Antonio L. Medina-Castillo

Characterization of supports activated with divinyl sulfone as a tool to immobilize and stabilize enzymes via multipoint covalent attachment. Application to chymotrypsin

Divinyl sulfone (DVS) has been used to activate agarose beads. The DVS activated agarose resulted quite stable in the pH range 5–10 at 25 °C under wet conditions, and can react rapidly with α-amides of Cys and His, at pH 5–10, with Lys mainly at pH 10 and with Tyr in a much slower fashion. After blocking with different nucleophiles, the support lost all reactivity, confirming that this protocol could be useful as an enzyme–support reaction end point. Then, chymotrypsin was immobilized on this support at pH 5, 7 and 10. Even though the enzyme was immobilized at all pH values, the immobilization rate decreased with the pH value. The effect of the immobilization on the activity depended on the immobilization pH, at pH 7 the activity decreased (to 50%) more than at pH 10 (by a 25%), while at pH 5 the immobilization has no effect. Then, the effect of blocking with different reagents was analyzed. It was found that blocking with ethylenediamine improved the enzyme activity by 70% and gave the best stability. The stability of all enzyme preparations improved when 24 h incubation was performed at pH 10, but the qualitative stabilization depended on the inactivation conditions. The analysis of the amino acids of the preparation immobilized at pH 10 showed that Lys, Tyr and Cys residues were involved in the immobilization, involving a minimum of 10 residues (glyoxyl agarose gave 4 Lys involved in the immobilization). The new preparation was 4–5 fold more stable than glyoxyl agarose preparation, considered a very stable one, and in some instances was more active than the free enzyme (170% for the enzyme immobilized at pH 10). Thus, DVS activated supports are very promising to permit the multipoint covalent attachment of enzymes, and that way to improve their stability.

Synthesis of a novel polyurethane-based-magnetic imprinted polymer for the selective optical detection of 1-naphthylamine in drinking water

The first polyurethane based magnetic-MIP for the selective detection of 1-naphthylamine (1-NA) in drinking water has been synthesised. The synthesis has been carried out in a two-step process: first,the incorporation of magnetite-coated-oleic acid nanoparticles (-Fe₃O₄-OA) into a lipophilic polymeric matrix (poly-MMA-co-EDMA) and second, the encapsulation of these magnetic seeds into the MIP structure by precipitation polymerisation. The mag-MIP was first RHTEM imaged showing a well-organised material with magnetite within the material and the imprinted polymer coating the magnetic core. Thereafter,it was evaluated by batch rebinding analysis and the derived Freundlich isotherm, calculating the number of binding sites (N(K(min)-K(max))=2.63 and 0.79 mmol g⁻¹, for mag-MIP and mag-NIP, respectively)and apparent average adsorption constant (K(K(min)-K(max))=3.31 and 3.06 mmol⁻¹, for mag-MIP and mag-NIP, respectively) showing a very effective imprinting process.We have also developed a magnetic optical sensor MIP by using an optical fiber coupled with a magnetic separator. An unexpected selectivity for 1-NA was revealed allowing the detection of this molecule in water, even in the presence of 4 structurally related compounds (2-naphthylamine, 1-naphthol, 2-naphthol and 1-naphthalenemethylamine), with a low limit of detection (LOD) = 18 ng mL⁻¹. Finally, we applied this new hybrid material to the analysis of 1-NA in tap and mineral waters, obtaining a 91.6%average recovery rate.

Polymer Nanocarriers for Dentin Adhesion

To obtain more durable adhesion to dentin, and to protect collagen fibrils of the dentin matrix from degradation, calcium- and phosphate-releasing particles have been incorporated into the dental adhesive procedure. The aim of the present study was to incorporate zinc-loaded polymeric nanocarriers into a dental adhesive system to facilitate inhibition of matrix metalloproteinases (MMPs)-mediated collagen degradation and to provide calcium ions for mineral deposition within the resin-dentin bonded interface. PolymP- n Active nanoparticles (nanoMyP) were zinc-loaded through 30-minute ZnCl2 immersion and tested for bioactivity by means of 7 days’ immersion in simulated body fluid solution (the Kokubo test). Zinc-loading and calcium phosphate depositions were examined by scanning and transmission electron microscopy, elemental analysis, and x-ray diffraction. Nanoparticles in ethanol solution infiltrated into phosphoric-acid-etched human dentin and Single Bond (3M/ESPE) were applied to determine whether the nanoparticles interfered with bonding. Debonded sticks were analyzed by scanning electron microscopy. A metalloproteinase collagen degradation assay was also performed in resin-infiltrated dentin with and without nanoparticles, measuring C-terminal telopeptide of type I collagen (ICTP) concentration in supernatants, after 4 wk of immersion in artificial saliva. Numerical data were analyzed by analysis of variance (ANOVA) and Student-Newman-Keuls multiple comparisons tests (p < .05). Nanoparticles were effectively zinc-loaded and were shown to have a chelating effect, retaining calcium regardless of zinc incorporation. Nanoparticles failed to infiltrate demineralized intertubular dentin and remained on top of the hybrid layer, without altering bond strength. Calcium and phosphorus were found covering nanoparticles at the hybrid layer, after 24 h. Nanoparticle application in etched dentin also reduced MMP-mediated collagen degradation. Tested nanoparticles may be incorporated into dental adhesive systems to provide the appropriate environment in which dentin MMP collagen degradation is inhibited and mineral growth can occur.

Design and synthesis by ATRP of novel, water-insoluble, lineal copolymers and their application in the development of fluorescent and pH-sensing nanofibres made by electrospinning

In this work, reverse Atom Transfer Radical Polymerization (ATRP) was used for synthesising novel water-insoluble lineal copolymers which were used for designing new fluorescent and pH-sensitive, nanostructured films based on nanofibre mats made by electrospinning. The copolymers are based on the copolymerization of fluorescein o-acrylate (fluorescent pH-sensitive monomer) with methyl methacrylate and hydroxyl ethyl methacrylate (principal framework of the copolymers) and 3-methacryloylaminopropyl-trimethylammonium chloride (positively charged monomer) or 2-acrylamido-2-methylpropane sulfonic acid (negatively charged monomer). The electrostatic properties of these copolymers were investigated, and perfectly adjusted to maintain the complete insolubility of these materials in aqueous media. The copolymers were also characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (1H-NMR) spectroscopy and by triple detection gel permeation chromatography (GPC). The synthesised copolymers are soluble in organic solvents such as tetrahydrofuran, acetone, and dimethylformamide. These facts make them highly versatile and very good candidates for the preparation of novel nanostructured, optical pH-sensitive sensing phases produced by different techniques such as electrospinning, spin coating, deep coating, spray dry, spray coating, etc., and with different structures, morphologies, and geometries for many different applications. To demonstrate these claims, we used these copolymers for providing highly fluorescent pH-sensitive nanofibres made by electrospinning. The selected flow rates and voltages of the electrospinning configuration allowed the collection of dry fibres in nonwoven mats. The nanofibres were characterised by scanning electron microscopy (SEM) and fluorescence microscopy and the fibre mats were successfully used for pH-monitoring in two different pH ranges (6.5–8 and 8–10) showing reversibility, high sensitive and low response times.

Novel optical sensing film based on a functional nonwoven nanofibre mat for an easy, fast and highly selective and sensitive detection of tryptamine in beer

In this paper, the combination of Solid Surface-Room Temperature Phosphorescence (SS-RTP) and nanotechnology has led to a new approach in the detection of biogenic amines in complex matrices. This novel approach allows, for the first time, the direct determination of the concentration of tryptamine in beers. The novelty of the proposed optical sensor resides in its simplicity, rapidity, absence of complex chromatographic separation, sample clean-up, preconcentration, and derivatization protocols. Therefore, this novel methodology simplifies and reduces considerably the time and cost of the analysis, resolving the two major problems of the determination of tryptamine in beer up to now: low sensitivity and matrix effects. The proposed sensor is based on a novel white, uncharged, and non-luminescent functional nonwoven nanofibre mat (Tiss®-Link) formed by hydrophilic nanofibres of 300 nm of diameter functionalized with a high concentration of active vinyl groups (330 µmol g(-1)). It is used to carry out a kinetically controlled covalent immobilisation of tryptamine via Michael type-reaction. The transduction of the sensor is phosphorescence; the covalently immobilized tryptamine is quantified by SS-RTP, obtaining a detection limit of 6 ng mL(-1) with short response times (15 min). The applicability of the sensor was demonstrated by analysing tryptamine in 10 different varieties of beers, obtaining recovery percentages close to 100%.

A semi-empirical model to simplify the synthesis of homogeneous and transparent cross-linked polymers and their application in the preparation of optical sensing films

We propose a simple, semi-empirical model based on Hansens solubility parameters for simplifying the synthesis and the optimization of homogeneous and transparent cross-linked polymers in order to obtain optical sensing films. More than 740 experiments were undertaken to demonstrate the reliability of the model and several applications are proposed. We have demonstrated that our model can help in the synthesis and optimization (percentage of cross-linker, changes in hydrophilicity, selection of porogens, quantity of template etc.) of homogeneous and transparent MIPs and NIPs (molecularly imprinted polymers) with VOCs; after the synthesis of 440 polymers in the homogeneity zone only 4.32% of them (19 samples out of 440) were heterogeneous. We suggest a role for its use in the development of novel polymeric resins for detecting volatile organic compounds in water by measuring intrinsic fluorescence, in simplifying the synthesis of imprinted polymers and in decreasing the number of experiments required to optimize optical sensing membranes. In addition, it might also be used for synthesizing and optimizing MIPs with a non-volatile template.

Synthesis and characterization of a molecularly imprinted polymer optosensor for TEXs-screening in drinking water

A molecularly imprinted polymer (MIP) was synthesized using toluene as template, and was implemented in a fluorescence optosensor (λ(exc)=260 nm, λ(em)=284 nm) for the screening of TEXs (toluene, ethylbenzene and xylenes) in drinking water. All the parameters which can affect the sensitivity and selectivity of the optical sensing phase, were carefully optimized. The screening test runs without the need for any pre-concentration step, thus rendering it suitable for routine use in water-quality-control laboratories. The test recognizes contaminated samples rapidly (81 s) and inexpensively with a cut-off level of 700 μg L(-1) ethylbenzene which corresponds with the maximum contaminant level (MCL) established by US Environmental Protection Agency (EPA) in drinking water. The threshold value of the screening test for the cut-off level was 8.27±0.57 a.u. (95% confidence level, n=10). The reliability of the screening test was 32% false positives and 0% false negatives for 50 samples, and its applicability has been demonstrated by analyzing 15 samples of mineral, tap and river waters obtaining 0% false negatives.

Ions-modified nanoparticles affect functional remineralization and energy dissipation through the resin-dentin interface

The aim of this study was to evaluate changes in the mechanical and chemical behavior, and bonding ability at dentin interfaces infiltrated with polymeric nanoparticlesstandard deviations and modes of failure are (NPs) prior to resin application. Dentin surfaces were treated with 37% phosphoric acid followed by application of an ethanol suspension of NPs, Zn-NPs or Ca-NPs followed by the application of an adhesive, Single Bond (SB). Bonded interfaces were stored for 24h, submitted to microtensile bond strength test, and evaluated by scanning electron microscopy. After 24h and 21 d of storage, the whole resin-dentin interface adhesive was evaluated using a Nano-DMA. Complex modulus, storage modulus and tan delta (δ) were assessed. AFM imaging and Raman analysis were performed. Bond strength was not affected by NPs infiltration. After 21 d of storage, tan δ generally decreased at Zn-NPs/resin-dentin interface, and augmented when Ca-NPs or non-doped NPs were used. When both Zn-NPs and Ca-NPs were employed, the storage modulus and complex modulus decreased, though both moduli increased at the adhesive and at peritubular dentin after Zn-NPs infiltration. The phosphate and the carbonate peaks, and carbonate substitution, augmented more at interfaces promoted with Ca-NPs than with Zn-NPs after 21 d of storage, but crystallinity did not differ at created interfaces with both ions-doped NPs. Crosslinking of collagen and the secondary structure of collagen improved with Zn-NPs resin-dentin infiltration. Ca-NPs-resin dentin infiltration produced a favorable dissipation of energy with minimal stress concentration trough the crystalline remineralized resin-dentin interface, causing minor damage at this structure.

Bioactive Polymeric Nanoparticles for Periodontal Therapy

Aims to design calcium and zinc-loaded bioactive and cytocompatible nanoparticles for the treatment of periodontal disease. Methods PolymP-nActive nanoparticles were zinc or calcium loaded. Biomimetic calcium phosphate precipitation on polymeric particles was assessed after 7 days immersion in simulated body fluid, by scanning electron microscopy attached to an energy dispersive analysis system. Amorphous mineral deposition was probed by X-ray diffraction. Cell viability analysis was performed using oral mucosa fibroblasts by: 1) quantifying the liberated deoxyribonucleic acid from dead cells, 2) detecting the amount of lactate dehydrogenase enzyme released by cells with damaged membranes, and 3) by examining the cytoplasmic esterase function and cell membranes integrity with a fluorescence-based method using the Live/Dead commercial kit. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests. Results Precipitation of calcium and phosphate on the nanoparticles surfaces was observed in calcium-loaded nanoparticles. Non-loaded nanoparticles were found to be non-toxic in all the assays, calcium and zinc-loaded particles presented a dose dependent but very low cytotoxic effect. Conclusions The ability of calcium-loaded nanoparticles to promote precipitation of calcium phosphate deposits, together with their observed non-toxicity may offer new strategies for periodontal disease treatment.

A novel optical biosensor for direct and selective determination of serotonin in serum by Solid Surface-Room Temperature Phosphorescence

This paper describes a novel biosensor which combines the use of nanotechnology (non-woven nanofibre mat) with Solid Surface-Room Temperature Phosphorescence (SS-RTP) measurement for the determination of serotonin in human serum. The developed biosensor is simple and can be directly applied in serum; only requires a simple clean-up protocol. Therefore it is the first time that serotonin is analysed directly in serum with a non-enzymatic technique. This new approach is based on the covalent immobilization of serotonin directly from serum on a functional nanofibre material (Tiss®-Link) with a preactivated surface for direct covalent immobilization of primary and secondary amines, and the subsequent measurement of serotonin phosphorescent emission from the solid surface. The phosphorescent detection allows avoiding the interference from any fluorescence emission or scattering light from any molecule present in the serum sample which can be also immobilised on the nanofibre material. The determination of serotonin with this SS-RTP sensor overcomes some limitations, such as large interference from the matrix and high cost and complexity of many of the methods widely used for serotonin analysis. The potential applicability of the sensor in the clinical diagnosis was demonstrated by analysing serum samples from seven healthy volunteers. The method was validated with an external reference laboratory, obtaining a correlation coefficient of 0.997 which indicates excellent correlation between the two methods.