Capillary Electrophoresis Laser-induced Fluorescence Research Articles

Two-Color Capillary Electrophoresis with On-Column Excitation and Wave-Guide Based Fluorescent Detection

An optical wave-guide based two-color capillary electrophoresis laser induced fluorescence (CE-LIF) instrument is described. The wave-guide based approach allows for on column excitation and detection with two-color discrimination. The instrument is designed to allow either electrokinetic or hydrodynamic injections. In its present configuration, the attainable limit of detection (LOD, S/N = 3) was 50 × 10−21 moles of fluorescein with a 488-nm excitation source. This study was designed to test the instrument design for applications in protein analyses. Fluorescent dyes with two different wavelengths were simultaneously separated and detected as were complexes formed by labeled antibodies to NF'B p65 and cdc2p34. Quantification of both proteins in THP-1 cell lysates performed using this approach illustrates a rapid screening application of this instrument.

Two-Color Capillary Electrophoresis with On-Column Excitation and Wave-Guide Based Fluorescent Detection

An optical wave-guide based two-color capillary electrophoresis laser induced fluorescence (CE-LIF) instrument is described. The wave-guide based approach allows for on column excitation and detect...

N-methyl-D-glucamine improves the laser-induced fluorescence capillary electrophoresis performance in the total plasma thiols measurement.

We describe an ultrarapid capillary electrophoresis with laser-induced fluorescence (CE-LIF) method for total plasma thiols measurement. Reduced thiols by 10% tri-n-butylphosphine (TBP) were derivatized in 10 min at room temperature with 5-iodoacetamidofluorescein (5-IAF) as fluorescent reagent. We show that CE-LIF allows a baseline separation of total plasma cysteinylglycine, homocysteine, cysteine, and glutathione in less than 5 min when N-methyl-D-glucamine in run buffer was added. CE was compared with high-performance liquid chromatography (HPLC) with fluorescence detection. The Bland-Altman test and Passing-Bablok regression demonstrates that the results obtained by CE-LIF and by HPLC are highly comparable. The simplified procedure of sample preparation, the short incubation and fast separation times, the high specificity, sensitivity and reproducibility, and the lower cost of analysis suggest that our proposed method can be considered valuable for the automation analysis in a routine laboratory.

Precise quantitation of 5α-reductase type 1 mRNA by RT-PCR in rat liver and its positive regulation by testosterone and dihydrotestosterone

The liver is a multifunctional organ responsible for steroid hormones catabolism. Thus, the enzymes responsible for steroid catabolism are located in the liver, including the steroid 5α-Reductase (5α-R) (EC 1.3.99.5) which catalyzes the conversion of compounds with Δ 4,5 double bonds such as testosterone (T) into their respective reduced derivatives such as dihydrotestosterone (DHT), which are more hydrosoluble, therefore facilitating their excretion. We present precise measurements of mRNA levels of steroid 5α-Reductase type 1 isozyme (5α-R1) in the liver of male rats with different androgen status, using a quantitative RT-PCR coupled to laser-induced fluorescence capillary electrophoresis (LIF-CE). By means of this technique, we demonstrate a high level of expression of the gene that encodes 5α-R1 isozyme in male rat liver, and both T and DHT exert a positive control on the genetic expression of liver 5α-R1 isozyme. Since DHT does not contain a Δ 4,5 double bond, our results raise the possibility that hepatic 5α-R type 1 not only participates in the catabolism of steroids with Δ 4,5 double bonds, but also in other physiological functions, perhaps in the masculinization of the external genitalia in males with 5α-R type 2 gene deficiency.

Determining eosin as a groundwater migration tracer by capillary electrophoresis/laser-induced fluorescence using a multiwavelength laser.

Measurements for determining of the path of groundwater migration remain an important tool in the overall assessment of environmental processes and transport of pollutants. This paper examines a multiwavelength laser for the determination of eosin, a groundwater tracer, using capillary electrophoresis/laser-induced fluorescence (CE-LIF) at excitation wavelength 514.5 nm. Eosin was one of four dyes used in a study of adjacent resource conservation and recovery act (RCRA) and Superfund sites (created by the comprehensive environmental response, compensation, and liability act) that routinely relied on spectrofluorimetry for determination as we have previously reported. However, the improved specificity of CE-LIF is further illustrated in this work applied to the analysis of adsorbent pads placed in monitoring wells after dye injection and flushing from injection wells. The multiwavelength laser provided the capability to analyze for several dyes with one laser. The advantages/disadvantages of CE-LIF versus spectrofluorimetry are discussed. Spectrofluorimetry is fast and sensitive and will likely continue to be the primary workhorse technique. CE-LIF could provide confirmation when greater specificity is needed in a regulatory context.

Subcellular Localization of Soybean 7S Globulin in HepG2 Cells and LDL Receptor Up-Regulation by Its α′ Constituent Subunit

The aims of this work were to monitor the subcellular localization of soybean 7S globulin in HepG2 cells and determine its interaction with cell protein components, by using laser-induced fluorescence capillary electrophoresis (LIF-CE). Furthermore, we evaluated in the same cell line the involvement of the alpha' constituent subunit from 7S globulin in the modulation of LDL catabolism. The results indicated a main fluorescein isothiocyanate-tagged 7S globulin (FITC-7S) component in the cytosolic fraction, that was not present in the nuclear compartment. The electrophoretic mobility of this tagged component suggested either a dissociation of the 7S oligomer or its partial intracellular degradation. Interactions of soybean 7S globulin with FITC-thioredoxin 1 and FITC-cyclophilin B, HepG2 cell membrane proteins, were demonstrated in in vitro assays. In a separate experiment with HepG2 cells, the ability of the alpha' subunit purified from soybean 7S globulin to modulate the activity of the LDL receptors was evaluated by tracking the uptake and degradation of labeled LDL. The up-regulation of LDL receptors by the alpha' subunit, as further confirmed by a LDL receptor promoter assay, was significantly greater than that found in the control cells. In conclusion, this study, while confirming our previous indirect evidence of the key role of alpha' subunit on the cell cholesterol homeostasis, reveals a potentially interesting association of soybean 7S globulin with proteins, such as thioredoxin 1 and cyclophilin B, that are involved in cell protection against oxidative stress.

A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene.

Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples.

A simple, rapid, and sensitive method for analysis of SYPRO Red labeled sodium dodecyl sulfate-protein complexes by capillary electrophoresis with laser-induced fluorescence.

We describe a segmental filling method for the analysis of SYPRO Red labeled sodium dodecyl sulfate (SDS)-proteins (SRSPs) by capillary electrophoresis-laser induced fluorescence (CE-LIF) with electroosmotic counterflow of poly(ethylene oxide) (PEO). It is shown that SDS and salt play a crucial role in determining the fluorescence intensity of the SRSP. Although the fluorimetric measurements reveal that the SRSPs fluoresce strongly in Tris-borate (TB) buffer containing 0.1% SDS and high concentrations of NaCl (100 mM), these conditions are not appropriate to CE in view of Joule heating. To overcome that impediment, we applied a plug of 0.1% SDS (1/5 to 1/3 of the injection volume) prior to injection of samples (0.64 microL) prepared in TB buffer containing 50 mM NaCl and SYPRO Red. When using a background electrolyte of 0.6% PEO in TB buffer containing NaCl, electroosmotic counterflow of the analytes allows one to concentrate large sample volumes (up to 1/3 of effective capillary length) in 21 min, with detection of 0.35 and 0.10 nM for bovine serum albumin and casein, respectively. With a linear dynamic range from 10 nM to 5 microM, this method provides the capability of determining the concentration of casein in cow's milk as 0.45 +/- 0.03 mM (n = 5).

Quantitation of mRNA levels of steroid 5α-reductase isozymes: a novel method that combines quantitative RT-PCR and capillary electrophoresis

A novel, accurate, rapid and modestly labor-intensive method has been developed to quantitate specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR). This strategy combines the high degree of specificity of competitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). The specific target mRNA and a mimic DNA fragment, used as an internal standard (IS), were co-amplified in a single reaction in which the same primers are used. The amount of mRNA was then quantitated by extrapolation from the standard curve generated with the internal standard. PCR primers were designed to amplify both a 185 bp fragment of the target cDNA for steroid 5α-reductase 1 (5α-R1) and a 192 bp fragment of the target cDNA for steroid 5α-reductase type 2 (5α-R2). The 5′ forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). Two synthetic internal standard DNAs of 300 bp were synthesized from the sequence of plasmid pEGFP-C1. The ratio of fluorescence intensity between amplified products of the target cDNA (185 or 192 bp fragments) and the competitive DNA (300 bp fragment) was determined quantitatively after separation by capillary electrophoresis and fluorescence analysis. The accurate quantitation of low-abundance mRNAs by the present method allows low-level gene expression to be characterized.

Separation and identification of zaleplon metabolites in human urine using capillary electrophoresis with laser-induced fluorescence detection and liquid chromatography–mass spectrometry

A capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection for the determination of the hypnotic drug zaleplon and its metabolites in human urine could be developed using carboxymethyl-β-cyclodextrin as a charged carrier. By the help of a complementary HPLC method coupled to mass spectrometry, three metabolites present in human urine could be identified as 5-oxozaleplon, 5-oxo- N-deethylzaleplon and 5-oxozaleplon glucuronide. N-Deethylzaleplon, a previously described zaleplon metabolite, as well as zaleplon itself could not be detected in human urine by the CE–LIF assay. The results were confirmed by spiking with reference compounds of the phase I metabolites. The metabolites differed very much concerning their fluorescence intensities, thus the 5-oxo metabolites present as lactam tautomer fluoresced tenfold lower than the unchanged drug zaleplon and its N-deethylated metabolite. The glucuronide of the 5-oxozaleplon, however, showed high fluorescence due to its lactim structure. Limits of quantification yielded by the CE–LIF assay including a ten-fold preconcentration step by solid-phase extraction were 10 ng/ml for zaleplon and N-deethylzaleplon and 100 ng/ml for 5-oxozaleplon and 5-oxo- N-deethylzaleplon.

Analysis of secretory immunoglobulin A in human saliva by laser-induced fluorescence capillary electrophoresis

The utility of capillary electrophoresis (CE) has been demonstrated for the analysis of secretory immunoglobulin A (sIgA) in human saliva. The amount of sIgA in saliva correlates with immune status. For detecting salivary sIgA, laser-induced fluorescence was conducted in this report for signal amplification. sIgA and anti-sIgA antibody were labeled with cyanine fluorescence (Cy5) for competitive immunoassay and non-competitive analysis, respectively. Cy5 was excited by He–Ne laser with a wavelength of 635 nm, with maximum emission at 670 nm. Migration time during electrophoresis depended on whether sIgA–Cy5 was mixed with antibody or anti-sIgA–Cy5 mixed with sIgA to form Ag–Ab complex. The results indicated that CE competitive immunoassay was effective for analyzing serum sIgA, but not for salivary sIgA. However, salivary sIgA can be analyzed by complex formation assay. The peak area of the complex was proportional to the amount of sIgA added. A standard linear regression curve was generated using purified sIgA. From this standard curve, the amount of sIgA from saliva of either normal or immunocompromised patients can be calculated from the Ag–Ab complex peak area.

Determination of amino acid neurotransmitters in cerebral cortex of rats administered with baicalin prior to cerebral ischemia by capillary electrophoresis–laser-induced fluorescence detection

An efficient, sensitive and rapid analysis of the amino acid neurotransmitters in the cerebral cortex of rats was developed by capillary electrophoresis with laser-induced fluorescence detection and fluorescein isothiocyanate (FITC) derivatization. This method was used to investigate the pharmacological effect of baicalin during cerebral ischemia. Different parameters which influenced derivatization and separation were optimized. The separation of amino acids was carried out in an uncoated fused-silica capillary (57 cm×75 μm I.D.) with a buffer of 15 m M borate at pH 9.2 and an applied voltage of 17.5 kV. The detection limits for six amino acids were in the range of 2.1×10 −11–6.3×10 −10 M. The changes in the level of amino acid neurotransmitters in brain cortex of three experimental rat groups were studied by this capillary electrophoresis–laser-induced fluorescence detection method. The results show that cerebral ischemia can cause a significant elevation in the concentrations of Glu, Asp, GABA, and Gly in cerebral cortex. Baicalin administration can attenuate the elevations of Glu and Asp induced by cerebral ischemia. This research demonstrates that baicalin may act as a neuroprotectant during cerebral ischemia.

Semiquantitative RT-PCR method coupled to capillary electrophoresis to study 5alpha-reductase mRNA isozymes in rat ventral prostate in different androgen status.

The development and growth of the prostate gland depends on androgen stimulation. Dihydrotestosterone (DHT) is the primary androgen responsible for prostate development and also for the pathogenesis of benign prostatic hyperplasia (BPH). The incidence of prostate cancer (PCa) and benign prostatic hypertrophy (BPH) continues to rise in the Western world. DHT is synthesized in prostate from circulating testosterone (T) through the action of 5alpha-Reductase (5alpha-R) (EC 1.3.99.5), which occurs as two isozymes, type 1 and type 2. Type-1 5alpha-R is widely distributed in the body, and type-2 5alpha-R is confined to androgen-dependent structures. Both types are expressed in the prostate: type-2 isozyme is implicated in BPH and PCa; type-1 isozyme is also increased in some prostatic adenocarcinomas. In recent years, various inhibitors of type-2 isozyme or of both type-1 and type-2 isozyme have been used in prostatic diseases. In this work we present measurements of mRNA levels of steroid 5alpha-R isozymes in the ventral prostate of rats of different androgen status. We used a novel method that combines the high specificity of semiquantitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). We demonstrated that T control the expression of 5alpha-R2 isozyme in rat prostrate. This approach could be of great value for the study of prostate diseases in humans and would allow study at the transcriptional level the effects of drugs that inhibit either or both of these isozymes.

One-second time resolution brain microdialysis in fully awake rats: Protocol for the collection, separation and sorting of nanoliter dialysate volumes

Capillary zone electrophoresis is capable of analyzing nanoliter volumes, reducing the challenge posed by brain microdialysis time resolution improvement to the management of nanoliter dialysate volumes. This fact has not been overlooked and 12- and 6-s time resolution microdialysis have been reported in anesthetized rats. However, behavioral experiments require fully awake and freely moving animals. To achieve high temporal resolution brain microdialysis in awake unrestrained rats, we have developed an online device that mixes the outflowing dialysate with fluorescein isothiocyanate and buffer within a 26-nl reactor. The mixture was continuously accumulated in a 99-μm-bore capillary tube. After the experiment the tube was cut into 4-mm pieces and the content of each piece (30 nl, equivalent to 1 s dialysate) was transferred to a test tube. After allowing 18 h for derivatization, the samples were diluted with water and injected into a capillary electrophoresis laser-induced fluorescence detection instrument. This protocol was tested first in an in vitro assay and proved to be capable of detecting glutamate concentration changes in only 1 s. For the in vivo assays, a probe was inserted into the primary somatosensory cortex of eight rats divided in two groups. One group was stimulated by gently moving its whiskers for 10 s. The other group had no whisker manipulation. Moving the whiskers released glutamate in the experimental group. The first and only change was observed at the 12th s. This method allows 1-s time resolution brain microdialysis in freely moving rats and multiple amino acid analysis every second during sensory perception or motor actions in behavioral experiments.

Separation and quantitation of phycobiliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection

The similar electrophoretic mobilities and sizes of several of the phycobiliproteins, which are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae, render their separation and quantitation a challenging problem. However, we have developed a suitable capillary electrophoresis (CE) method that employs a phytic acid–boric acid buffer and laser-induced fluorescence (LIF) detection with a single 594 nm He–Ne laser. This method takes advantage of the remarkably high quantum yields of these naturally fluorescent proteins, which can be attributed to their linear tetrapyrrole chromophores covalently bound to cysteinyl residues. As such, limits of detection of 1.18·10 −14, 5.26·10 −15, and 2.38·10 −15 mol/l were obtained for R-phycoerythrin, C-phycocyanin, and allophycocyanin proteins, respectively, with a linear dynamic range of eight orders of magnitude in each case. Unlike previously published CE–LIF methods, this work describes the separation of all three major classes of phycobiliproteins in under 5 min. Very good recoveries, ranging from 93.2 to 105.5%, were obtained for a standard mixture of the phycobiliproteins, based on seven-point calibration curves for both peak height and peak area. It is believed that this development will prove useful for the determination of phycobiliprotein content in naturally occurring cyanobacteria populations, thus providing a useful tool for understanding biological and chemical oceanographic processes.

Investigation of capillary electrophoresis-laser induced fluorescence as a tool in the characterization of sewage effluent for fluorescent acids: determination of salicylic acid.

The investigation of emerging contaminant issues is a proactive effort in environmental analysis. As a part of this effort, sewage effluent is of current analytical interest because of the presence of pharmaceuticals and their metabolites and personal care products. The environmental impact of these components is still under investigation but their constant perfusion into receiving waters and their potential effect on biota is of concern. This paper examines a tool for the characterization of sewage effluent using capillary electrophoresis-laser induced fluorescence (CE-LIF) with a frequency-doubled laser operated in the ultraviolet (UV). Fluorescent acidic analytes are targeted because they present special problems for techniques such as gas chromatography-mass spectrometry (GC-MS) but are readily accessible to CE-LIF. As an example of the application of this tool, salicylic acid is determined near the 100 ng/L (7 x 10(-10) M) level in sewage effluent. Salicylic acid is a metabolite of various analgesics. Relatively stable in the environment, it is a common contaminant of municipal sewage systems. Salicylic acid was recovered from freshly collected samples of the effluent by liquid-liquid extraction. Confirmation of identity was by electron ionization GC-MS after conversion of the salicylic acid to the methyl ester by means of trimethylsilyldiazomethane. CE-LIF in the UV has revealed more than 50 individual peaks in the extract and a background response that suggests a large and indeterminate number of additional compounds are present. These data together with complementary techniques provide information on the complexity and components in these effluent streams.

Multiplex Fluorescent RT-PCR to Quantify Leukemic Fusion Transcripts

The detection of chimeric transcripts derived from aberrant chromosomal fusion events provides an exceptionally valuable toolfor the diagnosis of leukemia. We have developed a simple, inexpensive, reproducible, and automated method to quantify RT-PCR products. Our approach utilizesfluorescent PCRfor the co-ampification of the specific fusion transcript with an internal control (HPRT). We have also combined the advantages of real-time quantitative PCR, namely continuous fluorescent detection of PCR products with the low cost of an endpoint assay by examining in a novel manner the amount offluorescent PCR product generated during the exponential phase of amplification. This has been achieved by using the automated loading and quantification capacity of a laser-induced fluorescence capillary electrophoresis system, the ABI PRIsMS 310A, so that we can effectively monitor amplification during the exponential phase cheaply, reproducibly, and in a sensitive manner. We have carefully verified our new technique using five leukemia cell lines, each expressing a differentfusion transcript. Specificity and reproducibility (cy within 10%) have been examined and demonstrate the excellent precision of our technology. The high sensitivity levels of at least 10(-4) to 10(-6) obtainedfor the serial dilutions of the five cell lines validate the choice of our fluorescent PCR as a comparable method to other more complicated and expensive methods. Our results have allowed us to quantify PCR products and the amount of chimeric mRNA originating from the translocation breakpoint. We demonstrate that our novelfluorescent method is useful to detect and quantify residual leukemic cells in patients undergoing therapy.

Individual molecules of thermostable alkaline phosphatase support different catalytic rates at room temperature.

Thermus thermophilus cells were grown at 70 degrees C, lysed and the lysate subjected to single molecule alkaline phosphatase assays, using a capillary electrophoresis laser-induced fluorescence detection-based method. The enzyme was found to be heterogeneous with respect to catalytic rate when assayed at room temperature. Turnover numbers ranged 12-fold, with an average of 400 +/- 200 reactions/min for the 80 molecules assayed.

Quantification of single nucleotide polymorphisms: a novel method that combines primer extension assay and capillary electrophoresis.

We present a novel method for accurate quantification of single nucleotide polymorphism (SNP) variants in transcripts and pooled DNAs in a one-tube reaction. Our approach is based on single- nucleotide primer extension (SNuPE) and laser-induced fluorescence capillary electrophoresis (LIF-CE), and takes advantage of distinct mobilities of SNuPE products with different nucleotides incorporated at their 3' ends. The method, called SNuPE-ONCE, was tested on two polymorphisms and five mutations that comprised the three most frequent ( approximately 70%) nucleotide changes in the human genome (C/T, A/G, and A/T). The usefulness of the method was demonstrated by analyzing nonsense-mediated mRNA instability in fibroblasts. Our data show 1) that the method provides highly reproducible relative allele frequencies (SD<0.017) with a good accuracy (e.g. for heterozygotes 0.500 +/- 0.036, P = 0.01), depending on the sequence and the proportion of the SNP variants in the sample, and 2) that relative allele frequencies as low as 1% can be detected quantitatively and unambiguously. Our assay relies on a CE instrument available in many laboratories and offers a useful method for quantitative SNP genotyping as well as for a variety of expression studies.

Detection of human immunodeficiency virus type 1 reverse transcriptase using aptamers as probes in affinity capillary electrophoresis.

An affinity capillary electrophoresis/laser-induced fluorescence (CE/LIF) assay was developed for direct and specific detection of reverse transcriptase (RI) of the type 1 human immunodeficiency virus (HIV-1) using fluorescently labeled single-stranded DNA aptamers as probes. The aptamer used (RT 26) is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-1 RT was readily formed, and calibration curves were linear up to 50 nM (6 microg/mL) HIV-1 RT concentration, with both the free probe and complex peak usable for analytical quantitation. Cell culture media (RPMI with 10% fetal bovine serum) interfered with the assay and aptamer-HIV-1 RT binding. Nonspecific binding was observed in low or undiluted culture, necessitating at least 100-fold dilution for analysis of raw culture samples.