A Biomimetic Platform to Study the Interactions of Bioelectroactive Molecules with Lipid Nanodomains

In this work, we developed a biomimetic platform where the study of membrane associated redox processes and high-resolution imaging of lipid nanodomains can be both performed, based on a new functional gold modification, l-cysteine self-assembled monolayer. This monolayer proved to be ideal for the preparation of defect-free planar supported lipid bilayers (SLBs) where nanodomains with height difference of ∼1.5 nm are clearly resolved by atomic force microscopy. Single and multicomponent lipid compositions were used, leading to the formation of different phases and domains mimicking the lateral organization of cellular membranes, and in all cases stable and continuous bilayers were obtained. These platforms were tested toward the interaction with bioelectroactive molecules, the antioxidant quercetin, and the hormone epinephrine. Despite the weak interaction detected between epinephrine and lipid bilayers, our biomimetic interface was able to sense the redox process of membrane-bound epinephrine, obtain its surface concentration (9.36 × 10^‑11 mol/cm² for a fluid bilayer), and estimate a mole fraction membrane/water partition coefficient (<i>K</i>_p) from cyclic voltammetric measurements (1.13 × 10⁴ for a fluid phase membrane). This <i>K</i>_p could be used to quantitatively describe the minute changes observed in the photophysical properties of epinephrine intrinsic fluorescence upon its interaction with liposome suspensions. Moreover, we showed that the lipid membrane stabilizes epinephrine structure, preventing its oxidation, which occurs in neutral aqueous solution, and that epinephrine partition and mobility in membranes depends on lipid phase, expanding our knowledge on hormone membrane interactions

Antibody Oriented Immobilization on Gold using the Reaction between Carbon Disulfide and Amine Groups and Its Application in Immunosensing

Carbon disulfide (CS₂) can spontaneously react with amine groups to form dithiocarbamates on gold surface, providing the possibility to immobilize some compounds with primary or secondary amine groups in one step. Using this principle, an immunosensor interface prepared for immunoglobulin G (IgG) sensing surface toward anti-IgG has been fabricated for the first time by simply immersing gold slides into a mixed aqueous solution of CS₂ and protein A, followed by incubation in immunoglobulin G solution. The reaction between CS₂ and protein A has been followed by UV–vis spectroscopy, whereas cyclic voltammetry has been employed in the characterization of the modified gold surface with CS₂ and protein A, both methods indicating that protein A immobilization is implemented by CS₂. Conventional ellipsometry, atomic force microscopy (AFM), as well as surface plasmon resonance (SPR) have been used to evaluate the specific binding of protein A with IgG and IgG with anti-IgG, revealing that IgG is specifically captured to form the biosensing interface, maintaining its bioactivity. Compared to direct adsorption of IgG on the gold surface, the IgG sensing surface constructed of CS₂ and protein A is far more sensitive to capture anti-IgG as its target molecule. In addition, the modified surface is proven to have good capability to inhibit nonspecific adsorption, as supported by control experiments using lysozyme and BSA. To conclude, antibody immobilization using this one-step method has potential as a simple and convenient surface modification approach for immunosensor development

Kinetics and Mechanism of the Thermal Dehydration of a Robust and Yet Metastable Hemihydrate of 4‑Hydroxynicotinic Acid

Hydrates are the most common type of solvates and certainly the most important ones for industries such as pharmaceuticals which strongly rely on the development, production, and marketing of organic molecular solids. A recent study indicated that, in contrast with thermodynamic predictions, a new hemihydrate of 4-hydroxynicotinic acid (4HNA·0.5H₂O) did not undergo facile spontaneous dehydration at ambient temperature and pressure. The origin of this robustness and the mechanism of dehydration were investigated in this work, through a combined approach which involved kinetic studies by thermogravimetry (TGA), crystal packing analysis based on X-ray diffraction data, and microscopic observations by hot stage microscopy (HSM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The TGA results indicated that the resilience of 4HNA·0.5H₂O to water loss is indeed of kinetic origin, c.f., due to a significant activation energy, <i>E</i>_a, which increased from 85 kJ·mol^–1 to 133 kJ·mol^–1 with the increase in particle size. This <i>E</i>_a range is compatible with the fact that four moderately strong hydrogen bonds (typically 20–30 kJ·mol^–1 each) must be broken to remove water from the crystal lattice. The dehydration kinetics conforms to the Avrami-Erofeev A2 model, which assumes a nucleation and growth mechanism. Support for a nucleation and growth mechanism was also provided by the HSM, SEM, and AFM observations. These observations further suggested that the reaction involves one-dimensional nucleation, which is rarely observed. Finally, a statistical analysis of Arrhenius plots for samples with different particle sizes revealed an isokinetic relationship between the activation parameters. This is consistent with the fact that the dehydration mechanism is independent of the sample particle size

Formation and Properties of Membrane-Ordered Domains by Phytoceramide: Role of Sphingoid Base Hydroxylation

Phytoceramide is the backbone of major sphingolipids in fungi and plants and is essential in several tissues of animal organisms, such as human skin. Its sphingoid base, phytosphingosine, differs from that usually found in mammals by the addition of a hydroxyl group to the 4-ene, which may be a crucial factor for the different properties of membrane microdomains among those organisms and tissues. Recently, sphingolipid hydroxylation in animal cells emerged as a key feature in several physiopathological processes. Hence, the study of the biophysical properties of phytosphingolipids is also relevant in that context since it helps us to understand the effects of sphingolipid hydroxylation. In this work, binary mixtures of <i>N</i>-stearoyl-phytoceramide (PhyCer) with palmitoyloleoylphosphatidylcholine (POPC) were studied. Steady-state and time-resolved fluorescence of membrane probes, X-ray diffraction, atomic force microscopy, and confocal microscopy were employed. As for other saturated ceramides, highly rigid gel domains start to form with just ∼5 mol % PhyCer at 24 °C. However, PhyCer gel-enriched domains in coexistence with POPC-enriched fluid present additional complexity since their properties (maximal order, shape, and thickness) change at specific POPC/PhyCer molar ratios, suggesting the formation of highly stable stoichiometric complexes with their own properties, distinct from both POPC and PhyCer. A POPC/PhyCer binary phase diagram, supported by the different experimental approaches employed, is proposed with complexes of 3:1 and 1:2 stoichiometries which are stable at least from ∼15 to ∼55 °C. Thus, it provides mechanisms for the in vivo formation of sphingolipid-enriched gel domains that may account for stable membrane compartments and diffusion barriers in eukaryotic cell membranes

Tip-Specific Functionalization of Gold Nanorods for Plasmonic Biosensing: Effect of Linker Chain Length

Gold nanorods are promising platforms for label-free biosensing. We have functionalized gold nanorods with biotin thiol linkers of increasing chain length and evaluated their ability in the molecular detection of streptavidin. We have found an unexpected effect of the increase in linker length, which resulted in a substantial improvement of the plasmon response at surface saturation. The plasmon peak shift increased from 5 to 14 nm, i.e., more than twice the response, between the short and long biotin linkers. This effect is observed only for site-selective tip functionalization, whereas for a full biotin coating there is no improvement observed with the linker length. The improved plasmon response for tip functionalization is attributed to low biotin coverage but is directed to the most sensitive regions, which, combined with a longer chain linker, reduces the steric hindrance for streptavidin binding on the rod’s surface. The model sensors were further characterized by measuring their dose–response curves and binding kinetic assays. Simulations of the discrete dipole approximation give theoretical plasmon shifts that compare well with the experimental ones for the long linker but not with those of the short linker, thus suggesting that steric hindrance affects the latter. Our results highlight the importance of specifically functionalizing the plasmonic hot spots in nanoparticle sensors with the adequate density of receptors in order to maximize their response

Exploiting the Therapeutic Potential of 8‑β‑d‑Glucopyranosylgenistein: Synthesis, Antidiabetic Activity, and Molecular Interaction with Islet Amyloid Polypeptide and Amyloid β‑Peptide (1–42)

8-β-d-Glucopyranosylgenistein (<b>1</b>), the major component of <i>Genista tenera</i>, was synthesized and showed an extensive therapeutical impact in the treatment of STZ-induced diabetic rats, producing normalization of fasting hyperglycemia and amelioration of excessive postprandial glucose excursions and and increasing β-cell sensitivity, insulin secretion, and circulating insulin within 7 days at a dose of 4 (mg/kg bw)/day. Suppression of islet amyloid polypeptide (IAPP) fibril formation by compound <b>1</b> was demonstrated by thioflavin T fluorescence and atomic force microscopy. Molecular recognition studies with IAPP and Aβ_1–42 employing saturation transfer difference (STD) confirmed the same binding mode for both amyloid peptides as suggested by their deduced epitope. Insights into the preferred conformation in the bound state and conformers’ geometry resulting from interaction with Aβ_1–42 were also given by STD, trNOESY, and MM calculations. These studies strongly support 8-β-d-glucopyranosylgenistein as a promising molecular entity for intervention in amyloid events of both diabetes and the frequently associated Alzheimer’s disease