A peptide-based coating toolbox to enable click chemistry on polymers, metals, and silicon through sortagging.

A versatile peptide-based toolbox for surface functionalization was established by a combination of a universal material binding peptide (LCI-anchor peptide) and sortase-mediated bioconjugation (sortagging). This toolbox facilitates surface functionalization either as a one- or a two-step strategy. In the case of the one-step strategy, the desired functionality was directly introduced to LCI. For the two-step strategy, LCI was modified with a reactive group, which can be further functionalized (e.g., employing "click" chemistry). Sortagging of LCI, employing sortase A from Staphylococcus aureus, was achieved with six different amine compounds: dibenzocyclooctyne amine, biotin-polyethylene glycol amine, Cyanine-3 amine, kanamycin, methoxypolyethylene glycol amine (Mn = 5000 Da), and 2,2,3,3,4,4,4-Heptafluorobutylamine. The purification of LCI-amine sortagging products was performed by a negative purification using Strep-tag II affinity chromatography, resulting in LCI-amine conjugates with purities >90%. For the two-step strategy, the LCI-dibenzocyclooctyne sortagging product was purified and enabled, through copper-free azide-alkyne "click" chemistry, universal surface functionalization of material surfaces such as polypropylene, polyethylene terephthalate, stainless steel, gold, and silicon. The click reaction was performed before or after surface binding of LCI-dibenzocyclooctyne. Finally, in the case of the one-step strategy, polypropylene was directly functionalized with Cyanine-3 and biotin-polyethylene glycol amine.

Rapid and Robust Coating Method to Render Polydimethylsiloxane Surfaces Cell-Adhesive.

Polydimethylsiloxane (PDMS) is a synthetic material with excellent properties for biomedical applications because of its easy fabrication method, high flexibility, permeability to oxygen, transparency, and potential to produce high-resolution structures in the case of lithography. However, PDMS needs to be modified to support homogeneous cell attachments and spreading. Even though many physical and chemical methods, like plasma treatment or extracellular matrix coatings, have been developed over the last decades to increase cell-surface interactions, these methods are still very time-consuming, often not efficient enough, complex, and can require several treatment steps. To overcome these issues, we present a novel, robust, and fast one-step PDMS coating method using engineered anchor peptides fused to the cell-adhesive peptide sequence (glycine-arginine-glycine-aspartate-serine, GRGDS). The anchor peptide attaches to the PDMS surface predominantly by hydrophobic interactions by simply dipping PDMS in a solution containing the anchor peptide, presenting the GRGDS sequence on the surface available for cell adhesion. The binding performance and kinetics of the anchor peptide to PDMS are characterized, and the coatings are optimized for efficient cell attachment of fibroblasts and endothelial cells. Additionally, the applicability is proven using PDMS-based directional nanotopographic gradients, showing a lower threshold of 5 µm wrinkles for fibroblast alignment.

Citrate and phosphate influence on green fluorescent protein thermal stability.

Protein structure and function can be regulated by no specific interactions, such as ionic interactions in the presence of salts. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. The aim of this study was to evaluate the thermal stability of GFP in the presence of different salts at several concentrations and exposed to constant temperatures, in a range of 70-95°C. Thermal stability was expressed in decimal reduction time. It was observed that the D-values obtained were higher in the presence of citrate and phosphate, when compared with that obtained in their absence, indicating that these salts stabilized the protein against thermal denaturation.

Effect of polyethylene glycol on the thermal stability of green fluorescent protein.

Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Some polymers, such as polyethylene glycol, are often used as modifiers of characteristics of biological macromolecules, to improve the biochemical activity and stability of proteins or drug bioavailability. The aim of this study was to evaluate the thermal stability of GFP in the presence of different PEG molar weights at several concentrations and exposed to constant temperatures, in a range of 70-95 degrees C. Thermal stability was expressed in decimal reduction time. It was observed that the D-values obtained were almost constant for temperatures of 85, 90, and 95 degrees C, despite the PEG concentration or molar weight studied. Even though PEG can stabilize proteins, only at 75 degrees C, PEG 600 and 4,000 g/mol stabilized GFP.