Isolation and Enrichment of Pathogens with a Surface‐Modified Aluminium Chip for Raman Spectroscopic Applications

We developed a Raman‐compatible chip for isolating microorganisms from complex media. The isolation of bacteria is achieved by using antibodies as capture molecules. Due to the very specific interaction with the targets, this approach is promising for isolation of bacteria even from complex matrices such as body fluids. Our choice of capture molecules also enabled the investigation of samples containing yet unidentified bacteria, as the antibodies can capture a large variety of bacteria based on their analogue cell wall surface structures. The capability of our system is demonstrated for a broad range of different Gram‐positive and Gram‐negative germs. Subsequent identification is done by recording Raman spectra of the bacteria. Further, it is shown that classification with chemometric methods is possible.     

Heart‐cut nano‐LC–CZE–MS for the characterization of proteins on the intact level

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed‐phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well‐suited geometries/dimensions. Here, a heart‐cut nano‐LC–CZE–MS setup was developed utilizing for the first time a mechanical 4‐port valve as LC–CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart‐cut transfer of individual LC peaks and subsequent CZE–MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower μg/mL range were determined, which are considerably lower compared to traditional CZE–MS. In addition, this study represents the first application of an LC–CE–MS system for intact protein analysis. The nano‐LC–CZE–MS system is expected to be applicable to various other analytical challenges.     

A study of interactions between bacteria and antibiotics by capillary electrophoresis

To assess the bacteria–antibiotic interactions in patients with postoperative wound infections, a simple electrophoretic test was performed. To estimate the effectiveness of the antibiotic therapy and to prepare 3‐day profiles of bacteria “quantity” in biological samples, CE was used. As our team demonstrated earlier, the method is easy and fast, sample pretreatment is not necessary, and it is characterized by high selectivity. Finally, the statistically optimal and significant results of the CZE test analysis for detection of Escherichia coli cells was established for migration time lower than 3.5 min. The obtained sensitivity and specificity amounted to 89.5 and 100%, respectively. It is the first application of CZE in the study of medical therapy.     

Highly sensitive detection of five typical fluoroquinolones in low‐fat milk by field‐enhanced sample injection‐based CE in bubble cell capillary

Fluoroquinolones are a group of synthetic antibiotics with a broad activity spectrum against mycoplasma, Gram‐positive, and Gram‐negative bacteria. Due to the extensive use of fluoroquinolones in farming and veterinary science, there is a constant need in the analytical methods able to efficiently monitor their residues in food products of animal origin, regulated by Commission Regulation (European Union) no. 37/2010. Herein, field‐enhanced sample injection for sample stacking prior the CZE separation was developed inside a bubble cell capillary for highly sensitive detection of five typical fluoroquinolones in bovine milk. Ethylenediamine was proposed as the main component of BGE for the antibiotics separation. The effect of BGE composition, injection parameters, and water plug length on the field‐enhanced sample injection‐based CE with UV detection was investigated. Under the optimized conditions, described field‐enhanced sample injection‐based CE‐UV analysis of fluoroquinolones provides LODs varying from 0.4 to 1.3 ng/mL. These LOD values are much lower (from 460 to 1500 times) than those obtained by a conventional CE in a standard capillary without bubble cell. The developed method was finally applied for the analysis of fluoroquinolones in low‐fat milk from a Swiss supermarket. Sample recovery values from 93.6 to 106.0% for different fluoroquinolones, and LODs from 0.7 to 2.5 μg/kg, were achieved. Moreover, the proposed ethylenediamine‐based BGE as volatile and compatible with MS system, enabled the coupling of the field‐enhanced sample injection‐based CE with a recently introduced electrostatic spray ionization MS via an iontophoretic fraction collection interface for qualitative fluoroquinolones identification.     

Capillary electrophoresis for fast detection of heterogeneous population in colistin‐resistant Gram‐negative bacteria

It has been shown that diverse strains of bacteria can be separated according to their characteristic surface properties by means of CE. We employed here this analytical technique to the study of colistin‐resistance in Gram‐negative bacteria, which involves the selection of mutants with modified outer membrane composition resulting in changes of surface cell properties. In the same way as with molecular entities, we performed firstly the validation of an ITP‐based CE method for three common pathogenic Gram‐negative bacteria namely Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Secondly, we compared the electrophoretic profiles of bacterial samples from a colistin‐susceptible clinical isolate of K. pneumoniae and from the corresponding colistin‐resistant derivative. By a simple CE run taking a few minutes, the coexistence of several bacterial subpopulations in the colistin‐resistant derivative was clearly evidenced. This work encourages further research that would allow applications of CE in clinical laboratory for a daily monitoring of bacterial population in cared patients when “last‐chance” colistin treatment is initiated against multidrug‐resistant bacteria.     

Preparation and evaluation of poly glycerol sebacate/poly hydroxy butyrate core‐shell electrospun nanofibers with sequentially release of ciprofloxacin and simvastatin in wound dressings

Biodegradable wound dressing of poly glycerol sebacate/poly hydroxy butyrate was fabricated via the coaxial electrospinning process. Simvastatin and ciprofloxacin were loaded in the core and shell of the fibers, respectively. Scanning electron microscopy and transmission electron microscopy images showed a uniform core/shell structure. Introducing drugs into the polymers would cause the dressing samples to become more hydrophilic and degradation to occur faster. Drugs release would face no interventions, in which, approximately 60% of ciprofloxacin was released during the first 24 hours. Simvastatin exhibited a slower and controlled release behavior, with its release peak recorded after 2 days. The drug‐containing samples showed a proper bactericidal activity against both Gram‐positive and Gram‐negative bacteria. It may be concluded that the drug‐laden wound dressing fabricated in this study is capable of releasing the 2 drugs sequentially and that it is the ideal conditions for controlling infections and reducing wound healing duration.     

Simultaneous quantification of energetically important metabolites in various cell types by CZE

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field‐enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on‐line pre‐concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.     

Separation of aromatic sulphonic acids by CZE in coated and non‐coated capillaries

Capillary zone electrophoresis (CZE), besides ion‐pairing mode HPLC and salting‐out HPLC, is well‐suited for the analysis of aromatic sulphonic acids widely used as intermediates in the production of synthetic dyes and optical brighteners. The separation selectivity in CZE of many aromatic acids, including positional isomers, can be controlled by the type and concentration of a cyclodextrin additive. The influence of the concentration of β‐cyclodextrin in the working electrolyte on the separation of the positional isomers of naphthalene (poly‐)sulphonic acids, and their amino‐ and hydroxy derivatives, by CZE was studied, both in non‐coated fused silica capillaries and in capillaries coated with polyacrylamide. The migration time scale was calibrated using 4‐alkylbenzenesulphonic acids as the calibration standards. Limiting mobilities of the free acid anions and of their complexes with β‐cyclodextrin were calculated and the effect of the inclusion guest‐host complex formation on the CZE separation was quantitatively characterized. The migration order in coated capillaries is reversed with respect to CZE in non‐coated fused silica capillaries, the separation selectivity is different and the separation of polysulphonic acids such as naphthalene tri‐ and tetrasulphonic acids is significantly accelerated.     

In-vitro antibacterial properties of crude aqueous and n-hexane extracts of the husk of Cocos nucifera

The increasing numbers of cases of antibiotic resistance among pathogenic bacteria such as Vibrio species poses a major problem to the food and aquaculture industries, as most antibiotics are no longer effective in controlling pathogenic bacteria affecting these industries. Therefore, this study was carried out to assess the antibacterial potentials of crude aqueous and n-hexane extracts of the husk of Cocos nucifera against some selected Vibrio species and other bacterial pathogens including those normally implicated in food and wound infections. The crude extracts were screened against forty-five strains of Vibrio pathogens and twenty-five other bacteria isolates made up of ten Gram positive and fifteen Gram negative bacteria. The aqueous extract was active against 17 of the tested bacterial and 37 of the Vibrio isolates; while the n-hexane extract showed antimicrobial activity against 21 of the test bacteria and 38 of the test Vibrio species. The minimum inhibitory concentrations (MICs) of the aqueous and n-hexane extracts against the susceptible bacteria ranged between 0.6-5.0 mg/mL and 0.3-5.0 mg/mL respectively, while the time kill study result for the aqueous extract ranged between 0.12 Log?? and 4.2 Log?? cfu/mL after 8 hours interaction in 1 x MIC and 2 x MIC. For the n-hexane extract, the log reduction ranged between 0.56 Log?? and 6.4 Log?? cfu/mL after 8 hours interaction in 1 x MIC and 2 x MIC. This study revealed the huge potential of C. nucifera extracts as alternative therapies against microbial infections.     

CZE study on adsorption processes of aliphatic and aromatic amines on PMMA chip

Adsorption processes on a PMMA chip linked with CZE separations of a group of 13 aliphatic and aromatic mono‐ and di‐amines were studied. Due to the lack of chromophores within aliphatic amines, contact conductivity detection implemented directly onto the chip was used for monitoring of cationic CZE separations. To prevent an adsorption of studied amines to the chip channels, the surface of PMMA chip was modified by dynamic coating. Different surface modifiers, such as aliphatic oligoamines (diethylenetriamine and triethylenetetramine), were added to the BGE solutions filling the chip channels. The effect of various concentrations of surface modifiers on peak profiles and separation parameters of amines was monitored. Of these, mainly, aliphatic di‐amines and aromatic mono‐amines adversely affected the CZE resolution of a whole group of analytes by their strong adsorption to the chip channels. A propionate BGE with pH 3.2 containing 100 μM triethylenetetramine and 25 mM 18‐crown‐6‐ether was found suitable for CZE resolution of 12 from a total of 13 amines studied. Simple dynamic modification of the surface of PMMA chip enabled fast (analysis time lasted 9 min), sensitive (sub‐μM LODs reached) and reproducible (1–3% RSD of the peak areas) CZE analysis of the aliphatic and aromatic amines.     

Characterization and separation of semiconductor quantum dots and their conjugates by capillary electrophoresis

Semiconductor quantum dots (QDs) are very important luminescent nanomaterials with a wide range of potential applications. Currently, QDs as labeling probes are broadly used in bioassays, including immunoassay, DNA hybridization, and bioimaging, due to their excellent physical and chemical properties, such as broad excitation spectra, narrow and size‐dependent emission profiles, long fluorescence life time, and good photostability. The characterization of QDs and their conjugates is crucial for their wide bioapplications. CE has become a powerful tool for the separation and characterization of QDs and their conjugates. In this review, some CE separation models of QDs are first introduced, mainly including CZE, CGE, MEKC, and ITP. And then, some key applications, such as the measurements of size, surface charge, and concentration of QDs and the characterization of QDs conjugates (e.g. QD–protein, QD–DNA, QD–small molecule), are also described. Finally, future perspectives are discussed.     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

Preparation of antimicrobial sutures by preirradiation grafting onto polypropylene monofilament

Antimicrobial sutures were prepared by the radiation grafting of acrylonitrile monomer onto polypropylene (PP) monofilament. The grafted sutures were subsequently hydrolyzed to transform nitrile groups into carboxylic groups for the immobilization of antimicrobial drug, tetracycline hydrochloride (TC). The modified sutures show continuous release of drug for a period of 4–5 days. The antimicrobial activity of the sutures was determined against both Gram positive and Gram negative bacteria by the zone of inhibition technique. Zone of inhibition was observed around the drug‐containing sutures in the plate inoculated with Escherichia coli (E. coli), Klebsiella pneumonea (K. pneumonea), and Staphylococcus aureus (S. aureus). The results of infection studies in albino rats against S. aureus showed no infection even after fourth postoperative day of surgery. This is because of the release of the TC drug at the site of injury, which inhibits the bacterial growth. Copyright © 2008 John Wiley & Sons, Ltd.     

Capillary electrophoresis of microbial aggregates

Uncontrolled aggregation of bacterial cells is a significant disadvantage of electrophoretic separations. Various aspects of the electrophoretic behavior of different strains of Gram‐positive Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus(1), and Micrococcus luteus bacteria and Gram‐negative Escherichia coli bacteria were investigated in this study. Our findings indicate that bacteria can be rapidly analyzed by CZE with surface charge modification by calcium ions (Ca²⁺). Bound Ca²⁺ ions increase zeta potential to more than 2.0 mV and significantly reduce repulsive forces. Under the above conditions, bacterial cells create compact aggregates, and fewer high‐intensity signals are observed in electropherograms. The above can be attributed to the bridging effect of Ca²⁺ between bacterial cells. CE was performed to analyze bacterial aggregates in an isotachophoretic mode. A single peak was observed in the electropherogram.     

Uses of ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate as a good separation electrolyte for direct electrophoretic separation of quaternary ammonium herbicides

This paper describes a new method for the direct separation of paraquat and diquat by CZE with ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate employed as reliable electrolyte. Several factors that affect the separation efficiency were investigated in detail. The experimental results indicated that the optimal running buffer consisted of 50 mM 1‐butyl‐3‐methylimidazolium hexafluorophosphate and 10% ethanol (pH 5.0), applied voltage was 15 kV, and temperature was kept at 30°C and baseline‐separation was achieved within 18 min for the analytes. The proposed method would be very useful and have wide use to monitor the residual level of such pollutants when combined with high‐sensitive detector and an excellent sample preconcentration technique with high enrichment factor in the future.     

Sensitive amperometric detection for capillary electrophoresis of phenol carbamates with in‐line thermal hydrolysis strategy

A strategy on amperometric detection for CZE of phenol carbamates as model analytes with a facile in‐line thermal hydrolysis was presented, in which a thermal hydrolysis, subsequent CZE separation and final column‐end amperometric detection were accomplished in an intact capillary. Key parameters of hydrolysis dynamics of carbamates and electrochemical detection of the hydrolysates were studied, as well as electrophoretic conditions. Under the optimal conditions, the capillary was utilized as chambers for in situ hydrolysis, CZE separation, and electrochemical detection. The successive separation of hydrolysates of five carbamates (propoxur, carbofuran, 3‐OH‐carbofuran, carbaryl and bendiocarb) were achieved within 17 min. Applied to vegetable samples, the recoveries of carbamates fortified at 0.02 and 0.05 mg/kg were ranging in 88–107.2 and 86.3–107.3%, respectively. The success in the implementation of such a scheme resulted in a simple instrument as compared with those current analytical methods with post‐column derivization or pre‐column hydrolysis, or online enrichment in chip, respectively. This protocol might possess a potential utility for the sensitive amperometric detection of phenol carbamates.     

Coupling porous sheathless interface MS with transient‐ITP in neutral capillaries for improved sensitivity in glycopeptide analysis

IgG antibodies are modulated in their function by the specific structure of the N‐glycans attached to their Fc (fragment crystallizable) portions. However, the glycosylation analysis of antigen‐specific IgGs is a challenging task as antibody levels to a given antigen only represent a fraction of the total IgG levels. Here, we investigated the use of a transient‐ITP (t‐ITP)—MS method for highly sensitive IgG1 glycosylation profiling as a complementary method to a high‐throughput nano‐RPLC‐MS method. It was found that t‐ITP‐CZE using neutrally coated separation capillaries with a large volume injection (37% of capillary volume) and interfaced to MS with a sheathless porous sprayer yielded a 40‐fold increase in sensitivity for IgG1 Fc glycopeptide analysis when compared to the conventional strategy. Furthermore, the glycoform profiles found with the t‐ITP‐CZE strategy were comparable to those from nano‐RPLC‐MS. In conclusion, the use of the highly sensitive t‐ITP‐CZE‐MS method will provide information on IgG Fc glycosylation for those samples with IgG1 concentrations below the LODs of the conventional method.     

Potential of Capillary Electrophoresis for the Profiling of Propolis

The usefulness of capillary electrophoresis (CE) with diode array detection for the profiling of Propolis, a hive product, is investigated. Water extracts of Propolis were analyzed with both capillary zone electrophoresis (CZE) at pH 7.0 and 9.3, and micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulfate at pH 9.3. Characteristic profiles were obtained and several organic acids and preservatives could be identified by means of library comparison of the recorded UV spectra combined with addition of reference compounds to the extracts. The selectivity of the CZE and MEKC system differed considerably but the information obtained with both methods was similar. The dry residues of the water extraction were extracted with ethanol-water (70 : 30, v/v) and analyzed with the MEKC system to enable the separation of the more hydrophobic constituents of the Propolis samples. Complex profiles containing various well separated peaks were obtained allowing the identification of some interesting flavonoids. On the basis of the recorded CZE and MEKC profiles, the Propolis samples could be divided into two clearly different groups which are probably from a different origin.     

Derivational,Structural, and Biological Studies of Some New Pyrazolyl,Isoxazolyl, Pyrimidinyl,Pyridazinyl, and Pyridopyridazinyl from 4‐Substituted Antipyrine

(1,5‐Dimethyl‐3‐oxo‐2‐phenyl‐2,3‐dihydro‐1H‐pyrazol‐4‐yl)carbono‐hydrazonoyl dicyanide was used as a key intermediate for the synthesis of novel pyrazole, isoxazole, pyrimidine, and pyridazine derivatives. The newly synthesized compounds were characterized by elemental analyses and spectral data (IR, ¹H‐NMR, ¹³C‐NMR, and mass spectra). The compounds were tested for their in vitro antibacterial activity against Gram‐positive bacteria as (Staphylococcus aureus and Bacillus subtilis ) and Gram‐negative bacteria (Pseudomonas aeruginosa and Escherichia coli ). The investigated compounds were tested against two strains of fungi Botrytis fabae and Fusarium oxysporum using diffusion agar technique. The biological results showed clearly that most of the synthesized compounds revealed mild to moderate activity against the used microorganisms.