Immobilizing redox enzymes at mesoporous and nanostructured electrodes

Three key challenges are stimulating intensive research in the development of productive direct electron transfer mode enzyme electrodes: proper enzyme orientation, high enzyme loading, and full retention of enzyme activity. In this review, we summarize some significant advances that have been reported in the last years on the design of mesoporous and nanostructured electrodes as enzyme scaffolds and of innovative methodologies for wiring enzymes to electrodes. Particular attention is given to investigations on physical factors that determine a favorable enzyme immobilization, to provide rational guidelines for the design of productive enzymatic electrodes. Finally, some emerging trends focused on the spatial organization of either single enzymes or enzyme cascades are also briefly addressed.     

Co-detection of Copper and Lead in Artisanal Sugarcane Spirit Using Caffeic Acid-modified Graphite Electrodes

Caffeic acid (CA)-modified graphite electrodes [GE/poly(CA)] was applied to the co-detection of copper and lead in artisanal sugarcane spirit using square-wave anodic stripping voltammetry (SWASV). Electrochemical and morphological studies were performed, and a mechanism for polymerization was proposed. Electropolymerization, SWASV, and analysis conditions parameters were optimized. Interferents, repeatability, reproducibility, and addition and recovery tests were carried out. GE/poly(CA) shows a linear range from 15 to 705 μg/L with a limit of detection of 3.01 μg/L for Pb(II) and 4.50 μg/L for Cu(II). Real samples of artisanal sugarcane spirit were used, and the electrochemical results were compared with atomic absorption spectroscopy experiments.     

Gelation Kinetics and Mechanical Properties of Thiol-Tetrazole Methylsulfone Hydrogels Designed for Cell Encapsulation

Hydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol-tetrazole methylsulfone (TzMS) reaction crosslinks star-poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell-adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG-4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture.     

The Role of Mechanical Properties and Structure of Type I Collagen Hydrogels on Colorectal Cancer Cell Migration

Mechanical interactions between cells and their microenvironment play an important role in determining cell fate, which is particularly relevant in metastasis, a process where cells invade tissue matrices with different mechanical properties. In vitro, type I collagen hydrogels have been commonly used for modeling the microenvironment due to its ubiquity in the human body. In this work, the combined influence of the stiffness of these hydrogels and their ultrastructure on the migration patterns of HCT-116 and HT-29 spheroids are analyzed. For this, six different types of pure type I collagen hydrogels by changing the collagen concentration and the gelation temperature are prepared. The stiffness of each sample is measured and its ultrastructure is characterized. Cell migration studies are then performed by seeding the spheroids in three different spatial conditions. It is shown that changes in the aforementioned parameters lead to differences in the mechanical stiffness of the matrices as well as the ultrastructure. These differences, in turn, lead to distinct cell migration patterns of HCT-116 and HT-29 spheroids in either of the spatial conditions tested. Based on these results, it is concluded that the stiffness and the ultrastructural organization of the matrix can actively modulate cell migration behavior in colorectal cancer spheroids.     

The Mechanical and Biological Performance of Photopolymerized Gelatin-Based Hydrogels as a Function of the Reaction Media

From the first experiments with biomaterials to mimic tissue properties, the mechanical and biochemical characterization has evolved extensively. Several properties can be described, however, what should be essential is to conduct a proper and physiologically relevant characterization. Herein, the influence of the reaction media (RM) and swelling media (SM)–phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) with two different glucose concentrations–is described in gelatin methacrylamide (GelMA) hydrogel mechanics and in the biological behavior of two tumoral cell lines (Caco-2 and HCT-116). All scaffolds are UV-photocrosslinked under identical conditions and evaluated for mass swelling ratio and stiffness. The results indicate that stiffness is highly susceptible to the RM, but not to the SM. Additionally, PBS-prepared hydrogels exhibited a higher photopolymerization degree according to high resolution magic-angle spinning (HR-MAS) NMR. These findings correlate with the biological response of Caco-2 and HCT-116 cells seeded on the substrates, which demonstrated flatter morphologies on stiffer hydrogels. Overall, cell viability and proliferation are excellent for both cell lines, and Caco-2 cells displayed a characteristic apical-basal polarization based on F-actin/Nuclei fluorescence images. These characterization experiments highlight the importance of conducting mechanical testing of biomaterials in the same medium as cell culture.     

Mesoporous Silica Nanoparticles Decorated with Carbosilane Dendrons as New Non‐viral Oligonucleotide Delivery Carriers

A novel nanosystem based on mesoporous silica nanoparticles covered with carbosilane dendrons grafted on the external surface of the nanoparticles is reported. This system is able to transport single‐stranded oligonucleotide into cells, avoiding an electrostatic repulsion between the cell membrane and the negatively charged nucleic acids thanks to the cationic charge provided by the dendron coating under physiological conditions. Moreover, the presence of the highly ordered pore network inside the silica matrix would make possible to allocate other therapeutic agents within the mesopores with the aim of achieving a double delivery. First, carbosilane dendrons of second and third generation possessing ammonium or tertiary amine groups as peripheral functional groups were prepared. Hence, different strategies were tested in order to obtain their suitable grafting on the outer surface of the nanoparticles. As nucleic acid model, a single‐stranded DNA oligonucleotide tagged with a fluorescent Cy3 moiety was used to evaluate the DNA adsorption capacity. The hybrid material functionalised with the third generation of a neutral dendron showed excellent DNA binding properties. Finally, the cytotoxicity as well as the capability to deliver DNA into cells, was tested in vitro by using a human osteoblast‐like cell line, achieving good levels of internalisation of the vector DNA/carbosilane dendron‐functionalised material without affecting the cellular viability.     

Light‐Directing Omnidirectional Circularly Polarized Reflection from Liquid‐Crystal Droplets

Constructing and tuning self‐organized three‐dimensional (3D) superstructures with tailored functionality is crucial in the nanofabrication of smart molecular devices. Herein we fabricate a self‐organized, phototunable 3D photonic superstructure from monodisperse droplets of one‐dimensional cholesteric liquid crystal (CLC) containing a photosensitive chiral molecular switch with high helical twisting power. The droplets are obtained by a glass capillary microfluidic technique by dispersing into PVA solution that facilitates planar anchoring of the liquid‐crystal molecules at the droplet surface, as confirmed by the observation of normal incidence selective circular polarized reflection in all directions from the core of individual droplet. Photoirradiation of the droplets furnishes dynamic reflection colors without thermal relaxation, whose wavelength can be tuned reversibly by variation of the irradiation time. The results provided clear evidence on the phototunable reflection in all directions.