ABSTRACT
The direct interaction of atmospheric pressure non-equilibrium plasmas with tyrosinase (Tyr) was investigated under typical conditions used in surface processing. Specifically, Tyr dry deposits were exposed to dielectric barrier discharges (DBDs) fed with helium, helium/oxygen, and helium/ethylene mixtures, and effects on enzyme functionality were evaluated. First of all, results show that DBDs have a measurable impact on Tyr only when experiments were carried out using very low enzyme amounts. An appreciable decrease in Tyr activity was observed upon exposure to oxygen-containing DBD. Nevertheless, the combined use of X-ray photoelectron spectroscopy and white-light vertical scanning interferometry revealed that, in this reactive environment, Tyr deposits displayed remarkable etching resistance, reasonably conferred by plasma-induced changes in their surface chemical composition as well as by their coffee-ring structure. Ethylene-containing DBDs were used to coat tyrosinase with a hydrocarbon polymer film, in order to obtain its immobilization. In particular, it was found that Tyr activity can be fully retained by properly adjusting thin film deposition conditions. All these findings enlighten a high stability of dry enzymes in various plasma environments and open new opportunities for the use of atmospheric pressure non-equilibrium plasmas in enzyme immobilization strategies.
ABSTRACT
The increase of antimicrobial resistance is challenging the scientific community to find solutions to eradicate bacteria, specifically biofilms. Light-Emitting Diodes (LED) represent an alternative way to tackle this problem in the presence of endogenous or exogenous photosensitizers. This work adds to a growing body of research on photodynamic inactivation using visible light against biofilms. Violet (400 nm), blue (420 nm), green (570 nm), yellow (584 nm) and red (698 nm) LEDs were used against Pseudomonas fluorescens and Staphylococcus epidermidis. Biofilms, grown on a polystyrene surface, were irradiated for 4 h. Different irradiance levels were investigated (2.5%, 25%, 50% and 100% of the maximum irradiance). Surviving cells were quantified and the inactivation kinetic parameters were estimated. Violet light could successfully inactivate P. fluorescens and S. epidermidis (up to 6.80 and 3.69 log10 reduction, respectively), while blue light was effective only against P. fluorescens (100% of maximum irradiance). Green, yellow and red irradiation neither increased nor reduced the biofilm cell density. This is the first research to test five different wavelengths (each with three intensities) in the visible spectrum against Gram-positive and Gram-negative biofilms. It provides a detailed study of the potential of visible light against biofilms of a different Gram-nature.
ABSTRACT
A general method to obtain the efficient entrapment of mixtures of glycoenzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glycoenzymes acting in series (trehalase, glucose oxidase, and horseradish peroxidase) have been coimmobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzyme's aggregation induced by the concanavalin A. The aggregation process has been monitored by dynamic light scattering technique, while both enzyme encapsulation efficiency and leakage have been quantified spectrophotometrically. Obtained data show an encapsulation efficiency above 95% and a negligible leakage from the beads when enzyme aggregates are larger than 300 nm. Operational stability of "as prepared" beads has been largely improved by a coating of alternated shells of polycation poly(diallyldimethylammonium chloride) and of alginate. As a test for the effectiveness of the overall procedure, analytical bioassays exploiting the enzyme-containing beads have been developed for the optical determination of glucose and trehalose, and limit of detection values of 0.2 and of 40 µM, respectively, have been obtained.
