Development of an enzyme immobilization platform based on microencapsulation for paper-based biosensors

Un papier bioactif est obtenu par la modification d’un papier en y immobilisant une ou plusieurs biomolécules. La recherche et le développement de papiers bioactifs est en plein essor car le papier est un substrat peu dispendieux qui est déjà d’usage très répandu à travers le monde. Bien que les papiers bioactifs n’aient pas connus de succès commercial depuis la mise en marche de bandelettes mesurant le taux de glucose dans les années cinquante, de nombreux groupes de recherche travaillent à immobiliser des biomolécules sur le papier pour obtenir un papier bioactif qui est abordable et possède une bonne durée de vie. Contrairement à la glucose oxidase, l’enzyme utilisée sur ces bandelettes, la majorité des biomolécules sont très fragiles et perdent leur activité très rapidement lorsqu’immobilisées sur des papiers. Le développement de nouveaux papiers bioactifs pouvant détecter des substances d’intérêt ou même désactiver des pathogènes dépend donc de découverte de nouvelles techniques d’immobilisation des biomolécules permettant de maintenir leur activité tout en étant applicable dans la chaîne de production actuelle des papiers fins. Le but de cette thèse est de développer une technique d’immobilisation efficace et versatile, permettant de protéger l’activité de biomolécules incorporées sur des papiers. La microencapsulation a été choisie comme technique d’immobilisation car elle permet d’enfermer de grandes quantités de biomolécules à l’intérieur d’une sphère poreuse permettant leur protection. Pour cette étude, le polymère poly(éthylènediimine) a été choisi afin de générer la paroi des microcapsules. Les enzymes laccase et glucose oxidase, dont les propriétés sont bien établies, seront utilisées comme biomolécules test. Dans un premier temps, deux procédures d’encapsulation ont été développées puis étudiées. La méthode par émulsion produit des microcapsules de plus petits diamètres que la méthode par encapsulation utilisant un encapsulateur, bien que cette dernière offre une meilleure efficacité d’encapsulation. Par la suite, l’effet de la procédure d’encapsulation sur l’activité enzymatique et la stabilité thermique des enzymes a été étudié à cause de l’importance du maintien de l’activité sur le développement d’une plateforme d’immobilisation. L’effet de la nature du polymère utilisé pour la fabrication des capsules sur la conformation de l’enzyme a été étudié pour la première fois. Finalement, l’applicabilité des microcapsules de poly(éthylèneimine) dans la confection de papiers bioactifs a été démontré par le biais de trois prototypes. Un papier réagissant au glucose a été obtenu en immobilisant des microcapsules contenant l’enzyme glucose oxidase. Un papier sensible à l’enzyme neuraminidase pour la détection de la vaginose bactérienne avec une plus grande stabilité durant l’entreposage a été fait en encapsulant les réactifs colorimétriques dans des capsules de poly(éthylèneimine). L’utilisation de microcapsules pour l’immobilisation d’anticorps a également été étudiée. Les avancées au niveau de la plateforme d’immobilisation de biomolécules par microencapsulation qui ont été réalisées lors de cette thèse permettront de mieux comprendre l’effet des réactifs impliqués dans la procédure de microencapsulation sur la stabilité, l’activité et la conformation des biomolécules. Les résultats obtenus démontrent que la plateforme d’immobilisation développée peut être appliquée pour la confection de nouveaux papiers bioactifs.Biosensing paper attracts increasing attention due to its benefits of being simple, visible, portable and useful for detecting various contaminants, pathogens and toxins. While there has been no bioactive paper commercialized since glucose paper strips developed in the fifties, many research groups are working to immobilize biomolecules on paper to achieve a bioactive paper that is affordable and has good shelf life. The goal of this research is to develop some highly useful bioactive paper that could, for example, measure blood glucose, or immediately detect and simultaneously deactivate pathogens such as neuraminidase and E.coli. Previously, bioactive paper was produced either through physically absorbing biorecognition elements or printing bio-ink onto paper substrate. Our methodology for fabrication of bioactive paper strips is compatible with existing paper making process and includes three procedures: the fabrication of microcapsules, enzyme or antibody microencapsulation, immobilization of enzymes or antibody-entrapped microcapsules into paper pulp. The first step, in fabricating of bioactive paper strips is to produce biocompatible and inexpensive microcapsules with suitable parameters. To do so, two types of microencapsulation methods were compared; the emulsion method and the vibration nozzle method accomplished with an encapsulator. The parameters for producing optimal microcapsules with both methods were studied. Factors that affect their diameter, wall thickness, shell pore size, encapsulation efficiency and membrane compositions were also discussed. By comparison, microcapsules prepared with poly(ethyleneimine) (PEI) by the emulsion method exhibit properties that were more suitable for enzyme encapsulation and paper making process, whereas the microcapsules prepared by the vibration nozzle method were too big to be immobilized within paper pulp, and had lower encapsulation efficiency, enzymatic activity and productivity. Thus the emulsion method was chosen for subsequent experiments such as enzyme and antibody microencapsulation and bacterial vaginosis (BV) paper preparation. Microcapsules made by the emulsion method were semi-permeable in that the diffusion of substrate and product molecules were allowed freely across the membranes but the encapsulated enzymes would be retained inside. Glucose oxidase from Aspergillus niger (GOx) and laccase from Trametes versicolor (TvL) microcapsules showed high encapsulation efficiency, but the encapsulation process caused a severe decrease in the specific activities of both enzymes. Results from circular dichroism (CD) studies, fluorescence properties, enzymatic activities of free enzymes and Michaelis-Menten behavior demonstrated that the Vmax decrease for GOx was due to the restriction of diffusion across microcapsule membranes with pore size less than 5 nm. The microencapsulation process improved the thermal stability of GOx but decreased that of laccase. Bioactive papers were fabricated either by incorporating microcapsules containing different enzymes or empty microcapsules soaked in substrate and enhancer solution into the paper pulp during the sheet making process. Both the GOx and the BV paper strips underwent a color change in the presence of glucose and potassium iodide, and sialidase from Clostridium perfringens respectively. Some preliminary studies on antibody sensitized microcapsules, in which antibody was either encapsulated within the PEI microcapsules or conjugated to its membranes, were also performed. Our objective was to establish an enzyme immobilization platform based on microencapsulation techniques for paper based biosensors. Even though our current studies only focused on the microencapsulation of two enzymes, TvL and GOx, as well as the bioactive paper preparation, a similar approach can be applied to other enzymes. We believe that this immobilization method can potentially be employed for bioactive paper preparation on an industrial scale

Evaluating causal effects on time-to-event outcomes in an RCT in Oncology with treatment discontinuation due to adverse events

In clinical trials, patients sometimes discontinue study treatments prematurely due to reasons such as adverse events. Treatment discontinuation occurs after the randomisation as an intercurrent event, making causal inference more challenging. The Intention-To-Treat (ITT) analysis provides valid causal estimates of the effect of treatment assignment; still, it does not take into account whether or not patients had to discontinue the treatment prematurely. We propose to deal with the problem of treatment discontinuation using principal stratification, recognised in the ICH E9(R1) addendum as a strategy for handling intercurrent events. Under this approach, we can decompose the overall ITT effect into principal causal effects for groups of patients defined by their potential discontinuation behaviour in continuous time. In this framework, we must consider that discontinuation happening in continuous time generates an infinite number of principal strata and that discontinuation time is not defined for patients who would never discontinue. An additional complication is that discontinuation time and time-to-event outcomes are subject to administrative censoring. We employ a flexible model-based Bayesian approach to deal with such complications. We apply the Bayesian principal stratification framework to analyse synthetic data based on a recent RCT in Oncology, aiming to assess the causal effects of a new investigational drug combined with standard of care vs. standard of care alone on progression-free survival. We simulate data under different assumptions that reflect real situations where patients' behaviour depends on critical baseline covariates. Finally, we highlight how such an approach makes it straightforward to characterise patients' discontinuation behaviour with respect to the available covariates with the help of a simulation study

2,4-Dinitro­benzaldehyde hydrazone

The title compound, C7H6N4O4, plays an important role in the synthesis of biologically active compounds. The planar hydrazone group is oriented at a dihedral angle of 8.27 (3)° with respect to the benzene ring. In the crystal structure, inter­molecular N—H⋯O and N—H⋯N hydrogen bonds link the mol­ecules

Influence of mixed-phase TiO2 on the activity of adsorption-plasma photocatalysis for total oxidation of toluene

Herein, the effects of different crystalline phases of TiO2 on the adsorption-plasma photocatalytic oxidation of toluene were investigated. First, photocatalysts loaded on a molecular sieve (MS) were characterised and the catalytic performance of toluene abatement was evaluated in a plasma system. The COx yield of the pure anatase (An) sample outperformed other samples in the adsorption-plasma photocatalytic oxidation process, especially for CO2 yield (69.1%). It was revealed that the highest space-time-yield of 2.35 gco(2)/Lcat.h was also achieved using plasma-An/MS. However, the highest total toluene abatement (99.5%) was achieved in the plasma-P25/MS system. The plasma-generated UV flux only played a minor role in photocatalyst activation because of the very low UV flux of 2.7 mu W/cm(2) generated by discharge. For the degradation pathway, compared with the plasma-MS system, byproducts of 1,3-Butadiyne (C4H2), guanidine, methyl-(C2H7N3) did not exist in the TiO2-assisted system, indicating a difference in the toluene degradation pathway. There were no obvious effects of different TiO2 samples on organic byproducts generation, and almost a complete mineralisation of all byproducts was observed after 30 min of treatment, with the exception of ethylamine (C2H7N) and acetaldehyde (C2H4O). Finally, a cycled adsorption-plasma study was conducted to reveal the sustainability of the process. A partial deactivation of plasma-An/MS with less than 7% decrease in CO2 selectivity after 7 cycles was revealed, which is a promising result for use in possible industrial applications

Novel regio- and stereoselective phosphonyl radical addition to glycals promoted by Mn(II)-air: syntheses of 1,2-dideoxy 2-C-diphenylphosphinylglycopyranosides

National Natural Science Foundation of China [21272219, 20972142]; State Key Laboratory of Bio-organic and Natural Products Chemistry, CAS [08417]1,2-Dideoxy-2-C-diphenylphosphinylglycopyranosides were first synthesized by the novel Mn(II)-air promoted reaction of diphenylphosphine oxide with various glycals in high yields with excellent regio- and stereoselectivities, which was clarified as a radical addition reaction controlled by the oxygen of vinyl ether

Effect of Gradually Decreasing Photoperiod on Immune Function in Siberian Hamsters

Animals usually use photoperiod as an important environmental cue to time the year. In terms of the winter immunocompetence enhancement hypothesis, animals in the non-tropical zone would actively enhance their immune function to decrease the negative influence of stressors such as low temperature and food shortage in winter. In the present study, we mimicked the transition from summer to winter by decreasing photoperiod gradually and examined the variations of immune repsonses in Siberian hamsters (Phodopus sungorus)  to test this hypothesis. Twenty two female adult hamsters were randomly divided into the control (12h light: 12h dark, Control, n=11) and the gradually decreasing photoperiod group (Experiment, n=11). In the experiment group, day length was decreased from 12 h: 12 h light-dark cycle to 8 h: 16 h light-dark cycle at the pace of half an hour per week. We found that gradually decreasing photoperiod had no effect on body composition (wet carcass mass, subcutaneous, retroperitoneal, mesenteric and total body fat mass) and the masses of the organs detected such as brain, heart, liver and so on in hamsters. Similarly, immunological parameters including immune organs (thymus and spleen), white blood cells and serum bacteria killing capacity indicative of innate immunity were also not influenced by gradually decreasing photoperiod, which did not support the winter immunocompetence enhancement hypothesis. However, gradually decreasing photoperiod increased phytohaemagglutinin response post-24h of PHA challenge, which supported this hypothesis. There was no correlation between cellular, innate immunity and body fat mass, suggesting that body fat was not the reasons of the changes of cellular immunity. In summary, distinct components of immune system respond to gradually decreasing photoperiod differently in Siberian hamsters