Immobilized capillary adenosine deaminase microreactor for inhibitor screening in natural extracts by capillary electrophoresis

Ji, Xiaowen; Ye, Fanggui; Lin, Pingtan; Zhao, Shulin

doi:10.1016/j.talanta.2010.06.029

Cited by 38 publications

(21 citation statements)

References 25 publications

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“…Recently, there has been a growing interest in utilizing immobilized-enzyme capillary microreactor for screening enzyme inhibitors by capillary electrophoresis (CE), [2][3][4][5][6] owing to the fact that the immobilized enzymes have many benefits over soluble enzymes in many laboratory-scale and biotechnology applications. In addition, magnetic carriers enable simple magnetic separation of immobilized enzymes, which offers a relatively simple technique for separating and reusing enzymes over a longer period.…”

Section: Introductionmentioning

confidence: 99%

Preparation of Magnetic Microsphere‐Gold Nanoparticle‐Immobilized Enzyme Batch Reactor and Its Application to Enzyme Inhibitor Screening in Natural Extracts by Capillary Electrophoresis

et al. 2017

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A novel strategy for screening the enzyme inhibitors from the natural products by capillary electrophoresis (CE) with a pre column enzyme batch reactor prepared by magnetic microsphere (MB)-gold nanoparticles (AuNPs) is reported. The enzyme batch reactor was prepared by immobilizing the enzymes on the MB conjugated AuNPs (MB-AuNPs). To demonstrate this strategy, xanthine oxidase (XOD) was employed as a model for the activity of the enzyme, inhibition study, and inhibitor screening. With the developed CE method, the enzyme activity was determined by the quantification of the peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of product in comparison to a reference electropherogram obtained in the absence of any inhibitor. A statistical parameter Z' factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be 0.7, implying a good accuracy. The screening of two natural extracts from Cortex Phellodendri and Rhizoma Galangae showed that they were positive for XOD inhibition by the present method. Using this immobilized enzyme technology combined with CE separation not only provides the advantages such as convenience, rapidity and low cost, but also provides a new platform for discovering enzyme-inhibitor drug lead compounds.

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Section: Introductionmentioning

confidence: 99%

Preparation of Magnetic Microsphere‐Gold Nanoparticle‐Immobilized Enzyme Batch Reactor and Its Application to Enzyme Inhibitor Screening in Natural Extracts by Capillary Electrophoresis

et al. 2017

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“…In the work by Ji et al. , ADA was immobilized using ionic binding with multiple layer‐by‐layer assembly. First the capillary inlet of 0.4 cm length was coated with PEI (30% w/v), subsequently a plug of the same length was injected containing 45.5 unit/mL ADA and 0.1 mg/mL sodium alginate.…”

Section: Applications Based On Pharmaceutical Relevancementioning

confidence: 92%

Immobilized‐enzyme reactors integrated with capillary electrophoresis for pharmaceutical research

Schejbal

Glatz

2017

J of Separation Science

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Enzymes play an essential role in many aspects of pharmaceutical research as drug targets, drug metabolizers, enzyme drugs and more. In this specific field, enzyme assays are required to meet a number of specific requirements, such as low cost, easy automation, and high reliability. The integration of an immobilized-enzyme reactor to capillary electrophoresis represents a unique approach to fulfilling these criteria by combining the benefits of enzyme immobilization, that is, increased stability and repeated use, as well as the minute sample consumption, short analysis time, and efficient analysis provided by capillary electrophoresis. In this review, we summarize, analyze, and discuss published works where pharmaceutically relevant enzymes were used to prepare capillary electrophoresis-integrated immobilized-enzyme reactors in an online manner. The presented assays are divided into three distinct groups based on the drug-enzyme relationship. The first, more extensively studied group employs enzymes that are considered to be therapeutic targets, the second group of assays present tools to assess drug metabolism and the third group assesses enzyme drugs. Furthermore, we examine various methods of enzyme immobilization and their implications for assay properties.

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“…Another suitable approach was the adsorption of either the enzyme [136][137][138][139][140][141] or enzyme previously immobilized to gold nanoparticles [142][143][144] to a capillary which had been coated by polybrene or poly(ethyleneimine). Alginate encapsulation of the enzymes prior to adsorption to the capillary has also been applied [145]. Furthermore, enzymes were entrapped in monolithic structures [146].…”

Section: Immobilized Enzyme Reactorsmentioning

confidence: 99%

“…The background electrolyte consisted of 10 mM aqueous ammonium bicarbonate, pH 8. The enzyme was [142] Adenosine deaminase Encapsulation in anionic alginate followed by adsorption to poly(ethyleneimine)-coated capillary Adenosine Enzyme kinetic data, Ki determination, screening of herbal extracts for inhibitory activity [145] Immobilization via glutaraldehyde to APTMS-functionalized capillary Adenosine, nucleoside prodrugs Enzyme kinetic data [132] Enzyme kinetic data, screening of herbal extracts for inhibitory activity [140] APTES 3-aminopropyl triethoxysilane, APTMS…”

Section: Immobilized Enzyme Reactorsmentioning

confidence: 99%

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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Immobilized capillary adenosine deaminase microreactor for inhibitor screening in natural extracts by capillary electrophoresis

Cited by 38 publications

References 25 publications

Preparation of Magnetic Microsphere‐Gold Nanoparticle‐Immobilized Enzyme Batch Reactor and Its Application to Enzyme Inhibitor Screening in Natural Extracts by Capillary Electrophoresis

Preparation of Magnetic Microsphere‐Gold Nanoparticle‐Immobilized Enzyme Batch Reactor and Its Application to Enzyme Inhibitor Screening in Natural Extracts by Capillary Electrophoresis

Immobilized‐enzyme reactors integrated with capillary electrophoresis for pharmaceutical research

Advances in Capillary Electrophoresis-Based Enzyme Assays

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