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The asymmetric flow field-flow fractionation (AF4) coupled on-line with elemental (inductively coupled plasma-mass spectrometry, ICP-MS) and molecular (fluorescence and UV) detection has been investigated as a powerful tool for the characterization of bioinorganic nano-conjugates. In this study, we described methods for the characterization of biotin-antibody complexes bioconjugated with streptavidin quantum dots (QDs-SA-b-Ab). Operating parameters of AF4 separation technique were optimized and two procedures are proposed using a channel thickness of 350 μm and 500 μm. The use of a 500 μm spacer allowed to achieve an efficient AF4 separation of the QDs-SA-b-Ab complexes from the excess of individual species used in the bioconjugation that was required for a proper characterization of the bioconjugates. Optimization of the AF4 allowed a separation resolution good enough to isolate the QDs-SA-b-Ab bioconjugates from the free excess of b-Ab and QD-SA. The efficiency of the bioconjugation process could be then calculated, obtaining a value of 86% for a 1 QDs-SA: 5 b-Ab bioconjugation ratio. In addition, sample recovery around 90% was achieved., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The synthesis and characterization of a novel water-compatible microsized material, based on fluorescent conjugated polymers (CPs), and its applicability for optical sensing of inorganic ions of environment interest (copper and cyanide) in water media is here described. Polyfluorene-based fluorescent CPs were synthesized and functionalized with imidazole moieties (selective recognition element) and a terminal double bond (covalently linked to an organic matrix) through a postfunctionalization strategy. Further, microspheres of the novel imidazole-functionalized fluorescent CPs, able to work in water media, were synthesized via a microemulsion and polymerization procedure. The synthesized imidazole-functionalized CP microspheres were then evaluated as fluorescence "turn-Off" sensing materials for Cu(2+) detection in aqueous media. Analyte detection was based on the quenching effect of the Cu(2+), selectively recognized by the imidazole group, on the polymer fluorescence emission. The developed optosensor exhibits a detection limit of 1 μg/L for the determination of Cu(2+) in water with a reproducibility of 4%. The synthesized microsized material was also evaluated for the "turn-on" optosensing of cyanide in water, measuring the recovery of the emission signal from the CP that has been previously deactivated by the presence of quencher species. The "turn-On" optosensor allows the selective determination of free cyanide in aqueous solution with high sensitivity (detection limit of 18 μg/L), obtaining a reproducibility of 2.9%. A high sample throughput (between 7 and 12 samples per hour) was achieved in both cases. Analytical applicability of the fluorescent CP microsphere materials has been successfully demonstrated by tap and mineral water analysis.

Herein, we demonstrate that both qualitative molecular and quantitative elemental data obtained from size exclusion chromatography coupled online for the first time to both molecular fluorescence and elemental mass spectrometry, respectively, turned out to be critical to evaluate the quality of coatings of quantum dots. Moreover, such an instrumental approach also allowed us to study quantitatively the appropriated bioconjugation of quantum dots to antibodies, a critical step for QDs future use in quantitative fluorescence immunoassays.

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.