Advances in immobilized enzyme microbioreactors in capillary electrophoresis

Iqbal, Jamshed; Iqbal, Shoaib; Müller, Christian

doi:10.1039/c3an00031a

Cited by 78 publications

(49 citation statements)

References 82 publications

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“…A review dedicated to such miniaturized systems and their applications in biochemical research has been published by Iqbal et al [125]. Furthermore, reviews were published on the use of IMERs in proteomics [126] and as microfluidic process reactors [127].…”

Section: Immobilized Enzyme Reactorsmentioning

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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Section: Immobilized Enzyme Reactorsmentioning

confidence: 98%

Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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“…Therefore, the aim of the present study was the development of a stable CE assay for the determination of the stereospecific activity of Msr enzymes. The general suitability of CE for enzyme assays due to the flexibility of the technique as well as low consumption of chemicals and enzyme has been summarized in the literature .…”

Section: Introductionmentioning

confidence: 99%

Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary

et al. 2013

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A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.

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“…As recently reviewed by J. Iqbal et al [123], this problem was solved in two different ways. First, the microreactor and separation capillaries exist as two distinct objects, while in the second, they are united in a single structure.…”

Section: Magnetic Beadsmentioning

confidence: 99%

“…The most important example in this field is the use of immobilized enzyme biomicroreactors in capillary electrophoresis [123,124], a well-known analytical technique where an electric field is applied to induce the separation of analytes on the basis of their electrophoretic mobility. Bio-microreactors for capillary electrophoresis are discussed separately in this paper because their preparation requires special approaches.…”

Section: Magnetic Beadsmentioning

confidence: 99%

“…When the two components are separated, both the reaction and separation conditions can be optimized independently, but the correct alignment of the microreactor with the separation capillary is critical. These types of system are mainly used in proteomics research using trypsin or pepsin for protein analysis and peptide mapping [107,125,126] and other appropriate enzymes for nucleic acids studies [123].…”

Section: Magnetic Beadsmentioning

confidence: 99%

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Enzymatic microreactors in biocatalysis: history, features, and future perspectives

Laurenti¹,

Vianna²

2016

Biocatalysis

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Microfluidic reaction devices are a very promising technology for chemical and biochemical processes. In microreactors, the micro dimensions, coupled with a high surface area/volume ratio, permit rapid heat exchange and mass transfer, resulting in higher reaction yields and reaction rates than in conventional reactors. Moreover, the lower energy consumption and easier separation of products permit these systems to have a lower environmental impact compared to macroscale, conventional reactors. Due to these benefits, the use of microreactors is increasing in the biocatalysis field, both by using enzymes in solution and their immobilized counterparts. Following an introduction to the most common applications of microreactors in chemical processes, a broad overview will be given of the latest applications in biocatalytic processes performed in microreactors with free or immobilized enzymes. In particular, attention is given to the nature of the materials used as a support for the enzymes and the strategies employed for their immobilization. Mathematical and engineering aspects concerning fluid dynamics in microreactors were also taken into account as fundamental factors for the optimization of these systems.

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Advances in immobilized enzyme microbioreactors in capillary electrophoresis

Cited by 78 publications

References 82 publications

Advances in Capillary Electrophoresis-Based Enzyme Assays

Advances in Capillary Electrophoresis-Based Enzyme Assays

Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary

Enzymatic microreactors in biocatalysis: history, features, and future perspectives

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