Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy

We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding eventsThis work was funded by the European Research Council ERC-AdG-340177 (3DNanoMech) grant to RG and by the Spanish Ministry of Economy (MINECO) through grants CSD2010-00024, MAT2013-44858-R to RG and BFU2011-30161- C02-01 and BFU2014-59009-P to VC.Peer reviewe

Advances in bimodal AFM imaging of molecules in Liquid

Conferencia invitada presentada en la 14th International Conference on Noncontact AFM, celebrada en Lindau (Alemania).Improving spatial resolution, data acquisition times and material properties imaging are some long established goals in atomic force microscopy (AFM). Currently, the most promising approaches to reach those goals involve the excitation and detection of several frequencies of the tip’s oscillation. Usually those frequencies are associated with either the higher harmonics of the oscillation or the eigenmodes of the cantilever. Bimodal AFM is an emerging multifrequency technique that is characterized by a high signal-to-noise ratio and the versatility to measure simultaneously different forces. The method is also compatible with molecular resolution imaging under the application of sub-50 pN peak forces.Peer Reviewe

Tumour heterogeneity in glioblastoma assessed by MRI texture analysis: a potential marker of survival

Objective: The main objective of this retrospective work was the study of three-dimensional (3D) heterogeneity measures of post-contrast pre-operative MR images acquired with T1 weighted sequences of patients with glioblastoma (GBM) as predictors of clinical outcome. Methods: 79 patients from 3 hospitals were included in the study. 16 3D textural heterogeneity measures were computed including run-length matrix (RLM) features (regional heterogeneity) and co-occurrence matrix (CM) features (local heterogeneity). The significance of the results was studied using Kaplan?Meier curves and Cox proportional hazards analysis. Correlation between the variables of the study was assessed using the Spearman?s correlation coefficient. Results: Kaplan?Meyer survival analysis showed that 4 of the 11 RLM features and 4 of the 5 CM features considered were robust predictors of survival. The median survival differences in the most significant cases were of over 6 months. Conclusion: Heterogeneity measures computed on the post-contrast pre-operative T1 weighted MR images of patients with GBM are predictors of survival. Advances in knowledge: Texture analysis to assess tumour heterogeneity has been widely studied. However, most works develop a two-dimensional analysis, focusing only on one MRI slice to state tumour heterogeneity. The study of fully 3D heterogeneity textural features as predictors of clinical outcome is more robust and is not dependent on the selected slice of the tumour

Standardized nanomechanical atomic force microscopy procedure (SNAP) for measuring soft and biological samples

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions

Método bimodal para cuantificar propiedades no topográficas en microscopía de fuerzas

Método bimodal para cuantificar propiedades no topográficas en microscopía de fuerzas, basado en modular en frecuencia y de forma simultánea al menos dos modos de vibración de la micropalanca de un microscopio, que emplea los desplazamientos de frecuencia de los modos excitados y los cambios en la fuerzas de excitación para cuantificar propiedades nanomecánicasPeer reviewedConsejo Superior de Investigaciones CientíficasA1 Solicitud de patente con informe sobre el estado de la técnic

Método bimodal para cuantificar propiedades no topográficas en microscopía de fuerzas

Método bimodal para cuantificar propiedades no topográficas en microscopía de fuerzas, basado en modular en frecuencia y de forma simultánea al menos dos modos de vibración de la micropalanca de un microscopio, que emplea los desplazamientos de frecuencia de los modos excitados y los cambios en la fuerzas de excitación para cuantificar propiedades nanomecánicasPeer reviewedConsejo Superior de Investigaciones CientíficasB1 Patente sin examen previ

Método bimodal para cuantificar propiedades no topográficas en microscopía de fuerzas

[EN] The invention relates to a bimodal method for quantifying non-topographical properties in force field microscopy, based on simultaneously modulating, in terms of frequency, at least two vibration modes of the microlever of a microscope, which uses the frequency shifts of the excited modes and the changes in the excitation forces to quantify nanomechanical properties.[FR] Méthode bimodale pour quantifier des propriétés non topographiques en microscopie à force, basée sur la modulation en fréquence et simultanément au moins deux modes de vibration du microlevier d'un microscope, qui utilise les déplacement de fréquence des modes excités et les changements dans les forces d'excitation pour quantifier des propriétés nanomécaniques.[Es] Método bimodal para cuantificar propiedades no topográficas en microscopía de fuerzas, basado en modular en frecuencia y de forma simultánea al menos dos modos de vibración de la micropalanca de un microscopio, que emplea los desplazamientos de frecuencia de los modos excitados y los cambios en la fuerzas de excitación para cuantificar propiedades nanomecánicas.Peer reviewedConsejo Superior de Investigaciones CientíficasA1 Solicitud de patente con informe sobre el estado de la técnic

Nanomechanical coupling enables detection and imaging of 5 nm superparamagnetic particles in liquid

We demonstrate that a force microscope operated in a bimodal mode enables the imaging and detection of superparamagnetic particles down to 5 nm. The bimodal method exploits the nanomechanical coupling of the excited modes to enhance the sensitivity of the higher mode to detect changes in material properties. The coupling requires the presence of nonlinear forces. Remarkably, bimodal operation enables us to identify changes of slowly varying forces (quasi-linear) in the presence of a stronger nonlinear force. Thus, unambiguous identification of single apoferritin (non-magnetic) and ferritin (magnetic) molecules in air and liquid is accomplished.We acknowledge the financial support from the Ministerio de Ciencia, Investigación e Innovación (MAT2009‐08650).Peer reviewe

Non-invasive Protein Structural Flexibility Mapping by Bimodal Dynamic Force Microscopy

PACS:87.15.-v, 62.25.-g, 62.30.+d, 68.37.PsMapping of the protein structural flexibility with sub-2-nm spatial resolution in liquid is achieved by combining bimodal excitation and frequency modulation force microscopy. The excitation of two cantilever eigenmodes in dynamic force microscopy enables the separation between topography and flexibility mapping. We have measured variations of the elastic modulus in a single antibody pentamer from 8 to 18 MPa when the probe is moved from the end of the protein arm to the central protrusion. Bimodal dynamic force microscopy enables us to perform the measurements under very small repulsive loads (30–40 pN).This work was funded by the Spanish Ministry of Science (MICINN) through Grants No. CSD2010-00024, No. MAT2009-08650, No. MAT2010-20843-C02, and the Comunidad de Madrid No. S2009/MAT-1467.Peer reviewe