A Gold Nanoparticles Enhanced Surface Plasmon Resonance Immunosensor for Highly Sensitive Detection of Ischemia-Modified Albumin

In this study a novel sensitive nanogold particle sensor enhancement based on mixed self-assembled monolayers was explored and used to construct a Surface Plasmon Resonance (SPR) immunosensor to detect Ischemia Modified Albumin (IMA). Compared with a direct binding SPR assay at a limit of detection (LOD) of 100 ng/L, gold nanoparticles (AuNPs) of 10 nm dramatically improved the LOD of IMA to 10 ng/L. Meanwhile, no interfering substance that may lead to false positive results was identified. These results suggested that the SPR biosensor presented superior properties, and provided a simple label-free strategy to increase assay sensitivity for further acute coronary syndrome (ACS) diagnosis

Evaluación del desempeño de sustitutos artificiales de tejido conectivo desarrollados con soportes multidireccionales y unidireccionales, en un modelo animal de heridas de piel de espesor total

La reparación de heridas cutáneas está mediada por factores de crecimiento y citoquinas que señalizan las células de la herida y del tejido sano circundante en cada una de las fases del proceso (inflamación, proliferación y remodelación). Estudios previos han mostrado que los sustitutos artificiales de tejido que contienen células, funcionan como sistemas de liberación de factores y citoquinas que intervienen en las primeras etapas del cierre de las heridas estimulando la formación de nuevo tejido y favoreciendo la regeneración sobre la reparación. Igualmente, que tejido conectivo artificial elaborado sembrando fibroblastos aislados de mucosa oral de conejo en soportes multidireccionales y unidireccionales de colágeno I, secretan en el medio de cultivo concentraciones diferentes de factores solubles importantes en el proceso de reparación de heridas. En este trabajo se comparó el desempeño de tejido conectivo autólogo artificial obtenido sembrando fibroblastos de piel en soportes con fibras y poros orientados multidireccionalmente (MTCA) y unidireccionalmente (UTCA) en un modelo lagomorfo de herida de espesor total de 4cm2 . Igualmente, se estandarizó una metodología de cuantificación de factores de crecimiento y citoquinas en el medio de cultivo de los tejidos artificiales mencionados y en los exudados de las heridas injertadas con ellos, basada en un biosensor óptico de plasmones de resonancia (SPR) y monocapas autoensambladas (SAM) de alcanotioles. Se cuantificaron 15 factores y citoquinas en los medios de cultivo y en los exudados tomados al tercer y sexto día post- cirugía, de las heridas injertadas y de las dejadas cerrar por segunda intención (control). A pesar de la cicatrización fibrosa de las heridas de piel característica del modelo lagomorfo, fue posible evaluar el desempeño como injerto de dermis autóloga artificial multi y unidireccional en heridas de espesor total. El conjunto de resultados obtenidos soporta la hipótesis de que el tejido conectivo autólogo actúa como un sistema de entrega de señales bioactivas cuyo perfil de concentracions cambia cuando las células se cultivan en soportes con diferente orientación de fibras y poros. Las observaciones clínicas, histológicas, histomorfométricas y las cuantificaciones de factores que modulan la cicatrización indican que los injertos elaborados con soportes multidireccionales mejoran el cierre de heridas de piel de espesor total en comparación al cierre de heridas injertadas con tejidos unidireccionales o dejadas cerrar por segunda intención.Abstract: The repair of skin wounds is mediated by growth factors and cytokines that signal the cells of the blood and surrounding healthy tissue in each of the phases of the process (inflammation, proliferation and remodeling). Previous studies have shown that the artificial substitutes of the tissue whit cells, work as delivery systems of growth factors and cytokines that mediate in the early stages of wound healing, stimulating the formation of new tissues and favoring regeneration over repair. Also, the artificial connective tissue produced by seeding fibroblasts isolated from rabbit oral mucosa on multidirectional and unidirectional scaffolds of collagen I, deliver different concentrations profiles of important growth factors and cytokines for wound healing in the culture media. In this work, the performance of artificial autologous connective tissue obtained by seeding skin fibroblasts in supports with multidirectionally oriented fibers and pores (MTCA) and unidirectionally (UTCA) in a lagomorph model with a full thickness of 4cm2 wound was compared. Also, methodology to quantify growth factors and cytokines in the culture medium of the artificial tissues and in the exudates of the wounds grafted with them, based on an optical resonance plasmon biosensor (SPR) and self-assembled monolayers ( SAM) of alkanethiols. Fifteen factors and cytokines were quantified in the culture media and in the exudates taken on the third and sixth day post-surgery, of the grafted wounds and those left closed by second intention (control). In spite of the fibrous healing of the wounds of the characteristic of the lagomorph model, it was possible to evaluate the performance as an artificial and unidirectional autologous dermal graft in full thickness wounds. The set of results obtained supports the hypothesis that the autologous connective tissue acts as a delivery system of bioactive signals whose concentration profile changes when the cells are grown in supports with different orientation of fibers and pores. Clinical, histological, histomorphometric observations and quantifications of factors that modulate healing, indicate that grafts made with multidirectional supports improve the healing of total thickness skin wounds compared to the wounds grafted with unidirectional tissues or left closed by secondary intentionDoctorad

Electrochemical and surface plasmon bioassays for circulating biomarkers

To address analytical detection needs, sensitive and selective assay methodologies are of great importance. Compared to simple buffer medium, a great challenge exists in detecting ultra-low levels of biomarkers in clinical matrices due to their inherent complexity and interferences posed by non-specific molecules. In addition, small molecules do not yield measurable assay signal changes compared to large biomolecules. My thesis research is focused on designing nano-biological interfaces to detect small and large molecules at low parts-per-billion and femto/picomolar concentrations in complex biofluids (serum and urine samples). Compared to harsh and tedious chemical carboxylation, non-covalent carboxylation of multiwalled carbon nanotubes by π-π stacking 1-pyrenebutyric acid retains the innate sp2 structure and electronic properties of the nanotubes and offers surface carboxyl groups for stable covalent amine coupling of a large amount of enzymes, thus improving the sensitivity of the assay. Chapter 2 demonstrates the first pyrenyl carbon nanostructure modified enzymatic bioelectrode for amperometric detection of urine formaldehyde at clinically relevant parts-per-billion levels with selectivity and wide dynamic range. Subsequently, we explored the low dielectric permittivity and intrinsic plasmonics of graphene for the detection of serum glutamic acid decarboxylase autoantibody (GADA). Graphene-based electrochemical immunosensing approach is advantageous due to its additional applicability for surface plasmon based validation and binding strength analysis with surface immobilized GAD-65 antigens (Chapter 3). My thesis focused on the third class of biomarkers, microRNAs, which are small oligonucleotides with 21-25 bases. To develop the microRNA assay with quantitative characterization, surface plasmon resonance imaging (SPRi) coupled with quartz crystal microbalance (QCM) was designed (Chapter 4). Gold nanoparticles (Au NPs) were linked to the oligonucleotides to increase the detection sensitivity upon hybridization with the selective capture oligonucleotide immobilized on the sensor surface with minimal non-specific signals. Often, cancer and other similar health disorders have been shown to be related to various types of biomarkers. Hence, in Chapter 5, we designed a multiplex assay platform for combined measurement of proteins and microRNAs. For this multiplex assay, we synthesized iron-gold bimetallic core/shell nanoparticles (Fe3O4@Au NPs) that displayed a greater plasmonic signal amplification than either Fe3O4 or Au NPs.Chemistr

Characterising nanoparticles in complex biological media

Progress in the application of nanotechnology within medicine has been limited in part due to the difficulties in understanding and predicting the behaviour of nanoparticles in complex biological media. How nanoparticles disperse in biological media and the interactions that occur at the bio-nano interface dramatically influence their subsequent biological function. Techniques to measure and monitor these behaviours are usually bulk techniques, however these can be limited by the more complex nature of biological environments which routinely contain a number of bio-macromolecules e.g. vitamins, proteins and salts. As such, high spatial resolution analysis through electron microscopy has been investigated as an alternative to more accurately characterise nanoparticles in biological fluids. However, electron microscopy itself can be limited by the ultra-high vacuum requirements which mean samples must be dried before analysis. This necessitates the development of in situ techniques in order to characterise nanoparticle suspensions in the representative, native hydrated state. Using cryogenic transmission electron microscopy (cryo-TEM) a sample can be analysed in the frozen, hydrated state. However, typically this is limited to imaging alone. In this thesis, the use of analytical scanning TEM (STEM) to characterise nanoparticle dispersions captured in a layer of vitreous ice is demonstrated using both STEM energy dispersive X-ray spectroscopy and electron energy loss spectroscopy under cryogenic conditions. A noticeable difference in damage to the surrounding vitreous ice was observed between conventional TEM (CTEM) and STEM where damage occurred at much higher electron fluences in STEM (<2000 e-/Å2) compared to CTEM (<100 e-/Å2). Applying these techniques to characterise nanoparticles dispersed in cell culture media showed that incorrect specimen preparation or analysis where a significant raise in pH level is caused can induce an artefactual, nanoscale, calcium phosphate-rich, amorphous coating on nanoparticles dispersed in cell culture media. Recommendations to prevent this are given which will prevent any specimen preparation artefacts that could drive alterations in the in vivo or in vitro function of nanoparticles. For nanoparticle dispersion analysis automated electron microscopy imaging and analysis of plunge frozen vacuum dried nanoparticle suspensions was shown to be a viable alternative to dynamic light scattering for quantification of nanoparticle agglomerates in biological dispersants. Using two simple freeware codes, CellProfiler and Ilastik automated image analysis was achieved and validated for both monodisperse and agglomerated nanoparticle systems. Finally, cellular uptake studies assessed the biological effect of two gold nanoparticles coating with PEG and either terminated with a positive NH2 or neutral OMe group, on blood cells. No significant differences in cell uptake, geno- and cyto- toxicity or immune response was observed for the two nanoparticle types. Protein corona analysis by sodium dodecylsulfate - polyacrylamide gel electrophoresis indicated a comparable hard corona composition for both particles. Preliminary work identified annular bright field-STEM as a potential pathway to image the protein corona. This characterisation indicated possible inter-particle variation in the presence of a corona around particles within an individual suspension. Overall, the results reported show in situ cryo-analytical S/TEM is a powerful tool to characterise nanoparticle dispersions in complex biological media in order to further the understanding of complex bio-nano interactions and nanoparticle dispersion behaviour that ultimately control biological function