In this study, 3D printing technology was exploited for the development of immobilized enzyme microreactors that could be used for biocatalytic processes in Deep Eutectic Solvent (DES)-based media. 3D-printed polylactic acid (PLA) microwell plates or tubular microfluidic reactors were modified with polyethylenimine (PEI) and lipase from Candida antarctica (CALB) was covalently immobilized in the interior of each structure. DESs were found to have a negligible effect on the activity and stability of CALB, and the system proved highly stable and reusable in the presence of DESs for the hydrolysis of p-nitrophenyl butyrate (p-NPB). A kinetic study under flow conditions revealed an enhancement of substrate accessibility in the presence of Betaine: Glycerol (Bet:Gly) DES, while the system was not severely affected by diffusion limitations. Incubation of microreactors in 100% Bet:Gly preserved the enzyme activity by 53% for 30 days of storage at 60 °C, while the buffer-stored sample had already been deactivated. The microfluidic enzyme reactor was efficiently used for the trans-esterification of ethyl ferulate (EF) with glycerol towards the production of glyceryl ferulate (GF), known for its antioxidant potential. The biocatalytic process under continuous flow conditions exhibited 23 times higher productivity than the batch reaction system. This study featured an effective and robust biocatalytic system with immobilized lipase that can be used both in hydrolytic and synthetic applications, while further optimization is expected to upgrade the microreactor system performance.

Nowadays, biosensors are gaining increasing interest in foods’ and beverages’ quality control, owing to their economic production, enhanced sensitivity, specificity, and faster analysis. In particular, colorimetric biosensors can be combined with color recognition applications on smartphones for the detection of analytes, rendering the whole procedure more applicable in everyday life. Herein, chitosan (CS) films were prepared with the deep eutectic solvent (DES) choline chloride/urea/glycerol (ChCl:U:Gly). Glucose oxidase (GOx), a widely utilized enzyme in quality control, was immobilized within CS films through glutaraldehyde (GA), leading to the formation of CS/GOx films. The optimized GOx concentration and DES content were determined for the films. Moreover, the effect of the pH and temperature of the glucose oxidation reaction on the enzymatic activity of GOx was studied. The structure, stability, and specificity of the CS/GOx films as well as the Km values of free and immobilized GOx were also determined. Finally, the analytical performance of the films was studied by using both a spectrophotometer and a color recognition application on a smartphone. The results demonstrated that the films were highly accurate, specific to glucose, and stable when stored at 4 °C for 4 weeks and when reused 10 times, without evident activity loss. Furthermore, the films displayed a good linear response range (0.1–0.8 mM) and a good limit of detection (LOD, 33 μM), thus being appropriate for the estimation of glucose concentration in real samples through a smartphone application.

In the field of biocatalysis, the implementation of sustainable processes such as enzyme immobilization or employment of environmentally friendly solvents, like Deep Eutectic Solvents (DESs) are of paramount importance. In this work, tyrosinase was extracted from fresh mushrooms and used in a carrier-free immobilization towards the preparation of both non-magnetic and magnetic cross-linked enzyme aggregates (CLEAs). The prepared biocatalyst was characterized and the biocatalytic and structural traits of free tyrosinase and tyrosinase magnetic CLEAs (mCLEAs) were evaluated in numerous DES aqueous solutions. The results showed that the nature and the concentration of the DESs used as co-solvents significantly affected the catalytic activity and stability of tyrosinase, while the immobilization enhanced the activity of the enzyme in comparison with the non-immobilized enzyme up to 3.6-fold. The biocatalyst retained the 100% of its initial activity after storage at −20 °C for 1 year and the 90% of its activity after 5 repeated cycles. Tyrosinase mCLEAs were further applied in the homogeneous modification of chitosan with caffeic acid in the presence of DES. The biocatalyst demonstrated great ability in the functionalization of chitosan with caffeic acid in the presence of 10% v/v DES [Bet:Gly (1:3)], enhancing the antioxidant activity of the films.

In the present work, methanolic extracts from thyme and dittany plants were prepared and characterized in terms of their polyphenolic content through analytical and spectrophotometric techniques. Rosmarinic acid, thymol and carvacrol were found to be the main components of the extracts, which were further biologically assessed for their antioxidant, anti-tyrosinase, anti-lipase and antibacterial activity against Gram-negative and Gram-positive bacteria. As found, thyme extracts exhibited superior antioxidant activity (SC₅₀ at 33.9 μg mL⁻¹), while dittany extracts inhibited the microbial growth to a great extent against Bacillus subtilis strain (MIC at 0.5 mg mL⁻¹) and E. coli strain (MIC at 2 mg mL⁻¹). Furthermore, the thyme extract was proven to strongly inhibit the activity of lipase from Candida rugosa (IC₅₀ at 63.9 μg mL⁻¹), comparable to the standard inhibitor orlistat, while its inhibitory effect against mushroom tyrosinase was weak. On the other hand, the dittany extract presented an inhibitory effect against the tested lipase (IC₅₀ over 500 μg mL⁻¹) and an activation effect against tyrosinase (at concentrations > 500 μg mL⁻¹). Additionally, molecular docking studies of the main compounds of the extracts showed that rosmarinic acid plays a crucial role on the inhibitory activity of the extracts against lipase, while thymol has a stronger effect on inhibiting tyrosinase. Furthermore, both extracts were employed in the preparation of gelatin-deep eutectic solvent (DES) hydrogels that were further studied for their antioxidant and antibacterial activity. The results showed that the incorporation of the extracts offered antibacterial properties to the biopolymer-based hydrogels and enhanced the antioxidant activity of gelatin up to 85%.

Biosensors play a central role in the early detection of abnormal glucose levels in individuals with diabetes; therefore, the development of less invasive systems is essential. Herein, a 3D-printed colorimetric biosensor combining microneedles and chitosan nanoparticles was developed for glucose detection in sweat using machine learning. Briefly, hollow 3D-printed polylactic acid microneedles were constructed and loaded with chitosan nanoparticles encapsulating glucose oxidase, horseradish peroxidase, and the chromogenic substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), resulting in the formation of the chitosan nanoparticle−microneedle patches. Glucose detection was performed colorimetrically by first incubating the chitosan nanoparticle−microneedle patches with glucose samples of varying concentrations and then by using photographs of the top side of each microneedle and a color recognition application on a smartphone. The Random Sample Consensus algorithm was used to train a simple linear regression model to predict glucose concentrations in unknown samples. The developed biosensor system exhibited a good linear response range toward glucose (0.025−0.375 mM), a low limit of detection (0.023 mM), a limit of quantification (0.078 mM), high specificity, and recovery rates ranging between 86–112%. Lastly, the biosensor was applied to glucose detection in spiked artificial sweat samples, confirming the potential of the proposed methodology for glucose detection in real samples.

Biocatalytic processes for the formation of bioactive compounds and biopolymer preparations that can be applied in pharmaceuticals and cosmetics are gaining increasing interest due to their safety and sustainability, relying on environmentally friendly approaches and biocompatible compounds. In this work, we investigate the implementation of various Deep Eutectic Solvents (DES) in the laccase-catalyzed oxidation of hydroxytyrosol (HT), aiming to produce its oligomer derivatives such as HT dimer and trimer. The composition of the reaction mixture in which the oligomers’ yield was the highest was 70% v/v Bet:PG (1:4 molar ratio). The oligomers formed were subsequently used for the non-enzymatic grafting of chitosan (CS) and the development of bioactive chitosan-based nanogels (NG). Grafted chitosan nanogels were prepared by ionic gelation using sodium tripolyphosphate (TPP) as a cross-linking agent. The functionalized chitosan was characterized using Fourier-Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy, while Scanning Electron Microscopy (SEM) was employed for nanogel characterization. Compared to unmodified chitosan nanogels, grafted chitosan nanogels exhibited almost ten-fold higher antioxidant activity and approximately 20% greater antibacterial activity.