Nanobiosensores y salud animal.

Los biosensores son dispositivos cuantitativos, semicuantitativos o analíticos, que contienen una biomolécula sensora capaz de convertir una señal biológica en una señal óptica o electroquímica. En los últimos años las nanopartículas de oro se han presentado como una interesante alternativa para la el biosensado de analitos debido a sus propiedades ópticas

Revisiting 30 years of biofunctionalization and surface chemistry of inorganic nanoparticles for nanomedicine

FP7-PEOPLE-2013-IOF, Project no. 626386 PEst-OE/SAU/U10009/2011-14 MAT2011-26851-C02-01In the last 30 years we have assisted to a massive advance of nanomaterials in material science. Nanomaterials and structures, in addition to their small size, have properties that differ from those of larger bulk materials, making them ideal for a host of novel applications. The spread of nanotechnology in the last years has been due to the improvement of synthesis and characterization methods on the nanoscale, a field rich in new physical phenomena and synthetic opportunities. In fact, the development of functional nanoparticles has progressed exponentially over the past two decades. This work aims to extensively review 30 years of different strategies of surface modification and functionalization of noble metal (gold) nanoparticles, magnetic nanocrystals and semiconductor nanoparticles, such as quantum dots. The aim of this review is not only to provide in-depth insights into the different biofunctionalization and characterization methods, but also to give an overview of possibilities and limitations of the available nanoparticles.publishersversionpublishe

Rescuing compound bioactivity in a secondary cell-based screening by using γ-cyclodextrin as a molecular carrier

This work is published under Creative Commons Attribution - Non Commercial (unported, v3.0) License.In vitro primary screening for identifying bioactive compounds (inhibitors, activators or pharmacological chaperones) against a protein target results in the discovery of lead compounds that must be tested in cell-based efficacy secondary screenings. Very often lead compounds do not succeed because of an apparent low potency in cell assays, despite an excellent performance in primary screening. Primary and secondary screenings differ significantly according to the conditions and challenges the compounds must overcome in order to interact with their intended target. Cellular internalization and intracellular metabolism are some of the difficulties the compounds must confront and different strategies can be envisaged for minimizing that problem. Using a novel screening procedure we have identified 15 compounds inhibiting the hepatitis C NS3 protease in an allosteric fashion. After characterizing biophysically the interaction with the target, some of the compounds were not able to inhibit viral replication in cell assays. In order to overcome this obstacle and potentially improve cellular internalization three of these compounds were complexed with γ-cyclodextrin. Two of them showed a five- and 16-fold activity increase, compared to their activity when delivered as free compounds in solution (while γ-cyclodextrin did not show antiviral activity by itself). The most remarkable result came from a third compound that showed no antiviral activity in cell assays when delivered free in solution, but its γ-cyclodextrin complex exhibited a 50% effective concentration of 5 µM. Thus, the antiviral activity of these compounds can be significantly improved, even completely rescued, using γ-cyclodextrin as carrier molecule.This work was supported by Spanish Ministerio de Ciencia e Innovación (BFU2010-19451 to AVC, PTA2009-2341-I to SV), Spanish Ministerio de Economía y Competitividad (BFU2013-47064-P to AVC), Spanish Ministerio de Educación, Cultura y Deporte (Grant FPU13/3870 to RCG), Miguel Servet Program from Instituto de Salud Carlos III (CP07/00289 to OA), Fondo de Investigaciones Sanitarias (PI10/00186 to OA, PI11/02578 to AL), grant ERC-Starting Grant (239931-NANOPUZZLE project to JML), Diputación General de Aragón (Grant B136/13 to RCG, Protein Targets Group B89 to AVC, Digestive Pathology Group B01 to OA, RCG, and AL, and Nanotherapy and Nanobiosensors Group E93 to JMF), Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd) to AL and OA, and Fondo Social Europeo to JMF.Peer reviewe

Tailoring the Synthesis and Heating Ability of Gold Nanoprisms for Bioapplications

The paper describes a novel and straightforward wet-chemical synthetic route to produce biocompatible single-crystalline gold tabular nanoparticles, herein called nanoprisms (NPRs) due to their characteristic shape. Besides the novelty of the method to produce NPRs with an unprecedented high yield, the synthesis avoids the use of highly toxic cetyltrimethylammonium bromide (CTAB), the most widely used surfactant for the synthesis of gold anisotropic nanoparticles such as nanorods or nanoprisms. The method presented here allows for tuning the edge length of NPRs in the range of 100–170 nm by adjusting the final concentration/molar ratio of gold salt and reducing agent (thiosulfate), while the thickness of NPRs remained constant (9 nm). Thus, the surface plasmon band of NPRs can be set along the near-infrared (NIR) range. The resulting NPRs were derivatized with heterobifunctional polyethylene glycol (PEG) and 4-aminophenyl β-d-glucopyranoside (glucose) chains to improve their stability and cellular uptake, respectively. The heating properties of colloidal solutions of NPRs upon 1064 nm light illumination were evaluated. As a proof of concept, the biocompatibility and suitability of functional NPRs as photothermal agents were studied in cell cultures. Due to their biocompatibility (avoiding CTAB), ease of production, ease of functionalization, and remarkable heating features, the NPRs discussed herein represent a significant advance in the biocompatibility of nanoparticles and serve as an attractive alternative to those currently in use as plasmonic photothermal agents

Applying the retro-enantio approach to obtain a peptide capable of overcoming the blood-brain barrier

The blood-brain barrier (BBB) is a formidable physical and enzymatic barrier that tightly controls the passage of molecules from the blood to the brain. In fact, less than 2% of all potential neurotherapeutics are able to cross it. Here, by applying the retro-enantio approach to a peptide that targets the transferrin receptor, a full protease-resistant peptide with the capacity to act as a BBB shuttle was obtained and thus enabled the transport of a variety of cargos into the central nervous system

Stiffness-dependent active wetting enables optimal collective cell durotaxis

The directed migration of cellular clusters enables morphogenesis, wound healing and collective cancer invasion. Gradients of substrate stiffness direct the migration of cellular clusters in a process called collective durotaxis, but the underlying mechanisms remain unclear. Here we unveil a connection between collective durotaxis and the wetting properties of cellular clusters. We show that clusters of cancer cells dewet soft substrates and wet stiff ones. At intermediate stiffness—at the crossover from low to high wettability—clusters on uniform-stiffness substrates become maximally motile, and clusters on stiffness gradients exhibit optimal durotaxis. Durotactic velocity increases with cluster size, stiffness gradient and actomyosin activity. We demonstrate this behaviour on substrates coated with the cell–cell adhesion protein E-cadherin and then establish its generality on substrates coated with extracellular matrix. We develop an active wetting model that explains collective durotaxis in terms of a balance between in-plane active traction and tissue contractility and out-of-plane surface tension. Finally, we show that the distribution of cluster displacements has a heavy tail, with infrequent but large cellular hops that contribute to durotactic migration. Our study demonstrates a physical mechanism of collective durotaxis, through both cell–cell and cell–substrate adhesion ligands, based on the wetting properties of active droplets

Spatially-Resolved EELS Analysis of Antibody Distribution on Biofunctionalized Magnetic Nanoparticles

Spatially resolved electron energy loss spectroscopy (SR-EELS) using scanning transmission electron microscope (STEM) allows the identification and determination of the spatial distribution of the components/elements of immuno-functionalized core–shell superparamagnetic magnetite nanoparticles. Here, we report that SR-EELS measurements allow the direct identification and study of the biological moieties (protein G and anti-HRP antibody) in complex bionanocarriers of relevance for biomedical applications. Our findings show that the biomacromolecules are located on specific areas on the nanoparticles’ surface. In addition, efficiency of this functionalization was evaluated by means of biochemical techniques

The intracellular number of magnetic nanoparticles modulates the apoptotic death pathway after magnetic hyperthermia treatment

Magnetic hyperthermia is a cancer treatment based on the exposure of magnetic nanoparticles to an alternating magnetic field in order to generate local heat. In this work, 3D cell culture models were prepared to observe the effect that a different number of internalized particles had on the mechanisms of cell death triggered upon the magnetic hyperthermia treatment. Macrophages were selected by their high capacity to uptake nanoparticles. Intracellular nanoparticle concentrations up to 7.5 pg Fe/cell were measured both by elemental analysis and magnetic characterization techniques. Cell viability after the magnetic hyperthermia treatment was decreased to <25% for intracellular iron contents above 1 pg per cell. Theoretical calculations of the intracellular thermal effects that occurred during the alternating magnetic field application indicated a very low increase in the global cell temperature. Different apoptotic routes were triggered depending on the number of internalized particles. At low intracellular magnetic nanoparticle amounts (below 1 pg Fe/cell), the intrinsic route was the main mechanism to induce apoptosis, as observed by the high Bax/Bcl-2 mRNA ratio and low caspase-8 activity. In contrast, at higher concentrations of internalized magnetic nanoparticles (1−7.5 pg Fe/cell), the extrinsic route was observed through the increased activity of caspase-8. Nevertheless, both mechanisms may coexist at intermediate iron concentrations. Knowledge on the different mechanisms of cell death triggered after the magnetic hyperthermia treatment is fundamental to understand the biological events activated by this procedure and their role in its effectiveness

Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA)

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence