A CE method based on a dual-enzyme co-immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co-immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. The enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed CE method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. 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 larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two-enzyme inhibition by the present method.

A facile protocol to prepare highly effective and durable in-line enzyme bioreactors inside capillary electrophoresis (CE) columns was developed. To demonstrate the methodology, Lglutamic dehydrogenase (GLDH) was selected as the model enzyme. GLDH was first immobilized onto 38 nm dia. gold nanoparticles (GNPs), and the functionalized GNPs were then assembled on the inner wall at the inlet end of the CE capillary treated with polyethyleneimine (PEI), producing an in-line GLDH bioreactor. Compared with a GLDH bioreactor prepared by immobilizing GLDH directly on PEI-treated capillary, the GNP-mediated bioreactor showed a higher enzymatic activity and a much better stability. The in-capillary enzyme bioreactor was proven very useful for screening of GLDH inhibitors deploying the GLDH-catalyzed α-ketoglutaric acid reaction. The screening assay was preliminarily validated by using a known GLDH inhibitor, perphenazine. A Z′ factor value of 0.95 (n = 10) was obtained, indicating the screening results were highly reliable. Screening of GLDH inhibitors present in medicinal plant extracts by the proposed method was demonstrated. Inhibition percentage was found to be 53% for radix scutellariae, 45% for radix codonopsis, 37% for radix paeoniae alba, and 0% for the other 22 extracts tested at a concentration of 0.6 mg extract /mL. KeywordsEnzyme bioreactor; capillary electrophoresis; gold nanoparticles; inhibitor screening; medicinal plant extracts Enzyme inhibitor screening is an effective approach to identify drug leads because many enzymes are involved in regulatory cellular processes and, thus become therapeutic drug targets [1][2][3]. High throughput screening (HTS) is the most commonly used method for screening of enzyme inhibitors [4][5][6]. It is based on colorimetric or fluorometric measurements carried out on multiwell microplates. It works perfectly with large libraries of pure compounds, but not applicable sometimes to assay complicate samples such as extracts of medicinal plants that may contain many UV-absorbing or fluorescent compounds [7][8]. Over the past years, search for enzyme inhibitors present in medicinal plants has been one of Corresponding author: Professor Yiming Liu, yiming.liu@jsums.edu, Professor Shulin Zhao, zhaoshulin001@163.com. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In an immobilization procedure via ionic binding, the solid support is first coated with a polyelectrolyte to obtain a suitably charged carrier surface (either positively or negatively depending on the predominant charge on the enzyme). The solid support with a charged surf...

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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.