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1.
Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 microM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2-2 microg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.  相似文献   

2.
An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5m-dCMP. After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%.  相似文献   

3.
Hsieh MM  Chang HT 《Electrophoresis》2005,26(1):187-195
On-line concentration and separation of biologically active amines and acids by capillary electrophoresis (CE) in conjunction with laser-induced fluorescence using an Nd:YAG laser at 266 nm under discontinuous conditions is presented. The suitable conditions for simultaneous analysis of amines and acids were: samples were prepared in a solution (pH* 3.1) consisting of 10 mM citric acid, 89% acetonitrile (ACN), and water; a capillary was filled with 1.5 M Tris-borate (TB) buffer (pH 10.0); and the anodic vial contained PTG10 buffer (pH* 9.0) that consists of 50 mM propanoic acid, Tris, 10% glycerol, and water. After injecting a large-volume sample, amines and acids were separately stacked at the front (cathodic side) and back (anodic side) of the acidic sample zone, mainly because of changes in their electrophoretic mobilities as a result of changes in pH, viscosity, and electric field when high voltage was applied. When the sample was injected at 15 kV for 360 s, the concentration limits of detection (LODs) for 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) were 0.27 and 0.31 nM, respectively, which are about 400- and 800-fold sensitivity improvements when compared to those injected at 1 kV for 10 s. For the analysis of amines, samples were prepared in 100 mM citric acid (pH* 1.8) containing 89% ACN and both the capillary and anodic vial were filled with 400 mM PTG20 (propanoic acid, Tris, 20% glycerol, and water) at pH* 4.5. Using a large injection volume (15 kV for 360 s), we achieved concentration LODs of 17 pM and 0.3 nM for tryptamine and epinephrine, which are about 5200- and 14,000-fold sensitivity improvements, respectively, in comparison with those injected at 1 kV for 10 s. The features of simplicity (no sample pretreatment), rapidity (12 min), and sensitivity for identification of amines and acids of interest in urine samples show diagnostic potential of the two approaches developed in this study.  相似文献   

4.
Serotonin or 5-hydroxytryptamine (5-HT) is a major neurotransmitter in the central nervous system. In this work, a method for analyzing 5-HT in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. A pH-mediated in-capillary preconcentration of samples was performed, and after separation by capillary zone electrophoresis, native fluorescence of 5-HT was detected by a 266 nm solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical bases. Separation of 5-HT was performed using a 80 mmol/L citrate buffer, pH 2.5, containing 20 mmol/L hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and +30 kV voltage. The detection limit was 2.5 x 10(-10) mol/L. This method allows the in vivo brain monitoring of 5-HT using a simple, accurate CE measurement in underivatized microdialysis samples.  相似文献   

5.
Kuo IT  Huang YF  Chang HT 《Electrophoresis》2005,26(13):2643-2651
This paper describes the analysis of biologically active amines by capillary electrophoresis (CE) in conjunction with laser-induced native fluorescence detection. In order to simultaneously analyze amines and acids as well as to achieve high sensitivity, 10 mM formic acid solutions (pH < 4.0) containing silica nanoparticles (SiNPs) were chosen as the background electrolytes. With increasing SiNP concentration, the migration times for seven analytes decrease as a result of increase in electroosmotic flow (EOF) and decrease in their electrophoretic mobilities against EOF. A small EOF generated at pH 3.0 reveals adsorption of SiNPs on the deactivated capillary wall. The decreases in electrophoretic mobilities with increasing SiNP concentration up to 0.3x indicate the interactions between the analytes and the SiNPs. Having a great sensitivity (the limits of detection at a signal-to-noise ratio (S/N) = 3 of 0.09 nM for tryptamine (TA)), high efficiency, and excellent reproducibility (less than 2.4% of the migration times), this developed method has been applied to the analysis of urinal samples with the concentrations of 0.50 +/- 0.02 microM, 0.49 +/- 0.04 microM, and 74 +/- 2 microM for TA, 5-hydroxytryptamine, and tryptophan, respectively. The successful examples demonstrated in this study open up a possibility of using functional nanoparticles for the separation of different analytes by CE.  相似文献   

6.
Summary A novel method of separating and detecting trace aluminum by capillary zone electrophoresis is described. Aluminum is reacted with lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzen-1-sulphonic acid] so that the complex can be selectively and sensitively detected by a laser-induced fluorescence detector after capillary electrophoretic separation. Using the proposed method, limits of detection in the sub parts per billion range are achieved. The technique is applied to the determination of aluminum in human serum.  相似文献   

7.
Py-1 and Py-6 are novel amino-reactive fluorescent reagents. The names given to them reflect that they consist of a pyrylium group attached to small aromatic moieties. Upon reaction with a primary amine there is a large spectral shift in the reagent, rendering them effectively fluorogenic. In this study, these reagents were used to label a test protein, (human serum albumin), and the sample was analyzed by capillary electrophoresis and laser-induced fluorescence detection. Detection limits after a 60 min labeling reaction at 22 degrees C (Py-1) and 50 degrees C (Py-6) were 6.5 ng/mL (98 pM) for Py-1 and 1.2 ng/mL (18 pM) for Py-6. Separation of immunoglobulin G (IgG), human serum albumin, lipase, and myoglobin after labeling with Py-6 were performed. The method was further modified to make it amenable to automation. Unlike many other amino reactive reagents used to label protein amino groups, reaction with Py-1 and Py-6 do not alter the charge of the protein and the advantage of this with respect to electrophoretic separations is discussed.  相似文献   

8.
Sluszny C  He Y  Yeung ES 《Electrophoresis》2005,26(21):4197-4203
A continuous-wave 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection of proteins in CE. The operating current and temperature of the LED were optimized in order to achieve high luminescence power. It was found that a forward current of 30 mA and a temperature of approximately 5 degrees C gave the best S/N. By using a set of two ball lenses to focus light from the LED, we achieved a spot of approximately 200 mum with a power of 0.1-0.2 mW on the detection window. Fluorescence was collected with a ball lens at 90 degrees angle through a bandpass filter onto a photomultiplier tube. In CZE an LOD of 20 nM for conalbumin was reached. In capillary gel electrophoresis all eight proteins from a commercial standard kit were detected with high S/N. For a 10 microg/mL total protein mixture, S/N was better than 3 for all proteins in solution. Further improvement in LOD should be possible on utilization of an LED with higher luminescence power.  相似文献   

9.
This paper reports the development of a method based on capillary electrophoresis with laser-induced fluorescence detection for the simultaneous determination of thiouracil (TU) and phenylthiouracil (PhTU) with high sensitivity (nanomolar range, i.e., attomoles detected). After derivatization with 5-iodoacetamidofluorescein, the analytes were separated by capillary zone electrophoresis using 20 mM phosphate buffer (pH 10.0) and quantified by fluorescence detection. The linearity range, precision, recovery, and detection limits were determined, and the method was shown to be applicable for the determination of TU and PhTU in spiked feed samples and urine.  相似文献   

10.
Huang Y  Duan J  Chen H  Chen M  Chen G 《Electrophoresis》2005,26(3):593-599
A capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the separation and detection of carnosine-related peptides (carnosine, anserine, and homocarnosine). A sensitive and fluorogenic regent, 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) was selected as a precapillary labeling reagent for imidazole dipeptides to form isoindole derivatives. The optimized molar ratio between CBQCA and peptide was found to be 75:1, and 50 mmol/L borate buffer (pH 9.2) was used for the derivatization in order to achieve good efficiency. Three imidazole dipeptides were baseline-separated within 20 min by using 112 mmol/L sodium borate (pH 10.4-10.8) as running buffer. Concentration detection limits (signal-to-noise ratios) for carnosine, anserine, and homocarnosine were 4.73, 4.37, and 3.94 nmol/L, respectively. This method has been applied to the analysis of human cerebrospinal fluid (CSF) and meat dry powder of pig and sheep. Recoveries were in the range of 82.9-104.8% for homocarnosine in CSF. For carnosine and anserine, the recoveries are 98.3% and 80.2% in meat dry powder of pig and 111.2% and 112.8% in meat dry powder of sheep, respectively.  相似文献   

11.
Orejuela E  Silva M 《Electrophoresis》2005,26(23):4478-4485
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%.  相似文献   

12.
Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.  相似文献   

13.
An ultrasensitive laser-induced fluorescence detector was used with capillary electrophoresis for the study of 5-carboxy-tetramethylrhodamine. The raw signal from the detector provided roughly three orders of magnitude dynamic range. The signal saturated at high analyte concentrations due to the dead time associated with the single-photon counting avalanche photodiode employed in the detector. The signal can be corrected for the detector dead time, providing an additional order of magnitude dynamic range. To further increase dynamic range, two fiber-optic beam-splitters were cascaded to generate a primary signal and two attenuated signals, each monitored by a single-photon counting avalanche photodiode. The combined signals from the three photodiodes are reasonably linear from the concentration detection limit of 3 pM to 10 μM, the maximum concentration investigated, a range of 3,000,000. Mass detection limits were 150 yoctomoles injected onto the capillary.  相似文献   

14.
We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.  相似文献   

15.
毛细管电泳荧光检测模式的研究进展   总被引:1,自引:0,他引:1  
毛细管电泳技术由于具有分析速度快、分离效率高、样品用量少、操作简单、抗污染能力强等特点,广泛的应用于环境监测、临床检验、生物样品分析等领域.荧光检测技术的引入使毛细管电泳在保持强大的分离能力的同时具有了较高的检测灵敏度.本文介绍了影响毛细管电泳-荧光检测灵敏度的两个重要因素(光源和检测模式),对目前常用的两种光源(激光和发光二极管)以及三种检测模式的光路结构(正交型、共线型和嵌入式光纤传导-柱内直接激发型)的设计进行了相应地比较.  相似文献   

16.
This work described a sensitive method for determination of metoprolol in rabbit plasma.The method involved purification by ultrafiltration,derivatization with fluorescein isothiocyanate,determination by capillary electrophoresis(CE) coupled with laser-induced fluorescence(LIF) detector.Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol in experimental condition.The assay had a wide range(2.0-500 ng/mL) of linearity and a detection limit of 0.8 ng/mL.The intra- and inter-day precisions were satisfactory with relative standard deviation(RSD) less than 10.0%and accuracy within 10.0%.This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood.  相似文献   

17.
Lin X  Zhu C  Hao A 《Electrophoresis》2005,26(20):3890-3896
The resolving ability of 2-O-(2-hydroxybutyl)-beta-CD (HB-beta-CD) with different degrees of substitution (DS = 2.9 and 4.0) as a chiral selector in CZE is reported in this work. Fourteen chiral drugs belonging to different classes of compounds of pharmaceutical interest such as beta-agonists, antifungal agents, ageneric agents, etc., were resolved. The effects of the DS of HB-beta-CD on separations were also investigated. The chiral resolution (R(s)) was strongly influenced by the concentrations of the CD derivative, the BGE, and the pH of the BGE. Under the conditions of 50 mmol/L Tris-phosphate buffer at pH 2.5 containing 5 mmol/L HB-beta-CD, all 14 analytes were separated. The very low concentration necessary to obtain separation was particularly impressive. The DS had a significant effect on the resolution of the chiral drugs and the ionic strength of the separation media; hence, the use of a well-characterized CD derivative is crucial.  相似文献   

18.
Zhang H  Jin W 《Electrophoresis》2004,25(3):480-486
A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.  相似文献   

19.
Rhodamine 110 (Rho110) has been used in the highly sensitive analysis of monosaccharides, as it reacts with the reducing carbonyl group of the saccharides. The monosaccharide derivatives were investigated by capillary electrophoresis with laser-induced fluorescence detection. The derivatization was performed at 90 °C for 30 min for all monosaccharides. The derivatized monosaccharides were separated using 200 mM borate (pH 10.5) as running buffer within 20 min. The fluorescence intensities of Rho110-derivatives were significantly decreased by the presence of excess reducing agent, but were greatly increased by the addition of potassium hexacyanoferrate(III). The concentration and mass detection limits for monosaccharides were in the range of 1.4–2.8 nM and 36–70 amol, respectively. We have applied this derivatization method to the analysis of the composition of monosaccharides in glycoproteins (ribonuclease B, fetuin, and erythropoietin) following their subjection to strong acid hydrolysis. The results from these analyses were in good agreements with the reported values established previously.  相似文献   

20.
A microfabricated, inexpensive, reusable glass capillary electrophoresis chip and a laser-induced fluorescence system were developed in-house for the rapid DNA-based analysis of genetically modified organisms (GMOs). The 35S promoter sequence of cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens were both detected since they are present in most genetically modified organisms. The detection of genetically modified soybean in the presence of unaltered soybean was chosen as a model. Lectin, a plant-specific gene, was also detected for confirmation of the integrity of extracted DNA. The chip was composed of two glass plates, each 25 x 76 mm, thermally bonded together to form a closed structure. Photomasks with cross-topology were prepared rapidly by using polymeric material instead of chrome plates. The widths of the injection and separation channels were 30 and 70 microm, respectively, the effective separation length 4.5 cm. The glass slide was etched to a depth of 30 microm for both the injection and separation channel. The cost of the chip was less than 1 $ and required 2 days for photomask preparation and microfabrication. The separation and detection of polymerase chain reaction-amplified NOS, 35S, and lectin sequences (180, 195, and 181 bp, respectively) was completed in less than 60 s. As low as 0.1% GMO content was detectable by the proposed system after 35 and 40 amplification cycles for 35S and NOS, respectively, using 25 ng of extracted DNA as starting material. This corresponds to only 20 genome copies of genetically modified soybean.  相似文献   

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