This thesis describes the synthesis and assembly of metal and semiconductor nanoparticles (NPs). The two research topics include i) hetero-assembly of metal and semiconductor NPs, ii) effect of ionic strength on homo-assembly of gold nanorods (GNRs). First, we present hetero-assembly of GNRs and semiconductor quantum dots (QDs) in a chain using biotin-streptavidin interaction. We synthesized alloyed CdTeSe QDs and modified them with mercaptoundecanoic acid to render them water-soluble and to attach streptavidin. We synthesized GNRs by a seed-mediated method and selectively modified the ends with biotin. Hetero-assembly of QDs and GNRs depended on the size, ligands, and ratio of QDs and GNRs. Second, we controlled the rate of homo-assembly of GNRs by varying the ionic strength of the DMF/water solution. The solubility of polystyrene on the ends of GNRs depended on the ionic strength of the solution, which correlated with the rate of assembly of GNRs into chains.

This study explored the imaging capability of our frequency-domain photoacoustic (FD-PA) system that utilizes correlation processing alias “photoacoustic radar” and ultrasonic phased array for imaging of soft tissues. The probe imaging capabilities were studied using tissue-mimicking phantoms, tissue samples ex vivo, blood vessels in a human wrist and a rat tumour model in vivo. Our experimental results have shown decent image correlation between our FD-PA and a clinical ultrasound modality. In comparison with ultrasound, we have also demonstrated strong potential of the FD-PA for deep (~15 mm) subsurface imaging with excellent contrast and high signal-to-noise ratio. Additionally, we assessed the potential of silica-coated super paramagnetic iron oxide nanoparticles (SPION) as a novel PA contrast agent. Detection of the nanoparticles up to 24 mm inside an optically tissue-like turbid media and about 5-fold PA signal amplification due to their presence in sheep blood (~1.4 mg/ml) are presented.

This thesis describes the synthesis and assembly of metal and semiconductor nanoparticles (NPs). The two research topics include i) hetero-assembly of metal and semiconductor NPs, ii) effect of ionic strength on homo-assembly of gold nanorods (GNRs). First, we present hetero-assembly of GNRs and semiconductor quantum dots (QDs) in a chain using biotin-streptavidin interaction. We synthesized alloyed CdTeSe QDs and modified them with mercaptoundecanoic acid to render them water-soluble and to attach streptavidin. We synthesized GNRs by a seed-mediated method and selectively modified the ends with biotin. Hetero-assembly of QDs and GNRs depended on the size, ligands, and ratio of QDs and GNRs. Second, we controlled the rate of homo-assembly of GNRs by varying the ionic strength of the DMF/water solution. The solubility of polystyrene on the ends of GNRs depended on the ionic strength of the solution, which correlated with the rate of assembly of GNRs into chains.

This study explored the imaging capability of our frequency-domain photoacoustic (FD-PA) system that utilizes correlation processing alias “photoacoustic radar” and ultrasonic phased array for imaging of soft tissues. The probe imaging capabilities were studied using tissue-mimicking phantoms, tissue samples ex vivo, blood vessels in a human wrist and a rat tumour model in vivo. Our experimental results have shown decent image correlation between our FD-PA and a clinical ultrasound modality. In comparison with ultrasound, we have also demonstrated strong potential of the FD-PA for deep (~15 mm) subsurface imaging with excellent contrast and high signal-to-noise ratio. Additionally, we assessed the potential of silica-coated super paramagnetic iron oxide nanoparticles (SPION) as a novel PA contrast agent. Detection of the nanoparticles up to 24 mm inside an optically tissue-like turbid media and about 5-fold PA signal amplification due to their presence in sheep blood (~1.4 mg/ml) are presented.

This work attempted to detect chemical warfare agent (CWA) simulants via electrochemistry utilizing two approaches. The first approach consisted of a ferrocene (Fc) amino acid derivative film on Au surfaces. The molecule [(BocHN)Fc(CO)CSA]2 was electrodeposited onto Au microelectrodes through a SAu bond. Once immobilized, the Fc amino acid derivative was Boc deprotected allowing for the amino group to react with the target molecule. Detection of the target simulant was monitored by cyclic voltammetry (CV) while following the formal potential of the Fc molecule, which is influenced by its immediate electronic microenvironment. Reaction with either 1 mM diethyl cyanophosphonate (DECP) or 2 chloroethyl ethyl sulfide (2 CEES), both effectively simulants for the CWAs Tabun nerve agent and blistering sulfur mustard respectively, was not observed. However, detection of 1 mM acetyl chloride was achieved by observing a potential anodic shift from 217 ± 6 mV, for the Boc deprotected form, to 388 ± 7 mV for the reacted state of the molecule. The lack of reactivity with the Fc amino acid system was hypothesized as a kinetic issue.<p> In the second approach, the electrochemistry of gas generated naked Ag nanoparticles (NPs) deposited on indium tin oxide covered glass plates is compared to bulk polycrystalline Ag. The nano specific electrochemistry of Ag NPs has been identified and includes the preferential formation of â oxides. In 100 mM KOH supporting electrolyte, disruption of â oxide formation is exploited to test for the presence of 1 mM DECP resulting in the dissolution of Ag via cyanide complexes leading to a CV signal decrease. While in 8.0 M KOH, â oxide formation is enhanced leading to testing capabilities for 1 mM 2 CEES resulting in the disappearance of the â oxide peak and the appearance of surface oxide peak during CV. Analogous electrochemistry is not observed on polycrystalline bulk Ag.

Dopamine is a neurotransmitter found in central nerve system which has a vital role for human health. Dopamine oxidation in body is an important issue since it may form reactive metabolites which can be toxic to the cell. Surface-enhanced Raman scattering (SERS) is currently recognized as one of the most sensitive spectroscopic tools, which can be exploited for ultrasensitive chemical and biological detection, addition to providing structural information on the systems of interest. SERS of dopamine displays three strong bands at 1269, 1331 and 1479 cm-1. These bands are the signature of dopamine molecule. The most intense band at 1479 cm-1 is contributed mainly from stretching of the carbon-carbon bond to which the oxygens are attached. A bidentate silver-dopamine complex or in general bidentate metal-dopamine complex formation is required for the SERS detection of dopamine and other catecholamines. In other words, for acquiring the characteristic dopamine SERS signature, both of the catechol oxygens should take a part in the adsorption of dopamine to the silver metal surface which is used as a SERS substrate. Therefore, the reactivity of different oxidation forms of dopamine for the formation of bidentate silver-dopamine complex was investigated by obtaining their SERS spectra and following the characteristic C-C ring vibration at 1479 cm-1. Dopamine oxidation was carried out electrochemically, utilizing platinum and silver electrodes as working electrode. Oxidation products formed were identified with UV-vis Spectrometer. Also, silver metal ions were used to oxidize dopamine, leading to formation silver nanoparticles. Dopamine functionalized silver nanoparticles were characterized by Scanning Electron Microscope, Transmission Electron Microscope, UV-vis Spectrometer. Surface- enhanced Raman spectra of polydopamine on the surface of synthesized silver nanoparticles and the electrodeposited dopamine on the porous surface of silver electrode were also obtained.

Nanoparticle-based drug delivery systems (NDDS) have emerged as a promising strategy for formulation of anticancer drugs due to their ability to passively target solid tumors via exploitation of the enhanced permeation and retention effect. In particular, nano-sized block copolymer micelles (BCMs) have proven to be a viable delivery vehicle for hydrophobic anticancer drugs. To further enhance the specificity of BCMs towards cancer cells, extensive research has been focused on the formulation of actively targeted BCMs with tumor cell binding antigens conjugated to their surface. However, the in vivo transport of passively and actively targeted BCMs has only been studied to a limited extent. This thesis explores the potential and limitations of passively and actively targeted BCMs, as NDDS for delivery to solid tumors. The in vivo transport of BCMs at the whole body, tumor, and cellular levels is investigated in human breast cancer xenografts. Overall, active targeting of BCMs with epidermal growth factor (EGF) as the tumor cell binding antigen was not found to alter the whole body clearance of the vehicles; however, particle size had a profound effect on their pharmacokinetics and biodistribution profiles. Both passively and actively targeted BCMs exhibited heterogeneous distribution throughout solid tumors, with preferential localization in the tumor periphery and/or highly vascularized regions. In addition, the BCMs were found to exhibit impaired tumor penetration due to limited mobility and/or the binding site barrier. Although active targeting increases the in vivo BCM cellular uptake, the BCMs largely remained in the extracellular compartment, indicating that incomplete BCM delivery to all tumor cells remains as a major biological barrier. Interestingly, EGF-conjugated BCMs induced a potent bystander effect in vitro as a result of the paradoxical apoptotic effect of EGF, which has the potential to treat nearby tumor cells that do not respond directly to BCM treatment in vivo. In this way, EGF-BCMs may be beneficial for rendering the aforementioned in vivo barriers such as limited tumor penetration, as well as heterogeneity in tumor vascularization and receptor expression.

There is an increased interest in understanding the toxicity and rational design of gold nanoparticles (GNPs) for biomedical applications in recent years. Such efforts warrant reliable, viable, and biofriendly synthetic methodology for GNPs with homogeneous sizes and shapes, particularly sizes above 30 nm, which is currently challenging. In the present study, an environmentally benign, biofriendly, singlestep/ single-phase synthetic method using dextrose as a reducing and capping agent in a buffered aqueous solution at moderate temperature is introduced. The resulting GNPs are near-spherical, stable, catalytically active, place exchangeable, and water-soluble within the size range of 10-120 nm. The added advantage of the biologically friendly reaction medium employed in this new synthetic approach provides a method for the direct embedment/integration of GNPs into biological systems such as the E. coli bacterium without additional capping ligand or surface modification processes.

En una primera parte de esta tesis fue desarrollado un nuevo electrodo a base de pasta de grafito-epoxi composite (GECE) conteniendo nitrato de bismuto [Bi(NO3)3] como precursor de bismuto incorporado [Bi(NO3)3-GECE)], como una posible alternativa para el análisis electroquímico por redisolución de metales pesados en cantidades traza. Los resultados claramente muestran las ventajas del Bi(NO3)3-GECE en combinación con la técnica de voltamperometría de redisolución anódica de onda cuadrada (SWASV) para la detección de metales pesados. Se llevaron a cabo medidas individuales y simultáneas de Pb y Cd y los resultados mostraron claramente las ventajas del Bi(NO3)3-GECE en combinación con la técnica SWASV para la detección de metales pesados. Con el uso del Bi(NO3)3-GECE construido se pueden realizar análisis rápidos y eficaces de iones de metal en cantidades traza como Pb y Cd entre otros en muestras ambientales de suelo, aguas naturales y aguas residuales. La ventaja inherente de la no necesidad de mercurio elimina muchas de las objeciones para el uso de métodos electroquímicos en la detección de tales especies en estos medios. Comparando el Bi(NO3)3-GECE con el electrodo de película de mercurio comúnmente usado y electrodo de película de bismuto desarrollado antes por nuestro grupo, el nuevo electrodo propuesto ofrece un notable funcionamiento en el análisis de metales pesados en cantidades traza, que puede ser de gran ventaja en electroquímica, contribuyendo a una aplicabilidad más amplia de técnicas electroquímicas por redisolución relacionadas con electrodos "sin mercurio". Además de aplicaciones ambientales el electrodo desarrollado basado en bismuto tendría interés especial para la aplicación en la detección de puntos cuánticos (QDs) basados en metales pesados. Tales aplicaciones están actualmente en proceso de estudio en nuestro grupo de investigación para la detección de ADN.Las otras partes de la tesis se dedican al desarrollo de nuevos sensores de ADN y proteínas basados en la misma técnica electroquímica de redisolución y el uso de nanopartículas de oro como marcas. Actualmente la detección electroquímica de secuencias de ADN específicas vía el evento de hibridación es una cuestión importante por lo cual diversas estrategias han sido propuestas.Genosensores electroquímicos de afinidad basados en el marcaje con nanopartículas de oro (AuNPs) y el uso de partículas paramagnéticas (MB) como plataforma para la inmovilización de la sonda de ADN de captura también han sido desarrollados en esta tesis a fin de demostrar la inducción magnética eficaz de un nuevo electrodo de grafito-epoxi composite-magnético (M-GECE) el cual fue construido también con pasta de grafito-epoxi composite con un pequeño imán de neodimio integrado.Todos los ensayos para la detección electroquímica de la hibridación del ADN desarrollados en esta tesis fueron basados en la detección directa de las marcas de AuNPs por medio de la técnica de voltametría de pulso diferencial (DPV) usando el M-GECE donde la intensidad de la corriente de la señal generada es directamente proporcional a la cantidad de ADN en la muestra. Como también ha sido demostrado, con el sensor de ADN asistido magnéticamente, el ADN analito condujo a una muy bien definida señal mientras que esencialmente ninguna señal fue observada para el ADN no complementario.Un nuevo inmunoensayo electroquímico sensible ha sido desarrollado, también basado en AuNPs como marca y MB como plataforma. El método fue evaluado para un inmunoensayo heterogéneo no competitivo de una IgG humana como proteína modelo. La detección electroquímica fue llevada a cabo en la misma forma que lo fue para ADN.La detección electroquímica de marcas de AuNPs en biosensores de afinidad usando métodos de redisolución permite el estudio detallado de la hibridación de ADN así como también inmuno-reacciones con interés en aplicaciones relacionadas con genosensores o inmunosensores. Los métodos electroquímicos usados para la detección de AuNPs como marca pueden ser muy prometedores tomando en cuenta su sensibilidad alta, límite de detección bajo, selectividad, simplicidad, bajo coste, y disponibilidad de instrumentos portátiles.Como conclusión final, las estrategias de análisis electroquímico de ADN y proteínas fueron demostradas con éxito y debido a los resultados prometedores su uso en muestras reales es viable. Tales biosensores de ADN e inmunosensores dan lugar a un enorme potencial de aplicación principalmente para diagnóstico clínico y monitoreo ambiental entre otros campos. / In the first part of this thesis a new graphite-epoxy composite electrode containing bismuth nitrate [Bi(NO3)3-GECE)], as built-in bismuth precursor as a possible alternative for electrochemical stripping analysis of trace heavy metals has been developed. Individual and simultaneous measurements of Pb and Cd were carried out and the results clearly showed the advantages of the Bi(NO3)3-GECE in combination with square wave anodic stripping voltammetry (SWASV) technique for heavy metals detection. Fast and effective analyses of trace metal ions such as Pb and Cd among others in environmental samples of soil, natural waters and effluents can be carried out by using the new Bi(NO3)3-GECE constructed. The inherent advantage of no necessity of mercury removes many of the objections for the use of the developed sensor. When comparing the Bi(NO3)3-GECE with the commonly used mercury film electrode and previously developed bismuth film electrode, the newly proposed electrode offers a remarkable performance in analysis of trace heavy metals, which can be advantageous in electrochemical, hence contributing to the wider applicability of electrochemical stripping techniques in connection with "mercury-free" electrodes. Beside environmental applications the developed bismuth based electrode would have special interest for application to heavy metal based quantum dots. Such applications are currently in the studying process at our research group for DNA detection.The other parts of the thesis are dedicated to the application of electrochemical stripping analysis in connection to gold nanoparticles for DNA and protein detection. Currently the electrochemical detection of specific DNA sequences via hybridization event is an important issue by which diverse strategies have been proposed. Affinity electrochemical genosensors based on labelling with gold nanoparticles (AuNPs) and the use of paramagnetic beads (MB) as platform for the immobilization of capture DNA probe have been also developed in this thesis in order to demonstrate the effective magnetic triggering of a new magnetic-graphite epoxy composite electrode (M-GECE) which was constructed with graphite-epoxy composite paste, with a small neodymium magnet integrated.All the assays for the DNA hybridization electrochemical detection developed in this thesis were based on the direct detection of AuNPs labels (anchored onto the M-GECE) by means of differential pulse voltammetry (DPV). The intensity of the generated current is directly proportional to the amount of DNA at the sample. As also has been demonstrated, with this magnetically assisted DNA sensor, target DNA leaded to very well defined signal whereas essentially no signal was observed for non-complementary DNA. By the other side a novel, sensitive electrochemical immunoassay has been also developed based in AuNPs as label and MB as platform. The method was studied and evaluated for a noncompetitive heterogeneous immunoassay of a human IgG as a model protein. The electrochemical detection was carried out in the same way that as for DNA.The electrochemical detection of AuNPs labels in affinity biosensors using stripping methods allows the detailed study of DNA hybridization as well as immunoreactions with interest in genosensor or immunosensor applications. The developed detection methodologies may be very promising taking into account their high sensitivity, low detection limit, selectivity, simplicity, low cost, and availability of portable instruments.As final conclusion, the DNA and protein electrochemical analysis strategies were successfully demonstrated and according to the promising results obtained its use for real samples is viable. Such DNA biosensors and immunosensors hold an enormous application potential principally for clinical diagnostic and environmental monitoring among other fields.

This PhD Thesis involves the development of a new synthetic protocol for iron oxide silica composite nanoparticles useful in MRI and magnetic hyperthermia. Our approximation combines the sol-gel chemistry and supercritical fluids to obtain biocompatible, spherical composite particles, with narrow particle size distribution. The composite particles showed very high values of relaxivity. We have also studied the dependence of the specific absorption rate with iron concentration. This manuscript is organized into seven chapters. Chapter 1 provides a general introduction to magnetic nanoparticles, their properties, synthesis, stabilization and applications, with special interest in the biomedical field. The aim of the chapter is to place the reader in the scientific context of the thesis. Chapter 2 describes the synthesis of nanoparticles by thermal decomposition of an iron complex, Fe(CO)5, and their characterization in solid and colloidal dispersion. Silica coating is a convenient approach to stabilize nanoparticles in a biocompatible way. In this thesis we have developed a new synthetic protocol to coat iron oxide nanoparticles with silica that combines the sol-gel chemistry and supercritical fluids. To understand and control the details of this procedure, we first synthesized nano- and microparticles of silica. They are described in Chapter 3. Chapter 4 includes the synthesis of the composite γ-Fe2O3@SiO2 nanoparticles. The core is composed of clustered iron oxide nanoparticles (described in Chapter 2) surrounded by a silica shell. Chapter 4 also describes the material characterization, with special emphasis on the magnetic properties, which will be exploited in the biomedical applications. We also succeeded in synthesizing composite ε-Fe2O3@SiO2 nanospheres from the previous γ-Fe2O3@SiO2 particles. The evaluation of the materials described in Chapters 2 and 4 as contrast agents for magnetic resonance imaging and as mediators for magnetic hyperthermia is included in Chapter 5. Chapter 6 lists the main conclusions derived from the present thesis. Finally, Chapter 7 gathers the annexes. It includes four publications arising from the main subject of this thesis, as well as five other publications and a patent on projects that I worked during the time that has lasted my doctoral thesis. The annexes also include a brief description of the experimental techniques used and protocols for sample preparation.