Search

A versatile and robust procedure is developed that allows the identification of individual target molecules using antibodies bound to a DeepTipTM functionalized atomic-force microscopy probe. The model system used for the validation of this process consists of a biotinylated anti-lactate dehydrogenase antibody immobilized on a streptavidin-decorated AFM probe. Lactate dehydrogenase (LDH) is employed as target molecule and covalently immobilized on functionalized MicroDeckTM substrates. The interaction between sensor and target molecules is explored by recording force–displacement (F–z) curves with an atomic-force microscope. F–z curves that correspond to the genuine sensor–target molecule interaction are identified based on the following three criteria: (i) number of peaks, (ii) value of the adhesion force, and (iii) presence or absence of the elastomeric trait. The application of these criteria leads to establishing seven groups, ranging from no interaction to multiple sensor–target molecule interactions, for which force–displacement curves are classified. The possibility of recording consistently single-molecule interaction events between an antibody and its specific antigen, in combination with the high proportion of successful interaction events obtained, increases remarkably the possibilities offered by affinity atomic-force microscopy for the characterization of biological and biomimetic systems from the molecular to the tissue scales. [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) is a powerful tool in Biology due to the possibility of identifying and locating single molecules at a subnanometer scale. Performing these experiments requires the usage of functionalized AFM tips to which sensing molecules can be attached. In this work the possibility of functionalizing AFM cantilevers and tips using the activated vapour silanization (AVS) process is explored. Functionalized thin films are deposited on commercial silicon nitride chips and their presence is assessed by measuring the variation in the resonance frequency of the cantilever. Subsequently, the presence and functionality of the films is studied through the covalent immobilization of a fluorescein-derived molecule to the amine reactive groups on the surface. With regard to its sensing ability, the functionalized tips are shown to sustain repetitive interactions with a model graphite substrate (HOPG) under relatively harsh conditions, while no apparent damage is observed in the tip. It is also found that the substrate-tip interaction may be modified by covalently attaching a molecule to the latter. Consequently, AVS functionalized AFM tips are found to be robust and reliable candidates for their use in the affinity (or chemical) atomic force microscopy (AF-AFM) mode. Unlabelled Image • Atomic force microscopy (AFM) cantilevers and tips were functionalized by activated vapor silanization (AVS). • The deposition of the functionalized layer was assessed through variations in the resonance frequency of the cantilever. • The reactivity of the amine groups in the functionalized layer was checked by forming covalent bonds with a fluorophore. • The stability of the functionalized film upon repeated interactions with a model HOPG substrate was established. • AVS functionalized AFM tips appear as promising candidates for their usage in affinity atomic force microscopy experiments. [ABSTRACT FROM AUTHOR]

Objective. To describe the selection of health care providers for a health need among the Mexican population in 2022. Materials and methods. We used the Encuesta Nacional de Salud y Nutrición Continua 2022. Results. From 24.6% of the population that reported a health need in the last three months, 44% received care in public health facilities. The use of private services was extensive but was higher for the population without social security. We found that 71% of the reasons for not selecting the facility of their affiliation is access and 21%, perceived quality. The reasons to select the facility where they received care included affiliation, distance and cost; quality aspects were rapid care and satisfaction with service. Conclusions. Improving access (distance, service hours, bureaucratic barriers) could help reduce the inequalities on health care access and out-of pocket expenditures associated with the use of private services, particularly among the population without social security. [ABSTRACT FROM AUTHOR]

Single molecule interactions between biotin and streptavidin were characterized with functionalized DeepTipTM probes and used as a model system to develop a comprehensive methodology for the high-yield identification and analysis of single molecular events. The procedure comprises the covalent binding of the target molecule to a surface and of the sensing molecule to the DeepTipTM probe, so that the interaction between both chemical species can be characterized by obtaining force–displacement curves in an atomic force microscope. It is shown that molecular resolution is consistently attained with a percentage of successful events higher than 90% of the total number of recorded curves, and a very low level of unspecific interactions. The combination of both features is a clear indication of the robustness and versatility of the proposed methodology. [ABSTRACT FROM AUTHOR]

The interaction between the plant lipid transfer protein Pru p 3 and phytosphingosine was assessed using an atomic force microscope. Phytosphingosine was covalently immobilized on DeepTipTM probes and Pru p 3 on MicroDeckTM functionalized substrates. Single-molecular interaction events between both molecules were retrieved and classified and the distribution for each one of the identified types was calculated. A success rate of over 70% was found by comparing the number of specific Pru p 3-phytosphingosine interaction events with the total number of recorded curves. The analysis of the distribution established among the various types of curves was further pursued to distinguish between those curves that can mainly be used for assessing the recognition between phytosphingosine (sensor molecule) and Pru p 3 (target molecule) in the context of affinity atomic force microscopy, and those that entail details of the interaction and might be employed in the context of force spectroscopy. The successful application of these functionalized probes and substrates to the characterization of the low-intensity hydrophobic interaction characteristic of this system is a clear indication of the potential of exploiting this approach with an extremely wide range of different biological molecules of interest. The possibility of characterizing molecular assembly events with single-molecule resolution offers an advantageous procedure to plough into the field of molecular biomimetics. [ABSTRACT FROM AUTHOR]

After an injury, the limited regenerative capacity of the central nervous system makes the reconnection and functional recovery of the affected nervous tissue almost impossible. To address this problem, biomaterials appear as a promising option for the design of scaffolds that promote and guide this regenerative process. Based on previous seminal works on the ability of regenerated silk fibroin fibers spun through the straining flow spinning (SFS) technique, this study is intended to show that the usage of functionalized SFS fibers allows an enhancement of the guidance ability of the material when compared with the control (nonfunctionalized) fibers. It is shown that the axons of the neurons not only tend to follow the path marked by the fibers, in contrast to the isotropic growth observed on conventional culture plates, but also that this guidance can be further modulated through the biofunctionalization of the material with adhesion peptides. Establishing the guidance ability of these fibers opens the possibility of their use as implants for spinal cord injuries, so that they may represent the core of a therapy that would allow the reconnection of the injured ends of the spinal cord. [ABSTRACT FROM AUTHOR]

High-performance regenerated silkworm (Bombyx mori) silk fibers can be produced efficiently through the straining flow spinning (SFS) technique. In addition to an enhanced biocompatibility that results from the removal of contaminants during the processing of the material, regenerated silk fibers may be functionalized conveniently by using a range of different strategies. In this work, the possibility of implementing various functionalization techniques is explored, including the production of fluorescent fibers that may be tracked when implanted, the combination of the fibers with enzymes to yield fibers with catalytic properties, and the functionalization of the fibers with cell-adhesion motifs to modulate the adherence of different cell lineages to the material. When considered globally, all these techniques are a strong indication not only of the high versatility offered by the functionalization of regenerated fibers in terms of the different chemistries that can be employed, but also on the wide range of applications that can be covered with these functionalized fibers. [ABSTRACT FROM AUTHOR]

The adhesion forces of cells to peptide-coated functionalized materials were assessed through the Single Cell Force Spectroscopy (SCFS) technique in order to develop a methodology that allows the fast selection of peptide motifs that favor the interaction between cells and the biomaterial. Borosilicate glasses were functionalized using the activated vapor silanization process (AVS) and subsequently decorated with an RGD- containing peptide using the EDC/NHS crosslinking chemistry. It is shown that the RGD-coated glass induces larger attachment forces on mesenchymal stem cell cultures (MSCs), compared to the bare glass substrates. These higher forces correlate well with the enhanced adhesion of the MSCs observed on RGD-coated substrates through conventional adhesion cell cultures and inverse centrifugation tests. The methodology based on the SCFS technique presented in this work constitutes a fast procedure for the screening of new peptides or their combinations to select candidates that may enhance the response of the organism to the implant of the functionalized biomaterials. [ABSTRACT FROM AUTHOR]