Identification of olive pollen allergens using a fluorescence‐based 2D multiplex method

Olive (Olea europaea L.) pollen is a major health concern in the Mediterranean countries and some olive growing regions in America and Australia. The molecular variability of pollen allergens constitutes a handicap for commercial extract standardization, which is the base of current diagnosis and vaccination procedures. In this paper, we report a time‐saving and plant material saving multiplex detection method for the rapid and simultaneous analysis of Ole e 1, Ole e 2, and Ole e 5 allergen polymorphism on a single blot. This method combines high‐resolution 2DE techniques with high‐sensitive fluorescence‐based detection methods. Using this strategy, we were capable to identify a higher number of allergen forms compared with classical 1D approach. The use of fluorescent probes and the increased resolution of 2D blots avoided overlapping effects, and allow estimating the amount of individual allergen forms. In addition, the pattern and identity of the IgE‐reactive proteins of either a population or individual patients allergic to olive pollen was also effortlessly determined in a single additional step. This flexible method might be extended to a higher number of olive allergens and cultivars, and is also applicable to other allergogenic plant species and sources.     

In‐capillary detection of fast antibody‐peptide binding using fluorescence coupled capillary electrophoresis

Herein, we report a technique for detecting the fast binding of antibody‐peptide inside a capillary. Anti‐HA was mixed and interacted with FAM‐labeled HA tag (FAM‐E₄) inside the capillary. Fluorescence coupled capillary electrophoresis (CE‐FL) was employed to measure and record the binding process. The efficiency of the antibody‐peptide binding on in‐capillary assays was found to be affected by the molar ratio. Furthermore, the stability of anti‐HA‐FAM‐E₄ complex was investigated as well. The results indicated that E₄YPYDVPDYA (E₄) or TAMRA‐E₄YPYDVPDYA (TAMRA‐E₄) had the same binding priorities with anti‐HA. The addition of excess E₄ or TAMRA‐E₄ could lead to partial dissociation of the complex and take a two‐step mechanism including dissociation and association. This method can be applied to detect a wide range of biomolecular interactions.     

Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Setup and detector performance

A lamp‐based fluorescence detection (Flu) system for CE was extended with a wavelength‐resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe‐Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut‐off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal‐intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE‐WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged‐polymer coated capillary. Using the 3‐D data set, signal averaging over time and emission‐wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.     

In‐capillary self‐assembly study of quantum dots and protein using fluorescence coupled capillary electrophoresis

As a vast number of novel materials in particular inorganic nanoparticles have been invented and introduced to all aspects of life, public concerns about how they might affect our ecosystem and human life continue to arise. Such incertitude roots at a fundamental question of how inorganic nanoparticles self‐assemble with biomolecules in solution. Various techniques have been developed to probe the interaction between particles and biomolecules, but very few if any can provide advantages of both rapid and convenient. Herein, we report a systematic investigation on quantum dots (QDs) and protein self‐assembly inside a capillary. QDs and protein were injected to a capillary one after another. They were mixed inside the capillary when a high voltage was applied. Online separation and detection were then achieved. This new method can also be used to study the self‐assembly kinetics of QDs and protein using the Hill equation, the K_D value for the self‐assembly of QDs and protein was calculated to be 8.8 μM. The obtained results were compared with the previous out of‐capillary method and confirmed the effectiveness of the present method.     

Surfactant‐based ionic liquids for extraction of phenolic compounds combined with rapid quantification using capillary electrophoresis

A rapid liquid phase extraction employing a novel hydrophobic surfactant‐based room temperature ionic liquid (RTIL), tetrabutylphosphonium dioctyl sulfosuccinate ([4C₄P][AOT]), coupled with capillary electrophoretic‐UV (CE‐UV) detection is developed for removal and determination of phenolic compounds. The long‐carbon‐chain RTIL used is sparingly soluble in most solvents and can be used to replace volatile organic solvents. This fact, in combination with functional‐surfactant‐anions, is proposed to reduce the interfacial energy of the two immiscible liquid phases, resulting in highly efficient extraction of analytes. Several parameters that influence the extraction efficiencies, such as extraction time, RTIL type, pH value, and ionic strength of aqueous solutions, were investigated. It was found that, under acidic conditions, most of the investigated phenols were extracted from aqueous solution into the RTIL phase within 12 min. Good linearity was observed over the concentration range of 0.1–80.0 μg/mL for all phenols investigated. The precision of this method, expressed as RSD, was determined to be within 3.4–5.3% range. The LODs (S/N = 3) of the method were in the range of 0.047–0.257 μg/mL. The proposed methodology was successfully applied to determination of phenols in real water samples.     

Metabolic profiling for the identification of Huntington biomarkers by on‐line solid‐phase extraction capillary electrophoresis mass spectrometry combined with advanced data analysis tools

In this work, an untargeted metabolomic approach based on sensitive analysis by on‐line solid‐phase extraction capillary electrophoresis mass spectrometry (SPE‐CE‐MS) in combination with multivariate data analysis is proposed as an efficient method for the identification of biomarkers of Huntington's disease (HD) progression in plasma. For this purpose, plasma samples from wild‐type (wt) and HD (R6/1) mice of different ages (8, 12, and 30 weeks), were analyzed by C₁₈‐SPE‐CE‐MS in order to obtain the characteristic electrophoretic profiles of low molecular mass compounds. Then, multivariate curve resolution alternating least squares (MCR‐ALS) was applied to the multiple full scan MS datasets. This strategy permitted the resolution of a large number of metabolites being characterized by their electrophoretic peaks and their corresponding mass spectra. A total number of 29 compounds were relevant to discriminate between wt and HD plasma samples, as well as to follow‐up the HD progression. The intracellular signaling was found to be the most affected metabolic pathway in HD mice after 12 weeks of birth, when mice already showed motor coordination deficiencies and cognitive decline. This fact agreed with the atrophy and dysfunction of specific neurons, loss of several types of receptors, and changed expression of neurotransmitters.     

Chip electrophoresis of gelatin‐based nanoparticles

Recently, biodegradable nanoparticles received increasing attention for pharmaceutical applications as well as applications in the food industry. With the current investigation we demonstrate chip electrophoresis of fluorescently (FL) labeled gelatin nanoparticles (gelatin NPs) on a commercially available instrument. FL labeling included a step for the removal of low molecular mass material (especially excess dye molecules). Nevertheless, for the investigated gelatin NP preparation two analyte peaks, one very homogeneous with an electrophoretic net mobility of μ = ?24.6 ± 0.3 × 10^?9 m²/Vs at the peak apex (n = 17) and another more heterogeneous peak with μ between approximately ?27.2 ± 0.2 × 10^?9 m²/Vs and ?36.6 ± 0.2 × 10^?9 m²/Vs at the peak beginning and end point (n = 11, respectively) were recorded. Filtration allowed enrichment of particles in the size range of approximately 35 nm (pore size employed for concentration of gelatin NPs) to 200 nm (pore size employed during FL labeling). This corresponded to the very homogeneous peak linking it to gelatin NPs, whereas the more heterogeneous peak probably corresponds to gelatin not cross‐linked to such a high degree (NP building blocks). Several further gelatin NP preparations were analyzed according to the same protocol yielding peaks with electrophoretic net mobilities between ?23.3 ± 0.3 × 10^?9 m²/Vs and ?28.9 ± 0.2 × 10^?9 m²/Vs at peak apexes (n = 15 and 6). Chip electrophoresis allows analyte separation in less than two minutes (including electrophoretic sample injection). Together with the high sensitivity of the FL detection – the LOD as derived for the first main peak of the applied dye from the threefold standard deviation of the background noise values 80 pM for determined separation conditions – this leads to a very promising high throughput separation technique especially for the analysis of bionanoparticles. For gelatin NP preparations, chip electrophoresis allows for example the comparison of preparation batches concerning the amount of NPs and gelatin building blocks as well as the indirect assessment of the degree of gelatin cross‐linking (from obtained FL signals).     

Detection of S‐adenosylhomocysteine and methylation index in blood by capillary electrophoresis

This work proposes an approach to the direct analysis of S‐adenosylhomocysteine (SAH) and the methylation index in blood using CE with UV detection (CE‐UV). After application of meglumine postinjection, we achieved SAH in‐capillary preconcentration in the HClO₄ extracts of erythrocytes, which improved the detection limit (S/N = 3) of SAH up to 3 fmol or 180 nmol/L at the injection volume of 50 nL, taking into account the sample dilution rate. CE‐UV was carried out in 30 mM glycine and 45 mmol/L HCl (pH ～1.8) at 17 kV in a capillary 48 cm in length and 50 μm id. Accuracy of the technique was 101% and reproducibility was about 12%.     

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.     

Ionic liquids in enhancing the sensitivity of capillary electrophoresis: Off‐line and on‐line sample preconcentration techniques

The popularity of ionic liquids (ILs) has grown during the last decade in enhancing the sensitivity of CE through different off‐line or on‐line sample preconcentration techniques. Water‐insoluble ILs were commonly used in IL‐based liquid phase microextraction, in all its variants, as off‐line sample preconcentration techniques combined with CE. Water‐soluble ILs were rarely used in IL‐based aqueous two phase system (IL‐ATPS) as an off‐line sample preconcentration approach combined with CE in spite of IL‐ATPS predicted features such as more compatibility with CE sample injection due to its relatively low viscosity and more compatibility with CE running buffers avoid, in some cases, anion exchange precipitation. Therefore, the attentions for the key parameters affecting the performance of IL‐ATPSs were generally presented and discussed. On‐line CE preconcentration techniques containing IL‐based surfactants at nonmicellar or micellar concentrations have become another interesting area to improve CE sensitivity and it is likely to remain a focus of the field in the endeavor because of their numerous to create rapid, simple and sensitive systems. In this article, significant contributions of ILs in enhancing the sensitivity of CE are described, and a specific overview of the relevant examples of their applications is also given.     

Sensitive determination of chloroanilines in water samples by hollow fiber‐based liquid‐phase microextraction prior to capillary electrophoresis with amperometric detection

A hollow fiber‐based liquid‐phase microextraction method has been developed for enrichment of trace chloroanilines in water samples. Target analytes including aniline, three mono‐chlorinated aniline isomers (o‐chloroaniline, m‐chloroaniline, and p‐chloroaniline) and four mono‐chlorinated methylaniline isomers (2‐chloro‐4‐methylaniline, 3‐chloro‐4‐methylaniline, 4‐chloro‐2‐methylaniline, and 5‐chloro‐2‐methylaniline) were determined by CE with amperometric detection after microextraction. Several factors that affect separation, detection, and extraction efficiency were investigated. Under the optimum conditions, eight aniline compounds could be well separated from other components coexisting in water samples within 25 min, exhibiting a linear calibration over three orders of magnitude (r > 0.998); the obtained enrichment factors were between 51 and 239, and the LODs were in the range of 0.01–0.1 ng/mL. The proposed method has been applied for the analyses of real environmental water and sewage samples with relative recoveries in the range of 83–108%.     

Using sheath‐liquid reagents for capillary electrophoresis‐mass spectrometry: Application to the analysis of phenolic plant extracts

The combination of CE and MS is now a widely used tool that can provide a combination of high resolution separations with detailed structural information. Recently, we highlighted the benefits of an approach to add further functionality to this well‐established hyphenated technique, namely the possibility to perform chemical reactions within the sheath‐liquid of the CE‐MS interface 1 . Apart from using hydrogen/deuterium exchange for online determination of numbers of exchangeable protons, the addition of DPPH? (2,2‐diphenyl‐1‐picrylhydrazyl) to the sheath‐liquid can be used as a fast screening tool for studying antioxidant characteristics of individual components. Such a CE‐MS methodology allows rapid and information‐rich analysis with minimal reagent and sample consumption to be performed. In the present work, we demonstrate the applicability of this approach for the characterization of phenolic plant extracts from the Labiatae family, namely Rosmarinus officinalis and Melissa officinalis. Using the described approach, a wide range of compounds (15 and 13 phenolic compounds, respectively) could be confidently identified using a combination of high resolution CE‐MS separations with implementation of online deuterium exchange and DPPH? reactions. These compounds included polyphenols, phenolic acids, and triterpene acids. In conjunction with online MS/MS experiments, extensive structural information for aglyconic and glycosylated antioxidants present in the extracts could be obtained using simple experimental changes, which can be carried out prior to the purchasing of expensive chemical standards or the time‐consuming preparative isolation of individual compounds.     

Co‐electroosmotic capillary electrophoresis of basic proteins with 1‐alkyl‐3‐methylimidazolium tetrafluoroborate ionic liquids as non‐covalent coating agents of the fused‐silica capillary and additives of the electrolyte solution

The paper reports the results of a study carried out to evaluate the use of three 1‐alkyl‐3‐methylimidazolium‐based ionic liquids as non‐covalent coating agents for bare fused‐silica capillaries and additives of the electrolyte solutions (BGE) for CE of basic proteins in the co‐EOF separation mode. The three ionic liquids are differentiated from each other by the length of the alkyl group on the imidazolium cation, consisting of either an ethyl, butyl or octyl substituent, whereas tetrafluoroborate is the common anionic component of the ionic liquids. Coating the capillary with the ionic liquid resulted in improved peak shape and protein separation, while the EOF was maintained cathodic. This indicates that each ionic liquid is effective at masking the protein interaction sites on the inner surface of the capillary, also when its adsorption onto the capillary wall has not completely neutralized all the negative charges arising from the ionization of the silanol groups and the ionic liquid is not incorporated into the BGE employed for separation. Using the coated capillaries with BGE containing the ionic liquid employed for the coating, at concentration low enough to maintaining the EOF cathodic, both peak shape and protein separation varied to different extents, based on the particular ionic liquid used and its concentration. Fast and efficient separation of the model basic protein mixture in co‐electroosmotic CE is obtained with the 1‐butyl‐3‐methylimidazolium tetrafluoroborate coated capillary and 100 mM acetate buffer (pH 4.0) containing 4.4 mM 1‐butyl‐3‐methylimidazolium tetrafluoroborate as the BGE.     

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.     

Triblock copolymer matrix‐based capillary electrophoretic microdevice for high‐resolution multiplex pathogen detection

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE‐SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) triblock copolymer as a sieving matrix for CE‐SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO‐PPO‐PEO copolymers, 255‐bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO‐PPO‐PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan? gel. Due to enhanced dynamic coating and sieving ability, PEO‐PPO‐PEO copolymer displayed fourfold enhancement of resolving power in the CE‐SSCP to separate same‐sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high‐resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO‐PPO‐PEO triblock copolymer an excellent matrix in the CE‐SSCP analysis on the microdevice.     

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.     

Assay of urinary 8‐hydroxy‐2′‐deoxyguanosine by capillary electrophoresis with spectrophotometric detection using a high‐sensitivity detection cell and solid‐phase extraction

A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z‐cell was devised for the assay of 8‐hydroxy‐2′‐deoxyguanosine (8OHdG) in human urine. Solid‐phase extraction (SPE) based on hydrophilic‐lipophilic‐balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused‐silica capillaries employing a Z‐cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10–1000 ng/mL; R² = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.     

Preparation of a dual‐enzyme co‐immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis

A CE method based on a dual‐enzyme co‐immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co‐immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. The enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed CE method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. A statistical parameter Z′ factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two‐enzyme inhibition by the present method.     

Determination of the enantiomeric and diastereomeric impurities of RS‐glycopyrrolate by capillary electrophoresis using sulfated‐β‐cyclodextrin as chiral selectors

A practical chiral CE method, using sulfated‐β‐CD as chiral selector, was developed for the enantioseparation of glycopyrrolate containing two chiral centers. Several parameters affecting the separation were studied, including the nature and concentration of the chiral selectors, BGE pH, buffer type and concentration, separation voltage, and temperature. The separation was carried out in an uncoated fused‐silica capillary of (effective length 40 cm) × 50 μm id with a separation voltage of 20 kV using 30 mM sodium phosphate buffer (pH 7.0, adjusted with 1 M sodium hydroxide) containing 2.0% w/v sulfated‐β‐CD at 25°C. Finally, the method for determining the enantiomeric impurities of RS‐glycopyrrolate was proposed. The method was further validated with respect to its specificity, linearity range, accuracy and precision, LODs, and quantification in the expected range of occurrence for the isomeric impurities (0.1%).