Advances in Capillary Electrophoretically Mediated Microanalysis for On‐line Enzymatic and Derivatization Reactions

Huang, Shengyun; Paul, Prasanta; Ramana, Pranov; Adams, Erwin; Augustijns, Patrick; Schepdael, Ann Van

doi:10.1002/elps.201700262

Cited by 38 publications

(19 citation statements)

References 87 publications

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“…In‐capillary assays require efficient mixing of substrate and enzyme solutions, which are injected as plugs. This can be achieved by longitudinal diffusion (at inlet reaction), by transverse diffusion of laminar flow profiles or by electrophoretic mixing (electrophoretically mediated microanalysis) as summarized, for example, in . In the present study, longitudinal as well as electrophoretic mixing was investigated.…”

Section: Resultsmentioning

confidence: 99%

“…In the case of β‐lactamases, various formats have been employed including photometric, biochemical, mass spectrometric or polymerase chain reaction‐based and other sequencing assays as summarized, for example, in . Due to the high efficiency and minute sample requirements, CE has been shown to be a powerful microanalytical technique for the determination of the activity of enzymes including the characterization of substrates and inhibitors . With regard to penicillinases, CE–MS has been used to identify β‐lactamase‐derived proteins .…”

Section: Introductionmentioning

confidence: 99%

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Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

et al. 2019

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Generic in‐capillary as well as offline CE‐based enzyme assays were developed for serine‐β‐lactamases and metallo‐β‐lactamases. The hydrolysis of benzylpenicillin to benzylpenicilloic acid was analyzed using 100 mM sodium phosphate solution, pH 6.0, as a background electrolyte. In‐capillary assays employed an uncoated as well as a polyethylene oxide‐coated capillary, while the offline assays employing long end and short end injection were performed in an uncoated capillary. Using procaine hydrochloride or 4‐hydroxybenzoic acid as internal standard, the respective assays were validated with regard to linearity, LOD and LOQ, repeatability, precision, and accuracy. The assays were applied to the determination of the Michaelis‐Menten parameters Km and Vmax of Bacillus cereus penicillinase as well as New Delhi metallo‐β‐lactamase 1 and Verona integrin‐encoded metallo‐β‐lactamase 2. Furthermore, the inhibition of the enzymes by irreversible and competitive inhibitors was evaluated. Comparable data were obtained with all assays. The use of a simple substrate ensured broad applicability to the various types of β‐lactamases.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

et al. 2019

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“…Online enzymatic CE assays comprise automatable analytical systems that consume only minute amounts of sample and reagents and are used, for example, within pharmacological chemistry to assess the enzyme activity and drug metabolism . The steps in a typical online assay are as follows: (1) consecutive hydrodynamic injection and mixing of enzyme and reactant solution plugs, (2) incubation of the reaction mixture, (3) voltage application and separation, and (4) detection of the reaction products.…”

Section: Input Parameters For Simulationsmentioning

confidence: 99%

Computer simulation of the enantioselective separation of weak bases in an online capillary electrophoresis based microanalysis configuration comprising sulfated cyclodextrin as selector

Mikkonen

Thormann

2018

Electrophoresis

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Computer simulation was utilized to characterize the electrophoretic processes occurring after reactant mixing in an online assay format used for monitoring the enantioselective N-demethylation of ketamine to norketamine in the presence of highly sulfated γ-cyclodextrin (HS-γ-CD). The incubated reaction mixture (at pH 7.4 and without chiral selector) is bracketed by a low pH BGE containing 2% HS-γ-CD as chiral selector, thereby forming a discontinuous buffer system. Upon power application, simulation provides insight into the formation of moving boundaries and new zones together with the prediction of the behavior of ketamine and norketamine enantiomers. The analytes first migrate cationically in a zone electrophoretic manner until they come in contact with HS-γ-CD upon which enantioseparation is initiated. Complexation has a focusing effect and the electrophoretic transport becomes reversed, that is, toward the anode. Simulation revealed that the initial conditions for the chiral separation, including buffer components concentrations, pH, and ionic strength, are different than those in the BGE. As a consequence thereof, the experimentally determined complexation parameters for the BGE were unable to correctly describe the migration behavior of the analytes in this column section. An increase in the input binding constants by a factor of two to four, as a result of the decreased ionic strength, resulted in simulation data that agreed with experimental findings.

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“…Capillary electrophoresis (CE) has become a routine analytical technique for analysis of both small and large analytes, owing to its several advantages like good separation efficiency, high sensitivity, diverse detection modes, fast analysis and extreme small sample consumption . It has also been extensively employed in enzymatic studies including enzyme assays, enzyme kinetics and enzyme inhibition . Another advantage of CE based assays is their suitability for compounds containing chromophores or phosphates that otherwise cannot be tested by spectrophotometric methods .…”

Section: Introductionmentioning

confidence: 99%

Development of a fast CE method for high throughput screening of ecto‐5′‐nucleotidase inhibitors

2018

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Overexpression of ecto-5'-nucleotidase is found to be linked to cancer progression and other diseases which makes screening of its inhibitors on demand. Unfortunately, there is scarcity of reported ecto-5'-nucleotidase inhibitors because of unavailability of simple, fast and efficient methods to study its inhibition. In this study, we have developed a very fast, reverse polarity capillary electrophoresis based method for screening of ecto-5'-nucleotidase inhibitors. Both EOF and electrophoretic mobility were maintained in same direction by dynamically coating lining of capillary and applying negative voltage, resulting in appearance of both adenosine monophosphate (AMP) and adenosine peaks in less than 2 min. Separations were performed in reverse polarity using -20 kV. Hexadimethrine bromide (HDB) was used as a buffer additive (0.025% in 40 mM borate buffer). An online sample stacking was attained by a special sweeping effect which enabled a long (65 s) injection time without compromise in peak shape. Using this newly developed method, both AMP and adenosine peaks were obtained with a baseline resolution, in less than two minutes. Method was validated by measuring k , and V values of human ecto-5'-nucleotidas and K value of standard inhibitor quercitin against this enzyme. Method is simple and fast and can be conveniently optimized for high throughput screening of ecto-5'-nucleotidase inhibitors.

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Advances in Capillary Electrophoretically Mediated Microanalysis for On‐line Enzymatic and Derivatization Reactions

Cited by 38 publications

References 87 publications

Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

Computer simulation of the enantioselective separation of weak bases in an online capillary electrophoresis based microanalysis configuration comprising sulfated cyclodextrin as selector

Development of a fast CE method for high throughput screening of ecto‐5′‐nucleotidase inhibitors

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