Light‐emitting‐diode‐induced chemiluminescence detection for capillary electrophoresis

In this work, an LED‐induced‐chemiluminescence (LED‐CL) system was developed to extend the application of CL detection in CE. In the LED‐CL, the analyte photooxidizes luminol under the irradiation of LEDs and generates CL. Taking the advantage of the small size nature of LEDs, the constructed photoreactor is greatly miniaturized, and especially suitable as a CE detector. The feasibility of the proposed detector was evaluated by detection of riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) after CE separation. Under the optimized conditions, the LODs for RF, FMN and FAD were 0.007, 0.02 and 0.1 μg/mL, respectively, better than those by UV detection. The RSDs were 3.4, 3.6 and 4.1% for 0.5 μg/mL RF, 2 μg/mL FMN and 5 μg/mL FAD, respectively. The LED‐CL detector features low cost, miniaturization, fast response, high sensitivity and good reproducibility.     

Quantification of riboflavin,flavin mononucleotide,and flavin adenine dinucleotide in mammalian model cells by CE with LED‐induced fluorescence detection

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein‐bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED‐induced fluorescence with limit of detections (LODs 0.5–3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1–14 amol/cell) and FAD (2.2–17.0 amol/cell) were the predominant flavins, while FMN (0.46–3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.     

亲水作用加压毛细管电色谱-激光诱导荧光检测法分析痕量核黄素类物质

一种新型的亲水作用毛细管电色谱(HI-CEC)整体柱被应用于加压毛细管电色谱-激光诱导荧光检测(pCEC-LIF)联用法对核黄素类物质的分离分析。采用自组装的pCEC-LIF系统,实现了对痕量核黄素(RF)、黄素单核苷酸(FMN)和黄素腺嘌呤二核苷酸(FAD)的快速分析。在最优的分离检测条件下,3种化合物在8.0 min内完全分离,RF、FMN和FAD的检出限(LOD, S/N=3)分别为5.0×10－11 mol/L、8.0×10－10mol/L和2.5×10－9mol/L,测定线性范围可达3个数量级,精密度良好。方法简便、全分析时间短、灵敏度和选择性高,血清样品分析实验结果良好,可望进一步应用于体液及细胞中核黄素类物质的痕量检测     

Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis

Simultaneous extraction, separation and quantitation of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in Chinese Hamster Ovary (CHO) cells were investigated. The separation of flavins and nicotinamide cofactors was performed by capillary electrophoresis with laser-induced fluorescence detection at the excitation wavelength of 325 nm. The separation protocol was established by investigating the excitation wavelength, high voltage and effects of buffer nature, pH and concentration. All endogenous fluorophores riboflavin, FAD, FMN, NADH and NADPH show wide linear range of quantitation. The limits of detection for the five compounds ranged from 4.5 to 23 nM. Extraction conditions were optimized for high-efficiency recovery of all endogenous fluorophores from CHO cells. To account for the complex matrix of cell extracts, a standard addition method was used to quantify FAD, FMN, NADH and NADPH in CHO cells. The quantitative results should be useful to reveal the metabolic status of cells. The protocols for extraction, separation and quantitation are readily adaptable to normal and cancer cell lines for the analysis of endogenous fluorophores.     

On-line wall-free cell for laser-induced fluorescence detection in capillary electrophoresis

A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm × 50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6 mm at one end of both 50 μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140 μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0 μmol/L. The column efficiency was in the range from 1.0 × 10⁵ to 2.5 × 10⁵ plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves.     

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.     

Altered flavin patterns in photobehavioral mutants of Phycomyces blakesleeanus.

Flavins were extracted from sporangiophores of the lower fungus Phycomyces blakesleeanus and identified by HPLC with fluorescence detection. In the wild-type strain NRRL1555 they were found to be present at the following concentrations: riboflavin (5.5 x 10(-6) M), flavin mononucleotide (FMN) (4.0 x 10(-6) M) and flavin adenine dinucleotide (1.4 x 10(-6) M). The HPLC elution profiles of the wild type were compared to a set of behavioral mutants (genotype mad) with specific defects in their light-transduction pathway. The photoreceptor mutants C109 (madB), C111 (madB) and L1 (madC) had normal amounts of flavins. The most prominent changes were found in single mutants with a defective madA gene which contained about 25% of riboflavin and about 10% of FMN and FAD normally found in the wild type. A hypertropic mutant with a defective madH gene contained instead 80% of riboflavin and 120% of FMN and FAD. The double mutant L52 (madA madC) and the triple mutant L72 (madA madB madC) had normal amounts of FAD and FMN. This indicates that the madC mutation, which itself causes loss of light sensitivity and which affects the near-UV/blue-light receptor (Galland and Lipson, 1985, Photochem. Photobiol. 41, 331-335) functions as a restorer of the flavin content in a genetic madA background. The double mutant L51 (madA madB) had about 40% of FMN and FAD, suggesting that the madB mutation functions as a partial restorer of flavin content. The photogravitropic thresholds (450 nm) reported for the wild type and the madA and madH mutants were positively correlated to the endogeneous concentrations of FMN and FAD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of electrochemical properties of FMN and FAD adsorbed on titanium electrode

The electrochemical properties (such as the values of the formal potentials, the dependence of the formal potentials on solution pH, the reversibility of the electrochemical process) of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) adsorbed on a titanium electrode were dependent on the electrolyte. The formal potentials of adsorbed FMN and FAD in phosphate, HEPES and PIPES buffers at pH 7 were similar to those for dissolved flavins (-460 to -480 mV vs. SCE) and changed linearly with a slope of about 52 mV per pH unit in the pH region 3 to 8. In TRIS buffer, the formal potentials of adsorbed FMN and FAD were also pH-dependent, however, with invariance in the pH range 4.5 to 5.5. In non-buffered solutions (KCl, LiCl, NaCl, CsCl, CaCl(2), Na(2)SO(4) at different concentrations), the electrochemical behavior of adsorbed FMN and FAD differed from that of dissolved flavins and was dependent on the electrolyte (especially at pH 4.5 and pH 5). Under certain conditions (electrolyte, concentration, pH), a two-step oxidation of FMN could be observed.     

Determination of picomolar levels of flavins in natural waters by solid-phase ion-pair extraction and liquid chromatography

A method is described for the rapid determination of flavins in sea water, based on solid-phase extraction followed by ion-pair high-performance liquid chromatography (HPLC) with fluorescence detection. Riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) and their photochemical breakdown products, lumiflavin, formylmethylflavin, and lumichrome can be determined with subpicomolar detection limits. The method was used at sea in the analysis of coastal and open ocean waters. In both environments, riboflavin, lumiflavin and lumichrome were routinely observed at concentrations in the picomolar range; lumiflavin and lumichrome were generally confined to the photic zone while riboflavin was present throughout the water column. Formylmethylflavin, FMN, and FAD were only occasionally observed; when present, these flavins were observed at consistently higher concentrations than riboflavin, lumiflavin and lumichrome.     

Oxygen uptake after electron transfer from amines, amino acids and ascorbic acid to triplet flavins in air-saturated aqueous solution

The photolysis of lumichrome, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) was studied in air-saturated aqueous solution at room temperature in the presence of appropriate electron donors: ascorbic acid, aromatic amino acids or amines, e.g. ethylenediaminetetraacetate (EDTA). The overall reaction is conversion of oxygen via the hydroperoxyl/superoxide radical into hydrogen peroxide. The quantum yield of oxygen uptake increases with the donor concentration, e.g. up to 0.3 for riboflavin, FMN or FAD in the presence of EDTA or ascorbic acid (0.3-10mM). The formation of H(2)O(2) is initiated by quenching of the acceptor triplet state by the electron donor and subsequent reaction of the semiquinone radical with oxygen. Specific properties of flavins are discussed including the radicals involved and the pH and concentration dependences. The quantum yield of photodegradation is low under air, but substantial under argon, where the major product absorbing in the visible spectral range is the corresponding hydroquinone.     

Light-triggered proton and electron transfer in flavin cofactors

The pH dependent behavior of two flavin cofactors, flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN), has been characterized using femtosecond transient absorption spectroscopy for the first time. The flavin excited state was characterized in three states of protonation (Fl(-), Fl, and FlH(+)). We found that Fl and Fl(-) exhibit the same excited state absorption but that the lifetime of Fl(-) is much shorter than that of Fl. The transient absorption spectrum of FlH(+) is significantly different from Fl and Fl(-), suggesting that the electronic properties of the flavin chromophore become appreciably modified by protonation. We further studied the excited state protonation of the flavin and found that the protonation sites of the flavin in the ground and excited state are not equivalent. In the case of FAD, its excited state dynamics are controlled by the two conformations it adopts. At low and high pH, FAD adopts an "open" conformation and behaves the same as FMN. In a neutral pH range, FAD undergoes a fast excited state deactivation due to the "stacked" conformer. The transition from stacked to open conformer occurs at pH ～ 3 (because of adenine protonation) and pH ～ 10 (because of flavin deprotonation).     

Ensemble and Single Molecule Studies on the Use of Metallic Nanostructures to Enhance the Intrinsic Emission of Enzyme Cofactors

We present a strategy for enhancing the intrinsic emission of the enzyme cofactors flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide (NADH). Ensemble studies show that silver island films (SIFs) are the optimal metal enhanced fluorescence (MEF) substrates for flavins and gave emission enhancements of over 10-fold for both FAD and FMN. A reduction in the lifetime of FAD and FMN on SIFs was also observed. Thermally evaporated aluminum films on quartz slides were found to be the optimal MEF substrate for NADH and gave a 5-fold increase in the emission intensity of NADH. We present finite-difference time-domain (FDTD) calculations that compute the enhancement in the radiated power emitting from an excited state dipole emitting in the wavelength range of NADH in close proximity to an aluminum nanoparticle, and a dipole emitting in the emission wavelength of flavins next to a silver nanoparticle. These calculations confirm that aluminum serves as the optimal MEF substrate for NADH and silver was the optimal MEF substrate for flavins. This is because the plasmon resonance properties of aluminum lie in the UV-blue regime and that of silver lie in the visible region. We also present the results of single molecule studies on FMN which show SIFs can both significantly enhance the intrinsic emission from single FMN molecules, significantly reduce their lifetimes and also significantly reduce FMN blinking. This is the first report of the observation of MEF from cofactors both at the ensemble and single molecule level. We hope this study will serve as a platform to encourage the future use of metallic nanostructures to study cofactors using their intrinsic fluorescence to directly monitor enzyme binding reactions without the need of extrinsic labeling of the molecules.     

Direct measurement by laser flash photolysis of intramolecular electron transfer in a two-domain construct of murine inducible nitric oxide synthase

Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.     

Inorganic oxidizer detection from propellants,pyrotechnics, and homemade explosive powders using gradient elution moving boundary electrophoresis

Advancement in rapid targeted chemical analysis of homemade and improvised explosive devices is critical for the identification of explosives-based hazards and threats. Gradient elution moving boundary electrophoresis (GEMBE), a robust electrokinetic separation technique, was employed for the separation and detection of common inorganic oxidizers from frequently encountered fuel-oxidizer mixtures. The GEMBE system incorporated sample and run buffer reservoirs, a short capillary (5 cm), an applied electric field, and a pressure-driven counterflow. GEMBE provided a separation format that allowed for continuous injection of sample, selectivity of analytes, and no sample cleanup or filtration prior to analysis. Nitrate, chlorate, and perchlorate oxidizers were successfully detected from low explosive propellants (e.g., black powders and black powder substitutes), pyrotechnics (e.g., flash powder), and tertiary explosive mixtures (e.g., ammonium nitrate- and potassium chlorate-based fuel-oxidizer mixtures). Separation of these mixtures exhibited detection without interference from a plethora of additional organic and inorganic fuels, enabled single particle analysis, and demonstrated semiquantitative capabilities. The bulk counterflow successfully excluded difficult components from fouling the capillary, yielding estimated limits of detection down to approximately 10 μmol/L. Finally, nitrate was separated and detected from postblast debris collected and directly analyzed from two nitrate-based charges.     

Quantitation of nicotinamide and serotonin derivatives and detection of flavins in neuronal extracts using capillary electrophoresis with multiphoton-excited fluorescence

Capillary electrophoresis (CE) with multiphoton-excited fluorescence detection (CE-MPE) allows low-background analysis of spectrally distinct fluorophores using a single long-wavelength laser. Extracts were prepared from immortalized rat raphe nuclei neurons, and were analyzed by CE-MPE. Native fluorescence was detected from reduced nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), riboflavin, serotonin, and 5-hydroxytryptophan (5HTrp). Quantitation of exogenous serotonin (taken up by cells) and endogenous NADH and 5HTrp was possible using internal standards or standard addition. This system should be useful to study monamine oxidase inhibitors (MAOIs) and selective serotonin reuptake inhibitors (SSRIs).     

KINETICS OF STACKING INTERACTIONS IN FLAVIN ADENINE DINUCLEOTIDE FROM TIME-RESOLVED FLAVIN FLUORESCENCE

The time dependence of the fluorescence of flavin adenine dinucleotide (FAD) was measured with a subnanosecond-resolving fluorometer. In contrast to the fluorescence decay of FMN, the decay of FAD was proved to be nonexponential. The time-dependent fluorescence of FAD can be interpreted by assuming an equilibrium between closed and open conformers in the ground state. The rate constant for folding in the excited state and the fluorescence lifetime of the intramolecular complex could be evaluated from analysis of the observed fluorescence decay. The results on FAD were compared to those on NADH obtained earlier.     

On-column trace enrichment by sequential frontal and elution electrochromatography II. Enhancement of sensitivity by segmented capillaries with z-cell configuration--application to the detection of dilute samples of moderately polar and nonpolar pesticides

An on-column trace enrichment method for capillary electrochromatography of dilute samples is described. It involves the sequential use of frontal and elution electrochromatography on a segmented capillary column comprising of two contiguous segments each packed with a different sorbent. While the entering segment is for preconcentration by frontal electrochromatography the second segment is much longer and is meant for separation of the enriched analytes in the subsequent elution electrochromatography step. The preconcentration segment is usually packed with a sorbent that affords the highest affinity towards the solutes of interest while the separation segment is packed with a stationary phase that exhibits the highest selectivity and separation efficiency for the analytes. The detection is performed in the UV using a z-cell configuration for achieving an increased path length for detection. The effectiveness of this on-column trace enrichment is demonstrated on dilute samples of moderately polar solutes (e.g., carbamate insecticides) and nonpolar solutes (e.g., pyrethroid insecticides). Under optimal frontal and elution electrochromatography conditions. 817- and 1100-fold sensitivity increase are achieved for permethrin (a pyrethroid insecticide) and methiocarb (a carbamate insecticide), respectively, with a UV detector. The method is demonstrated with real water samples (e.g., tap and lake water samples) spiked with carbamate and pyrethroid insecticides. The limits of detection for the pesticides achieved in tap and lake waters reached 10(-8) to 10(-9) M.     

Shah convolution differentiation Fourier transform for rear analysis in microchip capillary electrophoresis

This paper first reports the application of Shah convolution differentiation Fourier transform for rear analysis. Rear analysis eliminates the need to create a well-defined and reproducible sample plug, thus making the operation simpler. The number of solution reservoirs, for microchip capillary electrophoresis (CE), could be reduced from the usual four to three. Sample bias in CE could be avoided too. The separation channel was first filled with the fluorescent sample solution, and subsequently flushed out with the buffer. The rear of each analyte zone gives rise to its flight of sigmoid-shaped steps in the time-domain. The time-domain detector signal was first differentiated and then Fourier transform was performed. The Fourier transform results were represented in the form of a magnitude plot. It is proposed that this would be as equally applicable to other separation techniques (e.g., chromatography) and detection methods (e.g., absorption).     

A Homogenous Assay of FAD Using a Binding Between Apo‐Glucose Oxidase and FAD Labeled with an Electroactive Compound

A homogenous assay of FAD using a binding between glucose oxidase (apo‐GOD) and FAD labeled with an electroactive compound was developed. Because daunomycin is sensitively detected with voltammetry, daunomycin was connected to FAD with a cross‐linker. The peak current decreased due to the apo‐GOD‐labeled FAD binding. Competitive reaction to the apo‐GOD between FAD and the labeled FAD produces the increase of peak current. Accordingly, FAD is detected on the basis of the reaction. The merit of this method is that the influence from FMN and riboflavin in the measurement of FAD can be suppressed by the high selective binding.     

Supercritical fluid extraction as an on-line clean-up technique for determination of riboflavin vitamins in food samples by capillary electrophoresis with fluorimetric detection

An automatic method for the separation and determination of riboflavin (RF) vitamins (RF, flavin mononucleotide and flavin adenine dinucleotide) in food samples (chicken liver, tablet and powder milk) is proposed. The method is based on the on-line coupling of a supercritical fluid extractor (SFE) with a continuous flow-CE system with guided optical fiber fluorimetric detection (CF-CE-FD). The whole SFE-CF-CE-FD arrangement allowed the automatic treatment of food samples (clean-up of the sample followed by the extraction of the analytes), and the direct introduction of a small volume of the extracted plug to the CE-FD system for the determination of RF vitamins. Fluorescence detection introduced an appropriated sensitivity and contributed to avoid interferences of nonfluorescent polar compounds coming from the matrix samples in the extracted plug. Electrophoretic responses were linear within the 0.05-1 microg/g range, whereas the detection limits of RF vitamins were in the 0.036-0.042 microg/g range. The proposed arrangement opens up interesting prospects for the direct determination of polar analytes in complex samples with a good throughput and high level of automation.