A novel electrochemical platform based on mesoporous silica/titania and gold nanoparticles for simultaneous determination of norepinephrine and dopamine.

An amorphous and mesoporous silica/titania (SiTi) material was synthesized by sol-gel method and its surface was modified with gold nanoparticles (AuNP) previously stabilized in a chitosan solution. The presence of small AuNP, with diameter lower than 10 nm was confirmed by transmission electron microscopy (TEM) and UV-Vis spectroscopy. Carbon paste electrodes were prepared to test the electrochemical properties by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in [Fe(CN)6]3-/4- solution probe whereby the material silica-titania/gold nanoparticles (SiTi/AuNP) showed a huge improvement in the redox peak current and low charge transfer resistance. This electrode presented a good response for both norepinephrine and dopamine by means of square wave voltammetry (SWV) measurements; great sensitivity for both analytes, in an extensive linear range, was obtained. The limits of detection were 0.35 µmol L-1 and 0.57 µmol L-1 for norepinephrine and dopamine, respectively. Additionally, this electrode showed high selectivity for both analytes and it was applied in the simultaneous determination of norepinephrine and dopamine. The sensor was also tested in simulated biological fluids presenting a good recovery. The SWV electrochemical response of norepinephrine was also investigated in the presence of possible interferers such as uric acid, ascorbic acid and glucose and there was no significant interference. The prepared electrode also exhibits good reproducibility for norepinephrine detection, with relative standard deviation of 5.19%.

Designing a Support for Lipase Immobilization Based On Magnetic, Hydrophobic, and Mesoporous Silica.

A mesoporous, magnetic, and hydrophobic material was designed step by step to act as a support for lipase immobilization. Its pore size (8.0 nm) is compatible with the size of lipase from Thermomyces lanuginosus (TLL), and its hydrophobic surface (contact angle of a water drop = 125°) was planned to interact with lipase on its interfacially activated form (open conformation). The presence of magnetite particles provides magnetic retrieval of the material and enables recyclability of the biocatalysts. Regarding immobilization parameters, the hydrophobic support was tested in comparison to the unmodified hydrophilic support in phosphate buffer solution (50 mmol L-1, pH 7.5) at 25 °C. Hydrophobicity was found to be critical for the amount of immobilized TLL (immobilization yield of 97% versus 36% for the hydrophilic support), whereas the hydrophilic support favors the native conformational state and substrate access to the enzyme's catalytic site (specific activity of 5.7 versus 4.7 U g-1 for the hydrophobic support, even when it has higher TLL content). Therefore, the hydrophobic support immobilizes higher amounts of TLL and the hydrophilic support keeps the enzyme hyperactivated. Last, due to the stronger interactions of TLL with hydrophobic surfaces, the hydrophobic support offers better preservation of enzyme activity in repeated cycles (76% of activity retained after three cycles versus 50% for the hydrophilic support).

Immobilization of pectinase on chitosan-magnetic particles: Influence of particle preparation protocol on enzyme properties for fruit juice clarification.

Magnetic-chitosan particles were prepared following three different protocols enabling the preparation of particles with different sizes - nano (Nano-CMag, Micro (Micro-CMag) and Macro (Macro-CMag) - and used for pectinase immobilization and clarification of grape, apple and orange juices. The particle size had a great effect in the kinetic parameters, Nano-CMag biocatalyst presented the highest Vmax value (78.95 mg. min-1), followed by Micro-CMag and Macro-CMag, with Vmax of 57.20 mg.min-1 and 46.03 mg.min-1, respectively. However, the highest thermal stability was achieved using Macro-CMag, that was 8 and 3-times more stable than Nano-CMag and Micro-CMag biocatalysts, respectively. Pectinase immobilized on Macro-CMag kept 85% of its initial activity after 25 batch cycles in orange juice clarification. These results suggested that the chitosan magnetic biocatalysts presented great potential application as clarifying catalysts for the fruit juice industry and the great importance of the chitosan particles preparation on the final biocatalyst properties.

Magnetic biocatalysts of pectinase and cellulase: Synthesis and characterization of two preparations for application in grape juice clarification.

In the present study, we prepared two different magnetic biocatalysts of pectinase and cellulase: carrier-free magnetic CLEAs (CLEA-MP*) and immobilization on glutaraldehyde-activated magnetite (Enz-Glu-MP*). The biocatalysts were compared to their magnetic properties, immobilization parameters, stability and grape juice clarification. Enz-Glu-MP* presented higher magnetic properties than CLEA-MP*, whereas this presented higher surface area and pore volume. The KM of the enzyme immobilized on Enz-Glu-MP* was 25.65mM, lower in comparison to the CLEA-MP* (33.83mM). On the other hand, CLEA-MP* was the most active and stable biocatalyst, presenting higher recovered activity (33.4% of cellulase), higher thermal stability (2.39 stabilization factor) and improved reusability (8cycles). The integration of magnetic technology with enzymatic immobilization emerges as a possibility to increase the recover and reuse of biocatalysts for application in juice technology.

Highly stable novel silica/chitosan support for ß-galactosidase immobilization for application in dairy technology.

ß-d-Galactosidase is an important enzyme in the dairy industry, and the enzyme from the yeast Kluyveromyces lactis is most widely used. Here, we report immobilization of the enzyme on a silica/chitosan composite support, devised to have 10% and 20% chitosan (SiQT10 and SiQT20, respectively). Morphological and textural characterizations showed that chitosan is dispersed in micrometric regions in silica. For comparison, a silica organofunctionalized with 3-aminopropyltrimethoxysilane (SiO2aptms) was prepared. Performance of the biocatalysts was tested for lactose hydrolysis, and the enzyme immobilized in SiQT10 and SiQT20 composites showed higher efficiency (62% and 47%, respectively) compared with the enzyme in SiO2aptms. Operational stability in this system was evaluated for the first time. After 200 h of continuous use in a fixed-bed reactor, SiQT10 remained with approximately 90% activity. Thus, in addition to demonstrating compatibility for food processing, these results align the enzyme stabilization properties of chitosan with the mechanical resistance of silica.

Effects of immobilization, pH and reaction time in the modulation of α-, ß- or γ-cyclodextrins production by cyclodextrin glycosyltransferase: Batch and continuous process.

This study reports the immobilization of a ß-CGTase on glutaraldehyde pre-activated silica and its use to production of cyclodextrins in batch and continuous reactions. We were able to modulate the cyclodextrin production (α-, ß- and γ-CD) by immobilization and changing the reaction conditions. In batch reactions, the immobilized enzyme reached to maximum productions of 4.9mgmL-1 of α-CD, 3.6mgmL-1 of ß-CD and 3.5mgmL-1 of γ-CD at different conditions of temperature, pH and reaction time. In continuous reactor, varying the residence time and pH it was possible to produce at pH 4.0 and 141min of residence time preferentially γ-CD (0.75 and 3.36mgmL-1 of α- and γ-CD, respectively), or at pH 8.0 and 4.81min α- and ß-CDs (3.44 and 3.51mgmL-1).

Palladium(II) chemically bonded to silica surface applied to the separation and identification of polycyclic aromatic sulfur heterocycles in heavy oil.

Separation of polycyclic aromatic sulfur heterocycles among themselves and also from interferents in petrochemical matrices is a challenging task because of their low concentration, matrix complexity, and also due to the presence of polyaromatic hydrocarbons, as they present similar physico-chemical properties. Therefore, the objective of this work was preparation, characterization, and application of a stationary phase for separation of these compounds in a heavy gas oil sample and their identification by comprehensive two-dimensional gas chromatography. The stationary phase was prepared by grafting mercaptopropyltrimethoxisilane onto a silica surface, followed by palladium(II) chloride immobilization. Elemental analysis, thermogravimetry, nitrogen adsorption-desorption isotherms, infrared analysis, and scanning electron microscopy were performed to characterize this solid phase. Sulfur compounds were separated in an open column packed with the stationary phase and analyzed by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometric detection. The number of compounds tentatively identified was 314 and their classes were thiophenes, benzotiophenes, dibenzothiophenes, naphthothiophenes, benzonaphthothiophenes, and dinaphthothiophenes. Separation among sulfur compounds and polyaromatic hydrocarbons was successful, which is a difficult goal to achieve with the traditionally employed solid phases. Some recalcitrant compounds (dibenzothiophenes with substituents of two and four carbons) were fully separated and tentatively identified.

Comparison between pre-fractionation and fractionation process of heavy gas oil for determination of sulfur compounds using comprehensive two-dimensional gas chromatography.

The separation of the organic sulfur compounds (OSC) of petroleum or its heavy fractions is a critical step and is essential for the correct characterization of these compounds, especially due to similar physical and chemical properties of polycyclic aromatic sulfur heterocycles (PASH) and polycyclic aromatic hydrocarbons (PAH). This similarity results in coelutions among PAH and PASH and for this reason former steps of fractionation are required before gas chromatographic analysis. The objective of this study was to evaluate the potential of GC×GC for the separation and identification of OSC in a heavy gas oil sample without fractionation, after pre-fractionation in an alumina column and also after fractionation process. This last one was performed with a modified stationary phase manufactured and characterized in the laboratory, called Pd(II)-MPSG, where palladium is chemically linked to silica through mercaptopropyl groups. The fractions obtained from both procedures were analyzed by GC×GC/TOFMS, which was effective to separate and identify various classes of OSC. A hundred and thirty-five compounds were tentatively identified in the sample that was only pre-fractionated. However, when the fractionation was also performed with the Pd(II)-MPSG phase, a larger number of sulfur compounds were found (317). Results have shown that the analysis of a pre-fractionated sample by GC×GC/TOFMS is suitable when the goal is a general characterization of classes of compounds in the sample, while a more detailed analysis of PASH can be performed, using also the fractionation Pd(II)-MPSG phase. GC×GC/TOFMS played a major role in the comparison of samples obtained from pre-fractionation and fractionation steps due to its high peak capacity, selectivity, organized distribution of chromatographic peaks and resolution.