Investigating the effects of conductivity on zone overlap with EMMA: Computer simulation and experiment

Stahl, J.; Catherman, Adam D.; Sampath, Ranasinghe K.; Seneviratne, Chinthaka A.; Strein, Timothy G.

doi:10.1002/elps.201000451

Cited by 14 publications

(12 citation statements)

References 13 publications

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“…Consequently, a rapid migration of the ionic FP‐488, across this zone, results in a decrease in reaction efficiency. This behavior was also reported by Stahl et al . They suggested that low sample conductivity can be detrimental for on‐line reactions involving a neutral reactant.…”

Section: Resultssupporting

confidence: 80%

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

et al. 2013

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Electrophoretically mediated microanalysis was implemented for on-line fluorescence derivatization of ubiquitin (UBI), a potential biomarker of Alzheimer disease. Ubiquitin was labeled on its amino groups by the Fluoprobes® 488 N-hydroxysuccinimide. The pH of the BGE, the applied electric field, and the washing process of the capillary were optimized. The best derivatization yield was obtained at pH 9.5 under an electric field of ∼210 V/cm. The concentration and volume of the fluorescent dye as well as the sample medium and volume of injected UBI were also optimized. In order to improve further the LOD of UBI, electrophoretically mediated microanalysis was performed in a wider (100 μm id) and longer (110 cm) capillary. As expected, these capillary dimensions improved the analytical sensitivity of UBI when compared to the 50 μm id capillary. Under the optimized conditions, this analytical methodology allowed the analysis of free UBI in standard solution with a LOD of ∼15 nM. Finally, the on-line fluorescent derivatization of UBI was applied to the detection and the quantification of UBI in cerebrospinal fluid (CSF) samples. Due to the high salt concentration of biological samples, desalting of CSF was required prior to the CE analysis. Quantification of UBI in CSF either by a direct evaluation of peak areas or using the standard addition method was achieved.

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Section: Resultssupporting

confidence: 80%

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

et al. 2013

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“…Moreover, in the optimal conditions, the apparent velocities ( v ) of the different reactants were calculated as the effective length of detection (21.5 cm) over the corresponding analyte migration time. This can be tricky in this study because of the complexity of the injection sequence and the possible disparity of the conductivity throughout the capillary . In order to minimize the discontinuity of the electric field inside the capillary, the Tris/phosphoric acid/MgCl 2 buffer (pH 7.5 and ionic strength 20 mM) was used for electrophoretic separation of the reactants as well as for the incubation process.…”

Section: Resultsmentioning

confidence: 99%

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

Nehmé

Lafite

et al. 2013

J of Separation Science

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CE was used for the first time to study the two-substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine-5'-triphosphate were in-capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine-5'-triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero-potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine-5'-diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose-1,6-bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in-capillary mixed using an electrophoretically mediated microanalysis approach.

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“…anionic or cationic, does not matter but has to be taken into account when designing the injection sequence. It has been shown by experiments as well as computer simulation that sample zone conductivity can affect plug-plug mixing in EMMA [90]. Typically, the local electric fields in the reactant zones are significantly different from the average electric field along the entire capillary.…”

Section: In-capillary Enzyme Assaysmentioning

confidence: 98%

Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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IntroductionEnzymes catalyze metabolic reactions in cells regulating homeostasis, growth and reproduction of living organisms. Enzymes are also often involved in human disease. The analysis of the human genome has revealed that within the so-called druggable genome a significant portion (in some analyses more than half) of the potential drug targets are enzymes [1][2][3]. Consequently, enzymes are important targets in the development of new drugs. Moreover, enzymes play a role in the clinical diagnosis of diseases. Therefore, effective methods for characterization of enzymes as well as for the determination of their activity are important, for example, for the understanding of enzyme-mediated metabolic reactions or the identification of substrates and inhibitors for the treatment of human diseases.Capillary electrophoresis (CE) has been applied to enzyme analysis for more than 20 years since the first report by Banke et al. on an assay of alkaline protease [4]. Since then, all aspects of enzyme-related analysis have been studied by CE methods including the evaluation of enzymatic activity, enzyme kinetics, identification and characterization of enzyme inhibitors and activators as well as metabolic pathways as, for example, summarized in [5][6][7][8][9][10][11][12].Generally, CE-based enzyme assays can be divided in three groups, pre-capillary (offline) assays, in-capillary (online) assays and post-capillary assays. In the pre-capillary assays, all required components including enzyme, substrate, co-factors, etc., are mixed in a vial and incubated for an intended period of time. Subsequently, the reaction is quenched and an aliquot is subjected to CE analysis for the determination of substrate(s) and/or co-substrate(s) and/ or product(s). Multiple sampling or repeated sampling in combination with sequential runs for the determination of Abstract Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis-Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CEbased enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic a...

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Investigating the effects of conductivity on zone overlap with EMMA: Computer simulation and experiment

Cited by 14 publications

References 13 publications

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

On‐line capillary electrophoresis derivatization method for high sensitivity analysis of ubiquitin in filtered cerebrospinal fluid

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

Advances in Capillary Electrophoresis-Based Enzyme Assays

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