A PDMS sheath flow cuvette for high‐sensitivity LIF measurements in CE

A simple method for producing a sheath flow cuvette in PDMS suitable for post‐column detection in CE is described. Two types of cuvette were investigated. In the first, the sheath flow channel had a round cross‐section of approximately 635 μm diameter, whereas the second cuvette had a 300×300 μm² square channel. Both cuvettes produced laminar flows that ensheathed the separation capillary's effluent allowing sensitive fluorescence measurements. The elasticity of the PDMS allowed the 300×300 μm² square sheath flow channel to expand uniformly and accommodate the larger 330–340 μm od round separation capillary, producing a self‐aligning cuvette with robust mechanical properties. With this cuvette, linear calibrations of over five orders of magnitude and 15–30 zmol fluorescein detection limits were obtained for 12 and 50 μm id capillaries.     

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.     

毛细管电泳-473 nm固体激光诱导荧光检测系统的建立

以发射波长473nm的半导体激光泵浦固体激光器(LD DPSSL)为激发光源,研制了一种小型模块化激光诱导荧光检测器。以异硫氰酸荧光素(FITC)为荧光探针,毛细管电泳柱上检测(0.05mmi.d)评价了该体系,得到了5×10-12mol L的浓度检出限。利用该系统考察了氨基酸、实际样品中B族维生素的检测。     

Simultaneous determination of polyamines and acetylpolyamines in human urine by capillary electrophoresis with fluorescence detection

There has been evidence linking elevated polyamines (PAs) and acetylpolamines (AcPAs) level and cancer. So the simultaneous analysis of these compounds has become important task for cancer diagnosis and antitumor drug monitoring. A simple, fast and inexpensive CZE‐LIF method has been developed for the determination of cadaverine (CAD), putrescine (PUT), spermine (SPM), spermidine (SPD), acetylspermine (ASPM), and acetylspermidine (ASPD) in human urine using 4‐chloro‐7‐nitro‐2,1,3‐benzooxadiazole as a fluorescent reagent. Labeling reaction conditions were systematically investigated and were found to be 20 mM borate buffer at pH 7.4, labeling reaction time, and temperature were 10 min and 70°C, respectively. Under these optimized conditions the four PAs, two AcPAs and the internal standard were separated in 6 min. An Exactive‐MS with an ESI source was used for identification of the bis‐derivative of the ASPM. The method was validated in term of linearity, LODs, repeatability, intra‐ and interday assays, recovery, and selectivity. The LODs for CAD, PUT, SPM, SPD, ASPM, and ASPD were found to be 7.6, 10.0, 9.0, 8.8,7.8, and 3.3 nM, respectively. The method was successfully applied for the analysis of PAs and AcPAs in healthy human urine samples.     

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.     

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.     

Analysis of riboflavin in beer by capillary electrophoresis/blue light emitting diode (LED)-induced fluorescence detection combined with a dynamic pH junction technique

A simple, inexpensive and reliable method for the routine analysis of riboflavin in beer by capillary electrophoresis-light emitting diode (CE-LED) induced fluorescence detection is described. A simple and straightforward sample preparation is involved and the method is based on an inexpensive blue LED as the light source combined with an on-line sample concentration technique. For this detection system, using a normal micellar electrokinetic chromatography (MEKC), stacking-MEKC and dynamic pH junction techniques, the detection limits were found to be 480, 20 and 1 ng mL⁻¹, respectively. In addition, the number of theoretical plates for riboflavin was determined to be 3.8×10⁴ by means of a dynamic pH junction and this was improved to 3.2×10⁶ when the dynamic pH junction-sweeping mode was applied. The concentrations of riboflavin in 12 samples of different types of commercial beer were found to be in the range of 130-280 ng mL⁻¹.     

免疫毛细管电泳-激光诱导荧光分析DNA加合物的方法学研究

DNA加合物是一类重要的生物标志物,可应用于人体致癌物暴露监测、癌症风险评价和人群易感性研究。DNA加合物作为生物标志物的应用需要安全、灵敏、快速的先进分析技术。我们利用免疫毛细管电泳-激光诱导荧光分析,发展了高灵敏的DNA加合物分析方法和技术。本文主要介绍了相关的仪器研制及方法学研究。方法学研究涉及DNA加合物荧光探针的合成和表征、抗体与DNA加合物的相互作用及其结合计量学、抗原-抗体复合物的稳定化和DNA驱动电泳聚焦技术。     

Electrokinetic-driven microfluidic system in poly(dimethylsiloxane) for mass spectrometry detection integrating sample injection, capillary electrophoresis, and electrospray emitter on-chip

A novel microsystem device in poly(dimethylsiloxane) (PDMS) for MS detection is presented. The microchip integrates sample injection, capillary electrophoretic separation, and electrospray emitter in a single substrate, and all modules are fabricated in the PDMS bulk material. The injection and separation flow is driven electrokinetically and the total amount of external equipment needed consists of a three-channel high-voltage power supply. The instant switching between sample injection and separation is performed through a series of low-cost relays, limiting the separation field strength to a maximum of 270 V/cm. We show that this set-up is sufficient to accomplish electrospray MS analysis and, to a moderate extent, microchip separation of standard peptides. A new method of instant in-channel oxidation makes it possible to overcome the problem of irreversibly bonded PDMS channels that have recovered their hydrophobic properties over time. The fast method turns the channel surfaces hydrophilic and less prone to nonspecific analyte adsorption, yielding better separation efficiencies and higher apparent peptide mobilities.     

微流控芯片毛细管电泳激光诱导荧光检测法测定片剂中盐酸美西律的含量

建立了微流控芯片毛细管电泳激光诱导荧光检测法测定片剂中盐酸美西律含量的方法,对衍生条件和电泳条件进行了系统的考察。盐酸美西律经异硫氰酸荧光素(FITC)40℃衍生6h,以20 mmol/L硼砂为电泳缓冲溶液,进样30s后,分离电压2000V,可在1 min内完成一次检测。方法的检出限为0.022 mg/L、线性范围0.108～1.079 mg/L、相关系数0.994,加标回收率为99.7%～102.3%,方法适用于盐酸美西律的检测和质量控制。     

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.     

Light-emitting diode induced fluorescence (LED-IF) detection design for a pen-shaped cartridge based single capillary electrophoresis system

CGE is a well-established separation technique for the analysis of biologically important molecules such as nucleic acids. The inherent high resolving power, rapid analysis times, excellent detection sensitivity, and quantification capabilities makes this method favorable compared to conventional manual polyacrylamide and agarose slab gel electrophoresis techniques. In this paper we introduce a novel single-channel capillary gel electrophoresis system with LED-induced fluorescence detection also utilizing a compact pen-shaped capillary cartridge design for automatic analysis of samples from a 96-well plate. To evaluate the suitability of the system, 1000 genomic DNA(gDNA) samples were analyzed in gel filled capillaries and detected by the microball ended excitation and emission optical fiber based LED-induced fluorescence detection system. Excellent migration time reproducibility of RSD <0.75% was obtained over the course of 1000 runs. The system rapidly distinguished between intact and degraded gDNA samples, therefore provided important information if they could be used for downstream quantitative PCR processing where high-quality intact gDNA was key. We envision that this novel system design will rapidly find new applications in both research and clinical diagnostic laboratories as a highly sensitive and easy to use bio-analytical approach.     

Electrokinetic control of fluid flow in native poly(dimethylsiloxane) capillary electrophoresis devices

Capillary zone electrophoresis (CZE) devices fabricated in poly(dimethylsiloxane) (PDMS) require continuous voltage control of all intersecting channels in the fluidic network in order to avoid catastrophic leakage at the intersections. This contrasts with the behavior of similar flow channel designs fabricated in glass substrates. When the injection plugs are shaped by voltage control and leakage from side channels is controlled by the application of pushback voltages during separation, fluorescein samples give 64 200 theoretical plates (7000 V separation voltage, E = 1340 V/cm). Native PDMS devices exhibit stable retention times (+/- 8.6% RSD) over a period of five days when filled with water. Contact angles were unchanged (+/- 1.9% RSD) over a period of 16 weeks of dry storage, in contrast to the known behavior of plasma-oxidized PDMS surfaces. Electroosmotic flow (EOF) was observed in the direction of the cathode for the buffer systems studied (phosphate, pH 3-10.5), in the presence or absence of hydrophobic ions such as tetrabutylammonium or dodecyl sulfate. Electroosmotic mobilities of 1.49 x 10(-5) and 5.84 x 10(-4) cm2/Vs were observed on average at pH 3 and 10.5, respectively, the variation strongly suggesting that silica fillers in the polymer dominate the zeta potential of the material. Hydrophobic compounds such as dodecyl sulfate and BODIPY 493/503 adsorbed strongly to the PDMS, indicating the hydrophobicity of the channel walls is clearly problematic for CZE analysis of hydrophobic analytes. A method to stack multiple channel layers in PDMS is also described.     

Improved native UV laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices

Single cell analytics is a key method in the framework of proteom research allowing analyses, which are not subjected to ensemble-averaging, cell-cycle or heterogeneous cell-population effects. Our previous studies on single cell analysis in poly(dimethylsiloxane) microfluidic devices with native label-free laser induced fluorescence detection [W. Hellmich, C. Pelargus, K. Leffhalm, A. Ros, D. Anselmetti, Electrophoresis 26 (2005) 3689] were extended in order to improve separation efficiency and detection sensitivity. Here, we particularly focus on the influence of poly(oxyethylene) based coatings on the separation performance. In addition, the influence on background fluorescence is studied by the variation of the incident laser power as well as the adaptation of the confocal volume to the microfluidic channel dimensions. Last but not least, the use of carbon black particles further enhanced the detection limit to 25 nM, thereby reaching the relevant concentration ranges necessary for the label-free detection of low abundant proteins in single cells. On the basis of these results, we demonstrate the first electropherogram from an individual Spodoptera frugiperda (Sf9) cell with native label-free UV-LIF detection in a microfluidic chip.     

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2‐(diethylamino)ethanol and triethanolamine. A buffer solution containing 2‐(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene‐2,3‐dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4–28) of proteins, compared with conventional absorbance detection.     

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.     

Quantitative analysis of a ubiquitin‐dependent substrate using capillary electrophoresis with dual laser‐induced fluorescence

Protein degradation by the ubiquitin‐proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS‐dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)‐fusion of the ubiquitin substrate ribophorin 1 (GFP‐RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP‐RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris‐CHES buffer (pH 9.0) containing 10 mM SDS. GFP‐RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP‐RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP‐RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS‐dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.     

Comparative study between a commercial and a homemade capillary electrophoresis instrument for the simultaneous determination of aminated compounds by induced fluorescence detection

The performance of two capillary electrophoresis (CE) instruments, one commercial and one homemade device, were compared for the determination of derivatised aminated compounds with fluorescein isothiocyanate (FITC). The commercial CE system first uses an argon ion laser as excitation source; the homemade CE device uses an inexpensive blue-light-emitting diode (LED) as the light source and a charge-coupled device (CCD) as the detection system. After fine optimisation of several separation parameters in both devices, a co-electroosmotic flow CE methodology was achieved in coated capillary tubing with 0.001% hexadimetrine bromide (HDB), and 50 mmol L⁻¹ sodium borate at pH 9.3 with 20% 2-propanol for the determination of several amines and aminoacids. Analytical performances, applicability in beer samples and other aspects such as cost or potential for miniaturization have been compared for both devices.     

Indirect fluorescence detection of simple sugars via high-pH electrophoresis in poly(dimethylsiloxane) microfluidic chips

This article describes the successful electrophoretic separation of simple carbohydrates in a polymeric microfluidic chip. The device fabricated in poly(dimethylsiloxane) (PDMS) is found to be stable in high-pH solutions. This allows sugars to be separated electrophoretically at pH values at or above their pK(a) using indirect fluorescence detection. Signal-to-noise values greater than 10:1 were obtained using a mercury arc lamp excitation source and a fluorescein-containing mobile phase for the detection of sugars at concentrations as low as 5 mM. The results obtained compare favorably with published results for the same system using a traditional fused-silica capillary. Analysis of the data revealed a significant experimental sensitivity of the migration times measured in these PDMS devices, an aging effect that leads to considerable systematic drift over the course of a series of replicate measurements. These experiments highlighted the importance of the surface chemistry of PDMS, especially as it pertains to its ability to support stable electroosmotic flow within the separation device. Channel priming at high pH provides a necessary, but by itself insufficient, means by which this instability can be minimized.     

A simple and compact blue diode laser powered excitation source for fluorescence detection in capillary electrochromatographic microchip separation

A simple and compact fluorescence excitation source was prepared using a 405 nm blue laser diode module and characterized in capillary electrochromatographic or capillary electrophoretic microchip separation. An inexpensive blue laser diode module with a tiny focusing lens was simply mounted at the center of an aluminum block on a miniature linear motion guide for heat dissipation and position control. A slit unit has a series of fifteen laser-machined slits with 1 mm space along the direction of the separation channel of the microchip above this unit. The laser beam was focused through a slit with 50 μm width to the separation channel at the position of a desired length. Although the excitation source unit was connected to a simple current controlled power supply, it was stable with 0.1% drift per hour and 1.3% (1σ) fluctuation in intensity. This simple excitation source can be prepared easily with inexpensive minimum optical components and mounted with a microchip on the stage of an ordinary fluorescence microscope for daily separation studies using a CE or CEC microchip. The applicability of the excitation source was evaluated with FITC-amino acid derivative mixtures using a polymer based CEC microchip packed fully with submicron silica beads in its microchannel.