Very specific protein-salt interactions are involved in a multitude of biological phenomena such as protein folding/stability, enzymatic activity, and signal transduction events. In this work, we used two very simple, protein-mimic model biopolymers to obtain a better understanding of specific cation effects operating in aqueous protein environments. The two biopolymers used were Elastin-like Polypeptides (ELPs) and poly(N-isopropylacrylamide) (PNIPAM). ELPs are an especially an ideal model system as these polypeptides can be easily genetically engineered to observe the effect of specific amino acid residues and peptide chain length on these salt interactions. Both of these biopolymers are also highly thermoresponsive as their aqueous solutions undergo a hydrophobic collapse/aggregation induced phase transition process above a lower critical solution temperature (LCST). Thermodynamic measurements of these biopolymers were carried out under various salt solution conditions. Additionally, both of these biopolymers are suitable for making surface specific spectroscopic measurements. Vibrational sum frequeny spectroscopy (VSFS), a non-linear interface sensitive spectroscopic technique, was employed here to investigate biologically relevant cation interactions which occur at peptide/protein surfaces. First, the LCST response of a non-polar ELP and a neutral biopolymer, PNIPAM, was investigated in the presence of 12 different alkali, alkaline-earth metal and transition metal chloride salts. Even though the salt interactions for uncharged proteins are dominated by anions, subtle specific cation effects were also observed. The results followed a direct Hofmeister series for cations. Most alkali cations are excluded from the polar amide regions of proteins. More polarizable cations, however, can solvate the hydrophobic moieties and somewhat counter the salting-out effect of the chloride anion. More charged and hydrated ions like lithium and divalent cations showed a weak interaction to the amide moiety through their hydration shell. The role of acidic amino acid residues in inducing cation specificities was investigated using an aspartate-rich ELP system. Both thermodynamic and spectroscopic data conclusively proved that the negative charge on protein surfaces is the main driving force for cation partitioning and specificity under physiological relevant concentration regimes. Apparent binding constants of carboxylate moieties with cations were determined. This is the first quantitative and thoroughly systematic study of such biologically relevant cation-carboxylate interactions prevalent in enzyme active sites and protein surfaces.

Designing new materials with improved or tailored properties is one of the main goals of the chemists. Two common ways are mainly used to get a material with improved or new properties: blending or chemical synthesis. Chemical synthesis is an unlimited method to get new substances with well-defined properties even it is often time consuming and not seldom costly.A rheology study has shown a considerable increase in viscosity of 1% aqueous solutions, 40 and 120 times at 550C, for the graft copolymers with a composition 67 and 80% in PNIPAM respectively as temperature increases above 350C. This behaviour is also related with a critical concentration that has been found around 0.6% for the same copolymers. Fluorescence measurements have shown that this behavior is related with the hydrophobic character and aggregation of PNIPAM chains by increasing temperature over 350C. A dynamic light scattering study in dilute aqueous solutions by varying temperature could provide more information on the kind of the transition taking place and the nature of the particles formed as temperature increases. / Ο σχεδιασμός νέων υλικών με βελτιωμένες ιδιότητες είναι ένας από τους κύριους στόχους των χημικών. Δύο είναι οι πιο συνηθισμένοι τρόποι που χρησιμοποιούνται κυρίως για τη παραλαβή ενός υλικού με βελτιωμένες ή νέες ιδιότητες: ανάμειξη ή χημική σύνθεση. Χημική σύνθεση είναι η μέθοδος όπου λαμβάνονται νέα υλικά με καθορισμένες ιδιότητες αλλά έχει ως μειονέκτημα το κόστος και είναι συχνά χρονοβόρα.Μετρήσεις ρεολογίας έδειξαν μια αξιοσημείωτη αύξηση στο ιξώδες του 1% υδατικού διαλύματος, από 40 μέχρι 120 φορές στους 550C, για συστάσεις του εμβολιασμένου συμπολυμερούς σε PNIPAM 67 εως 80% αντίστοιχα, καθώς η θερμοκρασία αυξάνεται πάνω από τους 350C. Επιπλέον αυτή η συμπεριφορά σχετίζεται με μια κρίσιμη συγκέντρωση που βρέθηκε ίση με 0,6% για τα ίδια συμπολυμερή. Μετρήσεις φθορισμού έδειξαν ότι αυτή η συμπεριφορά σχετίζεται άμεσα με τον υδρόφοβο χαρακτήρα του PNIPAM καθώς η θερμοκρασία αυξάνεται πάνω από τους 350C. Μετρήσεις με δυναμική σκέδαση φωτός σε αραιά υδατικά διαλύματα σε διάφορες θερμοκρασίες μπορούν να δώσουν επιπλέον πληροφορίες για τη μετάπτωση που λαμβάνει χώρα και τη φύση των σωματιδίων όσο η θερμοκρασία αυξάνεται.

Cette thèse traite de deux projets en rapport avec la microfluidique. Le premier est consacré à la réalisation d'actionneurs à base d'hydrogel thermosensible. Avec une approche technologique basée sur le greffage et la structuration d'un hydrogel thermosensible sur des surfaces en verre, nous montrons comment un matériau capable de subir de fortes variations de volume peut être utilisé comme actionneur microfluidique dans les milieux aqueux. D'abord, en tant que vanne, nous démontrons que la stratégie de greffage des structures permet d'obtenir des actionneurs robustes et durables capables de réguler la circulation de fluides en micro-canaux avec une réponse rapide en miniaturisant la stimulation thermique. Ensuite, une autre configuration, dite de compartimentation libre, est mise en oeuvre et appliquée en biologies moléculaire et cellulaire. Le second est quant à lui dédié à l'étude du glissement par nanovélocimétrie en utilisant la technique de microscopie de fluorescence par réflexion totale interne. Cette technique permet de suivre les écoulement de nanoparticules à proximité immédiate de la paroi d'un micro-canal (dans les premiers 800nm de fluide). En utilisant les modèles de Langevin, nous avons été en mesure de prendre en compte différents artéfacts expérimentaux et les corriger. Nous confirmons l'absence de glissement de l'eau pure sur surface hydrophile et l'existence du glissement sur surface hydrophobe. Cette partie est traitée sous la forme d'une brève introduction, suivie d'un article publié dans Journal of Fluid Mechanics. / In this work, we have addressed two projects related to microfluidics.The first one is dedicated to the integration of hydrogel-based actuators in microsystems. Thanks toa technological approach based on the grafting and patterning of a thermosensitive hydrogel on glasssubstrates, we show how a material that can undergo large volume changes can be embedded as anactuator for aqueous solutions. First as a valve, we prove that the strategy consisting in grafting patternsof hydrogel enables us to obtain robust actuators able to regulate flows in micro-channels, and with afast response by miniaturising the thermal stimulation. Then we report another configuration called freecompartmentalization, applied to molecular and cellular biology. The second part of this manuscript deals with the study of slippage by nanovelocimetry using the totalinternal reflection fluorescence microscopy technique. This method enables us to follow fluorescent nanoparticlesflowing near the wall of a microchannel (within the first 800nm ). Using Langevin simulations,we have been able to take several experimental biases into account and correct them. We can confirmthe no-slip condition of water on hydrophilic surfaces and the actual slippage over hydrophobic surfaces.This part is made of a brief introduction followed by an publication in Journal of Fluid Mechanics.

PNIPAM (poly(N-Isopropylacrylamide)) is a well-known thermoresponsive polymer. Dissolved in water, it shows a structural change at 32 oC, above which the polymer folds together, and a phase separation occurs. The temperature where the polymer changes structure is known as the LCST (Lower Critical Solution Temperature), and can be modified by adding certain salts to the solution [1]. The mechanism by which the ionic components of the salts affect the LCST is not yet completely understood. The purpose of this master thesis is to study this mechanism. In order to investigate the mechanism, a combination of diffusion NMR and electrophoretic NMR was used, giving the effective charge per molecule which is directly proportional to the grade of association of ions to the polymer. The salts tested were: NaCl, NaClO4, NaSO4, NaI, NaSCN and CaCl2 from which the ClO4-, SCN-, and I- ions, as well as Cl- ions from CaCl2, were found to bind to PNIPAM.

No description available.

Microfluidic paper based analytical devices (microPADs) are a novel platform for point of care (POC) diagnostics. Limitations of reagent shelf life have been overcome with the introduction of reagent pencils as a method for solid-based reagent deposition. While useful, little work has been reported on the characterization and optimization of reagent pencils. Herein, an investigation on reagent pencil composition and efficiency is conducted via colorimetric release profile tests utilizing an erioglaucine disodium salt that yields a quantifiable blue colored product in the presence of water. Within this work, an investigation on the molecular weight dependence, polymer chain end functionality, and polymer-graphite ratio was conducted to determine the most desirable parameters in reagent pencil composition. Further, the effects of enzyme stability in the presence of poly(ethylene glycol) (PEG) is investigated. To show the versatility of reagent pencils, a novel reagent pencil incorporating a stimuli responsive polymer, poly(N-isporopylacrylamide) (PNIPAM) was developed. In this work, PNIPAM’s lower critical solution temperature (LCST) was manipulated with various salt solutions to control fluid flow both laterally and vertically through various microPAD designs. It was found that, while PNIPAM successfully blocked or retarded fluid flow in microPADs, the effect was limited when DI H2O wash solutions were run prior to salt solutions. To counteract this, PNIPAM was successfully covalently bound to alkene modified chromatography paper via thiolene click chemistry to reinforce solution wash tolerance.

<p> Poly(N-isopropylacrylamide) (PNIPAM) has attracted much attention as a thermo-responsive polymer. However, the molecular weight (MW) dependence of its phase transition temperature is still controversial. This situation is largely due to the difficulty in synthesizing narrow-disperse PNIPAM. We have addressed the challenge and developed an atom transfer radical polymerization (ATRP) method to prepare narrow-disperse PNIPAM with moderate to high conversions, using branched alcohols as solvents. Aqueous solutions of these narrow-disperse PNIPAMs showed a dramatic decrease of the phase transition temperature with increasing molecular weight, as measured by turbidimetry and differential scanning calorimetry. Four other series of narrow-disperse PNIPAM with well-controlled molecular weights and with end groups of varying hydrophobicity were also synthesized by ATRP using the corresponding initiators, which enabled us to resolve the MW and end group effects. All the four series of samples showed an inverse molecular weight (MW) dependence of their phase transition temperature. The magnitude of the MW dependence decreased when using more hydrophobic end groups. The end groups were observed to have effects on the cloud point temperature, on the shape of the cloud point curves, and on the enthalpy of the phase transition.</p> / Thesis / Master of Science (MSc)

Zwei Modellreaktionen wurden mit thermoresponsiven Nanoreaktoren untersucht. Die Reduktion von 4-Nitrophenol und von Kaliumhexacyanidoferrat(III) mit Natriumborhydrid. Die Nanoreaktoren bestehen aus einem Polystyrol Kern, umgeben von einer Hydrogel Schale aus Poly-(N-Isopropylacrylamid). Die Reaktionen werden auf der Oberfläche von Metall Nanopartikeln in der Hydrogel Schale katalysiert. In einer auf Gold- und Silberkatalysatoren fokussierten Literaturstudie zeigte sich, dass der geschwindigkeitsbestimmende Reaktionsschritt zwischen beiden Metallen variieren könnte. Kinetische Studien mit Silber haben gezeigt, dass ein erfolgreich auf Gold angewandtes Modell modifiziert werden muss um auf Silber anwendbar zu sein und haben gezeigt, dass sich die Kinetik der Reaktion auf beiden Metallen unterscheidet. Die weitere Analyse ergab die typische, nicht der Arrhenius Abhängigkeit folgende, Abhängigkeit der Reaktionsrate von der Temperatur und hat gezeigt, dass die Partitionierung der Reaktanden im Hydrogel für das kinetische Modell relevant ist. Die Reduktion von Kaliumhexacyanidoferrat(III) auf Gold hat gezeigt, dass elektrostatische Effekte hier eine maßgebliche Rolle spielen. Ein kinetisches Modell wurde erarbeitet, dass die relevanten Einflussfaktoren durch Hydrogel, Geometrie der Nanoreaktoren, diffusions- und elektrostatische Effekte miteinbezieht. Die gewonnenen Daten konnten mittels eines auf der Auswertung des stationären Zustands basierenden Modells erfolgreich gefittet werden. Hierbei wurde das komplexe Zusammenspiel von elektrostatischen Effekten, deren Abschirmung und Einfluss auf die Diffusion sowie die Reaktionsrate gezeigt. Mit wenigen physikalisch aussagekräftigen Fitparametern konnten alle beobachteten Effekte erfolgreich erklärt werden. Der Vergleich der Reduktion von 4-Nitrophenol und von Hexacyanidoferrat(III) zeigt hierbei die entscheidenden Faktoren sowohl für reaktions- als auch für diffusionskontrollierte Reaktionen in thermoresponsiven Hydrogelen. / Two model reactions were investigated with thermoresponsive core-shell nanoreactors, the reduction of 4-nitrophenol and of potassium hexacyanoferrate(III), both reduced with sodium borohydride. The nanoreactors comprise of a polystyrene core surrounded by a hydrogel shell of poly-N-isopropylacrylamide (PNIPAM) crosslinked with N,N’-methylenebisacrylamide. Metal nanoparticles are immobilized inside the hydrogel shell on the surface of which the model reactions are catalyzed. In the reduction of 4-nitrophenol, special emphasis is laid on the reduction on gold and silver catalysts. A literature review of mechanistic as well as kinetic studies reveals that the rate determining step may differ between the two catalyst metals. Kinetic investigations with a silver catalyst reveal that the kinetic model derived previously for gold catalysts needs to be modified for the kinetic analysis in this study, confirming a difference in the kinetics for both catalyst metals. The temperature dependent analysis reveals the typical non-Arrhenius dependency of the reaction rate and shows that the partition ratio of the reactants is relevant for the kinetics. The reduction of potassium hexacyanoferrate(III) on gold reveals that electrostatic effects play a major role in this reaction. A new kinetic model is derived, accounting the relevant influence factors of the hydrogel, the nanoreactor geometry, diffusional and electrostatic effects. With a stationary state approach the experimental data are fitted successfully, revealing the complex interplay of electrostatic effects, the screening thereof and the influence on diffusion and reaction rate. With only a few physically meaningful fit parameters all observed effects can be explained successfully. The comparison of the reduction of 4-nitrophenol and potassium hexacyanoferrate(III) highlights the decisive factors in both, reaction and diffusion controlled reactions inside thermoresponsive hydrogels.

This thesis is geared towards using hydrogel nanoparticles in various biotechnological applications. The polymer that was used in making these nanoparticles was poly(N-isopropylacrylamide), which is a thermoresponsive polymer. These particles were used in making fast responsive polymer films, which can be used in optics. It was observed that the rate of deswelling increased as the concentration of the nanoparticles in the film was increased. These particles were also used in making photoresponsive materials. In this case a photoresponsive dye (malachite green) was conjugated to these nanoparticles and in presence of light of appropriate wavelength the particles undergo a phase transition. A core/shell construct was synthesized where the core was composed of degradable cross-links and the shell of composed of non-degradable cross-links. The degradable cross-linker had vicinal diols, which can be cleaved by sodium periodate. Hence after degrading the core, hollow particles were obtained. Zwitterionic particles were made by incorporating a cationic and anionic comonomer. These microgels go from a positively charged state to zwitterionic to negatively charged state on increasing the pH. One of the important potential applications for these microgels is drug delivery. Microgels were used for targeting cancer cells. Folic acid was used as the targeting ligand. The microgels were conjugated with folic acid and were able to target cells that overexpress folate receptors. In one other application core/shell microgels were made which exhibit pore-size dependent permeation of proteins.

Thermo-responsive homopolymer poly(N-isopropylacrylamide), is a widely studied and used polymer. Our recent observations on thermal behavior of aqueous solutions of this polymer requires a short overview of existing results in order to understand the formation of different phases, both stable and unstable with the addition of hydrophilic Ionic liquids (ILs) 1-Butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), 1-Butyl-3-methylimidazolium acetate ([BMIM][OAc]) and 1-Butyl-3-methylimidazolium thiocyanate ([BMIM][SCN]) to the system. PNIPAM is soluble in cold water due to its inter- and intramolecular hydrogen bonding but phase separates upon heating at T > 32 , which is its lower critical solution temperature (LCST). PNIPAM exists in an expanded coil like conformation in water below its LCST which gives a transparent homogenous solution but at T > LCST it undergoes hydrophobic collapse marked by cloudiness of solution and conformational change from coil to globule state. All aqueous PNIPAM solutions undergo phase separation or cloud point transition at T > 32 , regardless of the molar mass of the polymer. Room temperature Ionic liquids (ILs) are unique designer fluids because of the novel physico-chemical properties arising from their structure, which have tremendous implications in the field of IL as solvents or co-solvents for polymeric solutes. During recent years a number of different imidazolium based ILs have also been tested for solubilization and stabilization of proteins as well as polymers due to hydrogen bond formation of the IL ions. Recent studies have shown that certain imidazolium based ILs can decrease the LCST of PNIPAM aqueous solution by hydrophobic collapse/aggregation of the PNIPAM chains, as well as some can induce an upper critical solution temperature (UCST) behavior of PNIPAM in neat IL solution. Even so, experimental studies of such phase transition/ instability of thermoresponsive polymer-IL systems has been a challenging task. In this research we have explored the critical solution temperature (CST) type phase behavior of multicomponent systems i.e. PNIPAM in solution media of water, neat IL and aqueous solutions of IL. The overall fundamental challenge is to understand how the interactions among the components control both structure and dynamics of PNIPAM network in solution. For example the disruption of hydrogen bonding or desolvation interactions between blocks of a PNIPAM molecule and solvent molecules in aqueous mixtures that lead to a LCST type transition at higher temperatures. Interestingly, it was found in our case that PNIPAM shows both LCST and UCST-type phase transition in some aqueous solutions of hydrophilic IL [BMIM][BF4]. It was found for the first time that this IL can influence the LCST type behavior of PNIPAM in aqueous solutions based on our visual and experimental cloud point (CP) observations. In our experiments the effect of the ILs [BMIM][BF4] and [BMIM][OAc] is qualitatively similar to influence of Kosmotropic salts on the LCST of aqueous PNIPAM solutions as predicted by the Hofmeister series.