Dynamic labeling of diagnostically significant microbial cells in cerebrospinal fluid by red chromophoric non-ionogenic surfactant for capillary electrophoresis separations

During bacterial infections of the central nervous system the number of microorganisms in the cerebrospinal fluid is often ranging from few up to hundreds of cells per milliliter. The electrophoretic techniques with the UV-detection reach a detection limit for whole cells of approximately 10⁷ cells per milliliter. The coupling of the filtration cartridge with capillary isoelectric focusing can improve the detection limit by four orders of magnitude. In order to improve the detection limit the red non-ionogenic surfactant 1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester, PAPAN 1000, has been prepared and used for the dynamic labeling of analytes before filtration of the sample with a concentration modulation in the analysis of proteins or microorganisms. Values of isoelectric points of labeled analytes have been calculated using pI markers detectable at 515 nm and have been found comparable with pI of the native compounds. Minimum detectable amounts of proteins and microorganisms were lower than nanograms and a hundred labeled cells, respectively. The introduced method, coupling of the filtration cerebrospinal fluid spiked with microorganisms and labeled by PAPAN, facilitates their rapid CIEF separation in the pH gradient pH range of 2–5 at their clinically important level 10¹ to 10² cells per milliliter.     

Rapid separation and identification of the subtypes of swine and equine influenza A viruses by electromigration techniques with UV and fluorometric detection

Influenza A is viral disease, which is a cause of yearly epidemics and, potentially, pandemics. The conventional techniques used today are equipment-demanding, time-consuming and laborious. Recently, we have confirmed that the capillary isoelectric focusing is a suitable fast alternative for the verifying of virus purity. In the wide pH gradient of pH range 2.0-7.5 the isoelectric points for subtypes of equine (H3N8) and swine (H1N2) influenza A viruses were determined approximately as 6.6 and 6.5, respectively. In this contribution we have verified these findings using different isolates of different viral subtypes of swine influenza, H1N1, H1N2, and of equine influenza, H3N8, H7N7, which were separated by capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) in the narrow pH gradient pH range from 6.0 to 7.0. It was found that the isoelectric points of different isolates and subtypes of equine and swine influenza are almost independent of their origin. The electromigration velocities of subtypes of equine or swine influenza viruses were dependent on the antigenic subtypes of their surface glycoproteins. The detection sensitivity of the influenza viruses labeled by the fluorescent non-ionogenic tenside based on poly(ethylene glycol)pyrenebutanoate for fluorometric detection was increased and down to ten labeled viruses were detected. The isoelectric points of the native and labeled equine and swine influenza A viruses and their subtypes do not differ. According to our experiments these methods appear to be useful for the fast preliminary differentiation of influenza viruses in future.     

The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing

Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface.     

Separation of attogram terpenes by the capillary zone electrophoresis with fluorometric detection

An original method based on capillary zone electrophoresis with fluorimetric detection has been developed for the determination of terpenic compounds. The method is based on the separation of a terpenes dynamically labeled by the non-ionogenic tenside poly(ethylene glycol) pyrenebutanoate, which was used previously for the labeling of biopolymers. The background electrolytes were composed of taurine-Tris buffer (pH 8.4). In addition to the non-ionogenic tenside aceton and poly(ethylene glycol) were used as the additives. The capillary zone electrophoresis with fluorometric detection at the excitation wavelength 335 nm and the emission wavelength 463 nm was successfully applied to the analysis of tonalid, cholesterol, vitamin A, ergosterol, estrone and farnesol at level of 10(-17) mol L(-1). Farnesol, is produced by Candida albicans as an extracellular quorum-sensing molecule that influences expression of a number of virulence factors, especially morphogenesis and biofilm formation. It enables this yeast to cause serious nosocomial infections. The sensitivity of this method was demonstrated on the separation of farnesol directly from the cultivation medium.     

CIEF separation,UV detection,and quantification of ampholytic antibiotics and bacteria from different matrices

The effect of antibiotics on the microbial cells and concentration of antibiotics in the human body is essential for the effective use of antimicrobial therapy. The capillary isoelectric focusing is a suitable technique for the separation and the detection of bacteria, and amphoteric substances from nature. However, the determination of isoelectric points of ampholytic antibiotics by conventional techniques is time consuming. For this reason, capillary isoelectric focusing seems to be appropriate as a simple and reliable way for establishing them. The separation conditions for the capillary isoelectric focusing of selected ampholytic antibiotics with known isoelectric points and pK _as, ampicillin (pI 4.9), ciprofloxacin (pI 7.4), ofloxacin (pI 7.1), tetracycline (pI 5.4), tigecycline (pI 9.7), and vancomycin (pI 8.1), were found and optimized in the suitable pH ranges pH 2.0–5.3, 2.0–9.6, and 9.0–10.4. The established values of isoelectric points correspond with those found in the literature except tigecycline. Its pI was not found in the literature. As an example of a possible procedure for direct detection of both ampholytic antibiotics and bacteria, Staphylococcus epidermidis, in the presence of culture media or whole human blood, was found. The changes of the bacterial cells after their treatment with tetracycline were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Capillary isoelectric focusing allows the fast and simple determination of isoelectric points of relevant antibiotics, their quantification from the environment, as well as studying their effectiveness on microorganisms in biological samples. Graphical Abstract

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Use of electrophoretic techniques and MALDI–TOF MS for rapid and reliable characterization of bacteria: analysis of intact cells, cell lysates, and “washed pellets”

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and “washed pellets”) were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of “washed pellets” and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI–TOF MS of “washed pellets” enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI–TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.     

Improving the detection limit in capillary isotachophoresis using asymmetric neutralisation reaction boundary

An online method involving transient electrokinetic dosing and ITP with neutralization reaction boundary (NRB) and/or carrier ampholyte-free isoelectric focusing (CAF IEF) was developed for the preconcentration, preseparation, and analytical determination of glyphosate in aqueous samples containing low concentrations of the analyte of interest. Various parameters were investigated in the framework of an optimization study with the aim of achieving the maximum concentration limit of detection (cLOD) decrease in minimum time. The proposed method used CAF IEF and/or ITP with NRB. The sample was dosed to the column on the stationary reaction boundary (CAF IEF) and/or moving reaction boundary (ITP with NRB), whereat a sharp pH step exists. Here, charge reversal was due to the ampholytes, and/or acid accumulation occurred because of charge loss. Similarly, the accumulated sample was mobilized with TE and analyzed using classical ITP in the second analytical column. Glyphosate (GLY), the analyte of interest, was chosen as a model substance for ITP with NRB and preconcentration as well as focusing preconcentration and CAF IEF using the asymmetric purpose-built NRB. On one side of the asymmetric boundary was the zone of acidic pH; while the opposite side comprised a neutral/basic non-conductive zone of the ampholyte—in this case, GLY. Such an arrangement enables the use of a lower pH on the acidic side, which allows the focusing of strongly acidic ampholytes and the accumulation of weak acids. The electrolyte composition and the dosing time were optimized, and a 14-fold accumulation was achieved in 25 min compared to that by classical ITP and a 180-fold accumulation was achieved through CAF IEF and preconcentration with a glyphosate sample. Both methods are simple and can be conducted using all commercial ITP systems.     

Recent developments in capillary and microchip electroseparations of peptides (2017–mid 2019)

The review presents a comprehensive survey of recent developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical and biochemical characterization of peptides since 2017 up to about the middle of 2019. Progress in the study of electromigration properties of peptides and in the methodology of their analysis (sample preseparation, preconcentration and derivatization, adsorption suppression, EOF control, and detection) are described. Advances in CE and CEC methods are demonstrated and their applications in the following areas are presented: qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. In addition, micropreparative separations and determinations of important physicochemical characteristics of peptides by CE and CEC methods are reported.     

Capillary isoelectric focusing of microorganisms in the pH range 2–5 in a dynamically modified FS capillary with UV detection

The isoelectric points of many microbial cells lie within the pH range spanning from 1.5 to 4.5. In this work, we suggest a CIEF method for the separation of cells according to their isoelectric points in the pH range of 2–5. It includes the segmental injection of the sample pulse composed of the segment of the selected simple ampholytes, the segment of the bioanalytes and the segment of carrier ampholytes into fused silica capillaries dynamically modified by poly(ethylene glycole). This polymer dissolved in the catholyte, in the anolyte and in the injected sample pulse was used for a prevention of the bioanalyte adsorption on the capillary surface and for the reduction of the electroosmotic flow. Between each focusing run, the capillaries were washed with the mixture of acetone/ethanol to achieve the reproducible and efficient CIEF. In order to trace of pH gradients, low-molecular-mass pI markers were used. The mixed cultures of microorganisms, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Candida glabrata, Candida tropicalis, CCM 8223, Proteus vulgaris, Klebsiela pneumoniae, Staphylococcus aureus CCM 3953, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224 and Staphylococcus epidermidis CCM 4418, were focused and separated by the CIEF method suggested here. This CIEF method enables the separation and detection of the microbes from the mixed cultures within several minutes. The minimum detectable number of microbial cells was less than 10³.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection

The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.     

Combination of micropreparative solution isoelectric focusing and high-performance liquid chromatography for differentiation of biofilm-positive and biofilm-negative Candida parapsilosis group from vascular catheter

This study utilizes the high-performance liquid chromatography technique in combination with the new micropreparative solution isoelectric focusing fractionation on non-woven fabric strip for the characterization and differentiation of biofilm-positive and biofilm-negative forms of Candida parapsilosis sensu stricto on the basis of the changes in the composition of their cell-surface. Treatment of yeasts by boiling in distilled water relased surface substances from yeasts cells. Consequently, the optimized procedure has been used for fast identification of the highly pathogenic biofilm-positive Candida parapsilosis group in real clinical material – sonicate from vascular catheters. Moreover, the capillary isoelectric focusing was used as supporting and control technique. Obtained results suggest that this new method can be used to distinguish between biofilm-positive and negative forms of Candida parapsilosis sensu stricto.     

Protease Inhibitors Purified from the Canola Meal Extracts of Two Genetically Diverse Genotypes Exhibit Antidiabetic and Antihypertension Properties

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC₅₀ values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.     

Sweeping and new on-line sample preconcentration techniques in capillary electrophoresis

Sweeping is a powerful on-line sample preconcentration technique that improves the concentration sensitivity of capillary electrophoresis (CE). This approach is designed to focus the analyte into narrow bands within the capillary, thereby increasing the sample volume that can be injected, without any loss of CE efficiency. It utilizes the interactions between an additive [i.e., a pseudostationary phase (PS) or complexing agent] in the separation buffer and the sample in a matrix that is devoid of the additive used. The accumulation occurs due to chromatographic partitioning, complexation or any interaction between analytes and the additive through electrophoresis. The extent of the preconcentration is dependent on the strength of interaction involved. Both charged and neutral analytes can be preconcentrated. Remarkable improvements—up to several thousandfold—in detection sensitivity have been achieved. This suggests that sweeping is a superior and general approach to on-line sample preconcentration in CE. The focusing mechanism of sweeping under different experimental conditions and its combination with other on-line preconcentration techniques are discussed in this review. The recently introduced techniques of transient trapping (tr-trapping) and analyte focusing by micelle collapse (AFMC) as well as other novel approaches to on-line sample preconcentration are also described.

Over 1000-fold protein preconcentration for microliter-volume samples at a pH junction using capillary electrophoresis

An effective protein preconcentration technique specifically designed for microliter-volume samples is presented. The preconcentration is based on the capturing of protein in its isoelectric point (pI) within an applied electric field, using a pH junction created by a discontinuous buffer system. The buffers were chosen to selectively preconcentrate proteins of neutral pI, myoglobin in this case, while removing other proteins with acidic or basic pIs. For the suppression of electro-osmotic flow (EOF) and protein adsorption, the capillary inner wall was modified with a zwitterionic phospholipid bilayer coating. A preconcentration factor of up to 1700 was obtained for a 1 microg/mL solution of myoglobin. The preconcentration was completed in approximately 20 min. Several sample introduction conditions were presented to accommodate sample volume from one to a few hundreds of microliters. The final volume of the preconcentrated sample band was estimated to be approximately 5 nL.     

Separation of phenotypically indistinguishable Candida species, C. orthopsilosis, C. metapsilosis and C. parapsilosis, by capillary electromigration techniques

At the current state of laboratory diagnostics, methods for fast identification of phenotypically indistinguishable species are difficult or inaccurate. An example is represented by Candida parapsilosis, which is the second most common yeast species isolated from bloodstream infections. C. parapsilosis comprises a complex of three genetically distinct groups. Genotypes II and III have been designated as the separate species Candida orthopsilosis and Candida metapsilosis, phenotypically indistinguishable. The considerable genetic variability of these newly described yeasts species has caused difficulties in the development of molecular techniques for their precise identification. Similarly, the detection of biofilm formation, which is considered as an important yeast virulence factor, is accompanied by difficulties. In this study we optimize the first precise and reproducible method for the separation and possible identification of C. orthopsilosis, C. metapsilosis and C. parapsilosis as well as the detection of their ability to form biofilm. The method is based on capillary isoelectric focusing and capillary electrophoresis with UV detection. In capillary isoelectric focusing, very narrow pH gradients were established. With such gradients, differences in isoelectric points of biofilm-negative and biofilm-positive species calculated from the migration times of the selected pI markers were below 0.03 pI units. In the capillary zone electrophoresis narrow zones of the cells of Candida species were detected with sufficient resolution. The values of the isoelectric point and the migration velocities of the examined species were independent on the origin of the tested strains. Capillary isoelectric focusing was examined also for the separation and detection of the cultivated biofilm-negative C. parapsilosis in the blood serum.     

Strategies for the on-line preconcentration and separation of hypolipidaemic drugs using micellar electrokinetic chromatography

Three strategies were investigated for the simultaneous separation and on-line preconcentration of charged and neutral hypolipidaemic drugs in micellar electrokinetic chromatography (MEKC). A background electrolyte (BGE) consisting of 20 mM ammonium bicarbonate buffer (pH 8.50) and 50 mM sodium dodecyl sulfate (SDS) was used for the separation and on-line preconcentration of the drugs. The efficiencies of sweeping, analyte focusing by micelle collapse (AFMC), and simultaneous field-amplified sample stacking (FASS) and sweeping, were compared for the preconcentration of eight hypolipidaemic drugs in different conductivity sample matrices. When compared with a hydrodynamic injection (5 s at 50 mbar, 0.51% of capillary volume to detection window) of drug mixture prepared in the separation BGE, improvements of detection sensitivity of 60-, 83-, and 80-fold were obtained with sweeping, AFMC and simultaneous FASS and sweeping, respectively, giving limits of detection (LODs) of 50, 36, and 38 μg/L, respectively. The studied techniques showed suitability for focusing different types of analytes having different values of retention factor (k). This is the first report for the separation of different types of hypolipidaemic drugs by capillary electrophoresis (CE). The three methods were validated then applied for the analysis of target analytes in wastewater samples from Hobart city.     

Velocity-difference induced focusing of xanthine and purine metabolites by capillary electrophoresis using a dynamic pH junction

Summary Velocity-difference induced focusing (V-DIF) of analytes by a dynamic pH junction represents a simple yet effective on-line preconcentration method to improve concentration sensitivity in capillary electrophoresis (CE). Differences in buffer type, pH and conductivity between sample and background electrolyte (BGE) segments of the capillary are properties used to optimize purine focusing within a multi-section electrolyte system. This method permits the injection of large volumes of sample (up to 450 nL or about 18% of capillary length), resulting in over a 50-fold improvement in sensitivity with baseline resolution. The limit of detection (S/N=3) for xanthine is determined to less than 4.0×10⁻⁸ M under optimum conditions when using UV detection. Analysis of micromolar amounts of xanthine in pooled urine is also demonstrated without sample pretreatment. A dual mechanism involving dynamic pH and isotachophoretic modes is proposed to enhance analyte focusing performance when employing buffer pH junctions based on different types of electrolyte co-ions.     

Peptidomic approach based on combined capillary isoelectric focusing and mass spectrometry for the characterization of the plasmin primary products from bovine and water buffalo beta-casein

The main peptides produced by hydrolysis of water buffalo beta-casein with plasmin were characterized by capillary electrophoresis and mass spectrometry and compared with their bovine homologous. A novel breakdown product arising from the hydrolysis of water buffalo beta-casein, originated by the presence of a plasmin-sensitive Lys bond at position 68 was identified, which was not present in bovine beta-casein. On the basis of this evidence, an improved procedure for the detection and the differentiation of the products of plasmin hydrolysis of bovine and water buffalo beta-casein by capillary isoelectric focusing was set-up. In the experimental conditions, the gamma-casein from the two species was efficiently separated. Comparison of the capillary electropherograms with those obtained by ultra-thin-layer isoelectric focusing, the reference method for routine analysis of plasmin digests of casein, suggests that capillary electrophoresis isoelectric focusing may constitute a successful alternative to the traditional slab gel electrophoresis analysis of plasmin digests of casein either for basic structural studies or for applications in the quality assessment of dairy products.