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Preparation of Magnetic Microsphere-Gold Nanoparticle- Immobilized Enzyme Batch Reactor and Its Application to Enzyme inhibitor screening in Natural Extracts by Capillary Electrophoresis

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Chinese Journal of Chemistry 2017年第6期35卷 943-948页

作者： Hengshan Wang Pingtan Lin Shulin Zhao Shuting Li Xin Lu Hong Liang State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources College of Chemistry and Pharmacy Guangxi Normal University Guilin Guangxi 541004 China

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 de- termined 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.

关键词： capillary electrophoresis inhibitor screening enzyme reactor xanthine oxidase

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screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor

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Acta Pharmaceutica Sinica B 2022年第7期12卷 3113-3123页

作者： Yuhong Cao Jiahao Fang Yiwei Shi Hui Wang Xiaofei Chen Yue Liu Zhenyu Zhu Yan Cao Zhanying Hong Yifeng Chai School of Pharmacy Second Military Medical UniversityShanghai Key Laboratory for Pharmaceutical Metabolites ResearchShanghai 200433China Zhejiang Institute for Food and Drug Control Hangzhou 310057China

P-glycoprotein(P-gp)highly expressed in cancer cells can lead to multidrug resistance(MDR)and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment.In this study,we established a label-free and detergent-free system combining surface plasmon resonance(SPR)biosensor with styrene maleic acid(SMA)polymer membrane proteins(MPs)stabilization technology to screen potential P-gp inhibitors.First,P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes(SMALPs).Following that,SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system,and the affinity between P-gp and small molecule ligand was determined.The methodological investigation proved that the screening system had good specificity and stability.Nine P-gp ligands were screened out from 50 natural products,and their affinity constants with P-gp were also determined.The in vitro cell verification experiments demonstrated that tetrandrine,fangchinoline,praeruptorin B,neobaicalein,and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin(Adr).Moreover,tetrandrine,praeruptorin B,and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp.This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system.SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp.The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

关键词： Styrene maleic acid P-Glycoprotein Surface plasmon resonance Membrane proteins inhibitor screening Affinity calculation Natural products Multidrug resistance

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Paper-based fluorescent devices for multifunctional assays:Biomarkers detection,inhibitors screening and chiral recognition

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Chinese Chemical Letters 2022年第9期33卷 4405-4410页

作者： Wang Li Xiaoyue Zhang Siqi Chen Yibing Ji Ruijun Li Department of Analytical Chemistry China Pharmaceutical UniversityNanjing 210009China Key Laboratory of Drug Quality Control and Pharmacovigilance Ministry of EducationNanjing 210009China

The development of a single analytical platform with different functions is highly desirable but remains a challenge at present.Here,a paper-based device based on fluorescent carbon dots(CDs)functionalized paper/MnO_(2)nanosheets(MnO_(2)NS)hybrid devices(PCD/NS)was proposed for single-device multi-function applications.MnO_(2)NS functioned as a fluorescence quencher of CDs and recognizer of H_(2)O_(2)released from the oxidase catalyzed system.Fluorescence recovery would occur after the decomposition of MnO_(2)NS induced by H_(2)O_(2),by which a simple and effective strategy could be developed for fluorescence monitoring multiplex biological events.Xanthine(XA)sensing,xanthine oxidase(XOD)inhibitors screening analysis and chiral recognition of glucose enantiomers were performed on PCD/NS to investigate the multifunctional application of the paper-based device.By means of PCD/NS,XA could be determined in the range of 0.1–40μmol/L with a low detection of limit of 0.06μmol/L.The IC_(50)value of allopurinol,the model inhibitor of XOD,was sensitively detected to be 7.4μmol/L.Glucose enantiomers were also recognized in terms of the specific fluorescence response to d-glucose.This work firstly presented a paper-based device capable of biomarkers detection,inhibitors screening and chiral recognition,which enlightened a promising strategy for the construction of multifunctional devices and hold the great potential application in clinical diagnosis and drug discovery.

关键词： Paper-based devices Multifunctional Xanthine inhibitor screening Chiral recognition

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