A gold nanoparticle-mediated enzyme bioreactor for inhibitor screening by capillary electrophoresis

Abstract

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...

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

“…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.…”

Immobilized‐enzyme reactors integrated with capillary electrophoresis for pharmaceutical research

“…Proof that a CE‐integrated IMER can be an interesting alternative to common microplate assays for novel targets is presented in a work by Zhao et al. . The authors describe an application of mammalian GLDH, which has been linked to recently discovered hyperinsulinism/hyperammonemia disorder.…”

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…[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). Alginate encapsulation of the enzymes prior to adsorption to the capillary has also been applied [145].…”

“…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…”