Recent advances in CE mediated microanalysis for enzymatic and derivatization reactions

Ramana, Pranov; Adams, Erwin; Augustijns, Patrick; Schepdael, Ann Van

doi:10.1002/elps.201500300

Cited by 19 publications

(18 citation statements)

References 54 publications

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“…Many complex reaction systems in nature require the participation of at least two different enzymes . However, most open‐tubular CE‐integrated IMERs are limited to the immobilization of single enzyme, which significantly confines their broad practical application.…”

Section: Introductionmentioning

confidence: 99%

Controllable and high‐performance immobilized enzyme reactor: DNA‐directed immobilization of multienzyme in polyamidoamine dendrimer‐functionalized capillaries

Shen

Zhou

et al. 2020

Electrophoresis

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In recent years, CE‐integrated immobilized enzyme reactors (IMERs) for single‐enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE‐integrated IMER, using DNA‐directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78–12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE‐integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE‐integrated IMERs are potentially useful in a wide range of biochemical research applications.

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Section: Introductionmentioning

confidence: 99%

Controllable and high‐performance immobilized enzyme reactor: DNA‐directed immobilization of multienzyme in polyamidoamine dendrimer‐functionalized capillaries

Shen

Zhou

et al. 2020

Electrophoresis

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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%

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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“…By combining biomolecular immobilization technology with biochemical microreactions, immobilized enzyme microreactors (IMERs) have several unique features for enzyme assays, such as reduced consumption of samples and reagents, reusability of enzyme, high catalysis efficiency, rapid analysis, and excellent stability . Thus, IMERs have been developed rapidly during the last decade, and widely applied in many research fields including affinity chromatography , medical diagnostics , bio‐processing/‐analysis/‐transformation , drug discovery , and so on.…”

Section: Introductionmentioning

confidence: 99%

Capillary electrophoresis‐integrated immobilized enzyme microreactor with graphene oxide as support: Immobilization of negatively charged L‐lactate dehydrogenase via hydrophobic interactions

Yin

Cui

et al. 2019

Electrophoresis

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We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE-integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L-lactate dehydrogenase (L-LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV-vis are used to characterize the fabricated CE-IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of interday and batch-to-batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five-day usage. The measured K m values of pyruvate and NADH of the immobilized L-LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π-π stacking is significant between the GO backbone and the aromatic residues of L-LDH and favorable to fabrication of CE-integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples.Abbreviations: GO, graphene oxide; IMER, immobilized enzyme microreactor; L-LDH, L-lactate dehydrogenase; NAD + , β-Nicotinamide adenine dinucleotide hydrate; NADH, β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate; PDDA, poly (diallyldimethylammonium chloride); UV-vis, ultraviolet-visible * These authors contributed equally in this work.Color online: See the article online to view Figs. 1-3 and 5 in color.

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Recent advances in CE mediated microanalysis for enzymatic and derivatization reactions

Cited by 19 publications

References 54 publications

Controllable and high‐performance immobilized enzyme reactor: DNA‐directed immobilization of multienzyme in polyamidoamine dendrimer‐functionalized capillaries

Controllable and high‐performance immobilized enzyme reactor: DNA‐directed immobilization of multienzyme in polyamidoamine dendrimer‐functionalized capillaries

Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

Capillary electrophoresis‐integrated immobilized enzyme microreactor with graphene oxide as support: Immobilization of negatively charged L‐lactate dehydrogenase via hydrophobic interactions

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