Artur J. Moro

On the Design of Fluorescent Ratiometric Nanosensors

Advances in nanoparticle technology have recently offered new tools to the bioanalytical field of research. In particular, new nanoparticle-based sensors have appeared able to give quantitative information about different species (ions, metabolites, biomolecules) in biosamples through ratiometric measurements. This article describes the methodologies developed so far in the design of such nanosensors. In particular, the different approaches to immobilize fluorescent chemosensor dyes to nanoparticles are presented. Concept designs of ratiometric nanosensors in terms of composition and architecture are also described and illustrated with examples taken from the literature.

Chemosensors Based on Molecularly Imprinted Polymers

A sensor is a device, which responds to a physical or chemical stimulus in order to produce a measurable detection signal or to control another operation [1]. Sensors are encountered in innumerable applications and have become an integral part of our day-to-day life. Examples of everyday use of sensors include a thermocouple, which responds to the change in temperature by an output voltage, or a touch-sensitive sensor of an interactive monitor screen. Basically, a sensor can respond, that is change its signal, to a single factor being sensed, i.e. either to the change of temperature or pressure in the above examples.

Magnetic and fluorescent core-shell nanoparticles for ratiometric pH sensing.

This paper describes the preparation of nanoparticles composed of a magnetic core surrounded by two successive silica shells embedding two fluorophores, showing uniform nanoparticle size (50-60 nm in diameter) and shape, which allow ratiometric pH measurements in the pH range 5-8. Uncoated iron oxide magnetic nanoparticles (∼10 nm in diameter) were formed by the coprecipitation reaction of ferrous and ferric salts. Then, they were added to a water-in-oil microemulsion where the hydrophilic silica shells were obtained through hydrolysis and condensation of tetraethoxyorthosilicate together with the corresponding silylated dye derivatives-a sulforhodamine was embedded in the inner silica shell and used as the reference dye while a pH-sensitive fluorescein was incorporated in the outer shell as the pH indicator. The magnetic nanoparticles were characterized using vibrating sample magnetometry, dynamic light scattering, transmission electron microscopy, x-ray diffraction and Fourier transform infrared spectroscopy. The relationship between the analytical parameter, that is, the ratio of fluorescence between the sensing and reference dyes versus the pH was adjusted to a sigmoidal fit using a Boltzmann type equation giving an apparent pK(a) value of 6.8. The fluorescence intensity of the reference dye did not change significantly (∼3.0%) on modifying the pH of the nanoparticle dispersion. Finally, the proposed method was statistically validated against a reference procedure using samples of water and physiological buffer with 2% of horse serum, indicating that there are no significant statistical differences at a 95% confidence level.

Magnetic core-shell fluorescent pH ratiometric nanosensor using a Stöber coating method

We describe the use of a modified Stöber method for coating maghemite (γ-Fe(2)O(3)) nanocrystals with silica shells in order to built magnetic fluorescent sensor nanoparticles in the 50-70nm diameter range. In detail, the magnetic cores were coated by two successive silica shells embedding two fluorophores (two different silylated dye derivatives), which allows for ratiometric pH-measurements in the pH range 5-8. Silica coated magnetic nanoparticles were prepared using maghemite nanocrystals as cores (5-10nm in diameter) coated by tetraethoxyorthosilicate via hydrolysis/condensation in ethanol, catalyzed by ammonia. In the inner shell was covalently attached a sulforhodamine B, which was used as a reference dye; while a pH-sensitive fluorescein was incorporated into the outer shell. Once synthesized, the particles were characterized in terms of morphology, size, composition and magnetization, using dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD) and vibrating sample magnetometry (VSM). TEM analysis showed the nanoparticles to be very uniform in size. Wide-angle X-ray diffractograms showed, for uncoated as well as coated nanoparticles, typical peaks for the spinel structure of maghemite at the same diffraction angle, with no structural changes after coating. When using VSM, we obtained the magnetization curves of the resulting nanoparticles and the typical magnetization parameters as saturation magnetization (M(s)), coercivity (H(c)), and remanent magnetization (M(r)). The dual-dye doped magnetic-silica nanoparticles showed a satisfactory magnetization that could be suitable for nanoparticle separation and localized concentration of them. Changes in fluorescence intensity of the pH indicator in the different pH buffered solutions were observed within few seconds indicating an easy accessibility of the embedded dye by protons through the pores of the silica shell. The relationship between the ratio in fluorescence (sensor/reference dyes) and pH was adjusted to a sigmoidal fit using a Boltzmann type equation. Finally, the proposed method was statistically validated against a reference procedure using samples of water and physiological buffer with 2% (w/v) of horse serum added, indicating that there are no significant statistical differences at a 95% confidence level.

Photocaged Competitor Guests: A General Approach Toward Light-Activated Cargo Release From Cucurbiturils

A general approach toward the light-induced guest release from cucurbit[7]uril by means of a photoactivatable competitor was devised. An o-nitrobenzyl-caged competitor is photolyzed to generate a competitive guest that can displace cargo from the host macrocycle solely based on considerations of chemical equilibrium. With this method the release of terpene guests from inclusion complexes with cucurbit[7]uril was demonstrated. The binding of the herein investigated terpenes, all being lead fragrant components in essential oils, has been characterized for the first time. They feature binding constants of up to 108  L mol-1 and a high differential binding selectivity (spanning four orders of magnitude for the binding constants for the particular set of terpenes). By fine-tuning the photoactivatable competitor guest, selective and also sequential release of the terpenes was achieved.

Gold(I)-complex–titania hybrid photocatalyst for hydrogen production

The integration of TiO2 with a AuI complex containing a thiocoumarin moiety resulted in a very efficient photocatalyst for the generation of H2. The molecular structure of the complex was preserved under the photoreaction owing to the strong AuI−S bond. The AuI complex played a determinant role in the photogeneration of H2 by accepting the photoinduced electrons originated in TiO2 upon light exposure. This is the first example of a AuI complex semiconductor hybrid photocatalyst. The rate of H2 generation under dynamic conditions from water/ethanol is approximately one order of magnitude superior on a metal basis to that obtained over conventional TiO2 decorated with Au metal nanoparticles.

Chemistry and Photochemistry of 2,6-Bis(2-hydroxybenzilidene)cyclohexanone. An Example of a Compound Following the Anthocyanins Network of Chemical Reactions

The kinetics and thermodynamics of the 2,6-bis(2-hydroxybenzilidene)cyclohexanone chemical reactions network was studied at different pH values using NMR, UV-vis, continuous irradiation, and flash photolysis. The chemical behavior of the system partially resembles anthocyanins and their analogue compounds. 2,6-Bis(2-hydroxybenzilidene)cyclohexanone exhibits a slow color change from yellow to red styrylflavylium under extreme acidic conditions. The rate constant for this process (5 × 10(-5) s(-1)) is pH independent and controlled by the cis-trans isomerization barrier. However, the interesting feature is the appearance of the colorless compound, 7,8-dihydro-6H-chromeno[3,2-d]xanthene, isolated from solutions of acid to neutral range, characterized by (1)H NMR and single crystal X-ray diffraction. Light absorption by 2,6-bis(2-hydroxybenzilidene)cyclohexanone solutions immediately after preparation exclusively results in cis-isomer as photoproduct, which via hemiketal formation yields (i) red styrylflavylium by dehydration under extremely acidic solutions (pH < 1) and (ii) colorless 7,8-dihydro-6H-chromeno[3,2-d]xanthene by cyclization in solutions of acid to neutral range.

Development of fluorescent thermoresponsive nanoparticles for temperature monitoring on membrane surfaces

In this work, tris(phenantroline)ruthenium(II) chloride (Ru(phen)3) was immobilized in silica nanoparticles prepared according to the Stöber method. Efforts were devoted on the optimization of the nano-thermometer in terms of size, polydispersity, intensity of the emission and temperature sensitivity. In particular, the immobilization of the luminophore in an external thin shell made of silica grown in a second step on bare silica nanoparticles allowed producing fluorescent monodisperse silica nanoparticles (420±20nm). A systematic study was addressed to maximize the intensity of the emission of the fluorescent nanoparticles by adjusting the concentration of Ru(phen)32+ in the shell from 0.2 to 24wt.%, whereas the thickness of the shell is affected by the amount of silica precursor employed. The luminescent activity of the doped nanoparticles was found to be sensitive to the temperature. In fact, the intensity of the emission linearly decreased by increasing the temperature from 20°C to 65°C. The thermoresponsive nanoparticles were functionalized with long aliphatic chains in order to obtain hydrophobic nanoparticles. The developed nanoparticles were immobilized via dip-coating procedure on the surface of hydrophobic porous membranes, such as Polyvinylidene fluoride (PVDF) prepared via Non-Solvent Induced Phase Separation (NIPS), providing local information about the membrane surface temperature.

Reversible Self-Assembly of Water-Soluble Gold(I) Complexes

The reaction of the gold polymers containing bipyridyl and terpyridyl units, [Au(C≡CC15H10N3)]n and [Au(C≡CC10H7N2)]n, with the water-soluble phosphines 1,3,5-triaza-7-phosphatricyclo[3.3.1.13.7]decane and 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane gives rise to the formation of four gold(I) alkynyl complexes that self-assemble in water (H2O) and dimethyl sulfoxide (DMSO), through different intermolecular interactions, with an impact on the observed luminescence displayed by the supramolecular assemblies. A detailed analysis carried out by NMR studies performed in different DMSO/deuterated H2O mixtures indicates the presence of two different assembly modes in the aggregates: (i) chain assemblies, which are based mainly on aurophilic interactions, and (ii) stacked assemblies, which are based on Au···π and π···π interactions. These different supramolecular environments can also be detected by their intrinsic optical properties (differences in absorption and emission spectra) and are predicted by the changes in the relative binding energy from density functional theory calculations carried out in DMSO and H2O. Small-angle X-ray scattering (SAXS) experiments performed in the same mixture of solvents are in agreement with the formation of aggregates in all cases. The aromatic units chosen, bipyridine and terpyridine, allow the use of external stimuli to reversibly change the aggregation state of the supramolecular assemblies. Interaction with the Zn2+ cation is observed to disassemble the aggregates, while encapsulating agents competing for Zn2+ complexation revert the process to the aggregation stage, as verified by SAXS and NMR. The adaptive nature of the supramolecular assemblies to the metal-ion content is accompanied by significant changes in the absorption and emission spectra, signaling the aggregation state and also the content on Zn2+.

Unusual Reduction Mechanism of Copper in Cysteine-Rich Environment

Copper-cysteine interactions play an important role in Biology and herein we used the copper-substituted rubredoxin (Cu-Rd) from Desulfovibrio gigas to gain further insights into the copper-cysteine redox chemistry. EPR spectroscopy results are consistent with Cu-Rd harboring a CuII center in a sulfur-rich coordination, in a distorted tetrahedral structure ( g∥,⊥ = 2.183 and 2.032 and A∥,⊥ = 76.4 × 10-4 and 12 × 10-4 cm-1). In Cu-Rd, two oxidation states at Cu-center (CuII and CuI) are associated with Cys oxidation-reduction, alternating in the redox cycle, as pointed by electrochemical studies that suggest internal geometry rearrangements associated with the electron transfer processes. The midpoint potential of [CuI(S-Cys)2(Cys-S-S-Cys)]/[CuII(S-Cys)4] redox couple was found to be -0.15 V vs NHE showing a large separation of cathodic and anodic peaks potential (Δ Ep = 0.575 V). Interestingly, sulfur-rich CuII-Rd is highly stable under argon in dark conditions, which is thermodynamically unfavorable to Cu-thiol autoreduction. The reduction of copper and concomitant oxidation of Cys can both undergo two possible pathways: oxidative as well as photochemical. Under O2, CuII plays the role of the electron carrier from one Cys to O2 followed by internal geometry rearrangement at the Cu site, which facilitates reduction at Cu-center to yield CuI(S-Cys)2(Cys-S-S-Cys). Photoinduced (irradiated at λex = 280 nm) reduction of the CuII center is observed by UV-visible photolysis (above 300 nm all bands disappeared) and tryptophan fluorescence (∼335 nm peak enhanced) experiments. In both pathways, geometry reorganization plays an important role in copper reduction yielding an energetically compatible donor-acceptor system. This model system provides unusual stability and redox chemistry rather than the universal Cu-thiol auto redox chemistry in cysteine-rich copper complexes.