Laser-induced fluorescence of formaldehyde hot bands in flames

Laser-induced fluorescence and excitation spectra of formaldehyde in the A-X 4(1)(0) band at 370 nm are recorded in the primary flame front of a Bunsen flame. An examination of partition functions shows that this excitation can minimize temperature bias for formaldehyde in situ diagnostic measurements.     

Laser-induced fluorescence of perylene in a microparticle suspension environment

Laser-induced fluorescence (LIF) is one of the most sensitive techniques for analysis of traces of polycyclic aromatic compounds in liquids. Application of this method to on-line monitoring requires solution of problems related to the presence of particulate materials. Thus, understanding the analytical effects associated with the suspension of microparticles is of considerable importance for both environmental and industrial applications. Here, LIF of perylene in acetonitrile solutions was studied for different light-scattering/-absorbing microparticle matrixes. With increasing suspension mass of efficiently light absorbing black-colored particles, the intensity of the associated LIF signals was found to obey an apparently exponential decrease. Their white-colored counterparts, however, have initially produced a sizable, ～20%, signal increase of the respective LIF responses. An exponential decrease then became predominant, too. A model that explains the observations in terms of absorption and scattering coefficients is developed and examined. The quantitative effect of the particulate mass is correctly reproduced by the model, as well as the laser wavelength dependence. A possible application of a calibrating algorithm is addressed.     

Laser-induced fluorescence ion diagnostics in light of plasma processing

A brief overview of non-perturbing light diagnostics is followed by recent examples of process plasma properties measured via laser-induced fluorescence (LIF), optical emission, and absorption spectroscopy. Examples include radical density measurement via absorption and emission spectroscopy. With LIF, examples show properties of ion beam etching sources and ion velocity angle variations in ICP sheaths near a process surface. Because of the wide range of process plasma parameters, appropriate choice of light diagnostics varies.     

Laser-induced fluorescence detection system for microfluidic chips based on an orthogonal optical arrangement

In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossed-channel configuration, with a 1.5-mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. A fluorescence collection angle of 45 degrees in the chip plane gave the best result, with a scattered light intensity 1/38 of that obtained at an angle of 90 degrees. Sodium fluorescein and fluorescein isothiocyanate-labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N = 3) of 1.1 pM fluorescein was obtained, which is comparable to that of optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness, and ease in building.     

Compact detector for proteins based on two-photon excitation of native fluorescence

A compact, inexpensive detector for proteins has been constructed based on two-photon excitation of fluorescence from phenylalanine, tyrosine, and tryptophan. The fluorescence was excited by a solid-state microchip laser operating at 532 nm. Detection limits for phenylalanine, tyrosine, and tryptophan were 62 microM, 2.0 microM, and 470 nM, respectively, in a volume of 3 fL. The detection limit for a test protein, bovine serum albumin, was 130 nM.     

Conjugated polyelectrolyte based real-time fluorescence assay for phospholipase C

A fluorescence turnoff assay for phospholipase C (PLC) from Clostridium perfringens is developed based on the reversible interaction between the natural substrate, phosphatidylcholine, and a fluorescent, water-soluble conjugated polyelectrolyte (CPE). The fluorescence intensity of the CPE in water is increased substantially by the addition of the phospholipid due to the formation of a CPE-lipid complex. Incubation of the CPE-lipid complex with the enzyme PLC causes the fluorescence intensity to decrease (turnoff sensor); the response arises due to PLC-catalyzed hydrolysis of the phosphatidylcholine, which effectively disrupts the CPE-lipid complex. The PLC assay operates with phospholipid substrate concentrations in the micromolar range, and the analytical detection limit for PLC is <1 nM. The optimized assay provides a convenient, rapid, and real-time sensor for PLC activity. The real-time fluorescence intensity from the CPE can be converted to substrate concentration by using an ex situ calibration curve, allowing PLC-catalyzed reaction rates and kinetic parameters to be determined. PLC activation by Ca2+ and inhibition by EDTA and fluoride ion are demonstrated using the optimized sensor.     

Monolithic capillary electrophoresis device with integrated fluorescence detector

A monolithic capillary electrophoresis system with integrated on-chip fluorescence detector has been microfabricated on a silicon substrate. Photodiodes in the silicon substrate measure fluorescence emitted from eluting molecules. The device incorporates an on-chip thin-film interference filter that prevents excitation light from inhibiting the fluorescence detection. A transparent AZO conducting ground plane is also used to prevent the high electric fields used for the separation from interfering with the photodiode response. Separations of DNA restriction fragments have been performed in these devices with femtogram detection limits using SYBR Green I intercalating dye.     

Laser-induced fluorescence of se, as, and sb in an electrothermal atomizer

Trace detection of Se, As, and Sb atoms has been performed by electrothermal atomization laser-induced fluorescence (ETA-LIF) approaches. Production of far-UV radiation necessary for excitation of As atoms at 193.696 nm and Se atoms at 196.026 nm was accomplished by stimulated Raman shifting (SRS) of the output of a frequency-doubled dye laser operating near 230 nm. Both wavelengths were obtained as second-order anti-Stokes shifts of the dye laser radiation and provided up to 10 μJ/pulse, which was shown through power dependence studies to be sufficient for saturation in the ETA. An excited-state direct line fluorescence approach using excitation at 206.279 nm was also investigated for the LIF detection of Se. High-sensitivity LIF of Sb atoms was accomplished using 206.833-nm excitation and detection at 259.805 nm. The accuracy of the ETA-LIF approaches was demonstrated by determining the As and Se content of aqueous reference samples. The limits of detection (absolute mass) were 200 fg by ground-state LIF and 150 fg by excited-state direct line fluorescence for Se, 200 fg for As, and 10 fg for Sb; these LODs compare favorably with results reported previously in the literature for ETA-LIF, GFAAS, and ICP-MS methods.     

Estrogen receptor binding assay method for endocrine disruptors using fluorescence polarization

A rapid, simple and nonhazardous assay method for endcrine disruptors was developed using an estrogen receptor (ER) and fluorescence polarization (FP). Among the fluorescent compounds, the 17alpha-fluorescein-labeled estradiol derivative was selected as the most suitable ligand for the ER binding assay, since it showed the highest affinity to ER. In the Scatchard plot analysis, its convex curve exhibited a positive cooperative binding, indicating the induction of a conformational change of the ER with the binding of the ligand to form a dimer and to increase the affinity for the additional ligand. On the basis of the Hill plot analysis, its dissociation constant and Hill coefficient were 10.4 nM and 1.63, respectively. A competitive binding assay with an unlabeled 17beta-estradiol (E2) yielded an IC50 value of 2.82 nM and a Hill coefficient of 1.67, thus providing a Ki value of 0.65 nM. In the same manner, the Hill coefficients for estrone, estriol, diethylstilbestrol, and tamoxifen were determined to be 0.99, 1.17, 1.59, and 2.44, respectively.     

Laser-induced fluorescence spectroscopy for phenol and intermediate products in aqueous solutions degraded by pulsed corona discharges above water

Laser-induced fluorescence (LIF) spectroscopy is introduced as an in situ diagnostic for phenol and intermediate products in an aqueous solution degraded by corona discharges. The complications that are inherent in applying LIF as a diagnostic for aqueous solutions are experimentally examined. The LIF intensities of phenol and the intermediate products are measured as a function of time. The absolute phenol concentration is determined. We confirm the applicability of LIF spectroscopy for monitoring phenol concentration during degradation.     

A new competitive fluorescence assay for the detection of patulin toxin

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expansum, but it has also been extracted from rotten fruits, moldy feeds, and stored cheese. Human exposure to patulin can lead to serious health problems, and according to a long-term investigation in rats, the World Health Organization has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. However, extensive protocols of sample cleanup are required prior to the analysis, and to accomplish it, expensive analytical instrumentation is necessary. An immunochemical analytical method, based on highly specific antigen-antibody interactions, would be desirable, offering several advantages compared to conventional techniques, i.e., low cost per sample, high selectivity, high sensitivity, and high throughput. In this paper, the synthesis of two new derivatives of patulin is described, along with their conjugation to the bovine serum albumin for the production of polyclonal antibodies. Finally, a fluorescence competitive immunoassay was developed for the on-line detection of patulin.     

Laser-induced fluorescence detection by liquid core waveguiding applied to DNA sequencing by capillary electrophoresis

A new laser-induced fluorescence detector for capillary electrophoresis (CE) is described. The detector is based on transverse illumination and collection of the emitted fluorescent light via total internal reflection along the separation capillary. The capillary is coated with a low refractive index fluoropolymer and serves as a liquid core waveguide (LCW). The emitted light is detected end-on with a CCD camera at the capillary exit. The observed detection limit for fluorescein is 2.7 pM (550 ymol) in the continuous-flow mode and 62 fM in the CE mode. The detector is applied to DNA sequencing. One-color G sequencing is performed with single-base resolution and signal-to-noise ratio approximately 250 for peaks around 500 bases. The signal-to-noise ratio is approximately 50 for peaks around 950 bases. Full four-color DNA sequencing is also demonstrated. The high sensitivity of the detector is suggested to partly be due to the efficient rejection of scattered laser light in the LCW. The concept should be highly suitable for capillary array detection.     

A comparative study of efficiencies for different detector geometries for source excited energy-dispersive X-ray fluorescence systems

Different geometries for source excited X-ray fluorescence analysis systems are compared concerning their efficiency. Results of calculations for the geometric excitation-detection efficiency are presented for the annular and central source geometries, as well as for the here proposed receded source geometry, as a function of D/R (detector window-to-target distance/detector radius). For both point and finite size sources the new receded source geometry is found to be superior, reaching efficiencies that are in general a factor of three better than those for the annular source.     

Coupling of solid-phase microextraction and capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection for protein analysis

A coupling method of solid-phase microextraction (SPME) and capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) was developed for the analysis of proteins. Unlike other liquid-phase separation methods and conventional CIEF, proteins are focused into stationary bands within a pH gradient in CIEF-WCID. Thus, CIEF-WCID is the most compatible liquid-phase separation method for coupling with SPME, which can effectively resolve the problems associated with the slow desorption kinetics of SPME in a liquid phase. By combining SPME and CIEF-WCID, the desorption time can be as long as necessary, allowing complete desorption without any band broadening and analyte carryover. By using this method, R-phycoerythrin in water can be extracted by SPME in 10 min, and subsequently analyzed by CIEF-LIF-WCID within 20 min, providing a limit of detection of 3.5 x 10(-12) M (S/N = 3). The feasibility of the SPME-CIEF-LIF-WCID method was demonstrated by extracting and analyzing extracellular phycoerythrins in cultured cyanobacteria samples. Extracellular phycoerythrins at the nanomolar level were extracted and analyzed in 30 min, while avoiding the interference of the cyanobacteria cells.     

Conjugated polyelectrolyte-based real-time fluorescence assay for alkaline phosphatase with pyrophosphate as substrate

The fluorescence of the anionic, carboxylate-substituted poly(phenylene ethynylene) polymer PPECO2 is quenched very efficiently via the addition of 1 equiv of Cu(2+). Addition of pyrophosphate (PPi) into the weakly fluorescent solution of PPECO2 and Cu(2+) induces recovery of the polymer's fluorescence; the recovery occurs because PPi complexes with Cu(2+), effectively sequestering the ion so it cannot bind to the carboxylate groups of the polymer. A calibration curve was developed that relates the extent of fluorescence recovery to [PPi], making the PPECO2-Cu(2+) system a sensitive and selective turn-on sensor for PPi. Using the PPECO2-Cu(2+) system as the signal transducer, a real-time fluorescence turn-off assay for the enzyme alkaline phosphatase (ALP) using PPi as the substrate is developed. The assay operates with [PPi] in the micromolar range, and it offers a straightforward and rapid detection of ALP activity with the enzyme present in the nanomolar concentration range, operating either in an end point or real-time format. Kinetic and product inhibition parameters are derived by converting time-dependent fluorescence intensity into PPi (substrate) concentration, thus allowing calculation of the initial reaction rates (v(o)). Weak, nonspecific fluorescence responses are observed concomitant to addition of other proteins to the assay solution; however, the signal response to ALP is demonstrated to arise from the ALP catalyzed hydrolysis of PPi to phosphate (Pi).