Jorge F. Fernández-Sánchez

Synthesis of caffeic acid molecularly imprinted polymer microspheres and high-performance liquid chromatography evaluation of their sorption properties

In the current work, a molecularly imprinted polymer (MIP) has been synthesised and used to enable the extraction of a naturally-occurring antioxidant from complex media. More specifically, we describe the first example of a caffeic acid (CA) MIP which has been synthesised in the form of well-defined polymer microspheres, and its use for the extraction of CA from fruit juice sample. The CA MIP was synthesised by precipitation polymerisation using 4-vinylpyridine as functional monomer, divinylbenzene-80 as crosslinker and acetonitrile:toluene (75/25, v/v) as porogen. The particle sizing and morphological characterisation of the polymers was carried out by means of scanning electron microscopy (narrow particle size distribution; ∼5 and 1.5 μm particle diameters for the MIP and NIP [non-imprinted polymer], respectively) and nitrogen sorption porosimetry (specific surface areas of 340 and 350 m(2)g(-1), and specific pore volumes of 0.17 and 0.19 cm(3)g(-1) for the MIP and NIP, respectively). The polymers were evaluated further by batch rebinding experiments, and from the derived isotherms their binding capacity and binding strength were determined (number of binding sites (N(K))=0.6 and 0.3 mmol g(-1) for the MIP and NIP, respectively, and apparent average adsorption constant (K(N))=10.0 and 1.6L mmol(-1) for the MIP and NIP, respectively). To evaluate the molecular recognition character of the MIP it was packed into a stainless steel column (50 mm × 4.6 mm i.d.) and evaluated as an HPLC-stationary phase. The mobile phase composition, flow rate, and the elution profile were then optimised in order to improve the peak shape without negatively affecting the imprinting factor (IF). Very interesting, promising properties were revealed. The imprinting factor (IF) under the optimised conditions was 11.9. Finally, when the imprinted LC column was used for the selective recognition of CA over eight related compounds, very good selectivity was obtained. This outcome enabled the direct extraction of CA in commercial apple juice samples with recoveries in excess of 81% and, rather significantly, without any need for a clean-up step prior to the extraction.

Novel luminescent Ir(III) dyes for developing highly sensitive oxygen sensing films

New sensing films have been developed for the detection of molecular oxygen. These films are based on luminescent Ir(III) dyes incorporated either into polystyrene (with and without plasticizer) or metal oxide, nanostructured material. The preparation and characterization of each film have been investigated in detail. Due to their high sensitivity for low oxygen concentration, the parameters p(O2) (S=1/2) and DeltaI(1%) have been also evaluated in order to establish the most sensitive membrane for controlling concentrations between 0 and 10% and low oxygen concentrations (lower than 1%), respectively. The results show that the use of nanostructured material increased the sensitivity of the film; the most sensitive membrane for controlling O(2) between 0 and 10% is based on N1001 immobilized in AP200/19 (k(sv)=2848+/-101 bar(-1) and p(O2) (S=1/2)=0.0006), and the complex N969 incorporated into AP200/19 seems to be the most suitable for applications in oxygen trace sensing (DeltaI(1%)=93.13+/-0.13%).

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.

The development of a MIP-optosensor for the detection of monoamine naphthalenes in drinking water.

To enhance the advantages of fluorescent flow-through sensing for drinking water we have designed a novel sensing matrix based on molecularly imprinted polymers (MIPs). The synergic combination of a tailor-made MIP recognition with a selective room temperature fluorescence detection is a novel concept for optosensing devices and is assessed here for the simple and selective determination of pollutants in water. We describe a simple approach to preparing synthetic receptors for monoamine naphthalene compounds (MA-NCs) using non-covalent molecular imprinting techniques and naphthalene as template. We examine in detail the binding characteristics of the imprinted polymer and describe the flow-through sensor of MA-NCs by solid-surface fluorescence. Its detection limits for recognizing 1-naphthylamine (1-NA) and 2-naphthylamine (2-NA) separately are 26 ngmL(-1) and 50 ngmL(-1), respectively, and it also determines 1-NA and 2-NA simultaneously with a detection limit of 45 ngmL(-1). All the instrumental, chemical and flow variables were carefully optimized and an interference study was carried out to demonstrate its applicability and selectivity. Finally, we applied it to the analysis of 1-NA and 2-NA in tap and mineral waters, obtaining a 98% average recovery rate.

Novel oxygen sensitive complexes for optical oxygen sensing

Novel optical sensing films for oxygen based on highly luminescent iridium (III) and ruthenium (II) complexes have been developed. These demonstrate excellent long-term photostability (several months) when incorporated into polystyrene membranes. The influence of different plasticizers on the specific luminescence quantum yield, the Stern-Volmer constant, the reversibility and the response time were evaluated. Additionally the sensing films can be sterilized by chemical cleaning and gamma-ray irradiation.

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.

Chemometric-assisted MIP-optosensing system for the simultaneous determination of monoamine naphthalenes in drinking waters.

In the present work a chemometric-assisted molecularly imprinted polymer (MIP)-fluorescence optosensing system has been developed for determining monoamines naphthalene compounds in drinking waters. The use of chemometrics for processing flow injection analysis with MIP fluorescence optosensor data allowed the simultaneous determination of the principal monoamine naphthalene compounds 1-naphthylamine (1-NA) and 2-naphthylamine (2-NA) even in presence of potential interferent 1-naphthalenemethylamine (1-NMA). Classical chemometrics tools such as partial least-squares (PLS-1), as well as second-order algorithms like multiway PLS (N-PLS) and unfolded PLS (U-PLS), were successfully applied, assisting fluorescence emission spectra at a fixed excitation wavelength or excitation-emission fluorescence matrices (EEM), respectively, when interferents are considered in the calibration set. The combinations of both N-PLS and U-PLS with residual bilinearization (RBL), achieving the second-order advantage, were satisfactory applied for the simultaneous determination of the main monoamine naphthalene compounds in drinking water, in the presence of a potential interferent without sample pretreatment, even when the later is not modeled in calibration set. Predictive ability, accuracy, figures of merit, as well as advantages and disadvantages of the different strategies were discussed.

A sensitive fluorescence optosensor for analysing propranolol in pharmaceutical preparations and a test for its control in urine in sport

We describe a simple and selective method for analysing propranolol and a sensitive test for its control in urine. A flow-through fluorescence optosensor based on on-line immobilization in a non-ionic-exchanger (Amberlite XAD-7) solid support in a continuous flow was used in both cases. Determination was made in 5 mM H(2)PO(4)(-)/HPO(4)(2-) buffer solution at pH 6 at a working temperature of 20 degrees C. Fluorescence intensities were measured at lambda(ex/em) = 300/338 nm with a response time of 80 s, thus obtaining a linear concentration range of between 0 and 250.0 ng ml(-1) with a detection limit of 1.3 ng ml(-1), an analytical sensitivity of 6.0 ng ml(-1) and a standard deviation of 2.40% at a 150 ng ml(-1) concentration level for propranolol. We also propose a test to detect propranolol in urine with a linear concentration range between 0 and 100.0 ng ml(-1), a detection limit of 0.2 ng ml(-1), an analytical sensitivity of 1.0 ng ml(-1), and a standard deviation of 0.84% at a 75 ng ml(-1) concentration level. The effect of proteins presents in urine samples were evaluated. The two proposed methods were satisfactorily applied to commercial formulations and urine samples respectively.

Direct observation of reversible electronic energy transfer involving an iridium center.

A cyclometalated iridium complex is reported where the core complex comprises naphthylpyridine as the main ligand and the ancillary 2,2′-bipyridine ligand is attached to a pyrene unit by a short alkyl bridge. To obtain the complex with satisfactory purity, it was necessary to modify the standard synthesis (direct reaction of the ancillary ligand with the chloro-bridged iridium dimer) to a method harnessing an intermediate tetramethylheptanolate-based complex, which was subjected to acid-promoted removal of the ancillary ligand and subsequent complexation. The photophysical behavior of the bichromophoric complex and a model complex without the pendant pyrene were studied using steady-state and time-resolved spectroscopies. Reversible electronic energy transfer (REET) is demonstrated, uniquely with an emissive cyclometalated iridium center and an adjacent organic chromophore. After excited-state equilibration is established (5 ns) as a result of REET, extremely long luminescence lifetimes of up to 225 μs result, compared to 8.3 μs for the model complex, without diminishing the emission quantum yield. As a result, remarkably high oxygen sensitivity is observed in both solution and polymeric matrices.

The development of solid-surface fluorescence characterization of polycyclic aromatic hydrocarbons for potential screening tests in environmental samples

This paper presents the characterization of polycyclic aromatic hydrocarbons (PAHs) in solid-surface fluorescence as the first step for obtaining new optical sensors for PAHs screening. The fluorescence properties of the EPA-PAHs (naphthalene, acenaphthene, acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, benzo[k]fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene, indeno [1,2,3-cd]pyrene, benzo[g,h,i]perylene and dibenzo[a,h]anthracene) on five types of solid-surfaces were evaluated. The experimental variables (pH and percentage of organic solvent in samples) were studied, obtaining different possibilities for making individual sensors for some of these PAHs and the best conditions for developing sensors for PAH screening were also studied.