Certain hydrogel forming de novo proteins that utilize different crosslinking methods are studied experimentally on a rheometer. The stress reaxation modulus of CRC, a telechelic, triblock protein, is shown to be that of a stretched exponential function with a value of β ≅ 0.5. The insertion of an integrin binding domain and changes in pH within the range 6.5–8.5 are shown not to significantly affect the resulting rheological behavior. A selective chemical crosslinker is used on CRC hydrogel systems and is shown to change the rheological behavior of the system to that of a combination of a chemically and physically crosslinked system. Chemically crosslinked hydrogels composed of W6, a wheat gluten-based protein, demonstrate a storage modulus weakly dependent on the angular frequency that is much greater than the loss modulus, with a modulus concentration dependence of c^9/4.

Ce travail de thèse a pour objectif l’étude de l’électrochimiluminescence (ECL) et son application pour le développement de nouvelles techniques analytiques. De par son importante sensibilité, l’ECL est une technique performante pour des applications telles que les diagnostics cliniques ou la chimie environnementale (présence d’agents contaminants dans l’eau ou la nourriture). L’immobilisation du luminophore ECL est réalisée généralement sur une phase solide. Cette étape constitue une phase essentielle pour obtenir une méthode d’immobilisation rapide, simple, flexible et efficace de ces luminophores ECL, tout en permettant une utilisation pour des systèmes variés. La première partie de ce travail présente l’optimisation de la déposition électrochimique de films de métallopolymère de ruthénium et son application pour la détection enzymatique. Un film d'épaisseur micrométrique de cet hydrogel redox a été préparé par voltamétrie cyclique. Cet hydrogel immobilisé a permis la détection d’un substrat modèle (le glucose) en utilisant l'enzyme glucose déshydrogénase. La seconde partie se concentre sur le développement d’une nouvelle méthode de photodéposition d’un polymère. Celle-ci permet l’immobilisation de centres actifs sur des régions sélectives. En utilisant les techniques de photolithographie, les figures du masque sont projetées sur la surface des électrodes. Cela permet la réalisation de spots micrométriques dont la taille, forme et épaisseur sont modulables. Les propriétés électrochimiques des films nanométriques obtenus sont comparables à ceux obtenues par électrodéposition. De même, les spectres ECL réalisés avec polymères immobilisés par ces deux stratégies sont identiques. Ces résultats montrent que les états excités induits lors de l’ECL sont identiques avec les deux techniques d’immobilisation. Le développement d'un tel procédé constitue une alternative prometteus pour la réalisation de réseaux de spots ECL différenciés et permettant la détection multipléxée par imagerie ECL. Dans la troisième partie de ce travail, nous avons associé la spectroélectrochimie et l'imagerie ECL pour contribuer à l’étude des mécanismes ECL au niveau d'une bille micrométrique fonctionnalisée par des complexes de ruthénium. En combinant microscopie de fluorescence et imagerie ECL, la distribution des sites électroactifs et des sites ECL a pu être mise en évidence. A partir de cette étude, nous pouvons clarifier les mécanismes conduisant à l'émission ECL au niveau de ces billes fonctionnalisées. / The main goal of this thesis was to study electrogenerated chemiluminescence (ECL) and its application in development of new analytical techniques. Due to its high sensitivity, ECL presents a powerful method for applications in clinical diagnostic and environmental chemistry (presence of contaminants in water or food). The immobilisation of an ECL luminophore is usually performed on a solid phase. This step is an essential point to obtain a technique for fast, simple, flexible and effective immobilisation of ECL luminophores with possibility of applications in various configurations. The first part of this work presents the optimisation of the electrochemical deposition of a ruthenium metallopolymer and its application in enzymatic detection. A redox hydrogel film with micrometric thickness was prepared using cyclic voltammetry. This immobilised hydrogel allows the detection of model substrate (glucose) using enzyme glucose dehydrogenase. The second part of this thesis is focused on the development of a new photodeposition method for the ECL polymer immobilisation. This method allows region-selective immobilisation of active centres. Using photolithographic methods, the figures from the mask are projected on the electrode surface. This allows the formation of micrometric spots which size, shape and thickness is modulated. Electrochemical properties of obtained nanometric films are comparable with those of electrodeposited films. Also, ECL spectra recorded with both immobilisation strategies are identical. It shows that the ECL excited state is the same. The obtained photopatterns were imaged using ECL. The development of such process presents an alternative for realisation of different ECL spot arrays and allows multiplexed detection by ECL imaging. In the third part of this work we have associated spectroelectrochemistry and ECL imaging to study the ECL mechanisms at the level of a single microbead, functionalised with ruthenium complex. Combining fluorescence microscopy and ECL imaging, the distribution of electroactive and ECL sites have been highlighted. From this study we can clarify the mechanism that leads to ECL emission at the level of functionalised beads.

Smart hydrogels are useful elements in microfluidic systems because they respond to environmental stimuli and are capable of storing reagents. We present here a concept of using hydrogels (poly(N-isopropylacrylamide)) as an interface between continuous and discontinuous microfluidics. Their swelling and shrinking capabilities allow them to act as storage elements for reagents absorbed in the swelling process. When the swollen hydrogel collapses in an oil-filled channel, the incorporated water and molecules are expelled from the hydrogel and form a water reservoir. Water-in-oil droplets can be released from the reservoir generating different sized droplets depending on the flow regime at various oil flow rates (dispensing functionality). Different hydrogel sizes and microfluidic structures are discussed in terms of their storage and droplet formation capabilities. The time behaviour of the hydrogel element is investigated by dynamic swelling experiments and computational fluid dynamics simulations. By precise temperature control, the device acts as an active droplet generator and converts continuous to discontinuous flows.

The aim of the doctoral thesis was to study advanced fluorescence techniques and its use in colloids or hyaluronan-surfactant systems and hydrogels based on hyaluronan, respectively. Steady-state and time-resolved fluorescence were used to study excited state proton transfer fluroescen probes in hyaluronan-surfactant systems to asses the influence of hyaluronan hydration to its interactions with oppositely charged surfactants. Firstly, different excited state proton transfer fluorescence probes were discussed to choose the most suitable candidate for next research. The influence of hyaluronan on inner environment of micells was determined based on the sensitivity of excited state proton transfer of chosen fluorescence probe 1-naphtol and, based on above mentioned experiments, the structure of hyaluronan hydration shell was discussed. The next part of doctoral thesis was focused on fluorescence lifetime correlation spectroscopy and on the development of method of nanorheology. Measured correlation functions were transformed to mean square displacement with developed MATLAB script. Firstly, the fluorescence method was compared with well described methods such as videomicrorheology and dynamic light scattering to asses the reliability of fluorescence correlation spectroscopy in microrheology. Secondly, nanorheology method was developed and its use in passive nanorheology of hyaluronan hydrogels was discussed. Based on mentioned experiments, the fluorescence correlation spectroscopy microrheology and nanorheology methods were optimized to use the methods in hydrogel research.

The thesis deals with the study of hydrophobic domains in phase separated hydrogels. Two types of polyelectrolytes (hyaluronan, dextran) and oppositely charged surfactants (carbethopendecinium bromide, sodium dodecylsulphate ) are used. Phase separated hydrogels are formed by electrostatic interactions. The target of the thesis is to study the polarity of environment in phase separated hydrogeles by fluorescence spectroscopy. Fluorescence measurements is performed by using pyrene, Nile red and laurdane as fluorescence probes. All probes are sensitive to the polarity of the environment. The results showed that probes can be expected in the polar environment of hydrogels.

This dissertation is focused on the controlled release of drugs from a biodegradable amphiphilic hydrogel based on hydrophobic poly(lactic acid), poly(glycolic acid) and hydrophilic poly(ethylene glycol) (PLGA-PEG-PLGA, ABA) and its modification with itaconic anhydride (ITA). The resulting ,-itaconyl(PLGA-PEG-PLGA) copolymer is referred to as ITA/PLGA-PEG-PLGA/ITA or ITA/ABA/ITA. Itaconic acid provides reactive double bonds and a functional carboxyl group at the ends of the PLGA-PEG-PLGA copolymer chain, thereby rendering the modified ITA/ABA/ITA copolymer less hydrophobic and offering the possibility of forming a carrier for hydrophilic drug substances. These functional copolymers are thermosensitive and change in the external environment (e.g. temperature) causes a sol-gel phase transition due to the formation of micellar structure. The bioactive substances can thus be mixed with a copolymer which is in a low viscous phase (sol phase) and subsequently the mixture can be injected into patient's body at the target site where it forms a gel at 37 °C. This hydrogel becomes a drug depot, which gradually releases the active substance. Prediction of the substance’s release profile from the hydrogel is an effective tool to determine the frequency of administration, potentially enhancing efficacy, and assessment of side effects associated with dosing. The analgesic paracetamol and the sulfonamide antibiotic sulfathiazole were used as model drugs, representing hydrophilic and hydrophobic substances, respectively. The active substances had a significant effect on the resulting hydrogel stiffness. Type of solvent, incubation medium and nanohydroxyapatite also influenced on the gel stiffness and subsequent stability of the hydrogel-drug system. Controlled release of drugs took place in simulated conditions of the human body. Verification of Korsmeyer-Peppas (KP) drug-release model is also discussed in this thesis. The KP model was found suitable for simulating the release of sulfathiazole from ABA and ITA/ABA/ITA hydrogels. On the contrary, the performance of KP model was not suitable for describing the release of paracetamol from the ABA hydrogels. Therefore, a new regression model suitable for both buffered simulated media and water has been proposed. The proposed model fitted better the release of both sulfathiazole and paracetamol from composite material prepared from ABA hydrogel and nanohydroxyapatite.

This thesis is focused on diffusion of dyes in biopolymer-based hydrogels. These hydrogels are based on interaction between biopolymer-like electrolytes with oppositely charged surfactants. When polyelectrolytes interact with oppositely charged surfactants, micelle-like nano-containers can be formed. These nano-containers are able of binding hydrophobic compounds. In this study, combination of modified dextran (diethylaminoethyl dextran) with positive charge and oppositely charged sodium dodecylsulphate as surfactant was used for preparation of hydrogels. Next type of hydrogel was based on hyaluronan and positive charged surfactant Septonex (carbethoxypendecinium bromide). As a diffusion probes in hydrogels dyes Nile red and ATTO 488 were used. The diffusion of these dyes from aqeous solutions of NaCl or surfactants into hydrogels was monitored in time. Transport of dyes into structure of hydrogels was characterized by diffusion coefficients and structural parameters of hydrogels.

The main aim of this thesis is the utilization of simple diffusion techniques for the study on transport properties of copper ions in the systems containing humic acids with respect to the other parameters, which can affect the process (the structure of diffusion environment, the interactions between transported specie and diffusion matrices, selective blocking of binding sites of humic acids). The first part of experimental works was focused on characterization of studied materials (humic acids, humic sol and humic hydrogel). The main part of the thesis was dealing with the optimization of simple diffusion techniques, which were suitable for the study on transport of copper ions in matrices containing humic acids, taking into account the mutual interactions between studied components in the system. The obtained diffusion characteristics were compared to the data determined using sorption experiments. Consequently, the minor goal of the experimental works of this thesis was also the assessment of the influence of basic physico-chemical parameters of studied materials on transport phenomenon.

Presented diploma thesis focuses on the study of diffusion of cupric ions in humic acid gels. A total of fifth different standards of humic acids and one sample humic acid prepared from same source as in bachelor's thesis, on which this thesis continues, were used for preparation solutions of humic acids. Thereafter, these solutions were used for preparation of agarose hydrogels, which were necessary in following diffusion experiments. The objective of the work was to compare transport properties of humic standards between themselves and then compare standards with humic acid prepared from same source as in bachelor's thesis. Measure, which was used to determine the transport properties, was comparison of effective diffusion coefficients. Method of instantaneous planar source diffusion was used to compare them. This method was based monitoring temporal evolution of diffusion profiles of cupric ions in humic hydrogels. Copper ions were selected as diffusing medium because of their high affinity and strong bonds to humic acids.

This diploma thesis deals with study of hydrogels formed by phase separation of hyaluronan with oppositely charged surfactants cetyltrimethylammonium bromide (CTAB) and Septonex. It follows the bachelor thesis and extends the knowledge about the detailed characterisation of the inner environment of the hydrogel by determining the diffusion behaviour of the fluorescent probes Atto 488 and Nile Red using fluorescence correlation spectroscopy (FCS) technique and its modified version dual-focus fluorescence correlation spectroscopy (2f-FCS). Compared results showed that both methods show similar values and probes specifically interact with CTAB but Atto 488 shows only weak interaction with Septonex compared to Nile Red. Additionally, these interactions were not affected by the molecular weight of hyaluronan. In conclusion, it was recommended to measure this type of hydrogel in a small depth of gel using a conventional method.