Inkjet Metrology: High-Accuracy Mass Measurements of Microdroplets Produced by a Drop-on-Demand Dispenser

We describe gravimetric methods for measuring the mass of droplets generated by a drop-on-demand (DOD) microdispenser. Droplets are deposited, either continuously at a known frequency or as a burst of known number, into a cylinder positioned on a submicrogram balance. Mass measurements are acquired precisely by computer, and results are corrected for evaporation. Capabilities are demonstrated using isobutyl alcohol droplets. For ejection rates greater than 100 Hz, the repeatability of droplet mass measurements was 0.2%, while the combined relative standard uncertainty (uc) was 0.9%. When bursts of droplets were dispensed, the limit of quantitation was 72 μg (1490 droplets) with uc = 1.0%. Individual droplet size in a burst was evaluated by high-speed videography. Diameters were consistent from the tenth droplet onward, and the mass of an individual droplet was best estimated by the average droplet mass with a combined uncertainty of about 1%. Diameters of the first several droplets were anomalous, but their contribution was accounted for when dispensing bursts. Above the limits of quantitation, the gravimetric methods provided statistically equivalent results and permit detailed study of operational factors that influence droplet mass during dispensing, including the development of reliable microassays and standard materials using DOD technologies

α-Aminoadipate pool concentration and penicillin biosynthesis in strains of <i>Penicillium chrysogenum</i>

Intracellular amino acid pools in four Penicillium chrysogenum strains, which differed in their ability to produce penicillin, were determined under conditions supporting growth without penicillin production and under conditions supporting penicillin production. A significant correlation between the rate of pencillin production and the intracellular concentration of α-aminoadipate was observed, which was not shown with any other amino acid in the pool. In replacement cultivation, penicillin production was stimulated by α-aminoadipate, but not by valine or cysteine. Exogenously added α-aminoadipate (2 or 3 mM) maximally stimulated penicillin synthesis in two strains of different productivity. Under these conditions intracellular concentrations of α-aminoadipate were comparable in the two strains in spite of the higher rate of penicillin production in the more productive strain. Results suggest that the lower penicillin titre of strain Q 176 is due to at least two factors: (i) the intracellular concentration of α-aminoadipate is insufficient to allow saturation of any enzyme which is rate limiting in the conversion of α-aminoadipate to penicillin and (ii) the level of an enzyme, which is rate limiting in the conversion of α-aminoadipate to penicillin, is lower in Q 176 (relative to strain D6/1014/A). Results suggest that the intracellular concentration of α-aminoadipate in strain D6/1014/A is sufficiently high to allow saturation of the rate-limiting penicillin biosynthetic enzyme in that strain. The basis of further correlation of intracellular α-aminoadipate concentration and penicillin titre among strains D6/1014/A, P2, and 389/3, the three highest penicillin producers studied here, remains to be established. Preliminary studies which attempted to explain the differences in intracellular α-aminoadipate concentrations in strains Q 176, D6/1014/A, and P2 in terms of differences in activities or kinetics of two enzymes of lysine biosynthesis (homocitrate synthase and saccharopine dehydrogenase) did not reveal differences in those enzymes among the three strains. </jats:p

Évaluation intégrée de la qualité d’un milieu récepteur basée sur des données en continue validées et la modélisation de la qualité

Colloque avec actes et comité de lecture. Internationale.International audienc

Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica.

To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) was expressed in P. pastoris. No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface

Superhydrophobic functionalization of cutinase activated poly(lactic acid) surfaces

Superhydrophobic materials have focused the interest of many researchers due to their potential in a wide spectrum of applications like microfluidics or biosensors in the biomedical field. Typically, the increased surface roughness at the micro or nano scale needed for superhydrophobic surfaces is achieved by coating of different substances, which in combination with a lower surface energy lead to Water Contact Angle (WCA) values greater than 150\uc2\ub0. Here, limited enzymatic surface hydrolyis poly(lactic acid) (PLA) was combined with spin coating of a steraic alkene ketene dimer (AKD) layer. The selective enzymatic hydrolysis creates, in a gentle and controlled way, new hydroxylic and carboxylic groups on the polymer surface without damaging the material bulk properties like alkaline treatment does. The creation of new hydrophilic surface groups lead to a significant increase in the hydrophilicity, decreasing the WCA to less than 30\uc2\ub0 while raising the roughness from an Rrmsof 50.5 to 90.8 nm concomittantly increasing the exposed surface vs. the projected one by 13.2%. Coupling of PLA hydroxy groups with AKD was demonstrated by using a PLA model substrate and subsequent identification of the reaction product via LC-TOF/MS. On the PLA film, FTIR based detection of the characteristic \uce\ub2-ketoester bond peak between the AKD and enzymatically generated hydroxy groups on the surface confirmed successful coupling. Scanning Electron Microscopy (SEM) & Atomic Force Microscopy (AFM) imaging confirmed the presence of fractal structures after curation of the enzymatically activated PLA film. The suitable size, 4.15 \uce\ubcm on the lateral dimension and 0.7 \uce\ubcm on height of the structures, together with the high density of these fractal structures lead to a superhydrophobic surface (WCA >150\uc2\ub0). This process represents an alternative to produce chemically inert superhydrophobic bio-based polyesters surfaces, by combining mild biocatalytic activation of a polyester film with non-toxic chemicals in an environmentally friendly manner

Modélisation numérique du comportement d'une particule sous champ électrique dans les microsystèmes (de la déformation au déplacement)

Un nouvel outil numérique pour prédire le mouvement et la déformation de particules sous champ électrique dans les microsystèmes biologiques est proposé. Ce moyen permettra à terme l'optimisation de labopuces dans les premières phases de conception. Le phénomène utilisé pour manipuler les particules est la diélectrophorèse associée au mouvement dû à la polarisation des diélectriques sous champ électrique non uniforme. L'outil numérique utilisé pour le suivi des interfaces mobiles sous champ électrique est la Méthode Intégrale aux Frontières (MIF). Les écoulements sont supposés irrotationnels. Par cet outil, nous avons réussi à retrouver les résultats de Taylor sur l'instabilité des gouttes sous champ uniforme. Les résultats du déplacement des gouttes sous champ non uniforme montrent un bon accord également entre les simulations et des modèles analytiques approchés. L'état de développement de l'outil MIF nous permet d'envisager son extension vers des problèmes électrohydrodynamiques encore plus riches.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF