5 research outputs found
Estudio de las interacciones de nanopartículas aniónicas de oro con ADN y sus monómeros constituyentes
En la presente Tesis Doctoral se desarrolla un estudio exhaustivo de la agregación de nanopartículas de oro (AuNPs) provocada por la interacción con las distintas unidades monoméricas del ADN, así como el efecto protector de ADN polimérico tanto de hebra doble como simple a la aglomeración inducida por una sal inerte. Se ha establecido un orden de afinidad para las nucleobases a partir de la determinación de valores de energías libres correspondientes al proceso de adsorción, obtenidos por tres procedimientos diferentes. Dicho estudio se ha extrapolado también a la adenosina y a la adenosina monofosfato, donde la agregación fue menor conforme aumentó el grado de complejidad del monómero; sin embargo, el no observarse aglomeración de nanoclusters no es sinónimo de no adsorción, hecho que se ha comprobado en el caso del nucleótido. La fuerza impulsora que diferencia el grado de agregación entre la adenosina neutra o cargada y el AMP es de carácter marcadamente electrostático.
Se ha desarrollado paralelamente un método colorimétrico de detección de ADN de cadena larga tomando como punto de referencia el color azul de la banda de plasmón correspondiente a nanopartículas agregadas. Para ello fue necesaria la optimización de la cantidad de sal a añadir para desestabilizar al coloide, el orden de adición de los diferentes componentes así como el tamaño de la nanopartícula. A diferencia de los oligonucleótidos, donde sólo hay protección contra la agregación inducida por un electrolito para los de cadena simple, se observó un aumento del grado de dispersión del sistema coloidal conforme aumentó la concentración del biopolímero. Así mismo, técnicas como la miscroscopía de fuerza atómica, medidas de viscosidad o de dispersión dinámica de luz, entre otras, confirman que la mera presencia de nanopartículas aniónicas provoca cambios estructurales en el polinucleótido. Estos cambios reducen la estabilidad del ADN, provocando una preferencia por interaccionar con los cationes sodios y protegiendo a las nanopartículas de la agregación. Por otro lado se han obtenido complejos ADN-nanopartículas similares en conformación y estructura a las histonas sin necesidad de funcionar la nanopartícula o dotarla de carga positiva, trabajando a diferentes temperaturas y corroborando a través de espectros de dicroísmo circular que el ADN mantiene una conformación similar a la nativa.In the present PhD. Thesis, a thorough study of gold nanoparticles’ (AuNPs) interaction, and the nanocluster aggregation induced by it, with different DNA monomeric units has been carried out. A protective effect of polymeric DNA, either double or single stranded, for inert salt-induced agglomeration has been found and explored. An affinity order has been established for the nucleobases based on the determination of their adsorption processes’ free energy values, calculated by three different methods. This study has been also extrapolated to adenosine and adenosine monophosphate (AMP), and a lesser aggregation grade was found as the complexity grade of the monomer increased. However, experimental results for adenosine prove that the absence of aggregation-induced color changes does not imply an absence of monomer-particle interactions. The reactivity differences between neutral or charged adenosine and AMP are distinctly charge-based.
A colorimetric method for the detection of long DNA molecules has been concurrently developed, taking as reference point the blue color of plasmon band of aggregated NPs. The optimization of added salt concentrations, as well as the addition order for the different components and the nanoparticle size, were key parts of this study. In contrast to oligonucleotides, where a degree of electrolyte-induced aggregation protection has only been described for single stranded DNA, in our case an increase of the dispersion grade of the colloidal system was observed when the biopolymer concentration rose. Techniques like atomic force microscopy, viscosity measures or light scattering dynamic, among others, support that the mere presence of anionic nanoparticles induces structural changes in the polynucleotide. These changes reduce DNA stability, and in turn increase its affinity for Na+ cations, reducing their concentration on the medium and protecting the anionic-protected particles from aggregation. Finally, DNA-nanoparticles complexes with remarkable similarities in both conformation and structure to histones have been obtained, without the need to functionalize or change the particle’s charge. This work has been carried out at different temperatures, and the DNA has been found to present a conformation not dissimilar to its native form by employing circular dichroism measurements
Encased Gold Nanoparticle Synthesis as a Probe for Oleuropein Self-Assembled Structure Formation
Stable oleuropein-coated gold nanoparticles in aqueous media were synthesized for the
first time. Oleuropein (OLE) concentration in the reaction medium was found to greatly influence
the outcome and stability of the resulting nanocolloid, with a marked decrease in particle size being
found for the more concentrated oleuropein solutions. The protection mechanisms involved in
the stabilized nanosystems were analyzed. Oleuropein self-assembled structures were found to
be formed at a concentration threshold of [OLE] > 5 × 10−5 M, and observed through the use of
CryoSEM imaging. Those structures were responsible for both the increased stability and the decrease
in size observed at the more concentrated solutions.Ministerio de Economía y Competitividad español-CTQ2016-78703-PFondo Europeo de Desarrollo Regional (FEDER) y el V Plan Propio de Subvenciones para Grupos Emergentes y la Oficina de Transferencia de Resultados de Investigación (OTRI) de la Universidad de Sevilla-2010/0000076
Decorating a single giant DNA with gold nanoparticles
We decorated a single giant DNA (1.66 × 105 base pairs) with gold nanoparticles through the simple procedure of mild warming, without denaturation of the DNA molecule. Single-molecule observation with fluorescence microscopy revealed that individual decorated DNA molecules stay in the bulk solution by avoiding aggregation and precipitation, and exhibit translational and conformational fluctuation, i.e., Brownian motion. An analysis of the intra-chain fluctuation of single DNA molecules revealed that the apparent spring constant and damping coefficient of a DNA chain increased by ca. 13- and 5-fold, respectively, upon decoration with gold nanoparticles. Observation by transmission electron microscopy revealed that gold nanoparticles were stably attached to the DNA skeleton. UV-visible measurements revealed the absence of any detectable change in surface plasmon resonance, suggesting that the gold nanoparticles assemble without the formation of a densely packed aggregate. CD measurements showed that the secondary structure of decorated DNA is still essentially the B-form.Spanish Ministerio de Economía y Competitividad CTQ2016-78703-PUniversidad de Sevilla 2010/00000762OTRI 2010/00000762JSPS KAKENHI 15H02121 2510301
CIELab chromaticity evolution to measure the binding free energy of non-colored biomolecules to gold nanoparticles
We explore the possibilities of the CIELab chromaticity system parameters' evolution in order to study the interaction between two non-colored biological reactants, lysozyme and adenosine, and the red-colored, non-functionalized citrate gold nanoparticles. By measuring and quantifying the AuNP color changes, which are strongly dependant on their aggregation state, binding free energies are obtained for the first time for these two systems. Results are confirmed via an alternate fitting method which makes use of deconvolution parameters from experimental absorbance spectra. Binding free energies obtained through the use of both means are in good agreement with each other
Colorimetric, naked-eye detection of lysozyme in human urine with gold nanoparticles
The stabilizing effect of lysozymes to salt addition over a gold colloid are exploited in order to detect lysozymes in human urine samples. The present research is aimed at the development of a fast, naked-eye detection test for urinary lysozymuria, in which direct comparison with a colorimetric reference, allows for the immediate determination of positive/negative cases. CIEL*a*b* parameters were obtained from sample absorbance measurements, and their color difference with respect to a fixed reference point was measured by calculating the ∆E76 parameter, which is a measure of how well the colors can be distinguished by an untrained observer. Results show that a simple and quick test can reliably, in less than 15 min, give a positive colorimetric response in the naked eye for concentrations of a urinary lysozyme over 57.2 µg/mL. This concentration is well within the limits of that observed for leukemia-associated lysozymurias, among other disorders.Universidad de Sevilla 2010/00000762, PP2016-5937, 2019/00000570, 2020/00001068, 2020/00001073Junta de Andalucía 2019/FQM-38