Screening of neuraminidase inhibitors from traditional Chinese medicines by integrating capillary electrophoresis with immobilized enzyme microreactor

Zhao, Haiyan; Chen, Zilin

doi:10.1016/j.chroma.2014.03.028

Cited by 57 publications

(35 citation statements)

References 31 publications

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“…In previous study, dual-wavelength detection was employed to eliminate the interference. 21 As seen in Fig. 7(b), when detected at 320 nm with a reference wavelength of 260 nm, the peaks of baicalein, baicalin, and vitexin disappeared while the peak height of 4-MU scarcely changed.…”

Section: E Inhibitors Screeningmentioning

confidence: 79%

“…In our previous work, an immobilized-enzyme microreactor (IMER) was fabricated for NAIs screening. 21 The IMER improved the stability of NA and reduced the consumption of NA, but required 3 days to prepare. In addition, it has a disadvantage of requiring a large consumption of substrate as each run in the screening uses a mixture of substrate and inhibitor.…”

Section: Screening Of Neuraminidase Inhibitors From Traditional Chinementioning

confidence: 99%

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Screening of neuraminidase inhibitors from traditional Chinese medicine by transverse diffusion mediated capillary microanalysis

Zhao

Chen

2014

Biomicrofluidics

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A transverse diffusion mediated capillary microanalysis method has been developed for screening of neuraminidase inhibitors from traditional Chinese medicine. The enzyme, substrate and inhibitors were sequentially injected, mixed efficiently by transverse diffusion of laminar flow profiles, then incubated and separated in the same capillary. To enhance the mixing efficiency of reactants, running buffer was injected by alternately applying +5 kPa and -5 kPa at the capillary inlet and the procedure was repeated three times. The capillary electrophoresis (CE) separation conditions and reactants mixing conditions were optimized. Dual-wavelength detection was employed to eliminate the interference with natural compounds. The method has been applied to determine the kinetics constant of neuraminidase and screen 12 compounds from traditional Chinese medicine. Four compounds have been found to be positive for enzyme inhibition. The results are in good agreement with those reported in the literature. The method realized the mixing of substrate and enzyme with identical electrophoretic mobility. This novel CE method was simple, rapid, economic, and fully automated. Therefore, it was appropriate for neuraminidase inhibitors screening and could be extended to other high-throughput screening of active components from traditional Chinese medicine.

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Section: E Inhibitors Screeningmentioning

confidence: 79%

Section: Screening Of Neuraminidase Inhibitors From Traditional Chinementioning

confidence: 99%

Screening of neuraminidase inhibitors from traditional Chinese medicine by transverse diffusion mediated capillary microanalysis

Zhao

Chen

2014

Biomicrofluidics

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“…In the majority of cases, the enzymes were immobilized via glutardialdehyde to aminopropyl-silanefunctionalized capillaries, e.g. [128][129][130][131][132][133][134][135]. 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).…”

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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“…The mechanism used to prevent cancer progression is to reduce the enzyme activity on the pathway that promotes cancer cell growth. In order to find a potent inhibitor, on-line analysis is generally adapted to screen the selected compounds, such as natural extracts [23,56,63,76] or traditional Chinese medicines [18,57,68,73]. In the process of screening the inhibition, the selected inhibitor is evaluated as the relative difference in the product peak area in the presence and in the absence of the inhibitor.…”

Section: Evaluating Enzyme Inhibitors Using Capillary Electrophoresismentioning

confidence: 99%

“…However, they are less stable than coatings based on covalent immobilization to the silica wall, such as silanol [57] and glutaraldehyde chemistry [78]. For example, one report utilizing electrostatic interactions noted that high voltage can lead to enzyme desorption from the wall [22].…”

Section: Enzyme Immobilization Techniquesmentioning

confidence: 99%

Advances in enzyme substrate analysis with capillary electrophoresis

Gattu

Crihfield

et al. 2018

Methods

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Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.

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Screening of neuraminidase inhibitors from traditional Chinese medicines by integrating capillary electrophoresis with immobilized enzyme microreactor

Cited by 57 publications

References 31 publications

Screening of neuraminidase inhibitors from traditional Chinese medicine by transverse diffusion mediated capillary microanalysis

Screening of neuraminidase inhibitors from traditional Chinese medicine by transverse diffusion mediated capillary microanalysis

Advances in Capillary Electrophoresis-Based Enzyme Assays

Advances in enzyme substrate analysis with capillary electrophoresis

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