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• A capillary electrophoresis method was developed for the rapid detection of E. coli. • The method based on online sample enrichment and offline signal amplification. • This method had a low detection limit of 3 CFU·mL−1 for E. coli. • The method was applied to bacterial detection in food samples. Efficient, accurate and economical detection of pathogenic bacteria is crucial in ensuring food safety and preventing foodborne illnesses. In this study, a capillary electrophoresis coupled laser-induced fluorescence assay (CE-LIF) was developed for the detection of Escherichia coli (E. coli) by detecting its specific DNA. The CE-LIF was assisted by both online enrichment and offline amplification to improve the detection sensitivity of bacterial DNA. Here the online amplification was performed by large volume sample stacking (LVSS), while the offline amplification was nicking endonuclease signal amplification (NESA). Under the optimal experimental conditions, the detection limit of bacterial target DNA was 2.5 fM, and the conversion concentration of E. coli was 3 CFU · mL−1. The method had been applied to the detection of commercially available skim milk samples with good results, which proved that it could be used as an effective tool for food and environmental bacteria monitoring. [ABSTRACT FROM AUTHOR]

• Fluorescence sensitization of flavonoids by forming flavonoids-tetraborate complexes. • Integrated strategy of derivatization & separation for CE-LIF analysis of flavonoids. • Non-destructive distinction of single seed for alfalfa and sweet clover via PCA. • Monitoring the substance metabolism at the level of single seed. Flavonoids are a class of natural polyphenolic compounds with great health benefits, and the development of methods for their analysis is of continuing interest. In this work, apigenin, kaempferol and formononetin were selected as the typical representatives of flavone, flavonol and isoflavone, three subclasses of flavonoids. Fluorescence studies revealed that tetraborate complexation could significantly sensitize the weak intrinsic fluorescence of flavonoids in solution, with a maximum of 137-fold for kaempferol. Subsequently, an integrated strategy of derivatization and separation was proposed for the universal analysis of flavonoids by capillary electrophoresis (CE) with 405 nm laser-induced fluorescence (LIF) detection. Using a running buffer consisting of 20 mM sodium tetraborate, 10 mM SDS and 10% methanol (pH 8.5), the dynamic derivatization was realized in the capillary, and the baseline separation was achieved within 10 min, with the detection limits of 0.92–35.46 nM (S/N=3) for the total of 9 flavonoids. The developed CE-LIF method was employed to the quantitative analysis of some flavonoids in Medicago sativa (alfalfa) plants and granulated alfalfa with the recoveries of 80.55–94.25%. Combined with the principal component analysis, the developed method was successfully applied to the non-destructive distinction of single seed for alfalfa and Melilotus officinalis (sweet clover), two forage grass seeds with very similar apparent morphology. Furthermore, this method was used to continuously monitor the substance metabolism during the soaking process at the level of single seed. [ABSTRACT FROM AUTHOR]

An aptamer-based microchip capillary electrophoresis coupled with laser induced fluorescence (MCE-LIF) detection method for fast determination of Aflatoxin B 1 (AFB 1 ) and Ochratoxin A (OTA) was developed. Aptamers that are specific to these two mycotoxins were first hybridized with their aptamer complementary oligonucleotides. The double strand DNA that comes in contact with mycotoxin-containing environment would be unwound into separate aptamer-mycotoxin complex and aptamer complementary single strand. Different types of oligonucleotides can be separated in MCE and detected under the aid of fluorescent dye SYBR gold in LIF detection unit. Under the optimal conditions, on-chip aptamer-mycotoxin conjugates analysis was achieved within 3 min with extremely low LODs (0.026 ng/mL for AFB 1 and 0.021 ng/mL for OTA). Specificity study indicated that other major mycotoxins would not cross-react with these two aptamers, demonstrating the good selectivity of the proposed method. Quantification of trace AFB 1 and OTA in real food samples was carried out and satisfactory recoveries were obtained. It is demonstrated that this method is fast, facile and specific for Simultaneous determination of trace AFB 1 and OTA from foodstuffs. [ABSTRACT FROM AUTHOR]

Curcuminoids, the major bioactive constituents of traditional medicine known as turmeric, have exhibited extensive therapeutic benefits. Excited by violet-blue light, curcuminoids can emit native fluorescence, making them possible to be detected with high sensitivity and specificity by laser-induced native fluorescence (LINF). Here, a commercial 445 nm laser diode was used as an excitation source to construct a confocal laser-induced fluorescence (LIF) detector and then a complete capillary electrophoresis (CE) system coupled with LIF detection was established. With three major curcuminoids, curcumin, demethoxy curcumin (DMC) and bisdemethoxy curcumin (BDMC) as target analytes, a micellar electrokinetic chromatography (MEKC) method was proposed using mixed micelles consisting of Triton X-100 and SDS to sensitize the native fluorescence of curcuminoids and enhance their separation efficiency. Fluorescence spectra revealed that the mixed micelles induced fluorescence synergism could enhance the signals of three curcuminoids by 77-, 57-, and 47-fold for curcumin, DMC, and BDMC. After systematic investigation, the optimal separation buffer for curcuminoids was chosen as follows: 20 mM Triton X-100, 20 mM SDS, 30% (v/v) methanol in 10 mM borax solution at pH 10.0. Under these conditions, a baseline separation of three curcuminoids was achieved within 10 min and the detection limits were found to be 4.1, 2.6, and 0.4 ng/mL for curcumin, DMC, and BDMC, respectively. Furthermore, the developed MEKC-LINF method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of three curcuminoids in turmeric, medicinal turmeric liniment, curry seasoning, and human urine samples. [ABSTRACT FROM AUTHOR]

• Fluorescence sensitization of SAs by forming SAs-borax-HP-β-CD ternary complex. • Integrated strategy of derivatization and separation for CE-LIF analysis of SAs. • Determining SAs in ginsengs and identifying species from electropherograms via PCA. • Monitoring SAB consumption in intervened reoxygenation by H/R model of H9c2 cells. Salvianolic acids (SAs) are a class of natural active substances that have been widely used in clinical treatment and food adjuvant therapy. In this work, we found that SAs could form the ternary complex with borax and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), thereby sensitizing their weak intrinsic fluorescence to maximized 92-fold for salvianolic acid B (SAB). The formation of ternary complex was dynamic and could complete once mixed, and the fluorescence intensity remained stable within 3 h. On this basis, an integrated strategy of derivatization and separation was proposed for the sensitive analysis of SAs using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. After the systematic investigation, a solution consisting of 20 mM borax and 20 mM HP-β-CD (pH 8.5) was selected as the running buffer. By the direct injection of SAs, the dynamic derivatization was realized in the capillary, and the baseline separation was achieved within 6 min, with the detection limits of 1.2-21.2 nM for four SAs (S/N=3). Then, the developed CE-LIF method was successfully applied to the quantitative analysis of SAs in four traditional ginsengs, including Salvia miltiorrhiza, Codonopsis pilosula, Panax quinquefolius and Panax ginseng with the recoveries ranging from 95.2% to 110.7%. Except for four target SAs, a large number of unknown electrophoretic peaks had also been observed in four ginsengs, and then were utilized for the identification of ginseng species via principal component analysis. Furthermore, a hypoxia/reoxygenation model was constructed using Rat cardiomyocyte H9c2 cells, and subsequently, the developed method was applied to continuously monitor the consumption of SAB in cell culture medium after its intervention. [ABSTRACT FROM AUTHOR]

• Overview of nanoscale electrophoresis and electrochromatography of DNAs, RNAs, and proteins. • Fabrication of nano-columns in different substrate materials including glass, quartz, PDMS, and thermoplastics (PMMA, COP, COC, etc.). Fabrication techniques presented include photolithography, high energy beam methods (electron beam lithography and focused ion beam milling), and replication-based techniques (nanoimprinting and injection molding). • Unique nanoscale phenomena, such as electrical double layer overlap, transverse electromigration, electroviscous effects, viscoelectric effects, and dielectrophorestic trapping. • Detection modalities for nanoscale electrophoresis and electrochromatography including laser-induced fluorescence, thermal lens spectroscopy, and resistive pulse sensing. Over the last 30-years, microchip electrophoresis and its applications have expanded due to the benefits it offers. Nanochip electrophoresis, on the other hand, is viewed as an evolving area of electrophoresis because it offers some unique advantages not associated with microchip electrophoresis. These advantages arise from unique phenomena that occur in the nanometer domain not readily apparent in the microscale domain due to scale-dependent effects. Scale-dependent effects associated with nanochip electrophoresis includes high surface area-to-volume ratio, electrical double layer overlap generating parabolic flow even for electrokinetic pumping, concentration polarization, transverse electromigration, surface charge dominating flow, and surface roughness. Nanochip electrophoresis devices consist of channels with dimensions ranging from 1 to 1000 nm including classical (1–100 nm) and extended (100 nm – 1000 nm) nanoscale devices. In this review, we highlight scale-dependent phenomena associated with nanochip electrophoresis and the utilization of those phenomena to provide unique biomolecular separations that are not possible with microchip electrophoresis. We will also review the range of materials used for nanoscale separations and the implication of material choice for the top-down fabrication and operation of these devices. We will also provide application examples of nanochip electrophoresis for biomolecule separations with an emphasis on nano-electrophoresis (nEP) and nano-electrochromatography (nEC). [ABSTRACT FROM AUTHOR]

We report a novel method for sequential on-line C-terminal sequencing of peptides, which combines carboxypeptidase Y (CPY) digestion with on-line derivatization and optically gated capillary electrophoresis with laser-induced fluorescence detection (OGCE-LIF). Various factors that may affect the C-terminal sequencing were investigated and optimized. High repeatability of on-line derivatization and the sequential OGCE-LIF assay of amino acids (AAs) was achieved with relative standard deviation (RSD) (n = 20) less than 1.5% and 3.2% for migration time and peak height, respectively. A total of 13 AAs was efficiently separated in the present study, indicating that the method can be used for sequencing of peptides consisting of the 13 AAs studied. Using two synthesized N-terminally blocked peptides as test examples, we show that the present method can on-line monitor the released AAs with a temporal resolution of 50 s during the entire CPY digestion process. The rates of AA release as a function of digestion time were easily measured; thus, the AA sequence of the peptide was determined with just one OGCE assay. Our study indicates the present approach is an effective, reliable, and convenient method for rapid analysis of the C-terminal sequence of peptides, with potential application in peptide analysis and proteome research. [ABSTRACT FROM AUTHOR]

Flow-gated capillary electrophoresis (CE) coupled with microdialysis has become an important tool for in vivo bioanalytical measurements because it is capable of performing rapid and efficient separations of complex biological mixtures thus enabling high temporal resolution in chemical monitoring. However, the limit of detection (LOD) is often limited to a micro- or nano-molar range while many important target analytes have picomolar or sub-nanomolar levels in brain and other tissues. To enhance the capability of flow-gated CE for catecholamine detection, a novel and simple on-line sample preconcentration method was developed exclusively for fluorescent derivatives of catecholamines that were fluorogenically derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The effective preconcentration coupled with the sensitive laser-induced fluorescence (LIF) detection lowered the LOD down to 20 pM for norepinephrine (NE) and 50 pM for dopamine (DA) at 3-fold of S/N ratio, and the signal enhancement was estimated to be over 100-fold relative to normal injection when standard analytes were dissolved in artificial cerebrospinal fluid (aCSF). The basic focusing principle is novel since the sample plug contains borate while the background electrolyte (BGE) is void of borate. This strategy took advantage of the complexation between diols and borate, through which one negative charge was added to the complex entity. The sample derivatization mixture was electrokinetically injected into a capillary via the flow-gated injection, and then NE and DA derivatives were selectively focused to a narrow zone by the reversible complexation. Separation of NE and DA derivatives was executed by incoming surfactants of cholate and deoxycholate mixed in the front BGE plug. This on-line preconcentration method was finally applied to the detection of DA in rat cerebrospinal fluid (CSF) via microdialysis and on-line derivatization. It is anticipated that the method would be valuable for in vivo monitoring of DA and NE in various brain regions of live animals on flow-gated CE or microchip platforms. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis-laser induced fluorescence (CZE-LIF) and microscale thermophoresis (MST) were used for the first time to study the inhibition of human neutrophil elastase (HNE). We recently studied HNE kinetics ( K m and V max ) by developing an in-capillary CZE-LIF assay based on transverse diffusion of laminar flow profiles (TDLFP) for reactant mixing. In this work, the former assay was adapted to monitor HNE inhibition. Two natural well known HNE inhibitors from the triterpene family, ursolic acid and oleanolic acid, were tested to validate the developed assay. Since the solubility of pentacyclic triterpenes in aqueous media where the enzymatic reaction will take place is limited, the effect of DMSO and ethanol on HNE was studied using microscale thermophoresis (MST). An agglomeration of the enzyme was revealed when preparing the inhibitor in 5% (v/v) DMSO. This phenomenon did not occur in the presence of ethanol. Therefore, ethanol was used as inhibitor solvent, at a limited percentage of 20% (v/v). In these conditions and after optimization of the TDLFP approach, the repeatability (RSD on migration times and peak-areas inferior to 2.2%) of the CZE-LIF assay and the sensitivity (LOQ of few nM) were found to be satisfactory for conducting inhibition assays. IC 50 values for ursolic and oleanolic acid were successfully determined. They were respectively equal to 5.62 ± 0.10 μM ( r 2 = 0.9807; n = 3) and to 8.21 ± 0.23 μM ( r 2 = 0.9887; n = 3). Excellent agreement was found between the results obtained by CE and those reported in literature which validates the developed method. Particularly, the CE-based assay is able to rank HNE inhibitors relative to each other. Furthermore, MST technique was used for evaluating HNE interaction with the ursolic acid. Up to 16 capillaries were automatically processed to obtain in one titration experiment the dissociation constant for the HNE-ursolic acid complex. K i was found to be 2.72 ± 0.66 μM ( n = 3) which is in excellent agreement with the value determined by CE enzyme inhibition studies ( K i = 2.81 μM) confirming the reliability of the developed CE assay and the competitive inhibition mode of ursolic acid. [ABSTRACT FROM AUTHOR]

In recent years, sphingolipidomics has emerged as an interesting omic science that encompasses the study of the full sphingolipidome characterization, content, structure and activity in cells, tissues or organisms. Like other omics, it has the potential to impact biomarker discovery, drug development and systems biology knowledge. Concretely, dietary food sphingolipids have gained considerable importance due to their extensively reported bioactivity. Because of the complexity of this lipid family and their diversity among foods, powerful analytical methodologies are needed for their study. The analytical tools developed in the past have been improved with the enormous advances made in recent years in mass spectrometry (MS) and chromatography, which allow the convenient and sensitive identification and quantitation of sphingolipid classes and form the basis of current sphingolipidomics methodologies. In addition, novel hyphenated nuclear magnetic resonance (NMR) strategies, new ionization strategies, and MS imaging are outlined as promising technologies to shape the future of sphingolipid analyses. This review traces the analytical methods of sphingolipidomics in food analysis concerning sample extraction, chromatographic separation, the identification and quantification of sphingolipids by MS and their structural elucidation by NMR. [ABSTRACT FROM AUTHOR]

Skin aging is a progressive process determining the ultimate skin appearance. Human neutrophil elastase (HNE) has been shown to play an important role in the degradation of the extracellular matrix. In order to assay HNE kinetics, a novel online capillary zone electrophoresis (CZE) assay has been developed in this study for the determination of the maximum velocity ( V max ) and of the Michaelis–Menten constant ( K m ) of HNE regarding several potential substrates. These assays are based on short-end injection to shorten analysis time, on transverse diffusion of laminar flow profiles (TDLFP) for in-capillary reactant mixing, and on UV or laser-induced fluorescence (LIF) detection. Kinetic constants for a referenced peptidic substrate were determined using not only online assays but also offline (pre-capillary) mode. The results obtained were cross compared and compared to the literature in order to validate the developed assays. The hydrolysis of three new potential fluorogenic substrates by HNE was also monitored. Two new peptidic substrates for HNE were identified through this study. K m values of these novel substrates were successfully determined using online CZE assay ( K m ∼0.07 mM). This value was in the same order of magnitude of that of the referenced substrate despite the presence of the labeling group 5-carboxyfluorescein (5-FAM). HNE activity has never been assessed using online CZE-based assay, neither with UV nor with LIF detection. The developed assay conducted with the new labeled substrates is particularly sensitive (LOQ of few nM), does not require the presence of micelles in the BGE (which is the case for the reference substrate) and only necessitates few nanoliters of reactants making it particularly adapted for screening studies. [ABSTRACT FROM AUTHOR]

Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5′- and 3′- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1 × 10 −19 mol in mass and 2.9 pM in concentration. [ABSTRACT FROM AUTHOR]

A composite 30-cm capillary was prepared. The head of the capillary was a 1.5-cm original and miniaturized aptamer-based monolithic affinity support that was in-line coupled to the end of the capillary used for capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The device was used for the preconcentration, separation and quantification of ochratoxin A (OTA) as a test solute. The 1.5-cm preconcentration unit consists of a fritless affinity monolithic bonded with 5′-SH-modified oligonucleotide aptamers. A vinyl spacer was used for thiol-ene photoclick chemistry with a 5 min irradiation at 365 nm. Photografting allowed to confine the binding reaction to the desired silica monolithic segment, upstream the empty section of the CE capillary using an UV mask. The photografting procedure was optimized preparing 10-cm capillary monoliths for nano-LC. The retention factors of cationic solutes in ion-exchange nano-LC allowed to follow the aptamer binding on the monolith. The reproducibility of the photografting process was satisfactory with inter-capillary variation lower than 10%. The aptamer bonding density can be increased by successive graftings of 100 μM aptamer concentration solution (5 pmol/cm/grafting). The optimal conditions to successfully perform the in-line coupling (preconcentration, elution and separation of OTA) with the composite capillary were adjusted depending on individual requirements of each step but also insuring compatibility. Under optimized conditions, OTA was successfully preconcentrated and quantified down to 0.1 pg (percolation of 2.65 μL of a 40 ng/L OTA solution). A quantitative recovery of OTA (93 ± 2%) was achieved in a single elution of 30 pg percolated OTA amount. The reproducibility of the overall process was satisfactory with a relative standard deviation lower than 10% with negligible non-specific adsorption. This device was applied for the preconcentration and analysis of OTA in beer and wine at the ppb level within a total analysis time of 30 min. [ABSTRACT FROM AUTHOR]

A new sensitive capillary electrophoretic method with laser-induced fluorescence (LIF) was developed for quantitation of glutathione (GSH) in biological samples. Eosin-5-maleimide was used to label the GSH molecule and the formed conjugate was separated in a 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid electrolyte at pH 7.0 in less than 3 min. The conjugate was detected with an in-house built LIF system, utilizing an inexpensive 515 nm diode laser module. Studies were performed to optimize the derivatization (the ratio of reagent to analyte, the reaction time, pH, etc.) and separation conditions. Sensitive detection of GSH at concentrations as low as 0.18 nM was obtained. The method was applied in the analysis of biological fluids (exhaled breath condensate, saliva) and was found to be suitable for determination of GSH in these samples at trace levels below 1 nM. To the best of our knowledge, this is the first report on determination of GSH in exhaled breath condensate by capillary electrophoresis (CE). [ABSTRACT FROM AUTHOR]

Highlights: [•] Development of CE with dual LIF detection method for the simultaneous detection of multiple miRNAs. [•] Determination and partial validation of three miRNAs in cells using CE with dual LIF detection system. [•] Simultaneous screening of three miRNAs in various lung cancer cell lines using CE with dual LIF detection system. [Copyright &y& Elsevier]

Highlights: [•] A new CZE/LIF method for analyzing proteins is demonstrated. [•] It is based on the bioconjugates of magnetic nanoparticles in CE. [•] C-reactive protein is used as a model analyte. [•] The process for optimizing the new method is described. [•] The quantitative analysis show good results. [Copyright &y& Elsevier]

Highlights: [•] Separation of amino acid enantiomers by a 532nm CAE-LIF scanner is presented. [•] CDs were studied and β-CD was effective for these enantiomers. [•] Additives were investigated and diamine was helpful for these enantiomers. [•] Optimum is 100mM Tris–borate buffer (pH 10), 2mM β-CD and 10mM hexamethylenediamine. [•] Determination of the enantiomeric excess of nonracemic alanine is demonstrated. [Copyright &y& Elsevier]

Abstract: With an increasing concern on food safety, fast screening of residues of widespread herbicides becomes necessary. Herein we report a microchip electrophoresis system with laser induced fluorescence (LIF) detection for rapid and sensitive analysis of glyphosate (GLYP) and glufosinate (GLUF) residues. Disposable cyclic olefin copolymer microchips and a low-cost LIF detector were employed to minimize the cost of the analysis. Systematic optimization of experimental conditions was performed to achieve highly efficient analysis. Under the selected condition, GLYP and GLUF were efficiently resolved from sample matrices with a buffer containing 10mmol/L borax and 2.0% (m/v) hydroxypropyl cellulose at pH 9.0. The number of theoretical plates of 1.0×106 m–1 was attained for both analytes. Derivatization at lower concentrations (<10μg/L) was also examined, successful detection of 0.34μg/L GLYP and 0.18μg/L GLUF was confirmed. The system was applied for the determination of both analytes in real samples without any preconcentration involved. Recoveries of GLYP and GLUF spiked in these samples were 84.0–101.0% and 90.0–103.0%, respectively. [Copyright &y& Elsevier]

Abstract: Over the past two decades, capillary electrophoresis (CE) has been the subject of extensive development and progress in various DNA based sieving electrophoresis applications, namely Sanger sequencing, forensic short tandem repeat (STR) analysis, clinical genotype screening (SNP), and phylogenetic fingerprinting. Yet, this trend has not been emulated in the RNA field. This review will highlight the development and key analysis parameters of RNA electrophoresis by CE and provide possible explanations for the low research interest in this area. [Copyright &y& Elsevier]

Abstract: Evidences accumulated over the past years that desmosines could be attractive indicators of elastic fibre degradation in Chronic Obstructive Pulmonary Disease have raised substantial interest with the development of reliable assays to measure their concentration in body fluids. It is a firm belief of researchers working in this field that accurate assessment of desmosine concentration would improve the understanding of elastin metabolism disorders and allow these cross-links to become a useful tool in the diagnosis and clinical management of these diseases. From among the variety of techniques available on the market, HPLC; CE and LC–MS have proved to be successful tools for measuring desmosines in biological fluids. However, differences in the analytical performance of methods may hinder the comparability of data, thus limiting the analytical strength and clinical utility of methods themselves. To address the relative contribution of different factors to the exact quantification of desmosines, the full potential of MEKC-LIF and LC–MS, the two systems that better than others offer more selective and sensitive detection for desmosine analysis, was studied on 56 urine samples. The results of this systematic comparative study underline the significant benefits of LC–MS over MEKC-LIF in terms of precision and sensitivity. Nevertheless, MEKC-LIF could be an attractive alternative in routine laboratories lacking the LC–MS instrumentation and skills to run these methods. [Copyright &y& Elsevier]

Abstract: Erythropoietin (EPO) is an important glycoprotein hormone. Recombinant human EPO (rhEPO) is an important therapeutic drug and can be also used as doping reagent in sports. The analysis of EPO glycoforms in pharmaceutical and sports areas greatly challenges analytical scientists from several aspects, among which sensitive detection and effective and facile sample preparation are two essential issues. Herein, we investigated new possibilities for these two aspects. Deep UV laser-induced fluorescence detection (deep UV-LIF) was established to detect the intrinsic fluorescence of EPO while an immuno-magnetic beads-based extraction (IMBE) was developed to specifically extract EPO glycoforms. Combined with capillary zone electrophoresis (CZE), CZE-deep UV-LIF allows high resolution glycoform profiling with improved sensitivity. The detection sensitivity was improved by one order of magnitude as compared with UV absorbance detection. An additional advantage is that the original glycoform distribution can be completely preserved because no fluorescent labeling is needed. By combining IMBE with CZE-deep UV-LIF, the overall detection sensitivity was 1.5×10−8 mol/L, which was enhanced by two orders of magnitude relative to conventional CZE with UV absorbance detection. It is applicable to the analysis of pharmaceutical preparations of EPO, but the sensitivity is insufficient for the anti-doping analysis of EPO in blood and urine. IMBE can be straightforward and effective approach for sample preparation. However, antibodies with high specificity were the key for application to urine samples because some urinary proteins can severely interfere the immuno-extraction. [Copyright &y& Elsevier]

Abstract: The methylation of the promoter region of DNA is an important regulatory mechanism for the downstream gene expression, and the extent of methylation has been linked to cancer formation. In this study, we report a simple method to screen for the degree of DNA methylation by combined bisulfite restriction analysis (COBRA) and capillary electrophoresis with laser-induced fluorescence (CE-LIF). After treating genomic DNA with sodium bisulfite, nested-PCR amplification and endonuclease (Taq I) digestion were performed. The digested DNA fragments were then separated by capillary electrophoresis using 1.5% poly(ethylene) oxide (M ave, 8,000,000g/mol) in the presence of electroosmotic flow. The improvement for DNA amplification using the nested PCR described here corresponded to approximately ten cells. In addition, the level of DNA methylation shown in the electropherograms obtained corresponded to the original percentage of DNA methylation from commercial available standard sample (0–100%). The electrophoretic patterns demonstrated that the six cancer cell lines tested displayed different degrees of DNA methylation and could be differentiated by hierarchical cluster analysis. Furthermore, the DNA methylation level was eliminated after treating the cells with an anti-cancer drug (5′-aza-2′-deoxycytidine). Together, these results suggest that CE-LIF is a potentially useful and cost-effective tool for cancer diagnosis or prognosis based on the heterogeneity in a patient''s DNA. [Copyright &y& Elsevier]

The adenosine triphosphate (ATP) binding-cassette (ABC) transporters are a superfamily of cellular proteins that have been partly implicated as a cause of multidrug resistance (MDR) in cancer cells. The ABC superfamily consists of P-glycoprotein, multidrug resistance-associated proteins (MRP) and breast cancer-related proteins, of which MRP is of particular interest because of its ability to efflux a broader range of substrates. Since MRP1 is the most prominent member of the MRP family, a simple technique is needed for its quantification. We developed a simple, fast (total analysis time of 3h) capillary electrophoresis immunoassay (CEIA) for the quantification of MRP1 in cancer cells. MRP1 antibody was labeled with fluorescein isothiocyanate. The labeled antibody was incubated with the cell lysate for a fixed interval (1h), after which the cell lysate mixture was directly injected into the capillary to separate the complex of MRP1 and its antibody from free antibody. The noncompetitive CEIA method had a limit of detection of 0.2nM and a good linear range (1.7–14.9×104 cells), and was fairly reproducible (RSD<10%). The results showed that two cell lines, A549 and RDES, expressed MRP1 in the absence of doxorubicin (DOX), with A549 registering a higher expression. Compared to DOX-free cancer cells, there was an acceleration of MRP1 expression during the 12h-exposure to DOX, after which the level of expression remained nearly constant as the intracellular accumulation of DOX decreased. The results obtained in this work indicate that the developed CEIA method is useful for relative quantification of MRPs in cancer cells. [Copyright &y& Elsevier]

Abstract: In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay''s linear range covers three orders of magnitude (1.0nM to 1.0pM) with a limit of detection (S/N=3) as low as 190fM (2.5zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs. [Copyright &y& Elsevier]

Abstract: Recombinant human erythropoietin-α (rHuEPO-α) has been widely used in clinic for anemia treatment. The detection and quantification of rHuEPO-α is essential for monitoring this widespread recombinant glycoprotein pharmaceutical. In this paper, we developed a new affinity probe capillary electrophoresis/laser-induced fluorescence (APCE/LIF) method for the detection of rHuEPO-α by using a specific single-stranded DNA aptamer probe for the first time. In this method, the complex of aptamer-rHuEPO-α and the free aptamer can be well separated and identified by their migration and fluorescence intensity after systematic optimization. The existence of sodium cation in the sample buffer and running buffer played a critical role for stabilizing complex and enhancing the separation efficiency, additionally, suitable high voltage and sample buffer additives were also important for improving the peak height of the complex. Under the optimized conditions, the method was successfully applied for the quantification of rHuEPO-α in physiological buffer, artificial urine and human serum. The linear range for rHuEPO-α was from 0.2 to 100nM and the limit of detection was 0.2nM (i.e. 7.4ng/mL). Further binding experiments using fluorescein isothiocyanate-labeled rHuEPO-α (F-rHuEPO-α) and N-deglycosylated F-rHuEPO-α demonstrated that the oligosaccharides moiety was of importance in the specific interaction between rHuEPO-α and its aptamer. [ABSTRACT FROM AUTHOR]

Abstract: In this work, a novel method combining constant pressure-assisted head-column field-amplified sample injection (PA-HC-FASI) with in-capillary derivatization was developed for enhancing the sensitivity of capillary electrophoresis. PA-HC-FASI uses an appropriate positive pressure to counterbalance the electroosmotic flow in the capillary column during electrokinetic injection, while taking advantage of the field amplification in the sample matrix and the water of the “head column”. Accordingly, the analytes were stacked at the stationary boundary between water and background electrolyte. After 600s PA-HC-FASI, 4-fluoro-7-nitro-2,1,3-benzoxadiazole as derivatization reagent was injected, followed by an electrokinetic step (5kV, 45s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 10min for derivatization reaction under 35°C, then the capillary temperature was cooled to 25°C and the derivatives were immediately separated and determined under 25°C. By investigating the variables of the presented approach in detail, on-line preconcentration, derivatization and separation could be automatically operated in one run and required no modification of current CE commercial instrument. Moreover, the sensitivity enhancement factor of 520 and 800 together with the detection limits of 16.32 and 6.34pg/mL was achieved for model compounds: glufosinate and aminomethylphosphonic acid, demonstrating the high detection sensitivity of the presented method. [Copyright &y& Elsevier]

Abstract: A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm×50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6mm at one end of both 50μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0μmol/L. The column efficiency was in the range from 1.0×105 to 2.5×105 plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves. [Copyright &y& Elsevier]

Abstract: A rapid method using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed to determine free and protein-bound glutathione (GSH) in human HepG2 hepatocarcinoma cells. The samples were derivatized with 5-iodoacetamidofluorescein (5-IAF), and analyzed at 22kV using sodium phosphate buffer (10mM, pH 11.4) and an uncoated 58cm×75μm I.D. fused silica capillary. The analysis time was less than 10min and N-acetylcysteine was used as internal standard. The derivatization conditions, such as reaction time, 5-IAF concentration, running buffer and cartridge temperature were optimized. Argon gas was used in the study to prevent the oxidization of GSH during sample preparation. The optimized method required only 30–40nl sample per analysis and was fast and sensitive. The method was applied to the analyses of HepG2 cells treated with the small metal chelating agent, pyrrolidine dithiocarbamate (PDTC). The results demonstrate that the amount of protein-bound GSH, which reflects the amount of protein S-glutathionylation, increased in a time-dependent manner upon cell treatment with PDTC, reaching a maximum of over 50% increase 2h post-PDTC. [Copyright &y& Elsevier]

Abstract: A screening test based on laser-induced fluorescence (LIF) and a method for individual identification – quantitation of aflatoxins (AFs) in olive leaves and drupes, based on chromatographic separation and triple-quad mass-spectrometry detection with electrospray ionization in positive mode, is here reported. The sensitivity and selectivity of both methods are enhanced by a preconcentration–cleanup step developed by a Prospekt station. The analysis frequency is at least 3.5 samples/h. The screening test makes able to detect the target analytes at concentrations of 0.7μg/kg without “false negatives”. The LC–MS/MS method provides limits of detection (LOD) and quantification (LOQ) ranging between 0.01–0.03 and 0.03–0.11μg/kg, respectively. The linear dynamic range is between LOQ–50μg/kg. The between-day precision, expressed as relative standard deviation, ranges between 0.97–2.86% and the within laboratory reproducibility, also expressed as RSD, between 1.63% and 4.84%. [Copyright &y& Elsevier]

Abstract: The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label α-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4×10−4 cm2 V−1 s−1. The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1% with the addition of each label. The signal increased linearly from 1.0nM to 100nM α-lactalbumin (r 2 =0.99), with a 3σ detection limit of 70pM. We then considered the separation of a mixture of ovalbumin, α-chymotrypsinogen A, and α-lactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank. [Copyright &y& Elsevier]

Abstract: The spectroscopic and electrophoretic properties of proteins labeled with Chromeo P503 were investigated. Its photobleaching characteristics were determined by continually infusing Chromeo P503-labeled α-lactalbumin into a sheath-flow cuvette and monitored fluorescence as a function of laser power. The labeled protein is relatively photo-labile with an optimum excitation power of about 2mW. The unreacted reagent is weakly fluorescent but present at much higher concentration than the labeled protein. The unreacted reagent undergoes photobleaching at a laser power more than an order of magnitude higher than the labeled protein. One-dimensional capillary electrophoresis analysis of Chromeo P503-labeled α-lactalbumin produced concentration detection limits (3σ) of 12pM and mass detection limits of 0.7zmol, but with modest theoretical plate counts of 17,000. The reagent was employed for the two-dimensional capillary electrophoresis analysis of a homogenate prepared from a Barrett''s esophagus cell line; the separation quality is similar to that produced by 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), a more commonly used reagent. [Copyright &y& Elsevier]

Abstract: This paper describes an automatic rapid approach for in-capillary derivatization of ephedrine (E) and pseudoephedrine (PE) and subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as fluorescent reagent. The unique feature of this method is the capillary being used as a small reaction chamber, in which the sample, derivatization buffer and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step (5kV, 15s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 1min for reaction, the derivatives were then immediately separated and determined. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated. Under these optimized conditions, a baseline separation of the two analytes was achieved within 10min and the derivatization concentration limits of detection were found to be 4.8ngmL−1 for E and 1.6ngmL−1 for PE, respectively. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of the two alkaloids in ephedra herb and its preparations. [Copyright &y& Elsevier]

Abstract: Bacterial counts provide important information during the processes such as pathogen detection and hygiene inspection and these processes are critical for public health and food/pharmaceutical production. In this study, we demonstrate the quantification of the number of bacterial cells based on the autofluorescence from the cell lysate on a microfluidic chip. We tested three model pathogenic bacteria (Listeria monocytogenes F4244, Salmonella Enteritidis PT1 and Escherichia coli O157:H7 EDL 933). In the experiment, a plug of ∼150pL containing lysate from 240 to 4100 cells was injected into a microfluidic channel with downstream laser-induced fluorescence detection under electrophoresis conditions. We found that the autofluorescence intensity increased with the number of cells almost linearly for all three bacteria. The autofluorescence remained a single peak when the cell lysate contained a mixture of different bacterial species. We also demonstrate a simple microfluidic device that integrates entrapment and electrical lysis of bacterial cells with fluorescence detection. Such a device can carry out the quantification of bacterial cells based on lysate autofluorescence without off-chip procedures. This study offers a simple and fast solution to on-chip quantification of bacterial cells without labeling. We believe that the method can be extended to other bacterial species. [Copyright &y& Elsevier]

Abstract: A new highly sensitive and compact 224nm laser-induced native fluorescence (LINF) detector was developed using a new generation of deep-UV laser and an innovating elliptical flow cell. The use of deep-UV excitation at 224nm allows to achieve fluorescence detection of an important range of molecules containing a single aromatic ring. The LINF detector was first evaluated in liquid chromatography. An improvement of a factor 500 over a conventional fluorimeter is reached with a limit of detection (LOD) of 1.5pmole for ibuprofen. LODs were in the nanomole range for phenylalanine and in the picomole range for tyrosine and tryptophan. The LINF detector is able to detect the same levels of peptides concentrations as an ESI-ion trap spectrometer used in scan mode. In this application, LINF outperforms the UV detection at 214 or 254nm and could be used with different additives with no noticeable effect on the detection. [Copyright &y& Elsevier]

Abstract: The application of quantum dots in capillary electrophoresis immunoassay was studied for the first time. Quantum dots were conjugated with antibody and subsequently tested by electrophoretic separation of free antibody and antibody–antigen complex. Antibody was fluorescently labeled by quantum dots via conjugation procedures and its electrophoretic characteristics were effectively modified due to the attachment of quantum dots. The determination of human IgM by direct CE based immunoassay could be easily achieved by simply changing the pH value of separation buffer. Polymer additive influenced the separation too but the effect was not as significant as buffer pH adjustment. Satisfactory separation of complex from free antibody could be achieved with 20mM sodium tetraborate as separation buffer, at pH 9.8. The immunoassay application of quantum dots in CE offers considerable advantages and can be readily applied to other large bio-molecules. [Copyright &y& Elsevier]

Abstract: We developed a rapid and sensitive method using in-capillary derivatization and laser-induced fluorescence (LIF) detection for the fully automated analysis of organophosphorus pesticides (OPPs), including glufosinate, aminomethylphosphonic acid (AMPA) and glyphosate by micellar electrokinetic chromatography (MEKC). The potential of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as in-capillary derivatization reagent is described for the first time. The unique feature of this MEKC method is the capillary being used as a small reaction chamber. In in-capillary derivatization, the sample and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step to enhance the mixing efficiency of analytes and reagent plugs in accordance with their different electrophoretic mobilities. Standing a specified time for reaction, the derivatives were then immediately separated and determined. Careful optimization of the derivatization and separation conditions allowed the determination of glufosinate, AMPA and glyphosate with detection limits of 2.8, 3.6 and 32.2ng/mL, respectively. These detection limits were comparable to those of 1.4, 1.9 and 23.8ng/mL obtained from conventional pre-capillary derivatization. Furthermore, repeatability better than 0.40% for migration time and 3.4% for peak area, as well as shorter migration time, was obtained. The method was successfully applied to the analysis of spiked river water sample with satisfactory results. [Copyright &y& Elsevier]

Abstract: Capillary isoelectric focusing (cIEF) can be a useful tool for the characterization and identification of microbes. Based on the whole column imaging detection (WCID) technique and using plant growth-promoting rhizobacteria (PGPR) as test microbes, we present a two-level cIEF characterization method for the characterization and identification of bacteria. Intact bacteria were first characterized according to their apparent isoelectric points measured by cIEF-WCID and then lysed bacteria were further characterized by cIEF profiling of the intracellular proteins. Cellular clustering was found to be the main experimental barrier for the characterization of intact bacteria. The addition of sodium chloride (100mM) to the sample mixture was found to be an effective way to reduce clustering. Due to the high efficiency and high resolution of cIEF-WCID, characterization of bacteria according to their intracellular proteins can be implemented simply and quickly without optimization of the experimental conditions. To improve the detection sensitivity with laser induced fluorescence (LIF)-WCID, the possibility to label bacteria with a non-covalent fluorescent dye, NanoOrange, was explored. [Copyright &y& Elsevier]

Abstract: Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with ≥1.5×106 molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5×106 MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5×106 MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles. [Copyright &y& Elsevier]

Abstract: The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by capillary electrophoresis in conjunction with laser-induced fluorescence (CE–LIF) detection using poly(ethylene oxide) (PEO) solution is described. The use of CTAB for improved separation reproducibility and efficiency of DNA has not been demonstrated although it is widely used for controlling the magnitude and direction of electroosmotic flow in CE. With increasing CTAB concentration, the interactions of DNA with ethidium bromide (EtBr) and with the capillary wall decrease. For the separation of DNA fragments with the sizes ranging from several base pairs (bp) to 2176bp, a polymer solution consisting of 0.75% poly(ethylene oxide), 100mM TB buffer (pH 8.0), 25μg/mL EtBr, and 0.36μg/mL CTAB is proper. Using the PEO solution, we separated a mixture of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BglI digest and pBR 328/HinfI digest) within 8min at −375V/cm, with the limit of detection of 2.0ng/mL based on the peak height for the 18-bp DNA fragment. The method is highly efficient (>106 plate/m), repeatable (RSD of the migration times <1.5%), and sensitive. In addition, it is convenient to fill a capillary (75μm in diameter) with such a low-viscosity PEO solution by syringe pushing. [Copyright &y& Elsevier]

Abstract: In this paper, we present a universal, highly efficient and sensitive method for the characterization of quantum dot (QD) bioconjugates based on capillary electrophoresis with laser-induced fluorescent (LIF) detection. We first prepared CdTe QDs in aqueous phase by a chemical route with mercaptopropionic acid as a ligand, and then were coupled to certain proteins using bifunctional linkage reagent or electrostatic attraction. The QD bioconjugates were characterized by capillary electrophoresis with LIF detection. We found that QD bioconjugates were efficiently separated with free QDs by the optimization of buffer pH. Furthermore, we found that ultrafiltration was an effective and simple approach to purify QD conjugates with bovine serum albumin (BSA). Due to their broad absorption spectra and size dependent emission wavelength tunability, QDs can be excited to emit different colour fluorescence using a single wavelength laser source, and therefore, we believe that CE with LIF detection will become a universal and efficient tool for the characterization of QD bioconjugates. [Copyright &y& Elsevier]

Abstract: Highly selective and sensitive assays are required for detection and quantitation of the small masses of DNA typically encountered in clinical and forensic settings. High detection sensitivity is achieved using fluorescent labeling dyes and detection techniques such as spectrofluorometers, microplate readers and cytometers. This work describes the use of a laser-induced fluorescence (LIF) detector in conjunction with a commercial capillary electrophoresis instrument for DNA quantitation. PicoGreen and YO-PRO-1, two fluorescent DNA labeling dyes, were used to assess the potential of the system for routine DNA analysis. Linearity, reproducibility, sensitivity, limits of detection and quantitation, and sample stability were examined for the two assays. The LIF detector response was found to be linear (R 2 >0.999) and reproducible (RSD<9%) in both cases. The PicoGreen assay displayed lower limits of detection and quantitation (20pg and 60pg, respectively) than the YO-PRO-1 assay (60pg and 260pg, respectively). Although a small variation in fluorescence was observed for the DNA/dye complexes over time, quantitation was not significantly affected and the solutions were found to be relatively stable for 80min. The advantages of the technique include a 4- to 40-fold reduction in the volume of sample required compared to traditional assays, a 2- to 20-fold reduction in the volume of reagents consumed, fast and automated analysis, and low cost (no specific instrumentation required). [Copyright &y& Elsevier]

Abstract: Erythrocytes were fused with liposome for intracellular derivatization of amino acids in cells. The fusion efficiency was evaluated with capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection. Reagent fluorescein isothiocyanate (FITC) was enveloped in liposomes and introduced into erythrocytes by fusion between liposomes and erythrocytes. The amino acids in the fused cells were derivated by the introduced FITC and the derivated amino acids were extracted for detection by capillary electrophoresis equipped with laser-induced fluorescence detector. The fusion conditions were investigated. It was found that incubation of liposome and erythrocytes in the presence of 13% polyethylene glycol 6000 (PEG 6000) for 15min produced the highest fusion efficiency and kept the erythrocytes stability. [Copyright &y& Elsevier]

Abstract: It was reported that a novel detection method, continuous wave (CW)-based multiphoton excitation (MPE) fluorescence detection with diode laser (DL), has been firstly proposed for capillary electrophoresis (CE). Special design of end-column detection configuration proved to be superior to on-column type, considering the detection sensitivity. Three different kinds of fluorescent tags that were widely used as molecular label in bio-analysis, such as small-molecule dye, fluorescent protein and nano particle or also referred to as quantum dot (QD), have been evaluated as samples for the constructed detection scheme. Quantitative analyses were also performed using rhodamine species as tests, which revealed dynamic linear range over two orders of magnitude, with detection limit down to zeptomole-level. Simultaneous detection of fluorescent dyestuffs with divergent excitation and emission wavelengths in a broad range showed advantage of this scheme over conventional laser-induced fluorescence (LIF) detection. Further investigations on CW-MPE fluorescence detection with diode laser for capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations of fluorescein isothiocyanate (FITC) labeled amino acids indicated good prospect of this detection approach in various micro or nano-column liquid phase separation technologies. [Copyright &y& Elsevier]

Abstract: A two-laser, two-color detector has been developed for the simultaneous detection of naturally occurring and recombinant (internal standards) cytokines within the same biological sample. The internal standards were labeled with Bimane and detected with a 408nm laser while the natural cytokines were labeled with AlexaFluor633 and detected with a 633nm laser. The two resulting electropherograms were plotted as overlaid traces and quantification of the natural materials determined by comparison with the standards. Using this system, recovery of all four cytokine standards was greater than 94% in both saline and cytokine-depleted plasma. These recoveries could be achieved with intra- and inter-assay coefficients of variance (c.v.) of less than 4.5 and 5.6, respectively. Application of this system to the examination of clinical samples demonstrated that measurement of the four pro-inflammatory cytokines could distinguish between normals, sub-clinical and clinical inflammation. An advantage of this approach is that direct calculation of unknowns by comparison to identical internal standards can shorten analytical time by eliminating the need for additional standard or calibration runs. [Copyright &y& Elsevier]

Abstract: We have demonstrated the analysis of aristolochic acids (AAs) that are naturally occurring nephrotoxin and carcinogen by capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs (OAAs), reduction of the analytes by iron powder in 10.0mM HCl is required prior to CE analysis. The reduced AAs (RAAs) exhibit fluorescence at 477nm when excited at 405nm using a solid-state blue laser. By using 50.0mM sodium tetraborate (pH 9.0) containing 10.0mM SDS, the determination of AA-I and AA-II by CE-LIF has been achieved within 12min. The CE-LIF provides the LODs of 8.2 and 5.4nM for AA-I and AA-II, respectively. The simple CE-LIF method has been validated by the analysis of 61 Chinese herbal samples. Prior to CE analysis, OAAs were extracted by using 5.0mL MeOH, and then the extracts were subjected to centrifugation at 3000rpm for 5min. After reduction, extraction, and centrifugation, the supernatants were collected and subjected to CE analysis. Of the 61 samples, 14 samples contain AA-I and AA-II, as well as 10 samples contain either AAI or AAII. The relative standard deviation (RSD) values of the migration times for AA-I and AA-II are less than 2.5% and 2.1% for three consecutive measurements of each sample. The RSD values for the peak heights corresponding to AA-I and AA-II in most samples are about 8.0% and 10.0%, respectively. The result shows that the present CE-LIF approach is sensitive, simple, efficient, and accurate for the determination of AAs in real samples. [Copyright &y& Elsevier]

Abstract: This paper describes a simple, sensitive, efficient, and rapid method for simultaneous analysis of biologically active amines and acids by capillary electrophoresis in conjunction with laser-induced native fluorescence detection (CE-LINF) using a diode pumped solid state nanolaser at 266nm. In order to optimize resolution of the amines that were prepared in 10.0mM formate-Tris (FT) solutions, 10.0mM FT solutions with and without containing halides were used to fill the capillary and reservoirs, respectively. The electrophoretic mobilities of tryptamine (TA) and serotonin (5-HT) at pH 4.0 decrease with the increase in halide concentration (0–10.0mM). Taken together with a great effect of iodide than other halides, we suggest that the formation of ion pairs is a main contributor for altering the migration of the amines. In order to simultaneously analyze the amines and their metabolites (acids) at low pH, a high bulk EOF is required. The analysis of 10 anlytes including amines and acids was completed within 12min by CE-LINF using a capillary treated with 0.5M NaOH and then filled with 10.0mM FT solutions (pH 4.0) containing 10.0mM KCl prior to analysis. The limits of detection for TA and 5-hydroxyindole-3-acetic acid (5-HIAA) are 0.12 and 6.0nM, respectively. The present method has been further validated by analyzing urine samples, with an RSD less than 3.1% (migration times) and 3.9% (concentration). [Copyright &y& Elsevier]

Effective removing of Joule heat in polymer-based microchip system is an important factor for high efficient separation because of lower heat conductivity of polymers than silica or glass. In this paper, a new kind of polydimethylsiloxane (PDMS) microchip electrophoresis system integrated with a laser-induced fluorescence detector has been successfully constructed on the basis of a commercial heat sink for computer CPU (central processor unit). Experimental results on separation current using high concentration running buffers demonstrated that heat dissipation of PDMS/PDMS microchip system was significantly improved. Furthermore, with this integrated system, theoretical plate number of fluorescein using 100mM phosphate-buffered saline + 1mM sodium dodecyl sulfate as running buffer was determined to be 2750 (for 2.5-cm separation channel, corresponding to 110,000/m). This high separation efficiency demonstrated that such heat sink-based polymer microchip system could be effectively applied for high-concentration buffers. [Copyright &y& Elsevier]

• High performance thin layer chromatography by picosecond laser induced fluorescence. • Densitometry method based on steady-state and time-resolved fluorescence detection. • Validation of the developed method and application on thioridazine photoproducts. • Laser-induced fluorescence chromatograms. • Fluorescence spectra and fluorescence lifetime of photoproducts. A densitometry method based on steady-state and time-resolved fluorescence assessments for thioridazine and its photoproducts applied on HPTLC plates has been developed. The excitation source was a picosecond diode laser emitting at 375 nm. This method was used for the analysis of the photoproducts resulted from thioridazine irradiation with 266 nm nanosecond-pulsed laser. The validation of the developed method was performed for thioridazine in terms of linearity, precision, limits of detection and quantification. Furthermore, analysis of the photoproducts of irradiated thioridazine was performed by steady-state and time-resolved fluorescence. The fluorescence spectra and fluorescence lifetime of each photoproduct were obtained and the horizontal chromatograms of fluorescence maxima were generated. [ABSTRACT FROM AUTHOR]