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.     

Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

A high‐sensitivity LIF detector with silver mirror coating detection window and small‐angle optical deflection from collinear system for CE

An LIF detector was integrated into a CE system based on silver mirror coating detection window and small‐angle optical deflection from collinear configuration. For this detection scheme, the incident light beam was focused on capillary through the edge of a lens, resulting in a small deflection angle that deviated 18° from the collinear configuration. Meanwhile, the excitation light and emitted fluorescence were effectively reflected by silver mirror coating at the detection window. The fluorescence was collected through the center of the same lens and delivered to a PMT in the vertical direction. In contrast to conventional collinear LIF detection systems, the fluorescence intensity was greatly enhanced and the background level was significantly eliminated. FITC and FITC‐labeled amino acids were used as model analytes to evaluate the performance with respect to design factors of this system. The limit LOD was estimated to be 0.5 pM for FITC (S/N = 3), which is comparable to that of optimized confocal LIF systems. All the results indicate that the proposed detection scheme will be promising for development of sensitive and low‐cost CE system.     

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye–DNA complexes may prevent from their wide applications in CE‐LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris‐glycine buffer and Tris‐glycine‐acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused‐silica capillary‐based CE‐LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium‐4‐pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4×10^?22 mol (150 molecules) in mass. We further demonstrate that the non‐gel sieving CE‐LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip‐based CE DNA analysis.     

Automated high‐speed CE system for multiple samples

A high‐speed CE system for multiple samples was developed based on a short capillary and an automated sample introduction device consisting of a commercial multi‐well plate and an x‐y‐z translation stage. The spontaneous injection method was used to achieve picoliter‐scale sample injection from different sample wells. Under the optimized conditions, a 40 μm‐long sample plug (corresponding to 78‐pL plug volume) was obtained in a 50 μm id capillary, which ensured both the high separation speed and high separation efficiency. The performance of the system was demonstrated in the separation of FITC‐labeled amino acids with LIF detection. Five FITC‐labeled amino acids including arginine, phenylalanine, glycine, glutamic acid, and asparagine were separated within 15 s with an effective separation length of 1.5 cm. The separation efficiency ranged from 7.96 × 10⁵/m to 1.12 × 10⁶ /m (corresponding to 1.26–0.89 μm plate heights). The repeatability of the peak heights calibrated with an inner standard for different sample wells was 2.4 and 2.7% (n = 20) for arginine and phenylalanine, respectively. The present system was also applied in consecutive separations of 20 different samples of FITC‐labeled amino acids with a whole separation time of less than 6 min.     

Efficient PKC inhibitor screening achieved using a quantitative CE‐LIF assay

An assay for protein kinase C delta (PKCδ) activity based on the quantification of a synthetic substrate using capillary electrophoresis with laser‐induced fluorescence detection was developed. The peptides labeled with fluorescein isothiocyanate F‐ERK (where ERK is extracellular signal‐regulated kinase) and the phosphorylated form, P‐F‐ERK, were utilized for the method development and validation. The migration time of F‐ERK and P‐F‐ERK were 6.3 ± 0.1 and 8.7 ± 0.2 min, respectively. LOD and LOQ values of F‐ERK were 2 and 6 ng/mL and those of P‐F‐ERK were 4 and 12 ng/mL. The correlation coefficients obtained from two standard curves were approximately 0.99. The reproducibility and accuracy of the method for F‐ERK ranged 1.5–4.7 and 86–109%, respectively, and those for P‐F‐ERK were 1.6–6.1 and 93–109%, respectively. The activity of PKCδ was studied in vitro using the human gastric cancer cell line MKN‐1. The use of PKCδ inhibitor candidates, including G?6983, bisindolylmaleimide II, staurosporine, and rottlerin in the assay resulted in IC₅₀ values of 50 nM, 15 nM, 795 nM, and 4 μM, respectively. Comparison of our assay with a commercial PKC kit revealed that our assay is more adaptable to differing enzyme isoforms. This method has potential for high throughput screening for kinase inhibitors as part of a drug discovery program.     

A new evaluation technique for the detection of impurities in purified proteins via CE with native UV‐LIF

An analytical methodology for quality control analyses of IgG and their impurities is presented using a new UV‐LIF (266 nm) detector inside the cassette of a CE instrument and its performance was evaluated. The observed sensitivity was very close to that obtained by silver staining of slab gels (LOD of 25 ng/mL), while the sensitivity of the analysis is 80 times better than with CE/UV absorption (214 nm). Examples of the analysis of pharmaceutical and other commercial IgGs are provided and the kinetics of the reduction of IgG by β‐mercaptoethanol is reported, demonstrating the ease of performing the analysis.     

A simple fabrication method for tapered capillary tip and its applications in high‐speed CE and ESI‐MS

Fabrication of capillaries with tapered tips is an important technique that is required in many analytical chemistry areas, such as ESI‐MS, CE, electrochemical analysis, and microinjection. This paper describes a simple and effective grinding‐based fabrication method for capillaries with tapered tips. A novel grinding mode utilizing the combination of rotation and precession of an elastic capillary was developed, which significantly improved the controllability to the grinding process as well as the capillary tip shape. The capillary was fabricated by fixing it in an electric drill installed perpendicularly, and grind the capillary tip rotated around its own axis as well as the drill axis on sandpapers. Compared with conventional fabrication techniques for capillary tips, the present method is easy to control the capillary tip shape in routine laboratories without the requirement of expensive equipments or poisonous reagent (e.g. hydrofluoric acid (HF) solution). Various capillaries with different tip diameters and tip taper angles could be fabricated using the present method with good controllability and reproducibility. These capillaries were applied in high‐speed CE and ESI‐MS analysis to demonstrate the feasibility and potential of this fabrication method.     

Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE‐dual‐LIF

Pt‐based anticancer drugs, such as cisplatin, are known to undergo several (bio‐)chemical transformation steps after administration. Hydrolysis and adduct formation with small nucleophiles and larger proteins are their most relevant reactions on the way to the final reaction site (DNA), but there are still many open questions regarding the identity and pharmacological relevance of various proposed adducts and intermediates. Furthermore, the role of buffer components or additives, which are inevitably added to samples during any type of analytical measurement, has been frequently neglected in previous studies. Here, we report on adduct formation reactions of the fluorescent cisplatin analogue carboxyfluorescein diacetate platinum (CFDA‐Pt) in commonly used buffers and cell culture medium. Our results indicate that chelation reactions with noninnocent buffers (e.g., Tris) and components of the cell culture/cell lysis medium must be taken into account when interpreting results. Adduct formation kinetics was followed up to 60 h at nanomolar concentrations of CFDA‐Pt by using CE‐LIF. CE‐MS enabled the online identification of such unexpected adducts down to the nanomolar concentration range. By using an optimized sample preparation strategy, unwanted adducts can be avoided and several fluorescent adducts of CFDA‐Pt are detectable in sensitive and cisplatin‐resistant cancer cell lines. By processing samples rapidly after incubation, we could even identify the initial, but transient, Pt species in the cells as deacetylated CFDA‐Pt with unaltered complexing environment at Pt. Overall, the proposed procedure enables a very sensitive and accurate analysis of low molecular mass Pt species in cancer cells, involving a fast CE‐LIF detection within 5 min.     

Boron‐chelating fluorescent probe (BOPB) in the red region combined with CE‐LIF for the detection of NO in mice liver

Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8‐(3’,4’‐Diamino phenyl)‐3,5‐(2‐hydroxyphenyl)‐dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE‐LIF detection. Therefore, BOPB was used in CE‐LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB‐T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H₃BO₃–NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10^?9–5.0 × 10^?7 M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization‐based CE‐LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.     

Enhancing resolution of free‐flow zone electrophoresis via a simple sheath‐flow sample injection

In this work, a simple and novel sheath‐flow sample injection method (SFSIM) is introduced to reduce the band broadening of free‐flow zone electrophoresis separation in newly developed self‐balance free‐flow electrophoresis instrument. A needle injector was placed in the center of the separation inlet, into which the BGE and sample solution were pumped simultaneously. BGE formed sheath flow outside the sample stream, resulting in less band broadening related to hydrodynamics and electrodynamics. Hemoglobin and C‐phycocyanin were successfully separated by the proposed method in contrast to the poor separation of free‐flow electrophoresis with the traditional injection method without sheath flow. About 3.75 times resolution enhancement could be achieved by sheath‐flow sample injection method.     

Total nucleotide analysis of Hydra DNA and RNA by MEKC with LIF detection and 32P‐postlabeling

The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity – fluorescence labeling of nucleotides followed by CE‐LIF detection and ³²P‐postlabeling. DNA of Hydra was digested either to deoxyribonucleoside‐5′‐monophosphates or deoxyribonucleoside‐3′‐monophosphates selectively conjugated with the fluorescent dye 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE‐LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5‐methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the ³²P‐postlabeling method.     

Dual‐enzyme,co‐immobilized capillary microreactor combined with substrate recycling for high‐sensitive glutamate determination based on CE

A new method for high‐sensitive determination of glutamate was developed and evaluated based on CE by using dual‐enzyme co‐immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co‐immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet‐visible spectroscopy, and fluorescence spectroscopy. The GDH‐GPT co‐immobilized capillary microreactor showed great stability and reproducibility. The apparent K_m for glutamate with GDH‐GPT coupled reaction was determined to be 0.61±0.06 mM but 2.56±0.24 mM when only GDH was immobilized. Glutamate determination was based on on‐column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co‐immobilized GDH‐GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7‐fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 μM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples.     

Frontal analysis in microchip CE: a simple and accurate method for determination of protein-DNA dissociation constant

Equilibrium constants, such as the dissociation constant (K(d)), are a key measurement of noncovalent interactions that are of importance for the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate CE method for the determination of K(d). Microchip CE coupled with LIF detection was used to determine K(d) of protein-DNA interactions using the FA method. A model system of IgE and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The K(d) of the IgE-aptamer pair was determined as 6 +/- 2 nM which is consistent with the reported results (8 nM).     

Influence of high‐conductivity buffer composition on field‐enhanced sample injection coupled to sweeping in CE

The aim of this work was to clarify the mechanism taking place in field‐enhanced sample injection coupled to sweeping and micellar EKC (FESI‐Sweep‐MEKC), with the utilization of two acidic high‐conductivity buffers (HCBs), phosphoric acid or sodium phosphate buffer, in view of maximizing sensitivity enhancements. Using cationic model compounds in acidic media, a chemometric approach and simulations with SIMUL5 were implemented. Experimental design first enabled to identify the significant factors and their potential interactions. Simulation demonstrates the formation of moving boundaries during sample injection, which originate at the initial sample/HCB and HCB/buffer discontinuities and gradually change the compositions of HCB and BGE. With sodium phosphate buffer, the HCB conductivity increased during the injection, leading to a more efficient preconcentration by staking (about 1.6 times) than with phosphoric acid alone, for which conductivity decreased during injection. For the same injection time at constant voltage, however, a lower amount of analytes was injected with sodium phosphate buffer than with phosphoric acid. Consequently sensitivity enhancements were lower for the whole FESI‐Sweep‐MEKC process. This is why, in order to maximize sensitivity enhancements, it is proposed to work with sodium phosphate buffer as HCB and to use constant current during sample injection.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.     

Fast screening of aflatoxins in dairy cattle feeds with CE‐LIF method combined with preconcentration technique of vortex assisted low density solvent–microextraction

Aflatoxin contamination in agricultural products poses a great threat to humans and livestock. The aim of this study was to establish a simple, rapid, highly sensitive, and inexpensive method for the simultaneous detection of aflatoxin B₁, B₂, G₁, and G₂ in agricultural products. We used a vortex assisted low density solvent–microextraction (VALDS‐ME) technique for sample preconcentration and sample detection was achieved with a CE‐LIF method. Aflatoxins were separated in an uncoated fused‐silica capillary with the MEKC mode and were excited by a 355 nm UV laser to produce native fluorescence for detection. The obtained LOD and LOQ for the four aflatoxins were in the range of 0.002–0.075 and 0.007–0.300 μg/L, respectively, and the analysis time was within 6.5 min. Using the established method, aflatoxins were screened in naturally contaminated dairy cattle feed samples including alfalfa, bran, and corn kernel. The result shows that the alfalfa and bran samples were contaminated with aflatoxins to varying degrees. Compared with other analytical techniques for aflatoxin screening in agricultural products, this CE‐LIF method combined with VALDS‐ME preconcentration technique is simple, rapid, highly efficient, and inexpensive.     

An in‐line SPE strategy to enhance sensitivity in CE for the determination of pharmaceutical compounds in river water samples

In this study, the suitability of solid‐phase extraction (SPE) coupled in‐line to CE with UV–Vis detection was evaluated for the preconcentration and separation of diluted solutions of five pharmaceuticals compounds: benzafibrate, piroxicam, diclofenac sodium, naproxen and clofibric acid. An SPE analyte concentrator containing Oasis^® HLB sorbent was constructed without frits and placed near the inlet end of the separation capillary. Different parameters such as sample pH, composition and volume of the elution plug and sample loading time were studied in order to obtain the maximum preconcentration factors. The LODs reached for standard samples were in the range 0.06–0.5 ng/mL with good reproducibility, and the developed strategy provides sensitivity enhancement factors around 14 000‐fold in peak area and 5900‐fold in peak height compared with the normal hydrodynamic injection. Finally, river water samples fortified with the pharmaceutical compounds were analyzed by the developed in‐line SPE‐CE‐UV method in order to show the potential of the methodology for the analysis of environmental aquatic samples. For these samples, high values of relative recoveries, between 73–107% and 79–103% for two concentration levels, 5 and 25 ng/mL, respectively, were obtained and LODs ranged between 0.19 and 1 ng/mL.     

A fluorescent derivatization method of proteins for the detection of low‐level impurities by microchip capillary gel electrophoresis

A novel pre‐chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water‐soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on‐chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver‐stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre‐column derivatization include a shorter analysis time and an increased robustness and ease of use.     

Optimization of urinary pteridine analysis conditions by CE‐LIF for clinical use in early cancer detection

The second leading cause of death in the US is cancer and early discovery of the disease has translated into reduced fatality rates. We have identified and performed a systematic investigation of a method for urinary pteridine analysis by using CE‐LIF, which is believed to possess the potential to diagnose the presence of cancer even earlier than existing methodologies. Through system enhancements, we have been able to improve the resolution of the two least resolved sets of peaks (6,7‐dimethylpterin versus 6‐biopterin and D ‐(+)‐neopterin versus 6‐hydroxymethylpterin) from 0.85 to 2.48 and 0.90 to 3.58, respectively. Additionally, we have discovered that the preparation of the urine samples in previous works was inadequate, and we have corrected the method to fully oxidize the pteridines in the urine, resulting in significantly less variability in quantification and greater ease of defining p‐values for healthy versus cancer patients. Finally, we have performed validation steps of spike and recovery and short‐term aging studies to demonstrate the method's robustness. As a result, we present an optimized and validated method ready for transfer from discovery phase to clinical trial that can potentially act as a non‐invasively pre‐screening test for cancer.