Fluorescent Photoproducts Research Articles

The Conversion and Degradation of Sulphaguanidine under UV and Electron Beam Irradiation Using Fluorescence.

The work presents a spectral-luminescent study of the sulfaguanidine transformation in water under a pulsed e-beam and UV irradiation of an UVb-04 bactericidal mercury lamp (from 180 to 275nm), KrCl (222nm), XeBr (282nm) and XeCl (308nm) excilamps. Fluorescent decay curves have been used in our analysis of the sulfaguanidine decomposition. The conversion of antibiotic under e-beam irradiation for up to 1min was more than 80%, compared with UV radiation: UVb-04-26%, XeBr - 20%. KrCl and XeCl - about 10%. At the end of 64min of irradiation with UVb-04 and XeBr lamps, the conversion was 99%. During irradiation with these lamps, sulfaguanidine almost completely decomposed and passed into the final fluorescent photoproducts. After e-beam irradiated at the end of 13min the decrease in sulfaguanidine was 93%. At the same time, the formation of sulfaguanidine transformation products was minimal compared to UV irradiation. The effect of UV irradiation and a powerful e-beam on the decomposition mechanisms of sulfaguanidine are significantly different, which is manifested in various changes in the absorption and fluorescence spectra.

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

Reaction monitoring of rose bengal photodegradation in alcohols using multivariate frequency-domain dynamic fluorescence

• Photodegradation of rose bengal (RB) exposed to simulated solar radiation evaluated. • Reaction progress monitored using matrix-formatted frequency domain fluorescence. • RB and photoproduct(s) spectra and kinetics resolved by multivariate data mining. • Facile RB dehalogenation in methanol; no photoproducts observed in cyclohexanol. • Multivariate approach insulates results from distortion by unknown concomitants. The photodegradation of rose bengal (RB) by simulated solar radiation in several air saturated alcohols, monitored using wavelength-resolved frequency-domain dynamic fluorescence measurements, is described. This work illustrates a novel strategy for characterizing photochemical reactions of fluorophores using a combination of reaction monitoring by multichannel frequency domain fluorescence measurements and data mining. Kinetic and mechanistic details of RB photodegradation in the selected alcohols are obtained by resolving the spectra and frequency domain decays of unreacted RB and its fluorescent photoproducts from the multivariate measurements. The analysis indicates that RB exhibits photochemical activities and photodegradation rates that are strongly dependent on solvent properties. An associated benefit of the data mining is the insulation of rate calculations from distortions produced by spectral overlap of undiscovered concomitants. This was most evident in RB photodegradation rates in methanol, which paradoxically, is a common solvent choice for photosensitizer efficiency measurements using RB as the standard reference. RB photodegradation in methanol was not only the fastest of all the solvents investigated at 10.0±1 μM/min, methanol supported the formation of three fluorescent photoproducts that had emission properties consistent with RB dehalogenation. In the remaining alcohols, the rates of RB degradation decreased with decreasing solvent relative polarity and increasing solvent viscosity and only one dehalogenated RB derivative was observed. In octanol, the photodegradation rate was faster than expected based on its dielectric constant at 0.249±0.006 μM/min. In cyclohexanol, no photoproduct formation was detected. These results suggest a role for diffusion control in photoinitiated RB dehalogenation.

Intrinsic fluorescence of UV-irradiated DNA

In this report, we show that UV irradiation of natural and synthetic DNA results in a fluorescent photoproduct. Calf thymus DNA, poly(dAdT)2, poly(dGdC)2 and poly(dA).poly(dT) in phosphate buffer at pH 7 were investigated with UV-Vis and fluorescence spectroscopy before and after irradiation with UV light from an Hg lamp. Exposure of calf thymus DNA and poly(dA).poly(dT) to UV light for select amounts of time resulted in a fluorescent photo-product. Excitation of the samples at 298 nm after irradiation with UV light produced an emission spectrum with a maximum at approximately 402 nm. The intensity of the emission increased with increasing exposure time to UV light prior to the fluorescence measurement. Poly(dAdT)2 shows some fluorescence after irradiation with UV light, but to a much lesser degree than calf thymus DNA and poly(dA).poly(dT). Poly(dGdC)2 shows no fluorescence after irradiation with UV light, however, the UV-Vis data shows that poly(dGdC)2 does undergo photochemical changes. The results show that under the conditions employed, fluorescence occurs when thymine and adenine base pairs are present and is enhanced when the thymine nucleobases are adjacent to each other on the same strand.

Mechanism of Cyanine5 to Cyanine3 Photoconversion and Its Application for High-Density Single-Particle Tracking in a Living Cell.

Cyanine (Cy) dyes are among the most useful organic fluorophores that have found a wide range of applications in single-molecule and super-resolution imaging as well as in other biophysical studies. However, recent observations that blueshifted derivatives of Cy dyes are formed via photoconversion have raised concerns as to the potential artifacts in multicolor imaging. Here, we report the mechanism for the photoconversion of Cy5 to Cy3 that occurs upon photoexcitation during fluorescent imaging. Our studies show that the formal C2H2 excision from Cy5 occurs mainly through an intermolecular pathway involving a combination of bond cleavage and reconstitution while unambiguously confirming the identity of the fluorescent photoproduct of Cy5 to be Cy3 using various spectroscopic tools. The carbonyl products generated from singlet oxygen-mediated photooxidation of Cy5 undergo a sequence of carbon-carbon bond-breaking and -forming events to bring about the novel dye-to-dye transformation. We also show that the deletion of a two-methine unit from the polymethine chain, which results in the formation of blueshifted products, commonly occurs in other cyanine dyes, such as Alexa Fluor 647 (AF647) and Cyanine5.5. The formation of a blueshifted congener dye can obscure the multicolor fluorescence imaging, leading to misinterpretation of the data. We demonstrate that the potentially deleterious photoconversion, however, can be exploited to develop a new photoactivation method for high-density single-particle tracking in a living cell without using UV illumination and cell-toxic additives.

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.

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 3ngmL-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.

Automated Photochemically Induced Method for the Quantitation of the Neonicotinoid Thiacloprid in Lettuce.

In this work, we present an automated luminescence sensor for the quantitation of the insecticide thiacloprid, one of the main neonicotinoids, in lettuce samples. A simple and automated manifold was constructed, using multicommutated solenoid valves to handle all solutions. The analyte was online irradiated with UV light to produce a highly fluorescent photoproduct (λexc/λem = 305/370 nm/nm) that was then retained on a solid support placed in the flow cell. In this way, the pre-concentration of the photoproduct was achieved in the detection area, increasing the sensitivity of the analytical method. A method-detection limit of 0.24 mg kg−1 was achieved in real samples, fulfilling the Maximum Residue Limit (MRL) of The European Union for thiacloprid in lettuce (1 mg kg−1). A sample throughput of eight samples per hour was obtained. Recovery experiments were carried out at values close to the MRL, obtaining recovery yields close to 100% and relative standard deviations lower than 5%. Hence, this method would be suitable for routine analyses in quality control, as an alternative to other existing methods.

Phenylalanine Photoinduced Fluorescence and Characterization of the Photoproducts by LC-MS.

Phenylalanine (Phe) is a direct precursor of tyrosine and several neurotransmitters. The accumulation of Phe in the brain generates serious and not recoverable pathologies in children. Early detection in newborns is fundamental to apply the appropriate therapy and avoid irreversible health problems. Although fluorescence is a sensitive and selective technique for the determination of amino acids, the fluorescent analysis of Phe is limited since it exhibits a very low fluorescence quantum yield; however, the fluorescence of Phe increases drastically under UV irradiation when a peroxide medium is used. The aim of this research was to analyze the effect of the UV-radiation on Phe aqueous-peroxide solutions and to study the influence of the chemical environment on the photoinducted fluorescence process. The nature and characteristics of the fluorescent photoproducts generated under off-line UV irradiation in hydrogen peroxide medium were achieved by high performance liquid chromatography (HPLC) using a spectrophotometer detector (DAD) coupled in series with a mass spectrometer (MS) or with a fast scan spectrofluorimetric detector (FSFD). Environmental characteristics such as pH, initial concentration of Phe, hydrogen peroxide amount and irradiation time were studied in order to establish their influence on the formation of each one of the photoproducts. As the formation of several highly fluorescent photoproducts has been confirmed, the possibility of designing a chromatographic system with a post-column on-line photoreactor is open. The measure of the total fluorescence signal generated from Phe at the optimized irradiation time, could be used for the determination, with high sensitivity, of the initial amount of Phe in aqueous media, such as human serum or environmental samples. These aspects are being studied at present. .

Determination of Chlorpyrifos by a Multicommutated Photochemically Induced Fluorescence Optosensor

We propose a multicommutated flow-through optosensor for the quantitation of the organophosphate pesticide chlorpyrifos in dried chili peppers. We used photochemically induced fluorescence detection to overcome the negligible native fluorescence of the target pesticide. Firstly, the photodegradation of chlorpyrifos was performed online with an ultraviolet lamp. Then, the fluorescent photoproduct was retained on a solid support (Sephadex QAE A-25) inside the flow-cell in order to record the analytical signal. The online photodegradation and preconcentration of the photoproduct on the solid support were critical for the automation, speed, and high sensitivity obtained. A QuEChERS procedure was selected for sample treatment, obtaining recovery yields close to 100%. The method proposed presents a quantitation limit of 18 mg kg−1 in real samples, hence fulfilling the maximum residue limit of Codex Alimentarius, 20 mg kg−1. Therefore, this approach offers a novel method for screening purposes in the agricultural and food industries.

Fluorescence analysis of polyaromatic hydrocarbon photodegradation in the presence of polypropylene microfibers.

In this study, degradation of naphthalene and anthracene in water using ultraviolet (UV) light in the presence of polypropylene microfibers (PPM) modified with TiO2 was investigated using fluorescence analysis. KrCl (λrad =222nm) and XeCl (λrad =308nm) excilamps were used in a photoreactor. Phototransformation was studied for compounds in the presence of PPMs modified with TiO2 particles. The results indicated that the toxicants concentration was reduced by two orders of magnitude in the presence of PPMs. This reduction was due to effective adsorption of naphthalene and anthracene from water onto the surface of the PPMs. Exposure to the toxicant-water-PPM system to UV light led to the formation of fluorescent photoproducts.

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 a new automatic on-site detector of pesticides in natural waters by photo induced fluorescence, application to three phenylurea and benzoylurea herbicide.

Prototypes of on-site automatic photo induced fluorescence detectors of pesticide in natural waters are set up and applied for the determination of the benzoyl- and phenylurea pesticides, namely fluometuron, monolinuron and diflubenzuron. As these pesticides present no native fluorescence the set up system use the photo conversion under UV irradiation of these pesticides into highly fluorescent photoproducts. A first system, called AUTOPIF, (evolution the commercial AQUAPOD system) is develop using a detection via a diode array spectrometer. To improve the sensitivity of the method, a second system, called AUTOPIF+, is developed with a more resolute spectrometer and an intensified CCD camera detection. Analytical applications were carried out in aqueous solution and detected on line with the AUTOPIF and AUTOPIF+ system. The calibration curves are linear over one order of magnitude, and the limits of detection are in the μg mL-1 range. The analytical performances of these methods for the determination of the three pesticides are satisfactory in comparison to other classical PIF methods published for the determination of phenylurea pesticides in aqueous solutions. Our results show that the AUTOPIF and AUTOPIF+ methods are versatile, sensible and can be easily applied as an alert system to detect pollutant residues in naturals waters over a threshold value.

Determination of nitenpyram and 6-chloronicotinic acid in environmental samples by ion chromatography coupled with online photochemically induced fluorescence detector.

A simple, cost-effective, sensitive, and quick method for the determination of nitenpyram and its metabolite 6-chloronicotinic acid in environmental samples was developed by coupling an ion chromatograph with a fluorescence detector and a post-column photochemical reactor. This developed analytical method involved a rapid sample extraction by modified and miniaturized quick, easy, cheap, effective, rugged, and safe method followed by isocratic ion chromatographic separation of nitenpyram and 6-chloronicotinic acid into an IonPac™ AS11-HC column protected by IonPac™ AG11A guard column by running 30mM NaOH + 10% acetonitrile mobile phase. A homemade post-column photochemical reactor was also integrated with the ion chromatographic system for online transformation of both analytes into their respective highly fluorescent photoproduct in basic media without using an extra pump. The developed method was validated by following SANTE/11945/2015 guidelines on analytical quality control and validation procedures. The method showed a good linear response (r>0.999), improved limit of detection (0.101-0.132μg/L), minimum or no matrix effect, excellent recoveries (90.2-100.10%) and relative standard deviations were found to be ≤6.50%.

Formation of hemoglobin photoproduct is responsible for two-photon and single photon-excited fluorescence of red blood cells

Two-photon excited fluorescence of red blood cells (RBC) has been reported to be applicable for their assessment in vitro and in vivo. The corresponding fluorescence emission was ascribed to hemoglobin (Hb), however, as Hb is essentially non-fluorescent at single-photon excitation, the mechanism of two-photon excited fluorescence of RBC remains debatable. Here we show that a fluorescent photoproduct, characterized by an ultrafast decay of excitation, is formed after irradiation of Hb with femtosecond laser pulses with ca. 8 · 10−5 quantum yield, and that it is also fluorescent at single-photon excitation. The formation of a similar photoproduct was also shown for Hb continuous wave irradiation with blue light with ca. 10−5 formation quantum yield. The kinetics of the Hb photoproduct formation and its spectral properties were investigated. The obtained results clarify the processes responsible for RBC fluorescence observed in two-photon microscopy experiments.

A photochemically induced fluorescence based flow-through optosensor for screening of nitenpyram residues in cruciferous vegetables

ABSTRACTOne of the most used agrochemicals in agricultural production, nitenpyram (NTP), has been determined by using a flow-through optosensing device based on Photochemically Induced Fluorescence detection. The combination of both methodologies allows, on one hand, a quick on-line photodegradation of NTP and, on the other hand, the preconcentration, quantification and desorption of the fluorescent photoproduct generated when retained on Sephadex QAE-A25 as solid support, which was monitored at 295 and 362 nm for excitation and emission, respectively. The proposed analytical method presents a detection limit of 500 pg mL−1 by using Multicommutated Flow Injection Analysis. Recovery experiments were carried out in different kinds of cruciferous vegetables at or below the MRL established in Japan, demonstrating that this method combines advantages of simplicity, high sensibility and high selectivity, fulfilling the requirements for its application in quality control. Results obtained in the analysis of real samples were in good agreement with those provided by a reference HPLC method.

Emissive Photoconversion Products of an Amino-triangulenium Dye.

Upon prolonged exposure to intense blue light, the tris(diethylamino)-trioxatriangulenium (A3-TOTA(+)) fluorophore can undergo a photochemical reaction to form either a blue-shifted or a red-shifted fluorescent photoproduct. The formation of the latter depends on the amount of oxygen present during the photoconversion. The A3-TOTA(+) fluorophore is structurally similar to rhodamine, with peripheral amino groups on a cationic aromatic system. The photoconversion products were identified by UV-vis absorption and steady-state and time-resolved fluorescence spectroscopy, and further characterized by HPLC, LC-MS, and (1)H NMR. Two reaction pathways were identified: a dealkylation reaction and an oxidation leading to formation of one or more amide groups on the peripheral donor groups. The photoconversion is controlled by the experimental conditions, in particular the presence of oxygen and water, and the choice of solvent. The results highlight the need to characterize the formation of fluorescent photoproducts of commonly used fluorescent probes, since these could give rise to false positives in multicolor/multilabel imaging, colocalization studies, and FRET based assays. Finally, an improved understanding of the photochemical reaction leading to bleaching of fluorescent dyes can lead to the creation of specific probes for fluorescence based monitoring of chemical reactions.

Determination of clothianidin in food products by using an automated system with photochemically induced fluorescence detection

A flow-through system based on the integration of solid-phase spectroscopic detection implemented with photochemically induced fluorescence (PIF) is proposed for the determination of clothianidin (a non-fluorescent neonicotinoid insecticide) through a multicommutated method. The pesticide is injected into the carrier stream (0.015molL−1C2H4O2/NaC2H3O2, pH=5.0) and flows towards a homemade photoreactor, which consists of a PTFE tubing loosely coiled around a low-pressure mercury lamp (15W). After the photochemical reaction of clothianidin, the generated fluorescent photoproduct is transported to a flow cell packed with Sephadex-SP C-25 where it is retained and monitored (λex=357nm/λem=418nm). The method presents a detection limit of 1.5ngmL−1, a sample throughput of 23h−1 and inter-day relative standard deviation lower than 3%. The described system has been satisfactorily applied to the determination of clothianidin in samples of drinking water, rice and honey. Taking into account that the maximum residue limit specified in the Codex Alimentarius Commission for rice grains is 0.5mgkg−1, recovery experiments have been carried out for clothianidin concentrations in the 0.3-10.0mgkg−1 range.

Development of an semi-automatic and sensitive photochemically induced fluorescence sensor for the determination of thiamethoxam in vegetables

The determination of thiamethoxam (TMX), a widely known neonicotinoid pesticide, by a multicommutated optosensing device implemented with photochemically induced fluorescence (PIF) has been developed. The combination of both methodologies allows, on one hand a quick on-line photodegradation of TMX and, on the other hand, the preconcentration, quantification and desorption of the fluorescent photoproduct generated once retained on C18 silica gel filling the flow-cell which was monitored at 353 and 407nm for excitation and emission wavelengths, respectively.The proposed analytical method presents a detection limit of 3.6ngmL−1 by using Multicommutated Flow Injection Analysis (MCFIA) as flow methodology. Recovery experiments have been carried out in different kinds of vegetables at levels same or below the legislated maximum residue limit, demonstrating that this method combines advantages such as simplicity, high sensibility and high selectivity, in addition to fulfill the requirements for its applications in quality control. The obtained results in the analysis of real samples were in good agreement with those provided by a reference liquid chromatography (HPLC) method.