Diode Laser-induced Fluorescence Research Articles

Dissolved organic matter in hand-dug well water as groundwater quality indicator: assessment using laser-induced fluorescence spectroscopy and multivariate statistical techniques

In groundwater, dissolved organic matter (DOM), a complex material, is a contaminant of concern owing to its ability to influence water quality and stimulate microbial metabolism. Using a 445-nm diode laser-induced fluorescence (LIF) spectroscopy, DOM contamination levels have been investigated of well water samples fetched from ten privately owned hand-dug wells during dry and wet seasons of 2016, 2017 and 2018, in Ghana. The results showed spatio-temporal heterogeneities in the LIF spectra, and the fluorescence intensity peaks were generally higher and broader during the wet season than the dry season. In this study, DOM fluorescence spectra at an emission wavelength band of 460–650 nm showed two distinct broad peak shoulders within 480–500 nm and 550–570 nm, engulfing the water Raman peak at 527 ± 2 nm for all the water samples studied. Furthermore, principal component analysis and cluster analysis were used to differentiate the 2016 water samples based on their DOM contamination levels. In each case, three groups or clusters were identified based on their similarities and dissimilarities. The study revealed humic DOM substances as the most typical well water fluorophores. Applying the K-nearest neighbour algorithm as a classifier method for the classification of 30 water samples studied in 2016, 16.7% (5/30) were classified as very good drinking water, 46.7% (14/30) as good, 26.7% (8/30) as fairly good, and 10% (3/30) as bad drinking water samples. In general, levels of dissolved organic matter contamination increased over the study period during the rainy seasons for wells situated in close proximity to septic tanks, refuse dumps, public toilets and in wetlands. Thus, in the study the fluorescence intensity depends on the sampling site and the season, and indicates the DOM contamination level.

Violet diode laser-induced chlorophyll fluorescence: a tool for assessing mosaic disease severity in cassava (Manihot esculenta Crantz) cultivars

Violet diode laser-induced chlorophyll fluorescence was used in agronomical assessment (disease severity and average yield per plant). Because cassava (Manihot esculenta Crantz) is of economic importance, improved cultivars with various levels of affinity for cassava mosaic disease were investigated. Fluorescence data correlated with cassava mosaic disease severity levels and with the average yield per plant.

Pressure Effect on the Velocity and Flux Distributions of Sputtered Metal Species in Magnetron Discharge Measured by Space‐Resolved Tunable Diode Laser Induced Fluorescence

AbstractThe pressure acts on the sputtered species resulting in their thermalization. Space‐resolved velocity distributions of titanium atoms sputtered in a direct‐current magnetron discharge were investigated using the tunable diode‐laser induced fluorescence technique (TD‐LIF). The titanium 3d24s2 − 3d2(3F)4s4p(1P0) transition centred at λ0 = 398.289 nm was used to excite neutral Ti atoms and to measure their velocity distribution profile through the TD‐LIF signal measured at λTD‐LIF = 736.31 nm. Two groups of metallic particles, namely energetic and thermalized atoms, could be clearly distinguished and the corresponding flux distributions along the laser beam direction were deduced. The paper discusses the relative importance of the energetic atoms versus the discharge pressure especially in the vicinity of the target.

Numerical simulation of the Zeeman effect in neutral xenon from NIR diode-laser spectroscopy

We present a numerical method for simulating neutral xenon absorption spectra from diode-laser spectroscopy of the Zeeman-split 6S′[1/2]→6P′[1/2] line at 834.682 nm-air in a galvatron’s plasma. To simulate the spectrum, we apply a Voigt profile to a spectrum of σ-transition lines of even- and odd-numbered isotopes computed from anomalous Zeeman and nonlinear Zeeman hyperfine structure theories, respectively. Simulated spectra agree well with Zeeman-split spectra measured from 30 to 300 G. A commercial nonlinear least-squares solver (LSQNONLIN) returns field strengths and translational plasma kinetic temperatures that minimize the error between simulated and experimental spectra. This work is a preamble to computing magnetic field topology and the speed distribution of neutral xenon particles in the plume of a Hall thruster from diode laser-induced fluorescence.

Microfluidic Capillary System for Immunoaffinity Separations of C-Reactive Protein in Human Serum and Cerebrospinal Fluid

A miniaturized system based on microfluidic capillaries is presented for point-of-care testing and clinical assessment. The approach relies on microsyringe pump-generated flow to deliver reagents and immunoaffinity chromatography to isolate the antigen from biological matrixes. Capillary sandwich immunoassays for C-reactive protein (CRP) were demonstrated in human serum and cerebrospinal fluid (CSF), which are relevant matrixes for cardiovascular disease risk and meningitis research, respectively. Capillaries packed with antibody-coated silica beads were used to capture CRP from the matrix and a second, dye-labeled antibody was introduced to form a sandwich complex. An acidic elution buffer dissociated the antibody-antigen complexes, and the labeled antibody was detected with diode laser-induced fluorescence. Four parameter logistic functions and % relative error plots were used to model and assess the data. The calibration ranges for CRP were 0.05-3.0 microg/mL in 1:10 diluted serum and 0.01-30 microg/mL in undiluted CSF. The microfluidic apparatus employed a flow rate of 2 microL/min and a sample injection volume of 250 nL. Since it was not necessary to reach antibody-antigen reaction equilibrium and the assay platform dimensions were minimal, run times were as short as 10 min.

Demonstration of a direct capture immunoaffinity separation for C-reactive protein using a capillary-based microfluidic device

C-reactive protein (CRP), a biomarker of inflammation and cardiovascular disease (CVD) risk assessment, was selected as a model antigen to demonstrate a direct labeling/direct capture immunoaffinity separation. The miniaturized device for immunoaffinity chromatography was constructed from two syringe pumps, a gradient mixing microchip, micro-injector with 250 nL capillary injection loop, a capillary column, and a diode laser-induced fluorescence detector fitted with a fused-silica capillary flow cell. Monoclonal anti-CRP was biotinylated and attached to 5.0 μm streptavidin-coated silica beads to make the solid support for separation columns. CRP in simulated serum matrix was fluorescently labeled in a one-step reaction and directly injected onto the immunoaffinity capillary. The purified antigen was then eluted in an acid gradient and measured. The antibody binding of CRP was evaluated in two physiological buffers, phosphate buffered saline (PBS) and Dulbecco's PBS (DPBS). A quadratic calibration model produced % relative errors of −15.9 to 12.6 for CRP concentration levels ranging from 0.47 to 95.0 μg/mL. The accuracy (% difference from nominal) and precision (% relative standard deviation) of replicate injections were within 17.0%. The limit of detection was 57.2 ng/mL and chromatographic run times were less than 10 min. The instrument design is simple, and potentially portable, while the assay procedure may be modified for other clinically relevant markers by changing the capture antibody.

Diode laser-induced fluorescence measurements of metastable argon ions in a magnetized inductively coupled plasma

Velocity distribution functions of metastable argon ions (3d′4F7∕2) have been measured to obtain metastable ion density and temperature by the diode laser-induced fluorescence (LIF) technique in magnetized inductively coupled plasma as a function of pressure, rf power, and magnetic field strength. Calculated density from a rate equation agrees with the trends observed in the experimental data. From the calculation, the metastable ion density should be over 107cm−3 to obtain a LIF signal. From a dc bias experiment, it is suggested that the spatial potential can be the dominant ion heating source, and a simple global model for ion temperature is constructed. In this model, approximately 0.01% and 10% of total spatial potential energy can contribute to ion and neutral temperatures, respectively. The measured ion temperature agrees with the calculation.

Rapid and sensitive determination of phosphorus-containing amino acid herbicides in soil samples by capillary zone electrophoresis with diode laser-induced fluorescence detection

A straightforward and sensitive method has been developed for the analysis of phosphorus-containing amino acid herbicides (glufosinate and aminomethylphosphonic acid, the major metabolite of glyphosate) in soil samples. For this purpose, the analytical features of two indocyanine fluorescent dyes, sulfoindocyanine succinimidyl ester (Cy5) and 1-ethyl-1-[5-(N-succinimidyl-oxycarbonyl)pentyl]-3,3,3,3-tetramethyl-indodicarbocyanine chloride, as labeling reagents for the determination of these herbicides by CZE with diode LIF detection were investigated. Practical aspects related to the labeling chemistry and CZE separation showed that the two probes behave similarly, Cy5 being the best choice for the determination of these herbicides on account of its higher sensitivity. The optimum procedure includes a derivatization step of the pesticides at 25 degrees C for 30 min and direct injection to CZE analysis, which is conducted within about 14 min using ACN in the running buffer. The lowest detectable analyte concentration ranged from 0.025 to 0.18 microg/L with a precision of 3.6-5.4%. These results indicate that indocyanine fluorescence dyes are useful as rapid and sensitive labels for the determination of these herbicides when compared with typical fluorescein dyes such as FITC and 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein, because they provide faster labeling reactions even at room temperature and the excess of reagent practically does not interfere the determination. Finally, the Cy5 method was successfully applied to soil samples without a preliminary clean-up procedure, and the herbicides were measured without any interference from coexisting substances. The recoveries of these compounds in these samples at fortification levels of 100-500 ng/g were 90-93%.

Determination of dye/protein ratios in a labeling reaction between a cyanine dye and bovine serum albumin by micellar electrokinetic chromatography using a diode laser-induced fluorescence detection.

The degree of labeling, i.e., dye/protein ratio (D/P) is important for characterizing properties of dye labeling with proteins. A method for the determination of this ratio between a fluorescent cyanine dye and bovine serum albumin (BSA), based on the separation of the labeling mixture using micellar electrokinetic chromatography with diode laser-induced fluorescence detection, is described. Two methods for the determination of D/P were examined in this study. In these methods, a hydrolysis product and impurities, which are usually unfavorable compounds that are best excluded for protein analysis, were utilized to determine the amounts of dye bound to BSA. One is a direct method in which a ratio of the peak area of BSA to the total peak area of all the products produced in the labeling reaction was used for determining the average number of dye molecules bound to a single BSA molecule. The other is an indirect determination, which is based on diminution of all peak areas related to the products except for the labeled BSA. These methods were directly compared by means of a spectrophotometric method. The experimental results show that the indirect method is both reliable and sensitive. Therefore, D/P values can be determined at trace levels using the indirect method.

Band broadening caused by the multiple labeling of proteins in micellar electrokinetic chromatography with diode laser-induced fluorescence detection

When a labeling reagent is used, in the determination of proteins by capillary electrophoresis with laser-induced fluorescence detection, the multiple labeling of proteins frequently occurs, which can degrade the separation efficiency. In order to understand the influence of the multiple labeling of proteins on separation efficiency, the band broadening caused by a labeling reaction between bovine serum albumin (BSA) and a cyanine fluorescent dye (Cy5) was investigated using micellar electrokinetic chromatography in conjunction with diode laser-induced fluorometry. With the aid of an internal standard, methylene blue, the height equivalent to the theoretical plate (HETP) ratio of BSA to methylene blue was used as an indicator for band broadening under optimum separation conditions. Labeling conditions, including reaction buffer pH, reaction time, and initial concentration of Cy5 to bovine serum albumin, were found to influence the HETP ratio. The separation efficiency for the labeled protein was degraded by experimental conditions employed in the labeling, which indicates an increase in the heterogeneity of the final products.

Micellar electrokinetic chromatography with diode laser-induced fluorescence detection as a tool for investigating the fluorescence labeling of proteins.

We describe the use of micellar electrokinetic chromatography with diode laser-induced fluorometry (MEKC-DLIF) as a tool to characterize a labeling reaction between sulfoindocyanine succinimidyl ester (Cy5), a cyanine fluorescent dye, and a model protein, bovine serum albumin (BSA). To decrease the influence of imprecise injection, methylene blue was added as an internal standard. Labeled BSA was completely separated from the unconjugated Cy5, and methylene blue under optimized conditions. A kinetic study of the reaction was performed by changing some parameters, such as reaction buffer pH, reaction buffer concentration and the initial concentration of BSA. A comparison between the current method and traditional methods was made.

Indocyanine green as a noncovalent, pseudofluorogenic label for protein determination by capillary electrophoresis.

Indocyanine green (ICG)--a negatively charged, polymethine dye--can interact noncovalently with proteins to form fluorescent complexes, with excitation and emission maxima near 780 and 820 nm, respectively. This behavior was realized utilizing either a 100 mM phosphate buffer or a 25 mM citric acid buffer, both at pH 3.1. The behavior of ICG under these conditions, termed pseudofluorogenic, rendered the dye suitable for use as a label for protein determination in capillary electrophoresis with diode laser-induced fluorescence detection (CE-LIF). To this end, pseudofluorogenic ICG was used both as an on-column label for human serum albumin (HSA) and as a precolumn label for a model mixture of proteins, including ribonuclease A, transferrin, and cytochrome c. These ICG-labeled proteins were successfully resolved in less than 11 min, with no interference from excess, unbound dye.

Capillary Electrophoresis Based Immunoassay for Monoclonal Antibody with Diode Laser Induced Fluorescence Detection

ABSTRACT A capillary electrophoresis based immunoassay (CEIA) for monoclonal antibody using diode laser induced fluorescence (LIF) detection was described. A direct assay for monoclonal anti-BSA in mouse serum was used as a model. BSA was labeled with Cy5 and used as the immunoreagent. The 635 nm line of a diode laser was used as the excitation source for LIF detection. The calibration curve for anti-BSA in mouse serum had a linear dynamic range of 4-40 nM. The concentration limit of detection (LOD) was 1.2 nM. Incubation time and CE conditions such as buffer concentration, pH and separation voltage were optimized, and the performances of different lasers as excitation sources were also compared.

Laser spectroscopy in analytical chemistry:

To date, lasers are widely accepted tools in analytical spectroscopy, involved in various stand-alone and hyphenated techniques. Furthermore, significant progress can be noted in this field. In this paper, first of all some laser characteristics are discussed. Subsequently, five selected topics are outlined to illustrate recent achievements and future developments: 1. Laser-induced fluorescence for detection in capillary electrophoresis, including the use of ultraviolet, continuous-wave lasers in combination with wavelength-resolved emission detection; the use of diode laser-induced fluorescence in the red region of the electromagnetic spectrum and the use of Ti:sapphire lasers for multiphoton-excited fluorescence detection. 2. Degenerate four-wave mixing for detection in liquid microseparation systems (based on the coherence of laser light). 3. Fluorescence line-narrowing spectroscopy for identification purposes, a cryogenic high-resolution molecular fluorescence technique with a high potential in environmental analysis. 4. Recent developments in Raman spectroscopy (including ultraviolet-Resonance Raman and hyphenation of liquid chromatography and Raman spectroscopy). 5. Use of lasers for sample introduction in inorganic analysis based on controlled material ablation.

Non-covalent labeling of human serum albumin with indocyanine green: a study by capillary electrophoresis with diode laser-induced fluorescence detection

Indocyanine green (ICG) is a negatively charged, water-soluble, tricarbocyanine dye used primarily for medical imaging. ICG is only weakly fluorescent in the near-infrared region in its free (unbound) state in dilute aqueous solution. However, when non-covalently bound to protein, its fluorescence is greatly enhanced, making it a candidate for diode laser-induced fluorescence (diode-LIF) detection of proteins in capillary electrophoresis (CE). This paper investigates the suitability of ICG as a fluorescent label for the separation and detection of human serum albumin (HSA) by CE with diode-LIF detection. Specifically, we have considered the separation conditions necessary to resolve free ICG from ICG–HSA complexes; the limits of detection for free and HSA-bound ICG; the stability of aqueous ICG and ICG–HSA solutions over time; and the stoichiometry of the ICG–HSA complex.

Characterization of TO-PRO-3 as an intercalator for double-stranded DNA analysis with red diode laser-induced fluorescence detection

We report the analysis of double-stranded DNA (dsDNA) with a blue intercalating dye, TO-PRO-3 (TP3) using a visible diode laser-induced fluorescence (LIF) detection. In the presence of single-stranded DNA (ssDNA), such as pd(A) 40–60 or pd(T) 20–40, TP3 adsorption maximum shifts slightly from 631 to 633 nm and, when intercalated to dsDNA, the absorption maxima shifts to 643 nm. TP3 itself does not fluoresce in the presence of eithet d(A)_ 18 or d(T) 18 alone, but the combination of both oliginucleotide species yielded intense fluorescence, about two orders of magnitude higher. The LIF detection was based on the use of a 2.5-mW diode laser emitting light at 635 nm and the fluorescence of the resulting dsDNA-bound TP3 was collected at 670 nm. The capillary electrophoretic (CE) separation of fragments from 72 to 1353 basepairs (bp) of ΦX174/ HaeIII digest were well-resolved within 10 min in the gel-buffer system with TP3. The application of TP3 as an intercalating dye for the analysis of dsDNA fragments produced by polymerase chain reaction (PCR) was examined. Excellent correlations between CE-LIF area and fragment size in basepairs were obtained with TP3 and ethidium bromide as the intercalating dyes. TP3-based chemistry along with the diode laser indiced fluorescence detection system is well-suited for rapid, high-sensitivity and automated DNA analysis of the PCR-amplified dsDNA products and DNA restriction fragments.

Visible diode laser-induced fluorescence detection of phenylacetic acid in plasma derivatized with Nile blue and using precolumn phase transfer catalysis.

This study reports the application of Nile blue (NB), a farred oxazine label, as a precolumn derivatization reagent for the measurement of free levels of phenylacetic acid (PAA) in plasma. The measurement of PAA in psychiatric populations is important because it provides a marker for 2-phenylethylamine (PEA), which has been implicated in the pathogenesis of schizophrenia and major depression. PAA was derivatized with NB through an amide linkage in the presence of 2-chloro-1-methylpyridinium iodide (carboxylic acid activator, CMP) and triethylamine (base catalyst, TEA), respectively. The formation of the NB-PAA derivative was confirmed using normal phase and reversed phase thin-layer chromatography, reversed phase liquid chromatography, and electrospray mass spectrometry. The formation of the NB-PAA derivative was optimized using a sequential single factor approach. The optimal conditions for the formation and chromatographic separation of the derivative were determined to be 8.0 nmol/mL NB, 390 nmol/mL CMP, 2 mumol/mL TEA, a reaction time of 45 min, and a reaction temperature of 25 degrees C. This derivatization scheme was performed in a phase transfer catalysis mode that enabled the simultaneous extraction, preconcentration, and derivatization of the analyte in a single step. The limit of derivatization of PAA was determined to be 1.0 x 10(-9) M in phosphate-buffered saline, a PAA-free matrix. This derivatization was limited not by the kinetics of the reaction but by the chromatographic separation of the derivative from a side reaction product. The method was used to estimate endogenous free levels of PAA in human plasma samples. The levels of PAA in four sources of plasma were determined to be within 30-70 ng/mL using the method of standard addition and reflected levels that have been reported in the literature. The limit of detection of the derivative was determined to be 7.33 x 10(-11) M using a laboratory-constructed HPLC-VDLIF detector.

Application of Rhodamine 800 for Reversed Phase Liquid Chromatographic Detection Using Visible Diode Laser-Induced Fluorescence

This study reports the application of rhodamine 800, a far-red dye, suitable for excitation using visible diode laser-induced fluorescence (VDLIF) detection. A reagent synthesized from rhodamine 800 was evaluated as a precolumn reagent for derivatization with amino-containing analytes. The derivative of this reagent with primary amine analytes showed a loss of fluorescence. Rhodamine 800 was then applied as a mobile phase additive in the indirect mode for quantitation of valproic acid in plasma using reversed phase HPLC in combination with VDLIF detection. A visible diode laser (output power 8.50 mW) temperature-tuned to oscillate at 674.70 nm was used as a light source for a laboratory constructed HPLC fluorescence detector. A liquid/liquid extraction procedure was applied to human blank plasma. The selectivity of this method was validated by demonstration of a lack of interfering peaks in extracts of plasma (n = 3 sources). A calibration curve for valproic acid between 40 and 200 μg/mL was shown to be linear (r = 0.9932). The recoveries of valproic acid at concentrations of 50 and 100 μg/mL were evaluated and determined to be 73 and 72%, respectively. The precision and accuracy (n = 5) of the assay was within 7.0% RSD and 8.0% difference from the spiked concentration, respectively. The limits of detection (S/N = 3) for extracted and unextracted valproic acid were 15.0 and 11.54 μg/mL, respectively. The theoretical (C(lim)) and practical (C(det)) limits of detection in the detector flow cell for unextracted valproic acid at a S/N = 1 were found to be within 15%.

An oxazine reagent for derivatization of carboxylic acid analytes suitable for liquid chromatographic detection using visible diode laser-induced fluorescence

This study reports the use of Nile Blue, an oxazine dye, as a derivatization reagent that fluoresces in the far-red spectral region and is suitable for derivatization with carboxylic-acid-containing analytes. Model carboxylic acid analytes such as benzoic acid, acetic acid, phenylacetic acid and hexanoic acid have been reacted as acid chlorides to form Nile Blue derivatives. The synthesis product of the Nile Blue benzoic acid derivative was confirmed using electrospray-mass spectrometry, infrared spectrometry, 1H and 13C nuclear magnetic resonance, reversed phase liquid chromatography (RP-HPLC), normal phase-thin layer chromatography, and spectral characterization. The synthesized Nile Blue derivatives, separated from reaction by-products with RP-HPLC, all demonstrated an approximately 10-fold drop in molar absorptivity and relative quantum yield. In addition, a 40 nm increase in Stokes shift was observed. A portion of the fluorescence was regained through post-column ionization of the Nile Blue benzoic acid derivative at pH 12. A RP-HPLC limit of detection of 88.25 fmol on column has been reported with conventional fluorescence detection-post-column ionization of the Nile Blue benzoic acid derivative. A limit of detection of 1.99 fmol on column (3.98 × 10 −11M) has been demonstrated for the Nile Blue benzoic acid derivative with the use of a laboratory-constructed visible diode laser fluorescence detector.

Column liquid chromatography with diode laser-induced fluorescence detection of carboxylic acids after pre-column derivatisation

Several pre-column approaches to fluorescence derivatisation of carboxylic acids, compatible with diode laser-induced fluorescence (DIO-LIF) detection, were compared. For this purpose red-absorbing labels containing hydrazide, bromoacetamide and iodoacetamide functionalities were studied. The sensitivity of DIO-LIF detection is illustrated by the (3–10) × 10 −12 M (75–250 amol) detection limit for various derivatised carboxylic acids. The real-life detection limit for the carboxylic acids is 3 × 10 −8 M , because derivatisation is not quantitative at lower concentrations. Derivatised n-alkylcarboxylic acids and non-steroidal anti-inflammatory drugs are easily separated by column liquid chromatography, despite the attachment of the bulky red-absorbing labels. The determination of the anti-inflammatory drug naproxen in saliva is shown as an example.