From a Sequential to a Continuous Approach for LVV-h7 Preparation during Enzymatic Proteolysis in a Microfluidic- Based Extraction Process

Intensification of process is increasingly interesting in the context of recovery of industrial wastes. Among these compounds, animal blood is underexploited although it is an important source of bioactive peptides. LVV-h7 (LVVYPWTQRF) is one of these bioactive peptides from bovine haemoglobin hydrolysate. Our innovative approach consists of a continuous process involving at microfluidic scale for enzymatic proteolysis of bovine haemoglobin by pepsin, selective extraction of LVV-h7 to an organic solvent during the enzymatic reaction, followed by a second extraction to an aqueous phase for organic solvent recycling. Thus, the obtainment of pure LVV-h7 peptide with an efficient methodology of extraction and solvent recycling was proved

BioMEMS aided design cold plasma : new approaches

La micro et la nanotechnologie a créé un bouleversement dans beaucoup de domaine tel que l’industrie ou la recherche. Pour la recherche, les enjeux économiques (quantité) et écologiques (déchets, risques chimiques) vont directement dans le sens de cette miniaturisation pour l’obtention de procédé sûr, propre et moins couteux. Cette thèse présente la mise en place d’un nouveau procédé de conception de BioMEMS assistée par plasma froid. L’objectif est le développement d’un microdispositif à partir d’un matériau non toxique, le Tetramethyldisiloxane (TMDSO), grâce à une technologie de dépôt de couche mince assisté par plasma, et intégrant une enzyme, pour la réalisation de réaction catalytique. Pour cela, un protocole d’immobilisation et d’intégration de l’enzyme, la β-galactosidase, a été développé afin de vérifier la capacité du TMDSO à retenir les enzymes et conserver sa fonction biologique. Ensuite, une évaluation de l’activité catalytique de l’enzyme immobilisée a été entreprise par la réalisation de réaction à l’échelle millifluidique, validant l’immobilisation ainsi que la biocompatibilité du ppTMDSO. Ensuite, un microréacteur à enzyme immobilisée a été réalisé, afin d’évaluer l’influence du passage à l’échelle microfluidique et de comprendre les phénomènes liés à la diffusion et la réaction des espèces au sein du dispositif. Enfin, la conception d’un microcanal en ppTMDSO et intégrant l’enzyme, a été réalisée afin de d’étudier la faisabilité d’une méthodologie « bio-integrante » pour la création d’un BioMEMS. L’utilisation d’une méthodologie bio-integrante peut être considérée comme une alternative prometteuse pour le développement de nouveaux outils de recherches.The micro and nanotechnology has created an upheaval in many field such as industry or research. For research, economic issues (quantity) and ecological (waste, chemical hazards) go straight in the direction of this miniaturization process for obtaining safe, clean and less expensive. This thesis presents the development of a new BioMEMS design process assisted by cold plasma. The objective is to develop a micro-device from a non-toxic material, tetramethyldisiloxane (TMDSO), through a plasma enhanced thin film deposition technology, and incorporating an enzyme, for carrying out catalytic reaction. For this, an immobilizer protocol and integration of the enzyme, β-galactosidase, was developed to verify TMDSO's ability to retain enzymes and retain its biological function. Then, an evaluation of the catalytic activity of the immobilized enzyme was carried out by carrying out the reaction millifluidic scale, validating the asset and the biocompatibility of ppTMDSO. Then, an immobilized enzyme microreactor was conducted to assess the influence of the transition to the microfluidic scale and understand the phenomena related to the diffusion and reaction of the species within the device. Finally, the design of a microchannel ppTMDSO and incorporating the enzyme, was conducted to study the feasibility of a "bio-integral 'methodology for establishing a BioMEMS. The use of a bio-integral method may be regarded as a promising alternative for the development of new research tools

Investigation of the Effect of Plasma Polymerized Siloxane Coating for Enzyme Immobilization and Microfluidic Device Conception

This paper describes the impact of a physical immobilization methodology, using plasma polymerized 1,1,3,3, tetramethyldisiloxane, on the catalytic performance of β-galactosidase from Aspergillus oryzae in a microfluidic device. The β-galactosidase was immobilized by a polymer coating grown by Plasma Enhanced Chemical Vapor Deposition (PEVCD). Combined with a microchannel patterned in the silicone, a microreactor was obtained with which the diffusion through the plasma polymerized layer and the hydrolysis of a synthetic substrate, the resorufin-β-d-galactopyranoside, were studied. A study of the efficiency of the immobilization procedure was investigated after several uses and kinetic parameters of immobilized β-galactosidase were calculated and compared with those of soluble enzyme. Simulation and a modelling approach were also initiated to understand phenomena that influenced enzyme behavior in the physical immobilization method. Thus, the catalytic performances of immobilized enzymes were directly influenced by immobilization conditions and particularly by the diffusion behavior and availability of substrate molecules in the enzyme microenvironment

Activity of enzymes immobilized on plasma treated polyester

International audienceThe aim of this study was to investigate the effect of 3 different surface plasma treatments on the immobilization of β-galactosidase on a fibrous PET nonwoven membrane. Two methods 1. Entrapment in a thin calcium alginate coating and 2. Direct sorption, were used to immobilize the enzyme. The three different plasma treatments for surface activation of PET nonwovens were: 1. Air atmospheric DBD plasma, 2. Cold remote plasma-CRP with 100% N2 and 3.- CRP with a mixture of N2/O2 gases. Plasma treatment of the PET fiber surface increased the quantity of immobilized enzyme using the entrapment method, and the degree of alginate film cross-linking highly influenced the enzyme activity. Highest enzyme activity was reached for the PET treated with air atmospheric plasma and cross-linked with 0.25 g/l of CaCl2. With the direct sorption method, greater amounts of enzyme were immobilized as compared to the entrapment method, but a considerable proportion of enzyme lost their catalytic activity. Only with the CRP N2/O2 plasma treatment, up to 90% of sorbed enzyme maintained their activity. Reusability study showed that for the optimized entrapment method, a progressive decrease in activity was observed after each use cycle. With the optimized sorption method using N2/O2 CRP plasma, no decrease in enzyme activity was detected, and the immobilized enzyme could be used over more than 15 cycles