Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis

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.     

Indirect laser-induced fluorescence detection for capillary electrophoresis using a frequency-doubled diode laser

A blue (452 nm) frequency-doubled diode laser with a quasi-cw optical output power of 10 microW is used for indirect laser-induced fluorescence detection in combination with the capillary electrophoretic separation of inorganic anions. As fluorescing probe ion the anion of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) was selected having an absorption maximum of 454 nm in alkaline medium. Employing a capillary coated with linear acrylamide, baseline separation of eight inorganic anions was possible within 5 min. With a separation buffer containing 50 micromol.L(-1) HPTS and 10 mmol.L(-1) lysine the limits of detection for sulfate, nitrite, nitrate, azide, thiocyanate, and chlorate were between 0.9 and 4.7 micromol.L(-1). Separation of chloride and sulfate was achieved by adding 0.25 mmol.L(-1) calcium hydroxide to the separation buffer. Inorganic anions in several mineral and tap water samples have been determined with the technique developed and results are compared to data obtained by ion chromatography in combination with conductivity detection after conductivity suppression.     

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.     

Pulsed multi-wavelength excitation using fiber-in-capillary light emitting diode induced fluorescence detection in capillary electrophoresis

A novel instrument was developed using a multi-wavelength pulsed LED array with in-column optic-fiber induced fluorescence detection by capillary electrophoresis. The light from 2 different wavelength LEDs (450 nm and 480 nm) was pulsed for short intervals at high intensity. The beam from each LED was collimated and reshaped with the gradient index (GRIN) lens group to achieve a highly effective coupling between LED light source and an optical fiber. The optical fiber was placed inside the capillary for in-capillary LED-induced fluorescence detection. The advantages of this system were validated by the simultaneous determination of vitamin B2 and fluorescein. Detection limits for vitamin B2 and fluorescein were estimated to be 5 nM and 0.29 nM (S/N = 3), respectively. The relative standard deviations (RSDs, n = 6) of the both compounds for migration time and peak area were better than 0.83%, 2.20% and 1.21%, 2.75%, respectively. The method was applied to the determination of vitamin B2 in commercial tablets and fluorescein in fluorescein sodium injection and the recoveries obtained were in the range of 96.6-102.0% and 99.9-102.8%, respectively. It was also applied to human serum, where the recoveries were found to be in the range of 94.4-97.0% and 92.6-96.4%, respectively. The system has been successfully applied in separation and determination of the both biological samples with acceptable analytical performance.     

Simplified sheath flow cuvette design for ultrasensitive laser induced fluorescence detection in capillary electrophoresis

We present a design for a sheath-flow cuvette that uses a relatively inexpensive quartz cuvette. The cuvette has a high optical quality square flow chamber that is fused to quartz tubes at each end. PEEK/TEFZEL fittings hold and seal the quartz flow chamber without putting strain on the cuvette. The performance of the cuvette is evaluated as a laser-induced fluorescence detector for capillary electrophoresis. The cuvette produces mass detection limits of 50 yoctomoles (30 copies) for 5-carboxyl tetramethylrhodamine (5 TAMRA SE) with a separation efficiency of 400,000 theoretical plates.     

A low-cost light-emitting diode induced fluorescence detector for capillary electrophoresis based on an orthogonal optical arrangement

In this work, a simple and low-cost miniaturized light-emitting diode induced fluorescence (LED-IF) detector based on an orthogonal optical arrangement for capillary electrophoresis (CE) was developed, using a blue concave light-emitting diode (LED) as excitation source and a photodiode as photodetector. A lens obtained from a waste DVD-ROM was used to focus the LED light beam into an ∼80 μm spot. Fluorescence was collected with an ocular obtained from a pen microscope at 45° angle, and passed through a band-pass filter to a photodiode detector. The performance of the LED-IF detector was demonstrated in CE separations using sodium fluorescein and fluorescein isothiocyanate (FITC)-labeled amino acids as model samples. The limit of detection for sodium fluorescein was 0.92 μM with a signal-to-noise ratio (S/N) of 3. The total cost of the LED-IF detector was less than $ 50.     

Design and evaluation of capillary coupled with optical fiber light‐emitting diode induced fluorescence detection for capillary electrophoresis

A new detector, capillary coupled with optical fiber LED‐induced fluorescence detector (CCOF‐LED‐IFD, using CCOF for short), is introduced for CE. The strategy of the present work was that the optical fiber and separation capillary were, in the parallel direction, fastened in a fixation capillary with larger inner diameter. By employing larger inner diameter, the fixation capillary allowed the large diameter of the optical fiber to be inserted into it. By transmitting an enhanced excitation light through the optical fiber, the detection sensitivity was improved. The advantages of the CCOF‐CE system were validated by the detection of riboflavin, and the results were compared to those obtained by the in‐capillary common optical fiber LED‐induced fluorescence detector (IC‐COF‐LED‐IFD, using COF for short). The LODs of CCOF‐CE and COF‐CE were 0.29 nM and 11.0 nM (S/N = 3), respectively. The intraday (n = 6) repeatability and interday (n = 6) reproducibility of migration time and corresponding peak area for both types of CE were all less than 1.10 and 3.30%, respectively. The accuracy of the proposed method was judged by employing standard addition method, and recoveries obtained were in the range of 98.0–102.4%. The results indicated that the sensitivity of the proposed system was largely improved, and that its reproducibility and accuracy were satisfactory. The proposed system was successfully applied to separate and determine riboflavin in real sample.     

Light-emitting diode-based indirect fluorescence detection for simultaneous determination of anions and cations in capillary electrophoresis

This report presents simultaneous analysis of cations and anions by capillary electrophoresis (CE) in conjunction with indirect fluorescence detection using a blue light-emitting diode (LED), based on the displacement of fluorescein with anionic EDTA-metal complexes and anions. A new focusing system combined with a plastic lens and a 40x objective was developed and used effectively to focus the diverging beam of the LED on the capillary. The optimum compositions for simultaneous analysis of metal ions and anions are the samples prepared in 5 mM borate, pH 9.2, containing 2 mM EDTA and the background electrolytes (BGEs) consisting of 5 mM borate buffer, 5 microM fluorescein, and 1 microM NaCl at pH 9.2. Using this pre-capillary complexation method, the analysis of a sample containing five metal ions and eight anions was accomplished in 8 min, with the relative standard deviation values for the migration times less than 2.0%. The peak heights against the concentrations of the metal ions and anions are linear in 10-1000 and 50-2000 microM, with correlation coefficients better than 0.998, and 0.982, respectively. The limits of detection at a signal-to-noise ratio 3 of up to 14.6 microM for formate and as low as 3.7 microM for Ni2+. The results of the analyses of pond water and a Chinese herbal soup present the advantages of this method, including simplicity, rapidity, reproducibility, and low costs.     

毛细管电泳光导纤维发光二极管诱导荧光检测法同时测定肾上腺素和多巴胺

采用自行设计、组装的毛细管电泳光导纤维发光二极管诱导荧光检测装置,建立了同时测定肾上腺素(EP)和多巴胺(DA)的方法。采用胶束电动色谱分离模式,通过优化分离电压、十二烷基硫酸钠(SDS)浓度、背景电解质浓度和pH等影响因素,在最佳实验条件下,EP和DA的线性范围分别为2.2×10-9~1.1×10-7mol/L和2.6×10-8~1.2×10-6mol/L,EP和DA的检测限(S/N=3)分别为1.2×10-9mol/L和1.1×10-8mol/L。该方法可应用于人血浆中EP和DA含量的测定。     

Immunoassay of P-glycoprotein on single cell by capillary electrophoresis with laser induced fluorescence detection

The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5-10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future.     

Immunoassays using capillary electrophoresis laser induced fluorescence detection for DNA adducts

Human DNA is exposed to a variety of endogenous and environmental agents that may induce a wide range of damage. The critical role of DNA damage in cancer development makes it essential to develop highly sensitive and specific assays for DNA lesions. We describe here ultrasensitive assays for DNA damage, which incorporate immuno-affinity with capillary electrophoresis (CE) separation and laser induced fluorescence (LIF) detection. Both competitive and non-competitive assays using CE/LIF were developed for the determination of DNA adducts of benzo[a]pyrene diol epoxide (BPDE). A fluorescently labeled oligonucleotide containing a single BPDE adduct was synthesized and used as a fluorescent probe for competitive assay. Binding between this synthetic oligonucleotide and a monoclonal antibody (MAb) showed both 1:1 and 1:2 complexes between the MAb and the oligonucleotide. The 1:1 and 1:2 complexes were separated by CE and detected with LIF, revealing binding stoichiometry information consistent with the bidentate nature of the immunoglobulin G antibody. For non-competitive assay, a fluorescently labeled secondary antibody fragment F(ab′)₂ was used as an affinity probe to recognize a primary antibody that was specific for the BPDE-DNA adducts. The ternary complex of BPDE-DNA adducts with the bound antibodies was separated from the unbound antibodies using CE and detected with LIF for quantitation of the DNA adducts. The assay was used for the determination of trace levels of BPDE-DNA adducts in human cells. Analysis of cellular DNA from A549 human lung carcinoma cells that were incubated with low doses of BPDE (32 nM–1 μM) showed a clear dose–response relationship. BPDE is a potent environmental carcinogen, and the ultrasensitive assays for BPDE-DNA adducts are potentially useful for monitoring human exposure to this carcinogen and for studying cellular repair of DNA damage.     

High sensitivity laser induced fluorescence detection in capillary zone electrophoresis

Laser induced fluorescence is used for the detection of labeled amino acids. A preliminary comparison is made of three fluorescence pre-column labeling reagents, ortho-phthaldialdehyde, naphthalene dicarboxaldehyde, and fluorescein isothiocyanate, and data on phenylalanine detection limits are given.     

On-capillary chemiluminescence detection for capillary electrophoresis with a single capillary.

On-capillary chemiluminescence detection for capillary electrophoresis with a single capillary was reported. A hole (about 30 microm diameter) was made on the capillary wall at about 50.5 cm from the inlet end. Hydrogen peroxide solution could enter the capillary from the hole, and mixed with luminol and copper(II) to produce chemiluminescence. The chemiluminescence was detected by a PMT under the hole. Several factors that influenced chemiluminescence intensity were investigated. The detection limits for luminol and N-(4-aminolbutyl)-N-ethylisoluminol (ABEI) were 1 x 10(-11) and 2 x 10(-10) mol L(-1), respectively. The method features simple construction and no dead volume.     

Novel wavelength-resolved fluorescence detection for a high-throughput capillary electrophoresis system under a diascopic configuration

This paper presents a novel method regarding a wavelength-resolved fluorescence detection scheme for high-throughput analysis of bio-samples in a micro-CE chip. Instead of using the conventional laser-induced fluorescence (LIF) microscope equipped with delicate spatial filters and complex control systems, this study adopts a hollow cone illumination generated using a dark-field condenser for exciting fluorescence in the microchannel and an ultraviolet-visible-near-infrared (UV-Vis-NIR) spectrometer for detecting the emission signals. Experimental results show that the proposed system is feasible for simultaneously detecting a mixed sample composed of Atto 610, Rhodamine B and fluorescein isothiocyanate (FITC) fluorescent dyes in a single test run. Furthermore, a mixed bio-sample composed of two mixed 16-mer single-stranded DNAs labeled with Cy3 and FITC fluorescent dyes is also successfully detected with the proposed system. The measured limit of detection (LOD) for detecting FITC of the proposed system can be as low as 5.4x10(-6)M (S/N=3). This proposed detection method has shown its potential on RNA identification and DNA sequencing applications.     

Study of single cells by using capillary electrophoresis and native fluorescence detection

Capillary electrophoresis is known for its compatibility with biological materials and with small samples. It is an ideal tool for the study of single biological cells. Either whole cells or the material secreted from cells can be quantified. By continuously flowing a chemical stimulant over an immobilized cell inside the entrance of the capillary, one can even record the temporal progression of cellular secretion with sub-second resolution. The use of native fluorescence detection in such experiments provides a sensitive, rapid, non-intrusive and quantitative probe of important biomolecules such as catecholamines and proteins.     

Multiphoton excitation fluorescence: a versatile detection method for capillary electrophoresis

Multiphoton excitation is a relatively old concept in quantum optics. But using multiphoton excitation fluorescence (MPEF) for bioanalysis is still in its infancy. Recently, MPEF has been introduced into the microseparation field, particularly CE, as a novel detection method. In this paper, MPEF detection for CE is reviewed, including MPEF fundamentals, approaches to achieving MPEF, detector configurations and applications in biological and environmental analyses. Emphasis will be placed on some recent advances of CE-MPEF in our laboratory. Challenges and future prospects are also discussed.     

Optical fiber light-emitting diode-induced fluorescence detection for capillary electrophoresis

A highly sensitive optical fiber light-emitting diode (LED)-induced fluorescence detector for CE has been constructed and evaluated. In this detector, a violet or blue LED was used as the excitation source and an optical fiber with 40 microm OD was used to transmit the excitation light. The upper end of the fiber was inserted into the separation capillary and was situated right at the detection window. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a cutoff filter before reaching the photomultiplier tube. Output signals were recorded and processed with a computer using in-house written software. The present CE/fluorescence detector deploys a simple and inexpensive optical system that requires only an LED as the light source. Its utility was successfully demonstrated by the separation and determination of amino acids (AAs) labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and FITC. Low detection limits were obtained ranging from 17 to 23 nM for NDA-tagged AAs and 8 to 12 nM for FITC-labeled AAs (S/N=3). By virtue of such valuable features as low cost, convenience, and miniaturization, the presented detection scheme was proven to be attractive for sensitive fluorescence detection in CE.     

Two-color excitation system for fluorescence detection in DNA sequencing by capillary array electrophoresis

Two computer-controlled galvanometer scanners are adapted for two-dimensional step scanning across a 96-capillary array for laser-induced fluorescence detection. 488 nm and 514 nm laser lines from the same Ar(+) laser were alternately coupled for two-color excitation in each capillary. The signal at a single photomultiplier tube is temporally sorted to distinguish among the capillaries and the excitation wavelengths. Based on the differences in absorption spectra for the dyes, the peak-height ratios in the 488 nm and 514 nm excitation electropherograms were used for peak identification for multiplexed capillary electrophoresis. Successful base calling for 24-capillary DNA sequencing was achieved to 450 bp with 99% accuracy. Advantages include the efficient utilization of light due to the high duty-cycle of step scan, good detection performance due to the reduction of stray light, ruggedness due to the small mass of the galvanometer mirror, low cost due to the simplicity of components and flexibility due to the independent paths for excitation and emission.     

Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment

Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detection was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3x bubble cell compared with a conventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL(-1) was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE.