Cyclodextrin-enhanced photo-induced fluorescence of tau-fluvalinate, molecular modelling of inclusion complexes and determination in natural waters.

The effect of adding organized supramolecular systems such as ß-cyclodextrin (ß-CD) and hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on the photochemically-induced fluorescence (PIF) spectral properties of tau-fluvalinate (TFV) in aqueous solutions was examined. The influence of pH, UV irradiation time and photoproduct stability on the cyclodextrin-enhanced photochemically-induced fluorescence intensity was also investigated. The spectral changes associated with the inclusion process yielded values for the formation constants of TFV inclusion complexes between 450 and 640 M-1, which were calculated using the nonlinear iterative regression approach least squares. In addition, host-guest interaction was clearly determined by PIF enhancement and a 1 : 1 stoichiometry was found for the ß-CD and HP-ß-CD complexes formed with TFV. The negative free energy (ΔG°) value indicated that the reaction of TFV with cyclodextrins was thermodynamically favorable. Furthermore, the structures of inclusion complexes of TFV with cyclodextrins were elucidated by 3-21G ab initio calculations. The limits of detection and quantification obtained ranged between 1.3 and 4.0 ng mL-1 and from 4.4 to 13.0 ng mL-1 in ß-CD and HP-ß-CD media, respectively. The analytical application in tap and river water samples yielded satisfactory mean recoveries ranging from 98.12 to 102.97%. Due to its sensitivity and ease of use, this method can be reliably applied to routine analysis.

Direct Spectrofluorimetric Method for the Analysis of Carbofuran and Fluometuron in Senegalese Natural Waters.

In this work, analytical study of carbofuran (CAF) and fluometuron (FLM) pesticides was carried out using direct spectrofluorimetric method in various solvents. Results showed that CAF and FLM are naturally fluorescent in all solvents under study including organic (MeOH, MeCN, DMF) and aqueous micellar one (CTAC, SDS, Brij-700). For the analysis of FLM, CTAC give the best fluorescence signal enhancement. Analytical performances, such as limit of detection (LOD) and quantification (LOQ) was evaluated after solvent optimization and were found to vary, respectively, between 0.1 and 11 ng mL- 1 and between 0.3 and 36.6 ng mL- 1. Analytical application in various environmental aqueous samples matrices (sea, tap, runoff and well waters) give satisfactory recovery rates in the limits of 73.7-113.7% for both pesticides. This method is described for its simplicity for routine analysis.

Analysis of non-native fluorescent pesticides by thermo-induced and photo-induced fluorescence, a comparative study.

This paper compares the performance of three analytical methods for the determination of pesticides in natural waters. As many pesticides are non-fluorescent, they are transformed into highly fluorescent by-products in two ways: elevated temperature in an alkaline medium (thermo-induced fluorescence - TIF); or UV irradiation in water (photo-induced fluorescence - PIF). The first method studied uses TIF, the second one uses PIF and the third one uses an automatic sampling and analysing PIF system. Analytical applications were carried out using the three methods for the determination of deltamethrin and cyhalothrin, pesticides widely used in Senegal. In both cases, the calibration curves obtained are linear without matrix effects, and the detection limits are good in the ng mL-1 range. It appears that the analytical performances of the automatic PIF method are better than the two others. The advantages and disadvantages of the three methods are then compared and discussed in term of analytical performance and usability.

High sensitivity on-site early warning system monitoring of pesticides by photo-induced fluorescence.

This paper describes the prototype of an on-site High Sensitivity Early Warning Monitoring System, using Photo-Induced Fluorescence, for pesticide monitoring in natural waters (HSEWPIF). To obtain a high sensitivity, the prototype was designed with four main features. Four UV LEDs are used to excite the photoproducts at different wavelengths and select the most efficient one. Two UV LEDs are used simultaneously at each wavelength to increase the excitation power and then the fluorescence emission of the photoproducts. High-pass filters are used to avoid the saturation of the spectrophotometer and to increase the signal-to-noise ratio. The HSEWPIF prototype also employs UV absorption to detect any occasional increase of suspended and dissolved organic matter, which could disrupt the fluorescence measurement. The conception of this new experimental set-up is explained and described, then analytical applications are carried out online for the determination of fipronil and monolinuron. We obtained a linear calibration range from 0 to 3 µg mL-1 with limits of detection of 1.24 ng mL-1 for fipronil and 0.32 ng mL-1 for monolinuron. A mean recovery of 99.2% for fipronil and 100.9% for monolinuron show that the method is accurate, moreover a standard deviation of 1.96% for fipronil and 2.49% for monolinuron show that the method is repeatable. Compared to other methods for the determination of pesticides by photo-induced fluorescence, the HSEWPIF prototype has good sensitivity with better limits of detection, and good analytical performances. These results show that HSEWPIF can be used for monitoring pesticide in natural waters to protect industrial facilities against accidental contamination.

Micellar enhanced photo-induced fluorescence and absorbance for the development of an on-site early warning water quality monitoring system for pesticides.

This paper describes two prototypes of an on-site Early Warning Water Quality Monitoring System (EWWQMS) for pesticide quantification in natural waters. As many pesticides are non-fluorescent, the EWWQMS setup uses UV photoconversion to form highly fluorescent photoproducts. To enhance sensitivity, the two prototypes use micellar-enhanced fluorescence with surfactant (cetyl trimethyl ammonium chloride) in aqueous solution. To improve specificity, four UV LEDs at different wavelengths then excite the fluorescent photoproducts. The EWWQMS prototypes also employ UV absorption for quantification of non-photosensitive pesticides. The first prototype detects the pesticides via a diode array spectrometer. The second system is developed with higher resolution spectrometer and an intensified CCD camera detection to improve the sensitivity of the method. These experimental set-ups are described, explained and tested. Analytical applications were carried out online in CTAC aqueous solution, for the determination of isoproturon, flufenoxuron and profenofos. The calibration curves obtained are linear over one order of magnitude, and the detection limits are in the ng mL-1 range. The analytical performances of these new methods are good compared with other published classical micellar enhanced photo-induced fluorescence methods for the determination of pesticides in aqueous solutions. Our results show that these EWWQMS prototypes can be used as a warning system to protect against pesticide contamination exceeding the threshold of treatment capabilities at industrial facilities using natural waters.

Alternative to High Pressure Mercury Vapor Lamp for Photo Induced Fluorescence Analytical Methods; Application to the Determination of Pesticides in Water.

The photo-induced fluorescence method is often applied to determine non-native fluorescent compounds. It typically uses UV irradiation from a high pressure mercury vapor discharge lamp to create photo-induced fluorescent compounds, which are then quantified by fluorescence spectroscopy. However, these mercury lamps require a high-voltage power supply and may accidentally induce electric shocks and the release of mercury vapors. As an alternative, we have evaluated in this technical note new UV-C germicidal lamps. These lamps exhibit a higher power at 254 nm and allowed us to obtain a far greater amount of photo-induced compounds in a shorter time. For the first time, this new irradiation system has been applied for the determination of pesticides in water and has shown a significant increase in the method sensitivity. These good results allowed us to conclude that the new UV-C lamps are a relevant alternative to high pressure mercury vapor discharge lamps for use with photo induced fluorescent methods.

Development of an on-site early warning water quality monitoring system for pesticide detection by absorption and photo-induced fluorescence.

This paper describes prototypes of an on-site early warning water quality monitoring system (EWWQMS) for pesticide quantification in natural waters by fluorescence and absorbance. As many pesticides are not naturally fluorescent, this EWWQMS uses UV irradiation to transform these compounds into highly fluorescent photoproducts and obtain sufficient sensitivity. To obtain a better specificity, the system uses four UV LEDs at different wavelengths to excite the fluorescent photoproducts. For pesticides that are not sensitive to photoconversion, the EWWQMS prototypes also use UV absorption for their quantification, thus offering a wider application range. A first system uses a diode array spectrometer for detection. A second system uses a higher resolution spectrometer and an intensified CCD camera detection to increase sensitivity. Analytical applications were conducted for the determination of fipronil, acetamipride, cyprodinil, trifluraline and pendimethaline in water using both the EWWQMS prototypes. The analytical performances of these new systems are good compared with other photo-induced fluorescence methods already published. Limits of detection without pre-concentration are in the range of 0.2 to 3 ng mL-1 and the recovery values range from 95 to 108%. These results show that the EWWQMS prototypes can be used as an alert system to protect industrial plants from pesticide contaminations that exceed the capabilities of their cleaning processes.

Analysis of Oxadiazon Herbicide in Natural Water Samples by a Micellar-enhanced Photo-induced Fluorescence.

A micellar-enhanced photo-induced fluorescence (ME-PIF) method was developed to analyze, for the first time, oxadiazole herbicide (namely oxadiazon) in natural water samples. Photo-conversion under UV irradiation of the herbicide into strongly fluorescent photoproducts was performed in aqueous solution and in the presence of two surfactants, cetyltrimethylammonium chloride (CTAC) or Tween 20, at micellar concentrations. The ME-PIF parameters were optimized. The ME-PIF method gave very good results with satisfactory analytical performance for the determination of a selected pesticide with concentration linear dynamic ranges of over one to two orders of magnitude. It yielded good reproducibility (RSD values of between 3.6 and 9.6%) in tap, river and sea water spiked samples, and the limits of detection were in the ng mL-1 range.

Combination of photoinduced fluorescence and GC-MS for elucidating the photodegradation mechanisms of diflubenzuron and fenuron pesticides.

Diflubenzuron (DFB) and fenuron (FEN) are benzoylurea and phenylurea pesticides, widely used in Senegal, that do not exhibit any natural fluorescence, but can be determined by means of photoinduced fluorescence (PIF) methods. Photodegradation of DFB and FEN yielded a number of fluorescent and non-fluorescent photoproducts. For both pesticides, at least 10 photoproducts were detected and identified by gas chromatography-mass spectrometry (GC/MS). To identify the formed fluorescent DFB and FEN photoproducts, their fluorescence spectra were compared with those of standard compounds, including phenol and p-hydroxyaniline.

Development of online automatic detector of hydrocarbons and suspended organic matter by simultaneously acquisition of fluorescence and scattering.

Permanent online monitoring of water supply pollution by hydrocarbons is needed for various industrial plants, to serve as an alert when thresholds are exceeded. Fluorescence spectroscopy is a suitable technique for this purpose due to its sensitivity and moderate cost. However, fluorescence measurements can be disturbed by the presence of suspended organic matter, which induces beam scattering and absorption, leading to an underestimation of hydrocarbon content. To overcome this problem, we propose an original technique of fluorescence spectra correction, based on a measure of the excitation beam scattering caused by suspended organic matter on the left side of the Rayleigh scattering spectral line. This correction allowed us to obtain a statistically validated estimate of the naphthalene content (used as representative of the polyaromatic hydrocarbon contamination), regardless of the amount of suspended organic matter in the sample. Moreover, it thus becomes possible, based on this correction, to estimate the amount of suspended organic matter. By this approach, the online warning system remains operational even when suspended organic matter is present in the water supply.

Biophysical properties of cationic lipophosphoramidates: Vesicle morphology, bilayer hydration and dynamics.

Cationic lipids are used to deliver genetic material to living cells. Their proper biophysical characterization is needed in order to design and control this process. In the present work we characterize some properties of recently synthetized cationic lipophosphoramidates. The studied compounds share the same structure of their hydrophobic backbone, but differ in their hydrophilic cationic headgroup, which is formed by a trimethylammonium, a trimethylarsonium or a dicationic moiety. Dynamic light scattering and cryo-transmission electron microscopy proves that the studied lipophosphoramidates create stable unilamellar vesicles. Fluorescence of polarity probe, Laurdan, analyzed using time-dependent fluorescence shift method (TDFS) and generalized polarization (GP) gives important information about the phase, hydration and dynamics of the lipophosphoramidate bilayers. While all of the compounds produced lipid bilayers that were sufficiently fluid for their potential application in gene therapy, their polarity/hydration and mobility was lower than for the standard cationic lipid - DOTAP. Mixing cationic lipophosphoramidates with DOPC helps to reduce this difference. The structure of the cationic headgroup has an important and complex influence on bilayer hydration and mobility. Both TDFS and GP methods are suitable for the characterization of cationic amphiphiles and can be used for screening of the newly synthesized compounds.

Physicochemical properties of cationic lipophosphoramidates with an arsonium head group and various lipid chains: A structure-activity approach.

We studied the physicochemical properties of some cationic lipophosphoramidates used as gene vectors in an attempt to better understand the link between the nature of the hydrophobic chain and both physico-chemical properties and transfection efficiency. These compounds have an arsonium head group and various chain lengths and unsaturation numbers. The synthesis of cationic phospholipids with oleic (Guenin et al., 2000 [1]; Floch et al., 2000 [2]) or linoleic (Fraix et al., 2011 [3]; Le Gall et al., 2010 [4]) chains has already been reported by our group and their efficiency as gene carriers has been demonstrated. Four new compounds were synthesized which incorporated either C14:0, C18:0, C20:4 or C20:5 chains. The membrane fluidity was studied by fluorescence anisotropy measurements. The fusion of liposomes and lipoplexes with membrane models was studied by Förster Resonant Energy Transfer. Finally, DNA condensation was studied and the lipoplexes were tested in vitro to quantify their transfection efficiency. From the results obtained on these cationic lipophosphoramidates series, we show that aliphatic chain length and unsaturation number have an important influence on liposomes physicochemical properties and transfection efficiency. However there is no direct link between fluidity and fusion efficiency or between fluidity and DNA condensation. Nevertheless, it seems that for best transfection efficiency the compounds need to combine the properties of fluidity, fusion efficiency and DNA condensation efficiency. This was the case for the C18:1 and C18:2 compounds.

Polyethylene glycol aggregates in water formed through hydrophobic helical structures.

The present study was aimed at elucidating the mechanism of aggregation in water of hydroxyl-terminated polyethylene glycol (PEG) of low molecular weight (600 g/mol). The results from fluorescence spectroscopy at different temperatures were consistent with surface tension measurements, suggesting aggregate formation. Indeed, the process of aggregation is accompanied by an increase in the fluorescence emission of a hydrophobic probe. So, PEG aggregates in the form of internal hydrated helices covered with CH(2) groups are shown to yield hydrophobic regions. These regions created upon PEG aggregation in water and at a temperature close to 35°C result from a balance between H bonding and entropic effects. By providing the first experimental evidence for hydrophobic mediation of aggregation with OH-terminated oxy-ethylene chains of low molecular weight, this study highlights their surfactant-like behaviour.

Direct laser photo-induced fluorescence determination of bisphenol A.

Classical photo-induced fluorescence methods are conducted in two steps: a UV irradiation step in order to form a photo-induced compound followed by its fluorimetric determination. Automated flow injection methods are frequently used for these analyses. In this work, we propose a new method of direct laser photo-induced fluorescence analysis. This new method is based on direct irradiation of the analyte in a fluorimetric cell in order to form a photo-induced fluorescent compound and its direct fluorimetric detection during a short irradiation time. Irradiation is performed with a tuneable Nd:YAG laser to select the optimal excitation wavelength and to improve the specificity. It has been applied to the determination of bisphenol A, an endocrine disrupter compound that may be a potential contaminant for food. Irradiation of bisphenol A at 230 nm produces a photo-induced compound with a much higher fluorescence quantum yield and specific excitation/emission wavelengths. In tap water, the fluorescence of bisphenol A increases linearly versus its concentration and, its determination by direct laser photo-induced fluorescence permits to obtain a low limit of detection of 17 µg L(-1).

Imaging time-resolved electrothermal atomization laser-excited atomic fluorescence spectrometry for determination of mercury in seawater.

In this study, direct determination of mercury at the nanogram per liter level in the complex seawater matrix by imaging time-resolved electrothermal atomization laser-excited atomic fluorescence spectrometry (ITR-ETA-LEAFS) is described. In the case of mercury, the use of a nonresonant line for fluorescence detection with only one laser excitation is not possible. For measurements at the 253.652 nm resonant line, scattering phenomena have been minimized by eliminating the simultaneous vaporization of salts and by using temporal resolution and the imaging mode of the camera. Electrothermal conditions (0.1 M oxalic acid as matrix modifier, low atomization temperature) have been optimized in order to suppress chemical interferences and to obtain a good separation of specific signal and seawater background signal. For ETA-LEAFS, a specific response has been obtained for Hg with the use of time resolution. Moreover, an important improvement of the detection limit has been obtained by selecting, from the furnace image, pixels collecting the lowest number of scattered photons. Using optimal experimental conditions, a detection limit of 10 ng L(-1) for 10 µL of sample, close to the lowest concentration level of total Hg in the open ocean, has been obtained.

Cationic lipo-thiophosphoramidates for gene delivery: synthesis, physico-chemical characterization and gene transfection activity--comparison with lipo-phosphoramidates.

The synthesis of cationic lipo-thiophosphoramidates, a new family of cationic lipids designed for gene delivery, is reported herein. This new class of lipids is less polar than its oxygenated equivalent the lipo-phosphoramidates. Fluorescence anisotropy and FRET were used to determine the fluidity and fusogenicity of the lipo-phosphoramidates 3a-b and lipo-thiophosphoramidates 7a-b. The determination of both the size and the zeta potential of the nano-objects (liposomes and lipoplexes) and the determination of the DNA binding ability of the liposomes have completed the physico-chemical characterizations of the cationic lipids studied. Finally, the cationic lipids 3a-b and 7a-c have been evaluated as synthetic vectors for gene transfection into a variety of mammalian cell lines. The lipo-thiophosphoramidate 7a proved to be an efficient and low toxicity synthetic vector even when used at low lipid to DNA charge ratios.

Spectroscopic properties and laser induced fluorescence determination of some endocrine disrupting compounds.

This work presents spectroscopic properties of some Endocrine Disrupting Compounds (EDCs), frequently found in food and in natural water. Studied molecules belong to the groups of phenolic and phthalate EDCs. In a first part, we have examined their absorption and fluorescence properties. Fluorescence emission wavelengths are about 300 nm for phenolic compounds and 360 nm for phtalate compounds; main excitation wavelengths being comprised between 210 nm and 230 nm. Fluorescence lifetimes measured are short (about 4 ns) and the fluorescence quantum yield has been determined. In a second part, to avoid the time consuming solvent extraction step, an analytical application to evaluate the performance of a direct analysis by laser induced fluorescence spectroscopy of ECDs traces in tap water and in raw water is presented. Good detection limits have been obtained, i.e.: 0.35 µg.L(-1) of chlorophenol in tap water, which are always lower than the reported Predictive Non Efficient Concentration (PNEC).

A study of marine pollution caused by the release of metals into seawater following acid spills.

This study examined the potential metal pollution induced by the accidental spill of different acids into seawater. The acids sink to the bottom according to their densities and subsequently react with marine sediments. The acids selected for this study were acetic, hydrochloric, nitric, sulfuric, and phosphoric acids; the metallic elements selected were Cr, Cu, Fe, Mn, Pb and Zn. The sediment was collected in Brest Harbour. The percentages of metals released from this sediment in the presence of various concentrations of acids in seawater were important; concentrations of approximately 7 mg L(-1) for Mn and 60 mg L(-1) for Zn were observed under our experimental conditions. We also examined the rate of release of these metals from the sediment into the seawater in the presence of the different acids and under different experimental conditions. We found that most of the metallic elements were released from the sediments into the seawater during the first fifteen minutes of exposure. After this time, a high degree of pollution was induced if acids leached into seawater were not rapidly diluted.

A novel cationic lipophosphoramide with diunsaturated lipid chains: synthesis, physicochemical properties, and transfection activities.

Cationic lipophosphoramidates constitute a class of cationic lipids we have previously reported to be efficient for gene transfection. Here, we synthesized and studied a novel lipophosphoramidate derivative characterized by an arsonium headgroup linked, via a phosphoramidate linker, to an unconventional lipidic moiety consisting of two diunsaturated linoleic chains. Physicochemical studies allowed us to comparatively evaluate the specific fluidity and fusogenicity properties of the liposomes formed. Although corresponding lipoplexes exhibited significant but relatively modest in vitro transfection efficiencies, they showed a remarkably efficient and reproducible ability to transfect mouse lung, with in vivo transfection levels higher than those observed with a monounsaturated analogue previously described. Thus, these results demonstrate that this diunsaturated cationic lipophosphoramidate constitutes an efficient and versatile nonviral vector for gene transfection. They also invite further evaluations of the transfection activity, especially in vivo, of gene delivery systems incorporating the lipid reported herein and/or other lipids bearing polyunsaturated chains.

Fluorescence study of lipid-based DNA carriers properties: influence of cationic lipid chemical structure.

We report here a study on the physicochemical properties of cationic phospholipids liposomes used for lipoplex formulation and DNA transfer. The original cationic phospholipids synthesized in our laboratory are first presented with the liposome formulation process. The second part deals with the liposomes fusogenic properties studied by fluorescence resonant energy transfer (FRET). The nature of the cationic polar head and the formulation with or without a neutral colipid have a great influence on the FRET signal. The third part reports the study of the viscosity of the liposome by fluorescence anisotropy measurements. It has been observed that the vectors having a saturated lipid chain exhibit a more pronounced anisotropy than those having unsaturated lipid chains. Finally, liposomes formed by a mixture of phospholipids and DC-Chol (a rigid lipid) leads to increase the anisotropy denoting a more rigid liposome.