the venous leg ulcer wounds: 61.4% of the 57 patients with venous leg ulcer achieved a relative wound area reduction of ≥ 40%, and 50.9% of the total 57 patients achieved a relative wound area reduction of ≥ 60%. Within the first 14 days, abundances of S100A8, S100A9, neutrophil elastase, matrix metalloproteinase-2, and fibronectin in venous leg ulcer exudates decreased significantly and remained stable, yet higher than in acute wounds. Interleukin-1β, tumour necrosis factor alpha, and matrix metalloproteinase-9 abundance ranges were similar in venous leg ulcers and acute wound fluids. Collagen (I) α1 abundance was higher in venous leg ulcer wound fluids and was not significantly regulated. Overall, significant biomarker changes occurred in the first 14 days before a clinically robust healing response in the venous leg ulcer cohort