Exploring quantitative cellular bioimaging and assessment of CdSe/ZnS quantum dots cellular uptake in single cells, using ns-LA-ICP-SFMS.

Quantitative bioimaging of Quantum Dots (QDs) uptake in single cells by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a challenging task due to the high sensitivity and high spatial resolution required, and to the lack of matrix-matched reference materials. In this work, high spatially resolved quantitative bioimaging of CdSe/ZnS QDs uptake in single HT22 mouse hippocampal neuronal cells and in single HeLa human cervical carcinoma cells is novelty investigated combining: (a) the use of a ns-LA-ICP-Sector Field (SF)MS unit with mono-elemental fast and sensitive single pulse response for 114Cd+; and (b) the spatially resolved analysis of dried pL-droplets from a solution with a known concentration of these QDs to obtain a response factor that allows quantification of elemental bioimages. Single cells and dried pL-droplets are morphologically characterized by Atomic Force Microscopy (AFM) to determine their volume and thickness distribution. Moreover, operating conditions (e.g. spot size, energy per laser pulse, etc.) are optimized to completely ablate the cells and pL droplets at high spatial resolution. Constant operating conditions for the analysis of the single cells and calibrating samples is employed to reduce potential fractionation effects related to mass load effects in the ICP. A number concentration of CdSe/ZnS QDs between 3.5 104 and 48 104 is estimated to be uptaken by several selected single HT22 and HeLa cells, after being incubated in the presence of a QDs suspension added to a standard cell culture medium. Mono-elemental bioimaging at subcellular resolution seems to show a higher number concentration of the CdSe/ZnS QDs in the cytosol around the cell nucleus.

Dynamic analysis of the photoenhancement process of colloidal quantum dots with different surface modifications.

Photoinduced fluorescence enhancement of colloidal quantum dots (QDs) is a hot topic addressed in many studies due to its great influence on the bioanalytical performance of such nanoparticles. However, understanding of this process is not a simple task, and it cannot be explained by a general mechanism as it greatly depends on the QDs' nature, solubilization strategies, surrounding environment, etc. In this vein, we have critically compared the behavior of CdSe QDs (widely used in bioanalytical applications) with different surface modifications (ligand exchange and polymer coating), in different controlled experimental conditions, in the presence-absence of the ZnS layer and in different media when exposed for long times to intense UV irradiation. Thus six different types of colloidal QDs were finally studied. This research was carried out from a novel perspective, based on the analysis of the dynamic behavior of the photoactivation process (of great interest for further applications of QDs as labels in biomedical applications). The results showed a different behavior of the studied colloidal QDs after UV irradiation in terms of their photoluminescence characteristics, potential toxicity due to metal release to the environment, nanoparticle stability and surface coating degradation.

Entrapment of quantum dots in sol-gel matrices to develop sensing material based on fluorescence resonance energy transfer.

A simple and general procedure is described for the development of fluorescence resonance energy transfer (FRET) systems by the co-immobilization in a sol-gel matrix of photoluminescent quantum dots with a given dye "acceptor".

Simple bio-conjugation of polymer-coated quantum dots with antibodies for fluorescence-based immunoassays.

A simple procedure for the bio-conjugation of amphiphilic polymer-coated quantum dots with antibodies is described. The formed bio-conjugates are purified from an excess of free antibodies and of free nanoparticles by size-exclusion HPLC and then characterized by fluorescence emission and MALDI-TOFMS. The applicability of the approach is demonstrated for aflatoxin B1 detection.

Direct and rapid discrimination of aflatoxigenic strains based on fibre-optic room temperature phosphorescence detection.

An innovative analytical methodology for the rapid identification of aflatoxin-producing moulds belonging to Aspergillus genus is presented here. The procedure is based on the measurement, using a fibre-optic luminometer, of the room temperature phosphorescence (RTP) emitted by aflatoxins produced by isolated aflatoxigenic strains, cultured in a special culture medium consisting of malt extract agar modified with beta-cyclodextrin and sodium deoxycholate for RTP induction. Unequivocal detection of the presence of aflatoxins in the culture medium is achieved within the first 36 h of incubation at 32 degrees C, owing to the selectivity and sensitivity of the RTP emission, as compared with the minimum of 72 h needed using a conventional microbiological method. In a first step, the capability of aflatoxin standard solutions to emit analytically useful RTP was evaluated. In this line all experimental conditions were optimised for in vitro induction of RTP from aflatoxins. In a second step, a simple analytical test was developed and it has been evaluated for the rapid identification of aflatoxigenic strains, as a discriminating assay from non-aflatoxigenic strains based on the measurement of experimental RTP emission observed. Confirmation of aflatoxin production on the studied culture plates was accomplished by means of an HPLC/fluorescence reference method.

Direct screening of tetracyclines in water and bovine milk using room temperature phosphorescence detection.

A fast and simple flow-through optosensor was designed and characterized for the direct screening of four tetracycline (TCC) antibiotics (tetracycline, oxytetracycline, chlortetracycline and doxycycline) in water and bovine milk samples. The proposed optosensor provides rapid binary yes/no overall responses, being appropriate for the screening of this family of antibiotics above or below a pre-set concentration threshold. The experimental set-up is based on a flow-injection manifold coupled on-line to a phosphorescence detector. Aliquots of the samples are pretreated with Eu(III) to form room temperature phosphorescent metal chelates and injected in the flow manifold. Those chelates are then on-line retained on a conventional flow-cell (packed with polymeric Amberlite XAD-4 particles) which is placed inside the cell holder of the phosphorimeter. After the emission is registered, the antibiotic-metal complexes are eluted from the packed resin with 1M HCl (for milk samples a second regeneration step, using methanol, should be performed). A sample throughput of about 20 samples per hour was obtained. Optimum experimental conditions include a pH 9, a Eu(III) concentration of 2 x 10(-4) M and 8 mM sodium sulphite as chemical deoxygenant. The phosphorescence emitted by the europium-TCC complexes was measured at 394 and 617 nm for excitation and emission wavelengths, respectively. The unreliability region, given by the probability of false positives and false negatives, respectively (set at 5% in both cases) was in the range between 0.2 and 11.6 nM for detection of tetracyclines in water samples (at a cut-off level of 4 nM) and in the range between 165 and 238 nM for detection of tetracyclines in milk (cut-off level fixed at the normative EU level of 200 nM). Finally, the applicability of the proposed screening optosensor was tested for the reliable control of tetracyclines in contaminated and uncontaminated water and milk samples.

Solid-supported room temperature phosphorescence from aflatoxins for analytical detection of Aspergillus spp. strains.

A simple, direct and rapid analytical methodology for the detection of aflatoxin producing Aspergillus spp. strains based on the measurement of room temperature phosphorescence from aflatoxins is presented here.

Iodinated molecularly imprinted polymer for room temperature phosphorescence optosensing of fluoranthene.

A novel molecularly imprinted polymer (MIP) of high interest for room temperature phosphorescence (RTP) sensing systems is described; the synthesized MIP contains iodine as internal heavy atom in the polymeric structure and its applicability for RTP sensing of fluoranthene at microg L(-1) levels is demonstrated.

Fluorescence optosensors based on different transducers for the determination of polycyclic aromatic hydrocarbons in water.

This paper presents the development of two optosensors for the determination of four polycyclic aromatic hydrocarbons (anthracene, benzo[a]pyrene, fluoranthene and benzo[b]fluoranthene) using a photomultiplier device and an intensified coupled charge device (ICCD) as optical transducers, respectively. These optosensors are based on the on-line immobilization of the analytes onto a non-ionic resin solid support (Amberlite XAD-4) in a continuous flow system, followed by the measurement of their native fluorescence. The determinations were performed using 15 mM H(2)PO(4)(-)/HPO(4)(2-) buffer solution at pH 7 and 25% 1,4-dioxane. Detection limits were 6.4 and 9.3 for ANT, 3.3 and 2.5 for BbF, 1.4 and 13.2 for FLT, and 1.7 and 7.8 for BaP using optosensor 1 or 2, respectively. Relative standard deviations were 7.9 and 6.7 for ANT at 50 ng mL(-1), 3.5 and 7.4 for BbF at 60 ng mL(-1), 3.6 and 8.9 for FLT at 50 ng mL(-1), and 6.7 and 11.6 for BaP at 50 ng mL(-1) using optosensor 1 or 2, respectively. Finally, a critical comparison between the two configurations based on different transducers (photomultiplier and ICCD) for resolving and simultaneously determining mixtures of the polycyclic aromatic hydrocarbons under study in water samples (tap and mineral waters) were carried out.

Determination of lead and mercury in sea water by preconcentration in a flow injection system followed by atomic absorption spectrometry detection.

The capabilities of three solid chelating reagents were compared for the preconcentration of lead and mercury in high salinity aqueous samples (sea waters). The tested materials were 7-(4-ethyl-1-methyloctyl)-8-hydroxiquinoline (Kelex 100) adsorbed on Bondapack C18 (Kelex-100/C18), 8-hydroxiquinoline immobilized on vinyl co-polymer Toyopearl gel (TSK) and the commercial polystyrene/DVB ion exchange resin with paired iminodiacetate groups (Chelex-100). The two metals preconcentration and final determination were carried out in a flow injection system, coupled on-line to an atomic absorption spectrometric detector. Analytes were preconcentrated in the minicolumn, packed with the materials under investigation, while elution was achieved by injection of 500 mul of an adequate mineral acid solution. The different packing materials and minicolumn designs have been evaluated in terms of sensitivity for simultaneous preconcentration of both metals in sea water. Regarding the solid support, the best results were obtained for the TSK solid phase. Concerning the minicolumn design, the behavior was different for lead and mercury. Lead was quantitatively eluted with 0.5 M HCl and best performance was achieved when packing the solid material in a minicolumn with relatively small volume (1 cm length and 2.5 mm i.d.). In the case of mercury, bigger minicolumn volumes (5.5 cm length and 5.0 mm i.d.) and mixtures, 2 M HCl+1 M HNO(3), were required for its quantitative recovery and elution. The system has been evaluated for quantitative determination of the two metals under study in different Asturian coastal aqueous samples.

Low-level mercury determination with thiamine by fluorescence optosensing.

A sensitive fluorescence optosensing method for the determination of Hg(II) in water samples is described. The method, using a flow injection technique, is based on the immobilization on a non-ionic-exchanger solid support (packed in a flow cell placed in a conventional fluorimeter) of the thiochrome formed by the oxidation of thiamine with Hg(II) in a continuous flow carrier at pH 8.1. Experimental parameters such as the solid support, the carrier pH, the thiamine concentration and the flow-rate were investigated to select the optimum operating conditions. The proposed optosensor showed a relative standard deviation of + 3.0% for ten replicates analysis of 100 ng ml(-1) of mercury(II). A detection limit of 3 ng ml(-1) for mercury(II) was achieved for 4-ml sample injections. A detailed study of interferences (possible elements present in natural waters) demonstrated that this optosensing method is virtually specific for this metal, because it allows the determination of mercury in the presence of relatively large amounts of other heavy metals and compounds present in natural waters, such as Mg(II) or Ca(II). The method was successfully applied to the determination of Hg(II) in spiked samples of mineral, tap and sea water.