Diode Laser-induced Fluorescence Detection Research Articles

Fiber optic illumination of a poly(dimethylsiloxane) sheath flow cuvette for diode laser induced fluorescence detection in capillary electrophoresis

A Tee configuration sheath flow cuvette with square cross-section channels has been produced in PDMS for CE detection. The output of a 1.4 W laser diode operating at 450 nm was focused onto the 300 μm core of a 370 μm od fiber optic whose end was inserted into one arm of the Tee for LIF. The optimal configuration had the fiber optic positioned 500 μm downstream from the intersection and the end of the 35 cm 50 μm id 365 μm od capillary just outside the intersection and in the leg of the Tee, resulting in a 90° configuration. Detection limits of 50 and 3 pM and linear calibrations of at least three orders of magnitude were obtained for Lucifer Yellow and fluorescein, respectively.

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%.

Fluorimetric studies and noncovalent labeling of protein with the near‐infrared dye HITCI for analysis by CE‐LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.

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.

Visible diode laser induced fluorescence detection of doxorubicin in plasma using pressurized capillary electrochromatography.

Pressurized capillary electrochromatography is a variant of capillary electrochromatography (CEC) in which the driving force is both electroosmotic and hydraulic. The inlet of the CEC capillary is pressurized using an HPLC pump, and an electric field is simultaneously applied. This work describes a method for the analysis of doxorubicin. Doxorubicin was reacted with Cy5.29.OSu in acetonitrile. The derivative was confirmed by RP-TLC. A CEC system equipped with a VDLIF detector was constructed and used to analyze the derivative. The reaction mixture was injected onto a capillary packed in-house with 3 microm C-18 Luna particles and separation was carried out at 25 kV using 70% acetonitrile/ 30% phosphate (10 mM, pH = 4.8) as the mobile phase. The derivatization reaction was optimized by the investigation of parameters such as reaction time, temperature and concentration of label in order to increase the yield of the derivative. The optimal conditions were determined to be 30 min, 80 degrees C and 50 nmol/mL, respectively. Doxorubicin was extracted from plasma using solid-phase extraction under alkaline conditions, derivatized and injected onto the CEC-VDLIF system. The selectivity of the assay was demonstrated by a lack of interfering peaks due to plasma constituents across the elution window of the derivative peak in blank plasma extracts (n = 6 sources). The limit of detection (LOD) of the assay in plasma calculated as 3 s(b)/m was determined to be 1.7 ng/mL. The precision of the assay determined at a concentration of 167.7 ng/mL (n = 5) was found to be within 7.04 %RSD.

Red diode laser induced fluorescence detection with a confocal microscope on a microchip for capillary electrophoresis

A highly sensitive laser induced fluorescence (LIF) detection system based on a 635 nm laser diode and cyanine-5 (Cy-5) dye, is described for use with a planar, microfluidic, capillary electrophoresis (CE) chip. The CE-chip is able to determine a protein biological threat agent simulant, ovalbumin (Ov), by performing an immunoassay separation of Cy-5 labeled anti-ovalbumin from its complex with Ov, in under 30 s. A confocal, epiluminescent detection system utilizing a photomultiplier tube gave optimum results with a 400 μm pinhole, an Omega 682DF22 emission filter, a 645DRLP02 dichroic mirror, a 634.54±5 nm excitation filter, and a Power Technology ACMO8 635 nm laser operated at 11.2 mW. Using this detector, a microchip CE device with a separation efficiency of 42 000 plates and an etch depth of 20 μm, gave a concentration detection limit of 9 pM Cy-5. This limit corresponds to the determination of 4560 injected molecules and detection of 900 of these molecules, given a probe volume of 1.6 pl and a probing efficiency of 20%.

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.

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.

Development of near-infrared fluorophoric labels for the determination of fatty acids separated by capillary electrophoresis with diode laser induced fluorescence detection†

Synthesis and characterization of a polymethine cyanine near-infrared (NIR) fluorophoric label for the derivatization and determination of fatty acids separated by capillary electrophoresis are described. The label contains an aromatic amine functionality, which is used to form a covalent linkage with the analyte. Various linking chemistries are explored, including direct amine–acid condensation using dicyclohexylcarbodiimide (DCC) as a carboxyl activating group. Spectrofluorimetry was used to probe the fluorescence efficiency of the label in order to assist in choosing a separation medium for capillary electrophoretic separation. A nonaqueous separation medium for capillary zone electrophoresis was used to provide high quantum efficiency for fluorescence and adequate solubility of fatty acid analytes. Diode laser-induced fluorescence detection following electrophoresis of a simple mixture of labeled fatty acids shows the applicability of this method to biologically relevant carboxylic acid analytes.

Visible diode laser induced fluorescence detection for capillary electrophoretic analysis of amantadine in human plasma following precolumn derivatization with Cy5.29.OSu

Visible diode laser induced fluorescence (VDLIF) detection (620–700 nm) has become important in bioanalysis due to the increased sensitivity and selectivity that can be achieved in biological matrices. A selective and sensitive capillary electrophoretic method employing VDLIF detection has been developed for the analysis of amantadine in plasma. Amantadine was extracted from plasma into toluene under alkaline conditions and the residue was derivatized with the far-red label Cy5.29.OSu. The reaction mixture was dried under nitrogen, reconstituted and then injected onto a laboratory constructed capillary electrophoresis system equipped with a laboratory constructed visible diode laser detector temperature tuned to oscillate at 647.8 nm. The selectivity of the technique was evaluated by demonstrating a lack of interfering peaks in extracts of blank plasma. A calibration curve ranging from 1.8 to 461.1 ng ml −1 was shown to be linear. The precision and accuracy of the assay ( n=6) were determined to be within 17% R.S.D. and 15% difference from the nominal concentration respectively. The limits of detection for unextracted amantadine and for amantadine from the extracted concentrate from plasma were determined to be 9.5 fmol and 115 amol respectively.

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%.

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.

Diode laser-induced fluorescence detection in capillary electrophoresis after pre-column derivatization of amino acids and small peptides

The use of diode laser-induced fluorescence (DIO-LIF) detection in the field of capillary electrophoresis (CE) is examined. A simple but sensitive detection system was constructed. The performance of the system was evaluated with respect to design factors and its sensitivity was compared with the theoretically achievable sensitivity. To enhance the applicability of direct DIO-LIF detection in CE, a derivatization method for amines was developed. A red-absorbing label, consisting of a dicarbocyanine fluorophore with a succinimidyl ester functionality, was synthesized for this purpose. After derivatization of 1 × 10 −6 M glycine, a detection limit of 0.1 amol was observed for the labeled glycine. Similar detection limits were observed for other amino acids. To show that derivatization preserves the separation efficiency of CE for the analytes examined, 18 amino acids and tyramine were separated with micellar electrokinetic chromatography after labeling. In addition, even labeled peptides, including structurally related enkephalin-type compounds, were separated from each other with zone electrophoresis. To test the applicability of the derivatization method to biological samples, tyramine was determined in urine before and after the consumption of cheese.

Visible Diode Laser-Induced Fluorescence Detection in Liquid Chromatography after Precolumn Derivatization of Amines

To fully exploit the attractive visible diode laser-induced fluorescence (LIF) detection technique in column liquid chromatography (LC), covalent labeling procedures need to be developed which are compatible with near-infrared fluorescence. For this purpose, several red-absorbing labels containing a single succinimidyl ester functionality were synthesized and used for the derivatization of primary and secondary amines. Oxazines/thiazines, squaraines, and dicarbocyanines were examined as redabsorbing fluorophores. The quality of the LC-diode LIF system for the analytes concerned is illustrated by the concentration detection limit of the labeled n-octylamine (a test compound), which was as low as 2×10 -12 M. As expected in view of the limited reactivity of the succinimidyl ester group, the analyte concentrations required to obtain quantitative reaction with the label were equal to or higher than 2×10 -8 M. To show the applicability of the method to biological samples, urine was spiked with 5×10 -7 M 1-adamantanamine, extracted, derivatized with a dicarbocyanine-based label, and analyzed by LC-diode LIF

Determination of the anticancer drug metabolite WR1065 using pre-column derivatization and diode laser induced fluorescence detection

A liquid chromatographic (LC) procedure using alumina as stationary phase in both the pre- and the analytical column, is reported for the determination of WR1065, the active metabolite of the amino- and thiol-containing anticancer drug WR2721. After pre-column derivatization of the thiol group, the analyte is determined by LC with diode laser induced fluorescence detection in the near-infrared. Selective removal of excess label is achieved by means of column switching; it allows the detection of 5 × 10 −9 M WR1065 in water and 10-fold diluted, deproteinated plasma samples. The detection limit is determined by the derivatization reaction and not by the fluorescence detection of the labelled analyte. Endogeneous thiols do not interfere.