Development of an Electrochemical Sensor for SARS-CoV-2 Detection Based on Loop-Mediated Isothermal Amplification.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused more than 6 million deaths all over the world, demonstrating the need for a simple, fast and cost-effective point-of-care (POC) test for the detection of the virus. In this work, we developed an electrochemical sensor for SARS-CoV-2 virus detection on clinical samples based on loop-mediated isothermal amplification (LAMP). With the development of this novel sensor, the time of each measurement is significantly reduced by avoiding the DNA extraction step and replacing it with inactivation of the sample by heating it at 95 °C for 10 min. To make the reaction compatible with the sample pre-treatment, an RNase inhibitor was added directly to the premix. The LAMP product was measured in a novel, easy-to-use manufactured sensor containing a custom-made screen-printed carbon electrode. Electrochemical detection was performed with a portable potentiostat, and methylene blue was used as the redox-transducing molecule. The developed sensor achieved a limit of detection of 62 viral copies and was 100% specific for the detection of the SARS-CoV-2 virus. The performance of the electrochemical sensor was validated with nasopharyngeal samples, obtaining a sensibility and specificity of 100% compared to the gold standard RT-PCR method.

Design and 3D printing of an electrochemical sensor for Listeria monocytogenes detection based on loop mediated isothermal amplification.

The aim of this work is the design and 3D printing of a new electrochemical sensor for the detection of Listeria monocytogenes based on loop mediated isothermal amplification (LAMP). The food related diseases involve a serious health issue all over the world. Listeria monocytogenes is one of the major problems of contaminated food, this pathogen causes a disease called listeriosis with a high rate of hospitalization and mortality. Having a fast, sensitive and specific detection method for food quality control is a must in the food industry to avoid the presence of this pathogen in the food chain (raw materials, facilities and products). A point-of-care biosensor based in LAMP and electrochemical detection is one of the best options to detect the bacteria on site and in a very short period of time. With the numerical analysis of different geometries and flow rates during sample injection in order to avoid bubbles, an optimized design of the microfluidic biosensor chamber was selected for 3D-printing and experimental analysis. For the electrochemical detection, a novel custom gold concentric-3-electrode consisting in a working electrode, reference electrode and a counter electrode was designed and placed in the bottom of the chamber. The LAMP reaction was optimized specifically for a primers set with a limit of detection of 1.25 pg of genomic DNA per reaction and 100% specific for detecting all 12 Listeria monocytogenes serotypes and no other Listeria species or food-related bacteria. The methylene blue redox-active molecule was tested as the electrochemical transducer and shown to be compatible with the LAMP reaction and very clearly distinguished negative from positive food samples when the reaction is measured at the end-point inside the biosensor.

Bioresorbable and Mechanically Optimized Nerve Guidance Conduit Based on a Naturally Derived Medium Chain Length Polyhydroxyalkanoate and Poly(ε-Caprolactone) Blend.

Severe peripheral nerve injuries represent a large clinical problem with relevant challenges such as the development of successful synthetic scaffolds as substitutes to autologous nerve grafting. Numerous studies have reported the use of polyesters and type I collagen-based nerve guidance conduits (NGCs) to promote nerve regeneration through critical nerve defects while providing protection from external factors. However, none of the commercially available hollow bioresorbable NGCs have demonstrated superior clinical outcomes to an autologous nerve graft. Hence, new materials and NGC geometries have been explored in the literature to mimic the native nerve properties and architecture. Here, we report a novel blend of a natural medium chain length polyhydroxyalkanoate (MCL-PHA) with a synthetic aliphatic polyester, poly(ε-caprolactone) (PCL), suitable for extrusion-based high-throughput manufacturing. The blend was designed to combine the excellent ability of PHAs to support the growth and proliferation of mammalian cells with the good processability of PCL. The material exhibited excellent neuroregenerative properties and a good bioresorption rate, while the extruded porous tubes exhibited similar mechanical properties to the rat sciatic nerve. The NGCs were implanted to treat a 10 mm long sciatic nerve defect in rats, where significant differences were found between thin and thick wall thickness implants, and both electrophysiological and histological data, as well as the number of recovered animals, provided superior outcomes than the well-referenced synthetic Neurolac NGC.

UV-Casting on Methacrylated PCL for the Production of a Peripheral Nerve Implant Containing an Array of Porous Aligned Microchannels.

Peripheral nerves are basic communication structures guiding motor and sensory information from the central nervous system to receptor units. Severed peripheral nerve injuries represent a large clinical problem with relevant challenges to successful synthetic nerve repair scaffolds as substitutes to autologous nerve grafting. Numerous studies reported the use of hollow tubes made of synthetic polymers sutured between severed nerve stumps to promote nerve regeneration while providing protection for external factors, such as scar tissue formation and inflammation. Few approaches have described the potential use of a lumen structure comprised of microchannels or microfibers to provide axon growth avoiding misdirection and fostering proper healing. Here, we report the use of a 3D porous microchannel-based structure made of a photocurable methacrylated polycaprolactone, whose mechanical properties are comparable to native nerves. The neuro-regenerative properties of the polymer were assessed in vitro, prior to the implantation of the 3D porous structure, in a 6-mm rat sciatic nerve gap injury. The manufactured implants were biocompatible and able to be resorbed by the host's body at a suitable rate, allowing the complete healing of the nerve. The innovative design of the highly porous structure with the axon guiding microchannels, along with the observation of myelinated axons and Schwann cells in the in vivo tests, led to a significant progress towards the standardized use of synthetic 3D multichannel-based structures in peripheral nerve surgery.

Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion.

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational-active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca(2+) resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.

Geometry sensing by dendritic cells dictates spatial organization and PGE(2)-induced dissolution of podosomes.

Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E(2) (PGE(2)). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE(2) causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE(2)-mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D and 3-D geometric cues control the spatial organization of podosomes. More importantly, our studies demonstrate the importance of substrate dimensionality in regulating podosome dissolution and suggest that substrate dimensionality plays an important role in controlling DC activation, a key process in initiating immune responses.