Biofunctional interfaces capable of anchoring biomolecules and nanoparticles of interest onto a platform are the key components of many biomedical assays, clinical pathologies, as well as antibacterial and antiviral surfaces. In an ideal biofunctional surface, bio-entities and particles are covalently immobilized on a substrate in order to provide robustness and long-term stability. Nonetheless, most of the reported covalent immobilization strategies incorporate complex wet-chemical steps and long incubation times hindering their implementation for mass production and commercialization. Another essential factor in the biointerface preparation, specially with regard to biosensors and diagnostic applications, is utilization of an efficient and durable blocking agent that can inhibit non-specific adsorption of biomolecules thereby enhancing the sensitivity of sensors by diminishing the level of background noise. Many of the commonly used blocking agents lack proper prevention of non-specific adsorption in complex fluids. In addition, most of these agents are physically attached to surfaces making them unreliable for long-term usage in harsh environments (i.e. where shear stresses above 50 dyn/cm2 or strong washing buffers are involved). This thesis presents novel and versatile strategies to covalently immobilize nanoparticles and biomolecules on substrates. The new surface coating techniques are first implemented for robust attachment of TiO2 nanoparticles onto ceramic tiles providing self-cleaning properties. Further, we utilize similar strategies to covalently immobilize proteins and culture cells in microfluidic channels either as a full surface coating or as micropatterns of interest. The new strategies allow us to obtain adhesion of ~ 400 cells/mm2 in microfluidic channels after only 1-day incubation, which is not achievable by the conventional methods. Moreover, we show the possibility of covalently micropatterning of biomolecules on lubricant-infused surfaces (LISs) so as to attain a new class of biofunctional LISs. By integration of these surfaces into a biosensing platform, we are able to detect interleukin 6 (IL-6) in a complex biofluid of human whole plasma with a limit of detection (LOD) of 0.5 pg.mL-1. This LOD is significantly lower than the smallest reported IL-6 LOD in plasma, 23 pg mL-1, using a complex electrochemical system. The higher sensitivity of our developed biosensor can be attributed to the distinguish capability of biofunctional LISs in preventing non-specific adhesion of biomolecules compared to other blocking agents. / Thesis / Doctor of Philosophy (PhD) / The key goal of this thesis is to provide new strategies for preparation of robust and durable biointerfaces that could be employed for many biomedical devices such as self-cleaning coatings, microfluidics, point-of-care diagnostics, biomedical assays, and biosensors in order to enhance their efficiency, sensitivity, and precision. The introduced surface biofunctionalization methods are straightforward to use and do not require multiple wet-chemistry steps and incubation times, making them suitable for mass production and high throughput demands. Moreover, the introduced surface coating strategies allow for creation of antibody/protein micro-patterns covalently bound onto a biomolecule-repellent surface. The repellent property of the surfaces is resulted from infusion of an FDA-approved lubricant into the surface of a chemically modified substrate. While the surface repellency can effectively prevent non-specific adhesion of biomolecules, the patterned antibodies can locally capture the desired analyte, making them a great candidate for biosensing.

Geometric modeling of biomolecules plays an important role in the study of biochemical processes. Many simulation methods depend heavily on the geometric models of biomolecules. Among various studies, shape analysis is one of the most important topics, which reveals the functionalities of biomolecules.

Biomolecular structure and interactions in aqueous environment are determined by a complicated interplay between physical and chemical forces including solvation, electrostatics, van der Waals forces, the hydrophobic effect and covalent bonding. Among them, electrostatics has been of particular interest due to its long-range nature and the tradeoff between desolvation and interaction effects [1]. In addition, electrostatic interactions play a significant role within a biomolecule as well as between biomolecules, making the balance between the two vital to the understanding of macromolecular systems. As a result, much effort has been devoted to accurate modeling and simulation of biomolecule electrostatics. One important application of this work is to compute the structure of electrostatic interactions for a biomolecule in an electrolyte solution, as well as the potential that the molecule generates in space. There are two valuable uses for these simulations. First, it provides a full picture of the electrostatic energetics of a biomolecular system, improving our understanding of how electrostatics contributes to stability, specificity, function, and molecular interaction [2]. Second, these simulations serve as a tool for molecular design, since electrostatic complementarity is an important feature of interacting molecules. Through examination of the electrostatics and potential field generated by a protein molecule, for example, it may be possible to suggest improvements to other proteins or drug molecules that interact with it, or perhaps even design new interacting molecules de novo [3]. There are two approaches in simulating a protein macromolecule in an aqueous solution with nonzero ionic strength. Discrete/atomistic approaches based on Monte-Carlo or molecular dynamics simulations treat the macromolecule and solvent explicitly at the atomic level. Therefore, an enormous number of solvent molecules are required to provide reasonable accuracy, especially when electric fields far away from macroscopic surface are of interest, leading to computational infeasibility. In this work, we adopt instead an approach based on a continuum description of the macromolecule and solvent. Although the continuum model of biomolecule electrostatics is widely used, the numerical techniques used to evaluate the model do not exploit fast solver approaches developed for analyzing integrated circuit interconnect. I will describe the formulation used for analyzing biomolecule electrostatics, and then derive an integral formulation of the problem that can be rapidly solved with precorrected-FFT method [4]. / Singapore-MIT Alliance (SMA)

abstract: The increasing pervasiveness of infections caused by multidrug-resistant bacteria (MDR) is a major global health issue that has been further exacerbated by the dearth of antibiotics developed over the past 40 years. Drug-resistant bacteria have led to significant morbidity and mortality, and ever-increasing antibiotic resistance threatens to reverse many of the medical advances enabled by antibiotics over the last 40 years. The traditional strategy for combating these superbugs involves the development of new antibiotics. Yet, only two new classes of antibiotics have been introduced to the clinic over the past two decades, and both failed to combat broad spectrum gram-negative bacteria. This situation demands alternative strategies to combat drug-resistant superbugs. Herein, these dissertation reports the development of potent antibacterials based on biomolecule-encapsulated two-dimensional inorganic materials, which combat multidrug-resistant bacteria using alternative mechanisms of strong physical interactions with bacterial cell membrane. These systems successfully eliminate all members of the ‘Superbugs’ set of pathogenic bacteria, which are known for developing antibiotic resistance, providing an alternative to the limited ‘one bug-one drug’ approach that is conventionally used. Furthermore, these systems demonstrate a multimodal antibacterial killing mechanism that induces outer membrane destabilization, unregulated ion movement across the membranes, induction of oxidative stress, and finally apoptotic-like cell death. In addition, a peptide-encapsulation of the two-dimensional material successfully eliminated biofilms and persisters at micromolar concentrations. Overall, these novel systems have great potential as next-generation antimicrobial agents for eradication of broad spectrum multidrug-resistant bacteria. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

This research investigates the interfacial interactions between biological entities and synthetic materials at two length scales: bulk and nanometer size. At the bulk scale, biomolecule adhesion is key for synthetic material incorporation in the body. Quantifying the adhesion strength becomes necessary. For the nanometer scale, the nanoparticles are generally delivered through the blood stream and their effect on the blood flow must be studied. An experimental approach was taken to study interaction at both material length scales. The cell/protein adhesion strength to bulk-sized materials was studied. The goal was to identify the most influential factor affecting adhesion: chemistry or surface roughness. The effects of nanoparticles on the viscosity of protein and amino acid solutions were measured. A statistical thermodynamic analysis was focused on the entropy change induced by the addition of gold nanoparticles to protein/amino acid solutions. Rheological studies were applied. A rheometer with a parallel plate was used to quantify the adhesion strength of cells and proteins to synthetic surfaces at the bulk scale. The adhesion strength depends on the applied shear stress and the radius of cells or proteins that remained attached to the surface after testing. At the nanometer scale, the viscosity of the nanoparticle enhanced protein or amino acid solutions were measured with a cone and plate. The adhesion studies were conducted with the following biological entities: fibroblasts, egg-white protein, and neurons. The fibroblast adhesion to poly(carbonate) and poly(methyl methacrylate) demonstrate fibroblasts are strongly attached to highly polar materials. Protein adhesion to titanium and chromium nitride coatings showed that chemical composition is the most influential factor. The neuron adhesion to poly-D-lysine coated glass demonstrated that neuron strengthening was due to an increase in adhesion molecules at the neuron/material interface. For nanoparticulates, it was found that the charged nanoparticles affect the protein and amino acid conformation and the potential energy of the solutions. Quantifying biomolecule adhesion to surfaces and predicting the behavior of nanoparticles inside a biological system are crucial for material selection and application. The major impact of this research lies in observing the interaction mechanisms at the interfaces of material-biological entities.

Numerous biological phenomena occur as a result of macromolecular interactions. Metal-ion-biomolecule binding account for a large portion of these reactions, and unsurprisingly, a vast amount of new research in this area is constantly emerging. Gaining insight into the characteristics that define these interactions; including equilibrium fluctuations, metal center formation, global stability perturbation, cooperativity, allostery, and site-specific binding are all significant. As with all chemical reactions, biological interactions are regulated by thermodynamics; and the development of novel tools and methods by which to study these interactions becomes highly relevant. In this dissertation, three systems involving macromolecular binding are studied using well established biophysical techniques in conjunction with a critical look at appropriate uses for mathematical modeling. The first system studied is that of the serpin plasminogen activator inhibitor-1 (PAI-1). PAI-1 is a protease inhibitor that specifically effects fibrinolysis, or the process that prevents the formation of blood clots, and misregulation of this enzyme leads to uncontrollable hemorrhaging. ITC was utilized to investigate the thermodynamics of copper binding to PAI-1. Human carbonic anhydrase II (CA) was the second system investigated. Studies were conducted on zinc(II) and copper(II) binding to CA, a metalloenzyme responsible for acid-base balances in the blood and the transport of carbon dioxide. Interestingly, CA binds two copper(II) ions, one at the active site, and one at a higher affinity N-terminal site. Temperature dependent ITC, CD and GdnHCl denaturation studies were performed to explore the impact of copper(II) binding, particularly at the higher affinity N-terminal site. Finally, protein binding to inorganic gold nanoparticles (AuNPs) was investigated. AuNPS are utilized in areas of diagnostics, biological sensing and drug delivery. We studied binding of nanoparticles to a set of six biologically relevant proteins; glutathione, wild-type GB3, K19C GB3 (a variant at position 19), bovine CA, bovine serum albumin, and fibrinogen. Nanoparticle-protein binding was monitored via UV-Visible extinction and polarized resonance synchronous spectroscopy (PRS2). The UV extinction maxima wavelength shifts were fit with two models, a Langmuir isotherm model and a mass action-derived model. The models fit the data equally well, however, they predict very different Kd values, specifically for smaller sized AuNPs.

No description available.

Le contrôle de la qualité des ressources en eau nécessite des outils de détection en continu et in situ. Dans ce contexte, les biocapteurs sont prometteurs dans le développement de nouveaux systèmes pour une détection précoce. On peut citer deux exemples dans des domaines aussi variés que la détection précoce de corrosion bactérienne ou la détection de la formation d'espèces biologiques responsables de maladies dégénératives.Ce travail propose la conception et la réalisation d'un biocapteur pour la détection de molécules d'intérêt biologiques. La réalisation de ce biocapteur est basée sur le dépôt de matériaux hybrides organiques/inorganiques à la surface d'un transducteur piézoélectrique.La première étape consiste à développer des matériaux fonctionnalisés innovants permettant l'encapsulation d'espèces biologiques. Pour cela deux voies ont été étudiées. La première passe par l'utilisation d'un acrylate type "acide aminé" le N-acryloyglycine (NAGly) permettant la synthèse de matériaux sous forme de film. La seconde utilise un autre type d'acrylate, le N-acryloxysuccinimide (NAS) couplé au 2-hydroxyethylacrylate (HEA) aboutissant un hydrogel fonctionnalisé. Les différentes mesures montrent que dans tous les cas, ces réseaux sont interpénétrés et permettent l'encapsulation de biomolécules.Pour la détection de ces espèces, un biocapteur piézoélectrique est développé dans la deuxième étape. Un dispositif expérimental développé au laboratoire assure la mesure et le suivi de l'évolution des propriétés viscoélastiques d'un matériau en contact avec un transducteur piézoélectrique. En effet, ces propriétés caractéristiques sont extraites à partir d'un modèle électrique original tenant compte simultanément des évolutions électriques et mécaniques du matériau. Ce capteur (transducteur + matériau déposé en surface) mis au contact avec les biomolécules permet leur détection et quantification.Les résultats montrent une corrélation entre le module visqueux du biocapteur et la concentration en biomolécules du milieu en contact. Cette corrélation est une première étape vers le développement d'un biocapteur piézoélectrique pour la détection et la quantification sélective de différentes espèces biologiques en solution. / The control of the quality of water resources requires tools for continuous and in-situ detection. In this context, biosensors are interesting in the development of new systems for early detection. For examples we can note the interest in the early detection of bacterial corrosion or the formation of biological species responsible of degenerative diseases.This work proposes the design and implementation of a biosensor for the detection of biological molecules. The realization of this biosensor is based on the deposition of organic/inorganic materials on the surface of a piezoelectric transducer.The first step is the development of innovative functionalized materials for encapsulation of biological species. For this, two approaches have been studied. The first involves the use of an "amino acid" acrylate, the N- acetylglycine (Nagly) for the synthesis of thin film. The second uses an other acrylate, the N- acryloxysuccinimide (NAS) copolymerized with 2- Hydroxyethyl acrylate (HEA) to form a functionalized hydrogel. The different measures show that in all cases, these networks are interpenetrating and allow the encapsulation of biomolecules.For the detection of these species, a piezoelectric biosensor is developed in the second step. An experimental device, developed in the laboratory, measures and monitors the evolution of the viscoelastic properties of a material in contact with a piezoelectric transducer. Indeed, these characteristics are extracted from an original electric model that take into account simultaneous electrical and mechanical changes in the material. This sensor (transducer and material deposited on the surface) in contact with biomolecules, enables the detection and quantification of these biomolecules.The results show a correlation between the viscous modulus of the biosensor and the concentration of biomolecules in contact. This correlation is a first step in the development of a piezoelectric biosensor for detection and selective quantification of different biological species in solution.

La tomographie par émission de positons (TEP) est une technique d'imagerie médicale largement utilisée en oncologie. Cette technique peut, par exemple, fournir des informations sur la localisation de tumeurs dans le corps humain, et le développement de nouvelles méthodes pour la production automatisée de radiotraceurs spécifiques reste un domaine d’actualité. Dans ce contexte, un nouveau type de précurseurs silylés pouvant être fluorés en dernière étape a été développé. La fluoration de ce nouveau motif imidazole-silylé par du [19F]F- puis par du [18F]F- a été validée sur différents bioconjugués. Le greffage de ce motif a ensuite été testé selon plusieurs méthodes. En particulier, une des voies de synthèse a pu montrer que la fluoration de bioconjugués portant ce motif et greffés sur une résine était possible au [19F]F-. Enfin, le produit radiomarqué au [18F]F- a pu être détecté au cours d’essais préliminaires, permettant ainsi de valider la stratégie choisie. / Positron Emission Tomography (PET) is a medical imaging technique widely used in oncology. For example, this technique can provide information on the localization of growing tumors in the human body, and the development of news methods for the automated production of specific radiotracers is still required. In this context, a new silylated precursor, which can be fluorinated in the last step, has been developed. The fluorination of this new imidazole-silylated unit with [19F]F- and with [18F]F- has been validated on various bioconjugates. The grafting of this moiety has been tested following different strategies. In particular, one of the synthetic pathway showed that the fluorination of bioconjugates with resin grafted moiety was possible with [19F]F-. Finally, the [18F] radiolabeled product was observed during preliminary [18F]F- fluorination experiments, thus validating the chosen strategy.

No description available.