Dissertations / Theses on the topic 'Conjugated Biomolecules'

Conjugated polyelectrolytes (CP) show interesting electrical and optical properties for organic electronics as well as for life science applications. Their possibilities of supramolecular assembly with nanowire like misfolded proteins, amyloids, as well as synthetic polypeptides or DNA forming conducting or luminescent nano composites is highly interesting as being a truly bottom up approach for fabrication of OLEDs, photovoltaic’s as well as logic devices. The conformation and aggregation dependent luminescence properties from the special class of CPs, Luminescent conjugated polyelectrolytes (LCP), have been utilised and developed as sensors to follow and study biomolecular interactions, DNA hybridisation, protein-protein interactions and staining of living cell cultures and tissue slides. In this thesis we are bringing the evolution a few steps further by applying new types of experimental techniques, such as light scattering and fluorescence correlation spectroscopy, combined with standard techniques as soft lithography and different spectroscopy techniques, to gain better knowledge of the optical behaviour of LCPs and their interactions with biomolecules. We explore the optical properties and vibronic transitions of LCPs; their ability of resonance energy transfer with LCPs indicating super lightning behaviour; the opposite fluorescence shift when interacting with α-helical rich polypeptides compared to earlier reports of interactions upon staining of β-rich amyloids; and the possibility of LCPs to influence protein aggregation as well as the possibility of fabricating biochips based on LCPs and soft lithography. Here we also show fundamental limitations to patterning using macromolecular fluids, of general relevance to soft lithography and nanoimprint lithography with low viscosity polymers.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.
Committee Chair: Dr. Uwe H.F. Bunz; Committee Member: Dr. Andrew Lyon; Committee Member: Dr. Laren Tolbert; Committee Member: Dr. Nicholas Hud; Committee Member: Dr. Sherry Michele Owen. Part of the SMARTech Electronic Thesis and Dissertation Collection.

This thesis is composed of two parts: Part I is focused on studying how quantum dot (QD) surface small-molecule capping ligands affect its Förster resonance energy transfer (FRET) with a fluorescent protein (FP); Part II is focused on developing an ultrasensitve DNA sensing technology by combining magnetic nanoparticle (MNP) and enzymatic signal amplification. Part I The FRET between a CdSe/ZnS core/shell QD capped with three different small-molecule ligands and a hexa-histidine (His6)-tagged FP (mCherry) has been studied. Results show that small-molecule ligands strongly affect the FRET behaviours between QD and FP. The QD-FP self-assembly process is fast (complete in minutes at low nM concentration), strong (with Kd ~ 1 nM), suggesting that the QD-His6-tagged biomolecule self-assembly is an effective approach for making compact QD-bioconjugates which may have a wide range of sensing and biomedical applications. PART II I have developed a facile, rapid, and sensitive DNA sensor by combining the efficient MNP-based target capture with the highly efficient signal amplification power of enzymes to achieve ultra-sensitivity. This sensor works efficiently in both pure buffer and complex media (such as 10% human serum in buffer). Moreover, this developed approach is able to quantitate two distinct DNA strands in a homogenous phase at the same time with a detection limit of ~5 pM. Second, I have developed a novel, highly sensitive and selective approach for label-free DNA detection and single-nucleotide polymorphism (SNP) discrimination by combining target-recycled ligation (TRL), MNP assisted target capture/ separation, and efficient enzymatic amplification. This approach possesses a detection limit of 600 fM unlabelled DNA targets and offers exquisitely high discrimination ratio (up to > 380 fold) between a perfect-match mutant and its single-base mismatch DNA target. Furthermore, it can quantitate the rare cancer mutant in large excesses of coexisting wildtype DNA down to 0.75%.

Biotemplating is the art of using a biological structure as a scaffold which is decorated with a functional material. In this fashion the structures will gain new functionalities and biotemplating offers a simple route of mass-producing mesoscopic material with new interesting properties. Biological structures are abundant and come in a great variety of elaborate and due to their natural origin they could be more suitable for interaction with biological systems than wholly synthetic materials. Conducting polymers are a novel class of material which was developed just 40 years ago and are well suited for interaction with biological material due to their organic composition. Furthermore the electronic properties of the conducting polymers can be tuned giving rise to dynamic control of the behavior of the material. Self-assembly processes are interesting since they do not require complicated or energy demanding processing conditions. This is particularly important as most biological materials are unstable at elevated temperatures or harsh environments. The main aim of this thesis is to show the possibility of using self-assembly to decorate a conducting polymer onto various biotemplates. Due to the intrinsic variety in charge, size and structure between the available natural scaffolds it is difficult, if not impossible, to find a universal method. In this thesis we show how biotemplating can be used to create new hybrid materials by self-assembling a conducting polymer with biological structures based on DNA, protein, lipids and cellulose, and in this fashion create material with novel optical and electronic properties.

Thesis (M.S.)--University of Missouri-Columbia, 2008.
"May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

The synthesis, photophysicochemcial properties, in vitro dark toxicity and photodynamic therapy (PDT) activities of different derivatives of zinc phthalocyanine (ZnPc) conjugates with biomolecules (folic acid, bovine serum albumin (BSA), ascorbic acid, uridine or spermine) and single-walled carbon nanotubes (SWCNTs) are presented in this work. The fluorescence quantum yields (ΦF) (Subscript F) of the ZnPc derivatives or ZnPc-biomolecule conjugates remained relatively the same as compared to the precursor Pcs. Slight increases were observed in the ΦF (Subscript F) values of conjugates containing substituents such as pyrene, folic acid or BSA with intrinsic fluorescence properties. The triplet quantum yield (ΦT ) (Subscript T) values for some ZnPc conjugates increases compared to the precursor ZnPcs due to extended π conjugation (for the conjugate with pyrene) and the presence of phenyl ring that support spin-orbit charge transfer intersystem crossing to triplet state. While some conjugates showed decreases in the ΦT (Subscript T) values compared to precursor ZnPcs due to the presence of substituents that could quench photo-excited state properties. The singlet oxygen quantum yield (ΦΔ ) values follow the trends of the triplet quantum yield values. The conjugates containing BSA also show increases in the ΦΔ values without corresponding increases in ΦT (Subscript T) values due to the ability of BSA to generate free radicals including singlet oxygen. The presence of SWCNTs decreases the photophysicochemcial properties of some ZnPc-SWCNT conjugates compared to the precursor ZnPcs due to photo-induced electron transfer from an excited Pc complex (electron donor) to SWCNTs (electron acceptor). However, increases were observed in some ZnPc-SWCNT conjugates as a result of fast charge recombination process due to highly short-lived radical ion pair produced. These phenomena affected the ΦF (Suscript F) values, ΦT (Suscript T) values, and the ΦΔ values. Increases or decreases in ΦT (Suscript T) values resulted in corresponding increases or decreases in ΦΔ values

In the last decade, conductive polymers have received great interest in both scientific and technological levels due to the electroactive properties exhibited by these materials. For this reason, the main aim of this Doctoral Thesis is to study the applications of some conducting polymers, mainly derived from polythiophene, in the field of biomedicine and biotechnology. According to the objectives proposed in this Document, different studies focused on the preparation and characterization of graft copolymers have been carried out using macromonomers formed by oligo-thiophenes to which PEG chains of well-defined molecular weight have been incorporated as substituents. After this, the behavior of these new polymers has been investigated in terms of cytotoxicity and biocompatibility using cell adhesion and cell proliferation assays. Then, the synthesis and characterization of conducting polymer-peptide hybrids have been developed. Initially, the methodology used for the preparation of these materials has been optimized using model systems in which the peptide has been replaced by an amino acid, which has been engineered by chemical similarity. After evaluate different synthetic approaches, hybrids constructed by combining the same conducting polymer and the RG*D peptide have been prepared, where RG*D is a sequence analogous to the RGD (Arg-Gly-Asp) in which the central Gly residue is replaced by the above mentioned engineered amino acids. The biocompatibility and the bioactivity of all these compounds have been examined. The last part of this thesis is oriented towards the encapsulation of small biomolecules in polymeric nanostructures that can be used as transport systems and controlled release systems when incorporated into the organism. The encapsulation of simple amino acids has been examined firstly, this research evolving towards the immobilization of linear and cyclic hydrophobic peptides. In all cases encapsulation was carried out by electrospraying or electrospinning.
En la última década, los polímeros conductores han despertado un gran interés tanto a nivel científico como tecnológico debido a las propiedades electroactivas que exhiben estos materiales. Por este motivo, en la presente Tesis Doctoral se han estudiado las aplicaciones de algunos de estos polímeros conductores, principalmente derivados del politiofeno, en el ámbito de la biomedicina y la biotecnología. Según los objetivos propuestos en esta Memoria, inicialmente se han realizado estudios relacionados con la preparación y caracterización de copolímeros de injerto empleando macromonómeros formados por oligotiofenos, a los que se les ha incorporado una cadena de polietilenglicol de peso molecular definido. Seguidamente se ha investigado su comportamiento mediante ensayos de citotoxicidad, adhesión celular y proliferación celular. A continuación, se ha realizado la síntesis y la caracterización de híbridos polímero conductor-péptido. Inicialmente, se ha optimizado el método de preparación de estos materiales empleando sistemas modelo, en los que el péptido ha sido re-emplazado por un amino ácido diseñado por similaridad química. Una vez evaluadas las diferentes estrategias sintéticas, se han obtenido híbridos formados por el mismo polímero y el péptido RG*D, donde RG*D es una secuencia análoga a la RGD (Arg-Gly-Asp) en la que el residuo central se ha sustituido por el amino ácido mencionado anteriormente. Para estos materiales se ha evaluado la biocompatibilidad y la bioactividad. La última parte de esta Tesis está orientada a la encapsulación de biomoléculas de pequeño tamaño en nanoestructuras poliméricas que pueden ser empleadas como sistemas transportadores y sistemas de liberación controlada cuando se incorporan al organismo. Los estudios se han iniciado encapsulando diferentes amino ácidos y se han acabado con péptidos hidrofóbicos lineales y cíclicos. En todos los casos la encapsulación se ha llevado a cabo mediante electrospraying o electrospinning.

Oxidative stress is a pathophysiological condition defined by an increased production of reactive oxygen species (ROS), which can result in the growth arrest of cells followed by cell disintegration or necrosis. A number of small molecule antioxidants (e.g. curcumin, quercetin and resveratrol) are capable of directly scavenging ROS, thereby short-circuiting the self-propagating oxidative stress state. However, poor solubility and rapid 1st pass metabolism results in overall low bioavailability and acts as a barrier for its use as a drug to suppress oxidative stress efficiently. To overcome this limitation, these small molecule antioxidants were covalently conjugated into poly(β-amino ester) (PβAE) cross-linked networks to formulate prodrug gel microparticles and nanoparticles (nanogels). Being hydrolytically degradable in nature, these PβAE crosslinked systems released antioxidants in their original structural form in a sustained controlled fashion. Both quercetin and curcumin-PβAE nanogels showed prolonged suppression of cellular oxidative stress induced by H2O2. Curcumin PβAE nanogels also demonstrated protection against mitochondrial oxidative stress induced by H2O2 and polychlorinated biphenyls. Curcumin-PβAE gel microparticles were also developed as a platform to treat oral mucositis through a local antioxidant delivery route. The same synthesis chemistry was transferred to formulate resveratrol PβAE gel microparticles for topical applications, to treat UV radiation induced oxidative stress. Both formulations showed suppression of induced oxidative stress. An in vivo trial with curcumin-PβAE microparticles further showed relatively reduced the severity of induced oral mucositis (OM) in hamster check pouch as compared to placebo.

abstract: Fluorescence spectroscopy is a powerful tool for biophysical studies due to its high sensitivity and broad availability. It is possible to detect fluorescence from single molecules allowing researchers to see the behavior of subpopulations whose presence is obscured by “bulk” collection methods. The fluorescent probes used in these experiments are affected by the solution and macromolecular environments they are in. A misunderstanding of a probe’s photophysics can lead researchers to assign observed behavior to biomolecules, when in fact the probe is responsible. On the other hand, a probe’s photophysical behavior is a signature of the environment surrounding it; it can be exploited to learn about the biomolecule(s) under study. A thorough examination of a probe’s photophysics is critical to data interpretation in both cases and is the focus of this work. This dissertation investigates the photophysical behavior of symmetric and asymmetric cyanines in a variety of solution and biomolecular environments. Using fluorescent techniques—such as time-correlated single photon counting (TCSPC) and fluorescence correlation spectroscopy (FCS)—it was found that cyanines are influenced by the local environment. In the first project, the symmetric cyanines are found to be susceptible to paramagnetic species, such as manganese(II), that enhance the intersystem crossing (ISC) rate increasing triplet blinking and accelerating photobleaching. Another project found the increase in fluorescence of Cy3 in the protein induced fluorescence enhancement (PIFE) technique is due to reduced photoisomerization caused by the proximity of protein to Cy3. The third project focused on asymmetric cyanines; their photophysical behavior has not been previously characterized. Dy630 as a free dye behaves like Cy3; it has a short lifetime and can deactivate via photoisomerization. Preliminary experiments on Dy dyes conjugated to DNA show these dyes do not photoisomerize, and do not show PIFE potential. Further research will explore other conjugation strategies, with the goal of optimizing conditions in which Dy630 can be used as the red-absorbing analogue of Cy3 for PIFE applications. In summary, this dissertation focused on photophysical investigations, the understanding of which forms the backbone of rigorous fluorescent studies and is vital to the development of the fluorescence field.
Dissertation/Thesis
Doctoral Dissertation Chemistry 2017

Thesis (Ph. D.)--Florida State University, 2008.
Advisor: Geoffrey Strouse, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Mar. 12, 2009). Document formatted into pages; contains xi, 86 pages. Includes bibliographical references.

博士
國立臺灣師範大學
化學系
99
The applications of nanomaterials-biomolecular conjugates in DNA detection and RNA interference (RNAi) have led to ever-growing developments in the past decades. In this work, we have developed a new technique used fluorescent silica nanotubes for simple and sensitive DNA detection. The quantum dots embedded silica nanotubes (QD-SNTs) were fabricated by a sol-gel reaction using anodic aluminum oxide (AAO) as a template. The fluorescent QD-SNTs of different colors were then immobilized with single stranded DNA and used as nanoprobes to detect dye-labeled target DNA in a solution phase. The optical and structural properties of QD-SNTs nanoprobes were examined using photoluminescence spectroscopy, confocal microscopy and transmission electron microscopy (TEM). The obvious color change of the QD-SNTs nanoprobes was observed by eyes under a simple microscope after the successful detection with target DNA. The quantitative analyses indicated that ~100 attomole of target DNA in one nanoprobe can generate the distinguishable and observable color change. The detection results also demonstrated that our assay exhibited high specificity, high selectivity and very low non-specific adsorption. Our simple DNA assay based on QD-SNTs nanoprobes is expected to be quite useful for the needs of fast DNA screening and detecting applications. Furthermore, we study the kinetics of siRNA-based gene-silencing by gold nanoparticles-siRNA conjugates mediation. The kinetics factors relative to the concentrations of conjugates, dosing times of conjugates and cell doubling time were studied separately. The resulting duration time of silence (TE, a period of time that EGFP expression level was regulated down to 50 % or less) by three variables mentioned above were also investigated. We found that TE showed natural logarithm relationship with the concentrations of conjugates. But for other two variables, dosing times of conjugates and cell doubling time, TE showed linear relationship with both of them. Based on these relationships, an expected siRNA-mediated gene expression level can be designed. For the need of researches and treatments, prolonged TE can be achieved by varying concentrations and/or dosing times of conjugates. Besides, choosing of model cell (relative to cell doubling time) may be helpful for the purpose of prolonged TE. Importantly, according to these relationships, the possibility of trial and error in RNAi based applications can be much reduced. Our study holds great potential for RNAi-based therapeutic applications.

博士
國立交通大學
材料科學與工程學系奈米科技碩博士班
99
Protein surface recognition provides an appealing tool to regulate protein–protein interactions and enzymatic activities in the field of biological science. We are interested in using nanoparticle (NP) to bind enzyme surfaces through multivalent interactions and then engineer the protein properties. In this study, we have investigated the activity of the enzyme–NP conjugates and demonstrated that adsorbing enzyme onto NPs significantly increased its enzymatic activity. We ascribe this event of the enzymatic reactions by kinetic and thermodynamic studies, which provide a way of understanding and predicting the catalytic behaviors of the enzyme-functionalized NPs. In addition, NPs are excellent systems for modeling enzymes’ surfaces because they can be readily fabricated on size scales comparable with those of their biomolecular targets. Therefore, we were curious to study whether varying the dimensions of the NPs would affect their catalytic reactions. We have developed a series of kinetic experiments to systematically analyze the NP size–dependent enzymatic activities, and have developed a model to explain the phenomenon. Kinetic studies revealed that association of enzyme with NPs did not influence the turnover number, but smaller NPs did promote the catalytic efficiency of enzyme by increasing its kinetic affinity. A shielding model, based on diffusion–collision theory, explains the correlation between the size effects and the kinetic responses of the enzyme–NP conjugates. This size-effect model provides chemical and physical meaning, leading to the observed substrate specificities and catalytic constants. From the combined kinetic and theoretical investigation of enzyme bound to NPs, we found that these conjugates acted as a controllable and efficient factor for modulating the activity of the enzyme. In nature, controllable modulation of enzyme activity is a potent means of regulating several cellular processes (e.g., signal transduction, biosynthesis, metabolism). The modulation of biocatalytic behavior is an attractive feature for exploitation in the field of nanobiotechnology.

For any drug candidate to be approved by the U.S. Food and Drug Administration, it must meet strict standards for safety and efficacy. While the field of nuclear medicine is over 100 years old, traditional methods such as external beams or systematic administration have rarely met these standards or have limited application. Ligand-targeted therapy and diagnostics, or “theranostics,” has emerged in the past several decades as an exciting field that offers new possibilities to design drugs that are both safe and effective. When applied to nuclear medicine, the field of ligand-targeted radioactive theranostics is younger still, with many critical lessons being discovered and applied currently. This dissertation outlines the necessary principles of radioactive theranostic drug design, then demonstrates the application of several more recent techniques to improve both the efficacy and safety of radioactive theranostics targeting two high priority oncological targets: fibroblast activation protein alpha and folate receptor.