Adsorption of proteins at interfaces has an important role in biotechnological and pharmaceutical applications. Indeed, several undesirable processes are related to protein adsorption, as for example: fouling of contact lenses, clotting on blood contacting devices, triggering inammation around articial organs, diminished circulation time of therapeutic proteins and drug bearing liposomes. Neutral water soluble polymers, such as poly(ethylene glycol) (PEG), are used to repress protein adsorption: by coating the surface with a polymer brush, a "cushion" is created between the protein and the surface, that can reduce, or even completely repress the adsorption. Understanding the mechanism that inhibits the adsorption at interfaces is an active field of research, and could lead to relevant improvements in biomaterials performances and design. A clear understanding of the mechanism of protein adsorption onto polymer brushes is still missing. The first models describing the interactions of a polymer brush with adsorbing particles predicted two adsorption modes: primary adsorption at the grafting surface, and secondary adsorption at the outer edge of the brush (occurring for large cylindrical proteins). Primary adsorption can be repressed by increasing the grafting density of the brush, and secondary adsorption by increasing its thickness, in agreement with the experiments reported in the literature. But experimental evidences (a maximum in the adsorbed amount observed for long brushes) suggested then the existence of a third mode: ternary adsorption within the brush itself, due to attractive interactions between the protein and the brush. Standard techniques can in general only probe the total adsorbed amount. The aim of this work was to separate primary and ternary adsorption isotherms, by using neutron reectivity and deuterated proteins. As neutrons interact differently with hydrogen and deuterium atoms, the contrast between the hydrogenated brush and the deuterated protein is high enough to separate the two contributions. We studied the adsorption of deuterated myoglobin on PEG brushes with different degrees of polymerisation (N = 56, 146 and 770), and as a function of the area per grafted chain. The contribution of primary and ternary adsorption was separated for the different systems, and the adsorbed amount was extracted and the adsorption isotherms compared to the theoretical predictions. The ability to distinguish between the different adsorption modes, and the quantification of their relative contribution to the overall amount of adsorbed proteins, represents a major advance in optimising surface properties. In particular, the occurrence of ternary adsorption onto PEG brushes affects their status as tool for repressing protein adsorption. L’adsorption de protéines aux interfaces a un rôle important pour certaines applications pharmaceutiques ou biotechnologiques. En effet, plusieurs processus indésirables sont liés à l’adsorption de protéines, par exemple l’encrassement de lentilles de contact, la coagulation dans des appareils contenant du sang, l’inflammation d’organes artificiels ou encore la diminution du temps de circulation dans le corps de protéines ou liposomes thérapeutiques. Certains polymères, tels que le polyéthylène glycol (PEG), sont utilisés pour réprimer l’adsorption de protéines : en greffant une brosse de PEG sur la surface, une couche est créée entre la protéine et celle-ci qui diminue, voire même réprime complètement l’adsorption. Comprendre le mécanisme qui entrave l’adsorption aux interfaces est un sujet de recherche actif, qui pourrait mener à des améliorations significatives dans la conception de biomatériaux. À ce jour, la compréhension du mécanisme d’adsorption de protéines sur des brosses de polymère n’est pas claire. Les premiers modèles décrivant les interactions entre brosses de polymères et particules adsorbantes prédisaient deux modes d’adsorption : l’adsorption primaire sur la surface de greffage, et l’adsorption secondaire à l’extérieur de la brosse (pour les grandes protéines cylindriques uniquement). L’adsorption primaire peut-être réprimée en augmentant la densité de greffage de la brosse, et l’adsorption secondaire en augmentant son épaisseur, en accord avec les expériences reportées dans la littérature. Mais d’autres évidences expérimentales (un maximum dans la quantité adsorbée observé pour les brosses longues) ont ensuite suggéré l’existence d’un troisième mode : l’adsorption ternaire à l’intérieur même de la brosse, due aux interactions attractives entre la protéine et la brosse. Les techniques standards peuvent en général mesurer la quantité adsorbée totale. Le but de ce travail était de séparer les isothermes d’adsorption primaire et ternaire, en utilisant la réflectivité de neutrons et des protéines deutérées. Comme les neutrons interagissent différemment avec les atomes d’hydrogène ou de deutérium, le contraste entre la brosse hydrogénée et la protéine deutérée est ainsi suffisant pour séparer les deux contributions. Nous avons étudié l’adsorption de myoglobine deutérée sur des brosses de PEG avec différents degrés de polymérisation (N = 56, 146 and 770), en fonction de l’aire par chaîne Σ. La contribution des adsorptions primaire et ternaire put être séparée pour les différents systèmes, et les quantités adsorbées extraites pour finalement comparer les isothermes d’adsorption aux prédictions théoriques. La possibilité de distinguer les différents modes d’adsorption, et la quantification de leur contribution relative à la quantité totale de protéines adsorbées représente une avancée majeure dans l’optimisation des propriétés des surfaces. L’adsorption ternaire dans les brosses de PEG en particulier remet en question leur utilisation pour réprimer l’adsorption de protéines.

Hematopoietic stem cells (HSCs) are commonly used in the treatment of blood cancers, like leukemia, and other cancers where radiation or chemotherapy damages the native HSC population. The development of a novel system to study and maintain HSCs ex vivo would give researchers and clinicians the ability to investigate the basic biological processes of HSCs, improve current treatment regimens, and explore their use in new therapies. The work in this thesis focuses on the development of a synthetic PEG hydrogel scaffold that accurately mimics aspects of the HSC microenvironment and can expand clinically relevant HSC populations. PEG hydrogel well surfaces were covalently functionalized with bioactive factors known to be critical in controlling HSC fate in vivo. In initial studies, 32D cells, a myeloid progenitor, were cultured in the wells for 6 days. On surfaces with the adhesive RGDS peptide sequence, 32D cell adhesion increased concurrently with RGDS surface concentrations. With the immobilization of two niche cytokines, SCF and SDF1α, onto hydrogel surfaces, 32D cells demonstrated significant increases in adhesion and spreading. These results confirmed that hematopoietic cell behavior could be controlled through the design of the bioactive PEG scaffold. In studies with a primary hematopoietic cell population (c-kit + , lin - ), the effects of bioactive molecules on cell expansion and differentiation were investigated after 2 weeks in culture. The adhesive peptides sequences, RGDS and CS1, and four niche proteins, SCF, SDF1α, JAG1, and IFNγ, were covalently tethered to hydrogel well surfaces. Primary cells proliferated significantly on gels containing SCF and IFNγ though only SCF was capable of preventing HSC differentiation. Cells cultured on surfaces functionalized with JAG1 and SDF1α did not proliferate extensively, but they were able to maintain primitive HSC populations. Primary c-kit + cells were also encapsulated within biodegradable PEG hydrogels and cultured for 2-5 weeks. Cells remained viable for 5 weeks in culture, and preliminary results indicated minimal cell differentiation. In this work, biomimetic PEG hydrogels were successfully employed to expand HSC populations in both two and three dimensions. The ability to generate large populations of primitive HSCs ex vivo has broad clinical and research implications.

Cell adhesion is the first step of cell response to materials and the extracellular matrix (ECM), and is essential to all cell behaviours such as cell proliferation, differentiation, migration and apoptosis for anchor-dependent cells. Therefore, studies of cell attachment have important implications to control and study cell behaviours. During many developed techniques for cell attachment, the manipulation of surface chemistry is a very important method to control initial cell attachment. To control cell adhesion on a two-dimensional surface is a simple model to study cell behaviours, and is a fundamental topic for cell biology, tissue engineering, and the development of biosensors. From the engineering point of view, the preparation of a material with controllable surface chemistry can help studies of cell behaviours and help scientists understand how surface features and chemistry influence cell behaviours. During the fabrication, the challenge is to create a surface with heterogeneous surface properties in the micro scale and subsequently to guide cell initial adhesion. In order to control cell adhesion in a spatial and temporal manner, a photochemical method to control surface chemistry was employed to control the surface property for cell adhesion in this project. Two photocleavable derivatives of the nitrobenzyl group were tried on two types of surfaces: a model self-assembled monolayer (SAM) with alkanethiol-gold surface and biodegradable chitosan. Reactive functional groups on two different surfaces can be inactivated by covalent binding with these photocleavable molecules, and light can be further introduced into the system as a stimulus to recover their reactivity. By simply applying a photomask with diffe

This thesis develops a novel photopatterning strategy for biomimetic scaffolds that enables spatial and biochemical control of engineered cellular architectures, such as the microvasculature. Intricate tools that allow for the three dimensional (3D) manipulation of biomaterial microenvironments will be critical for organizing cellular behavior, directing tissue formation, and ultimately, developing functional therapeutics to treat patients with critical organ failure. Poly(ethylene glycol) (PEG) based hydrogels, which without modification naturally resist protein adsorption and cellular adhesion, were utilized in combination with a two-photon laser patterning approach to covalently immobilize specific biomolecules in custom-designed, three-dimensional (3D) micropatterns. This technique, known as two-photon laser scanning lithography (TP-LSL), was shown in this thesis to possess the capability to micropattern multiple different biomolecules at modular concentrations into a single hydrogel microenvironment over a broad range of size scales with high 3D resolution. 3D cellular adhesion and migration were then explored in detail using time-lapse confocal microscopy to follow cells as they migrated along micropatterned tracks of various 3D size and composition. Further, in a valuable modification of TP-LSL, images from the endogenous microenvironment were converted into instructions to precisely direct the laser patterning of biomolecules within PEG-based hydrogels. 3D images of endogenous microvasculature from various tissues were directly converted into 3D biomolecule patterns within the hydrogel scaffold with precise pattern fidelity. While tissue engineers have previously demonstrated the formation of vessels through the encapsulation of endothelial cells and pericyte precursor cells within PEG-based hydrogels, the vessel structure had been random, uncoordinated, and therefore, ultimately non-functional. This thesis has utilized image guided TP-LSL to pattern biomolecules into a 3D structure that directs the organization of vessels to mimic that of the endogenous tissue vasculature. TP-LSL now stands as a valuable tool to control the microstructure of engineered cellular architectures, thereby providing a critical step in the development of cellularized scaffolds into functional tissues. Ultimately, this thesis develops new technologies that advance the field of regenerative medicine towards the goal of engineering viable organs to therapeutically treat the 18 patients who die every day waiting on the organ transplant list.

Abstract Aqueous solution containing tin chloride as precusor was traditionally added with NH3(aq) to promote hydrolysis and hence condensation. This results in a particulate sol which possesses little viscosity and the aggregation of precusor particles makes the subsequcently spin-coated thin film very rough in the surface and poorly-adhered with the substrate. One objective of this work is to improve the film quality by refluxing the sol to reduce precursor aggregation, enhance hydrolysis and promote HCl(g) evaporation. Experimtntal results show that, after refluxing the sol with DI-water or methanol as solvent, one obtains better films when basic sol (NH3(aq) added) and SnCl2 precursor is used instead of acidic sol (HCl(aq)added) and SnCl4 precursor. Moreover, to further reduce the effect of Cl¡Ð ion in aggregation and increase viscosity, ethylene glycol was used as solvent and two-stage heating-stirring of the sol in 80 oC and 130 oC ~150 oC was carried out to promote generation of H2O(g) and HCl(g). The evaporation of H2O(g) and HCl(g) enhances the polymerization of precursor and increase the viscosity of the sol. The aggregation caused by Cl¡Ð ions is thus reduced due to the steric effect present in the polymerical sol. XRD, SEM, FT-IR , TGA and DSC were used to examine the evolution from sol to films. FT-IR results show that absorbtion peaks of the xerogel appear at 636 cm-1(O-Sn-O) and 500 cm-1 (Sn-O). XRD results of the calcined (4 hr) powders show that rutile (SnO2) crystallization starts at 200 oC for that derived from the SnCl2-containing sol while powder derived from the SnCl4-containing sol starts crystallization at 250 oC. However, grain growth is faster in powder derived from SnCl4-containing sol as their XRD peaks become sharper than that corresponding to SnCl2 precursor as calcination temperature is raised. Based on the examination of the evolution process, it is concluded that SnCl2 polymerizes in ethylene glycol as a one dimensional chain while SnCl4 forming a 3-D network after polymerizing in ethylene glycol.

Studies of biomolecular interactions are of interest for several reasons. Beside basic research, the knowledge gained from such studies is also very valuable in for example drug target identification. Medical care is another area where biomolecules may be used as biomarkers to aid physicians in making correct diagnosis. In addition, the highly specific interactions between antibodies and almost any substance opens up the possibilities to design systems for detection of trace amounts of both biological and non-biological substances within environmental restoration, law enforcement, correctional care, customs service and national security. A biochip, which contains a biologically active material, offers a means of monitoring the molecular interactions in the above applications in a sensitive and specific manner. The biochip is a key component of a biosensor, which also includes components for transforming the interaction events into a human-readable signal. This thesis describes the use of poly(ethylene glycol) (PEG) in biochip design. Two different approaches are presented, the first based on ethylene glycol (EG)-containing alkyl thiol self-assembled monolayers (SAMs) on flat gold and the second on photo-induced graft copolymerisation of PEG-containing methacrylate monomers onto various substrates. The former is a two dimensional system where EG-terminated thiols are mixed with similar thiols presenting tail groups that mimic the explosive substance 2,4,6-trinitrotoluene (TNT). In an immunoassay, the detection limit for TNT was determined to fall in the range 1-10 µg/L. In the second approach, a branched three dimensional biosensor matrix (hydrogel) is proposed. The carboxymethylated (CM) dextran matrix, which is commonly used within the biosensing community, is not always ideal for studies of biointeractions, due to the non-specific binding frequently encountered in work with complex biological solutions and various proteins. To employ PEG, which displays a low non-specific binding of such species, is therefore an interesting option worth investigating. The use of a branched graft polymerised PEG matrix in biosensor applications is novel as compared to previous reports which have focused on linear PEG chains. The latter approach provides, at maximum, one functional group, per surface anchoring point, for immobilisation of sensor elements. Thus, it has the inherited disadvantage that it limits the number of available immobilisation sites. The present PEG matrix contains a large number of functional groups, for immobilisation of sensor elements, per grafting site and offers the potential of improved response upon binding to the analyte as demonstrated in a series of successful sensor experiments. Furthermore, the nature of the process enables easy preparation of matrix patterns and gradients. In a PEG matrix gradient, protein permeability is studied and the capabilities of immobilising proteins are demonstrated. By combining the patterning technique with different monomers in a two-step process, an inert platform, lacking chemical attachment sites, is provided with arrays of spots (with immobilisation capabilities), which are conveniently addressed via microdispensing and used for biosensor purposes. The EG-terminated thiols present another means of generating such inert platforms, a route which is also investigated. To further explore the sensor quality of these spots, the concepts of patterning and gradient formation are combined and studied. / Det är intressant att studera biomolekylära interaktioner av många anledningar. För att kunna bedriva framgångsrik läkemedelsutveckling är det oerhört viktigt att känna till hur olika molekyler samverkar i människokroppen. Inom sjukvården kan biomolekyler användas som biomarkörer, då närvaro av dem eller förändringar av deras koncentrationer är kopplade till sjukdomstillstånd, och därmed hjälper läkaren att ställa rätt diagnos. Dessutom kan de mycket specifika interaktionerna mellan antikroppar och (i princip) valfri substans användas för detektion av spårämnen vid miljösaneringsarbete, gränskontroller, polisarbete, fängelser och arbete med nationell säkerhet. Den här avhandlingen beskriver hur polymeren polyetylenglykol (PEG) kan användas vid design av biochip. Ett biochip är en liten anordning, som kan användas för att detektera specifika molekyler med hjälp av en biologisk interaktion. Traditionellt har PEG använts inom biomaterialsektorn, men återfinns även i hygienartiklar som tvål och tandkräm. Ett annat användningsområde är konservering av bärgade träskepp och i en del litiumjonbatterier ingår PEG som en komponent. Dessutom pågår utveckling av PEG-innehållande skyddsvästar. I det här arbetet används PEG framför allt på grund av sin förmåga att minimera ospecifik inbindning av proteiner, som utgör en stor del av gruppen biomolekyler, till ytor på biochip. Två olika typer av ytbeläggningar, som innehåller den här polymeren, har använts. Den första typen ger mycket tunna (~0.000003 mm), tvådimensionella filmer medan den andra ger en något tjockare (~0.00005 mm), tredimensionell struktur (matris). De tvådimensionella filmerna har använts för att utveckla en sprängämnesdetektor med mycket hög känslighet (detektionsgräns mellan 1-10 ppb). En viktig beståndsdel i detta system är antikroppar riktade mot sprängämnet trinitrotoluen (TNT). Den tredimensionella matrisen är mer generell och kan användas för att studera många olika molekylära interaktioner. Tillverkningsmetoden av matrisen är baserad på belysning med ultraviolett ljus och är därmed lämpad för att skapa mönstrade ytor. Genom att blockera delar av ljusflödet begränsas tillväxten av matrisen till de belysta delarna. På så sätt har bland annat så kallade mikro-arrayer, bestående av mikrometerstora (tusendels millimeter) strukturer i ett regelbundet mönster, tillverkats. Tekniken tillåter även tillverkning av gradienter, där matrisens tjocklek varierar längs med provet, genom att belysa olika delar av provytan olika länge. Genom att undersöka dessa gradienter har information om matrisens genomsläpplighet för proteiner kunnat extraheras. Gradientkonceptet har även kombinerats med mikro-arraytillverkningen och gett möjlighet att studera interaktioner mellan flera olika modellproteiner och deras motsvarande antikroppar i olika tjocka matriser på en och samma yta. Det finns ett stort antal sätt att utnyttja interaktionerna mellan olika molekyler på ett biochip. Ett tilltalande tillvägagångssätt är exempelvis att i en mikro-array binda in olika molekyler som kan fånga kliniskt intressanta biomolekyler, i syfte att skapa en hälsoprofil. Ett sådant biochip skulle ge möjlighet att parallellt detektera eller bestämma koncentrationen av ett stort antal biomolekyler i till exempel en droppe blod. På så sätt kan en diagnos snabbt ställas, kanske till och med utan att patienten behöver uppsöka sjukvården. Den utvecklade PEG-matrisen har god potential att fungera i en sådan applikation.

Currently, cancer is the second leading cause of death in the United States. Conventional cancer treatment includes chemotherapy, radiation, and surgical resection, but unfortunately, all of these methods have significant drawbacks. Hyperthermia, the heating of cancerous tissues to between 41 and 45°C, has been shown to improve the efficacy of cancer therapy when used in conjunction with irradiation and/or chemotherapy. In this work, a novel method for remotely administering heat is presented. This method involves heating of tumor tissue using hydrogel nanocomposites containing magnetic nanoparticles which can be remotely heated upon exposure to an external alternating magnetic field (AMF). The iron oxide nanoparticles contained in the hydrogel nanocomposites are able to heat via an AMF due to Brownian and Neel relaxation processes. The administration of hyperthermia via hydrogel nanocomposites allows for local delivery of heat to tumor tissue while also providing a drug depot to deliver chemotherapeutic agents. Both in vivo and in vitro studies have demonstrated that numerous chemotherapeutic agents, when used in conjunction with hyperthermia, show improved efficacy in treating cancer Various magnetic hydrogel nanocomposites were synthesized and characterized for this work including poly(ethylene glycol) (PEG)-based hydrogels, which were studied due to their inherent biocompatibility and “stealth” properties, as well as, poly(β-amino ester) (PBAE)-based hydrogels which have tailorable degradation properties. The PEG hydrogels were investigated for their temperature-responsiveness swelling, mechanical strength, heating capabilities, biocompatibility, ability to kill M059K glioblastoma cells via thermoablation, and the ability to deliver paclitaxel, a chemotherapeutic agent. PBAE hydrogels were also characterized for their degradation and swelling properties, ability to heat upon exposure to an AMF, biocompatibility, mechanical strength, and ability to deliver paclitaxel in a controlled fashion. Additionally, multiple cancer cell lines were exposed to a combination of paclitaxel and heat (at 42.5 °C) in vitro and it was shown that A539 lung carcinoma cells exhibit higher cytotoxicity when exposed to both heat and paclitaxel than either treatment alone. Overall, magnetic hydrogel nanocomposites are promising materials that can be utilized for the multi-modality treatment of cancer through the synergistic delivery of both heat and chemotherapeutic agents.

Protein PEGylation (covalent attachment of poly(ethylene glycol) or PEG to proteins) is an excellent example of a drug delivery system that improves pharmacokinetics and pharmacodynamic properties of therapeutics. However, although PEGylation is clinically proven and attracts both scientific and commercial interest, the technique is associated with many process constraints, in particular related to controlling the number of conjugated PEG chains. A novel, solid-phase PEGylation methodology was attempted to overcome the drawbacks of the commonly used solution-phase methods for preparing PEGylated products. The solid-phase PEGylation methodology involved conjugating protein onto a tethered PEG derivative attached onto a solid matrix, followed by hydrolytic cleavage of the PEG chain from the solid matrix under mild conditions to yield PEGylated protein in free solution. PEGs with molecular weights (MWs) 2000 and 4000 Da were used and a heterobifunctional PEG derivative, α-(β-alanine)-ω-carboxy PEG, with a cleavable β-alanine ester terminal was prepared for surface grafting and protein conjugation. The amine terminal of this PEG derivative was used for grafting PEG onto carboxy functionalized hydrophilic Sephadex and hydrophobic polystyrene derivatives. The free carboxyl terminal was used for protein conjugation via amine coupling. A kinetic study of PEG-surface grafting was performed to understand the influence of a number of parameters on the PEG surface concentration and its conformation, including temperature, reaction time, nature of the matrix, solvent and base, and MW of PEG. PEG grafted matrices were characterized using various surface characterization tools including Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Higher PEG grafting was observed with polystyrene matrices (up to 0.3 mmol/g) than either of the Sephadex derivatives (less than 0.15 mmol/g) using both molecular weights. Detailed surface characterization using XPS studies showed a layer thickness of 11.87 nm was achieved with polystyrene matrices using 4000 Da PEG derivatives after a grafting period of 72 hours at 40°C, indicating the presence of brush conformations for the grafted PEGs. In contrast, mushroom conformations were observed for PEG molecules grafted on both carboxymethyl and carboxypentyl Sephadex derivatives after the same reaction period, with a layer thickness of 2.62 nm and 4.14 nm respectively. Optimized PEG grafting and hydrolysis conditions were developed for solid-phase protein PEGylation using Cytochrome c as a model protein. The presence of PEGylated species were detected by size exclusion chromatography (SEC) from Sephadex derivatives but were absent when using polystyrene matrices. Both Sephadex derivatives gave mainly multi-PEGylated species with poor yields, in place of the expected mono-PEGylated products. A solution-phase PEGylation using the same PEG derivatives was performed successfully and various PEGylated species were identified and characterized using SEC and gel electrophoresis, based on their viscosity radius. An examination of the surface characteristics of the PEG-grafted was carried out by XPS, showing that protein conjugation was greatly influenced by surface force interactions, which depended on the PEG grafting densities and the nature of the solid matrices. Finally, fluorescent images obtained using confocal microscope with fluorescein isothiocyanate labelled Cytochrome c provided supporting evidence regarding the factors that constrained the solid-phase PEGylation process.

The aim of this thesis is the study of protein resistant oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs) using in situ techniques, such as neutron reflectivity (NR), polarisation modulation infrared spectroscopy (PMIR) and small-angle x-ray scattering (SAXS). In order to elucidate the mechanisms that lead to the nonfouling properties of these SAMs, the SAM-water, protein-protein and protein-SAM interactions have been studied separately. NR measurements, focused on the solid-liquid interface between OEG SAMs and water, show clear evidence of an extended layer with reduced density water. The reduction in density is up to 10% compared to the bulk value, and extends up to 5 nm into the bulk. The effective area (density reduction x length) of this reduced density water layer did not significantly change when the temperature was reduced to 5°C. In a complementary study, the interaction of water with protein-resistant HS(CHV₂)₁₁(OCH₂CH₂)₃OMe monolayers was examined using in and ex situ PMIR. In particular, shifts in the position of the characteristic C-O-C stretching vibration were observed after the monolayers had been exposed to water. The shift in frequency increased when the SAM was observed in direct contact with a thin layer of water. It was found that the magnitude of the shift also depended on the surface coverage of the SAM. These results suggest a rather strong interaction of oligo(ethylene glycol) SAMs with water and indicate the penetration of water into the upper region of the monolayer. These findings indicate the presence of a tightly bound water layer at the SAM-water interface. Further NR studies of the interface between OEG SAMs and a highly concentrated protein solution revealed an oscillating protein density profile. A protein depleted region of about 4-5 nm close to the SAM was followed by a more densely populated region of 5-6 nm. These oscillations were then rapidly damped out until the bulk value was reached. The influence of temperature and salt concentration on the protein density profile was small, indicating a rather minor contribution of electrostatic interactions to the protein repulsive force. SAXS measurements of OEG coated gold colloids mixed with proteins in solution did also not show any pronounced salt concentration dependence of the colloid-protein interaction. The strong association of water with the SAM and the layer of tightly bound water, together with the lack of electrostatic repulsion, suggest that the adsorption of proteins is energetically hindered by the presence of a strongly bound hydration layer.

Semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs) have gained tremendous attention in the last decade as a result of their size-dependent spectroscopic properties. These nanoparticles have been a subject of intense study to bridge the gap between macroscopic and atomic behavior, as well as to generate new materials for novel applications in therapeutics, biological sensing, light emitting devices, microelectronics, lasers, and solar cells. One of the most promising areas for the use of these nanoparticles is in biotechnology, where their size-dependent optical properties are harnessed for imaging and sensing applications. However, these nanoparticles, as synthesized, are often not stable in aqueous media and lack simple and reliable means of covalently linking to biomolecules. The focus of this work is to advance the progress of these nanomaterials for biotechnology by synthesizing them, characterizing their optical properties and rendering them water-soluble and functional while maintaining their coveted optical properties. QDs were synthesized by an organometallic chemical procedure that utilizes coordinating solvents to provide brightly luminescent nanoparticles. The optical interactions of these QDs were studied as a function of concentration to identify particle size-dependent optimal concentrations, where scattering and indirection excitation are minimized and the amount light observed per particle is maximized. Both QDs and AuNPs were rendered water-soluble and stable in a broad range of biologically relevant conditions by using a series of ligands composed of dihydrolipoic acid (DHLA) appended to poly(ethylene glycol) methyl ether. By studying the stability of the surface modified AuNPs, we revealed some interesting information regarding the role of the surface ligand on the nanoparticle stability (i.e. solubility in high salt concentration, resistance to dithiothreitol competition and cyanide decomposition). Furthermore, the nanoparticles were functionalized using a series of bifunctional ligands that contain a dithiol group (DHLA) for surface binding, a PEG segment to instill water-solubility and a terminal functional group for easy bioconjugation (i.e. NH 2 , COOH, or biotin). Finally, a sensing application was demonstrated to detect the presence of microbial DNA (unmethlylated CpG) by using Toll-like receptor 9 proteins as the recognition components and the QDs as the transduction elements via Förster Resonance Energy Transfer.