Selective recognition and imaging of bacterial model membranes over mammalian ones by using cationic conjugated polyelectrolytes.

The development of new tools for the detection and fluorescence imaging of bacteria is of great interest in clinical diagnosis and food and environmental safety. In this work, we have explored the ability of two cationic fluorene-based conjugated polyelectrolytes, HTMA-PFP and HTMA-PFNT, emitting in the blue and red spectral regions respectively, to selectively label bacterial over mammalian cells. With this end in view, vesicles with lipid compositions mimicking those of bacterial or mammalian membranes were used as model membranes to explore the interaction of the polyelectrolytes with both systems in samples containing either a single type of vesicle or a mixture of both. Changes in the intrinsic fluorescence of HTMA-PFP and HTMA-PFNT were used to quantify the affinity of these polyelectrolytes for the model lipid membranes, while quenching experiments were employed to evaluate their selectivity to each lipid system. In addition, fluorescence microscopy experiments were performed to check the ability of polyelectrolytes to label the vesicles without affecting their integrity. Results showed that both polyelectrolytes rapidly label the model vesicles but they preferentially bind to those mimicking bacterial membranes, HTMA-PFNT being much more selective to this type of membranes than HTMA-PFP. Preliminary experiments with living bacteria and mammalian cells support this conclusion, showing that in samples with both types of cells together, HTMA-PFNT only images the bacterial cells, thus evidencing its potential use for the selective recognition and imaging of bacterial presence.

Synthesis of a new fluorescent conjugated polymer microsphere for chemical sensing in aqueous media.

A novel conjugated polymer microsphere of high value for fluorescent sensing in aqueous media has been synthesized. New conjugated polymers were functionalized in the side chain with imidazole moieties (recognition element) and a terminal double bond (covalently linked to an organic matrix) through a post-functionalization strategy.

Development of a dual-analyte fluorescent sensor for the determination of bioactive nitrite and selenite in water samples.

Nitrite and selenium are two bioactive compounds found in the environment which show beneficial effects for health at low levels but have toxic effects at higher doses. Consequently, quantification of both analytes in water samples results of great interest in areas such as biomedicine, food technology and environmental analysis. In a recent paper, we immobilized the inclusion complex formed between 2,3-diaminonaphthalene (DAN) and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) in a sol-gel matrix, in order to prepare a highly sensitive reagentless fluorescence-based sensor for the specific measurement of nitrite. Here we have explored the possibility of using the sol-gel immobilized complex to quantify selenite (Se (IV)), the more toxic form of selenium, as well as to act as a dual-analyte chemical sensor for simultaneous quantification of both nitrite and selenite in aqueous samples. Results show that (a) inclusion of DAN in HP-beta-CD and its subsequent immobilization in a sol-gel matrix do not modify the reactivity of DAN against selenite, (b) the reaction product formed (4,5-benzopiazselenol) remains into the cyclodextrin increasing considerably its fluorescence quantum yield and avoiding, therefore, its extraction into organic solvents, (c) the developed sensor can detect selenite concentrations at submicromolar level with a minimum detection limit of 13 nM, (d) the immobilized system is able to simultaneously quantify nitrite and selenite at submicromolar concentrations in natural water samples with no further sample pre-treatment.

Multienzymatic system immobilization in sol-gel slides: fluorescent superoxide biosensors development.

One of the potential areas of research in the development of biosensors is the production of analytical devices based on the use of immobilized multienzymatic systems. In this work, we report the development of three analytical systems for superoxide radical detection using sol-gel technology to immobilize enzyme systems. These systems are based on the connected reactions of three enzymes (xanthine oxidase, superoxide dismutase and horseradish peroxidase) coupled to the probe Amplex red. The difference between these three systems lies in the immobilization of two or three enzymes into a single or in different sol-gel slides. We check the potential use of each designed systems to quantify superoxide radical and potential evaluation of radical scavenging properties of several antioxidant compounds.

Immobilization and characterization of 2,3-diaminonaphthalene/cyclodextrin complexes in a sol-gel matrix: a new fluorimetric sensor for nitrite.

The aromatic diamino compound 2,3-diaminonaphthalene (DAN) has been extensively used to detect and quantify nitrite ions in biological and environmental samples. We have immobilized the DAN reagent in a porous silicate glass matrix, via previous incorporation of the dye in HP-beta-CD. Changes in fluorescence intensity were used to characterize the inclusion complexes and determine the association constant and stoichiometry of the process. Fluorescence spectrum of these complexes was also used to monitor their immobilization within the sol-gel matrix. Reactivity of the immobilized complexes was evaluated with increasing concentrations of nitrite up to 10 microM (with a detection limit around 20 nM). Results show that sol-gel immobilization does not modify the reactivity of the dye against nitrite and serves to prepare a highly sensitive ready to use fluorescence-based sensor for the specific measurement of nitrite at submicromolar concentrations with no further sample pretreatment.

In situ electrochemical fluorescence studies of PPV.

Conjugated phenylene-vinylene polymers are widely used in organic light-emitting and photovoltaic devices. The comprehension of the optical properties upon charge injection is of crucial importance for the improvement of such organoelectronic devices. The processes of electrochemical doping, electrolyte diffusion, and degradation have been studied by cyclic voltammetry and chronoamperometric methods. Kinetic studies by in situ fluorescence spectroscopy have been used for the determination of the mobility of charge carriers in the polymer making used of electrochemical Stern-Volmer analysis. The mobility of holes for MDMO-PPV measured by this method was 2.5 x 10(-7) cm2 V s(-1). Non-Faradic variations of the fluorescence after doping-dedoping cycles have been related to morphological changes in the polymeric layer. The evolution of the fluorescence obeys a first-order kinetics law, similarly to the trend of the variation of volume during gel shrinking.

Charge transport in luminescent polymers studied by in situ fluorescence spectroscopy.

Modulation of the photoluminescence of poly-[2,7-(fluorene)-1,4-(phenylene)] can be attained by reversible electrochemical modification of the conjugated chain (p- or n-doping). Controlled injection of charge quenches the fluorescent emission of the conjugated polymer. The injection of holes completely eliminates the emission, while the electrons only quench up to one-third of the initial fluorescence of the polymer. Analogous quenching effects have been previously reported for solid-state organoelectronic devices. Electrochemical Stern-Volmer plots permit the estimation of the relative mobility of charge carriers in the polymer layer. The mobility of holes is 1 order of magnitude higher that the mobility of electrons, as determined by this method.

Holography as a technique for the study of photopolymerization kinetics in dry polymeric films with a nonlinear response.

A method is reported that makes use of holography to study the kinetics of the radical photopolymerization of acrylamide in a polyvinyl alcohol when the Kogelnik theory is applied. A mechanism of unimolecular termination by the radicals that initiate the polymerization reaction is postulated to calculate the quantum yield, the molar-extinction coefficient, the index of refraction, and the thickness of the film. The conversion percentage of monomers is obtained along with the ratio of rate constants of the mechanism of polymerization from the nonlinear fit of the transmittance curves, their angular response, and the temporal evolution of diffraction efficiency. Compared with previous holographic techniques, this method has the advantage of predicting these chemical parameters using all the data points of the temporal diffraction efficiency variation rather than being restricted to the linear zone of these curves. In this way the whole reaction process, not just the initial process, is taken into account.

Theoretical and experimental study of the bleaching of a dye in a film-polymerization process.

The quantum efficiency and the molar-absorption coefficients of different phenothiazine dyes are obtained by means of fitting the experimental data of transmittance as a function of time. An analytical expression for the intensity transmitted in a photopolymerizable holographic material is obtained, and good agreement between theory and experience is also achieved. The analysis of these parameters is of fundamental quantities in the photochemical characterization of holographic recording materials.

Optimization of an acrylamide-based dry film used for holographic recording.

A study of the optimization and the characteristics of a dry film photopolymerizable recording material is presented. The effects of intensity, the thickness, and the variation of the concentration of each component have been studied. Diffraction efficiencies of 80%, with energetic sensitivities of 40 mJ/cm(2), have been obtained in photosensitive films of a 35-mum thickness with a spatial frequency of 1000 lines/mm.