• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Recent advances in screening of enzymes inhibitors based on capillary electrophoresis

    2018-08-22 06:41:22MengxiaChengZilinChen
    Journal of Pharmaceutical Analysis 2018年4期

    Mengxia Cheng,Zilin Chen

    Key Laboratory of Combinatorial Biosynthesis and Drug Discovery,Ministry of Education,and Wuhan University,School of Pharmaceutical Sciences,Wuhan 430071,China

    Keywords:Capillary electrophoresis Enzyme inhibitor screening Pre-capillary enzyme assays Electrophoretically mediated microanalysis Immobilized enzyme microreactor

    ABSTRACT Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening.This review gives an overview on the recent advances in the developments and applications of capillary electrophoresis in the field of enzyme inhibitor screening.The period covers 2013 to 2017.Both the pre-capillary enzyme assays and in-capillary enzyme assays which include electrophoretically mediated microanalysis(EMMA)and immobilized enzyme microreactor(IMER)are summarized in this article.

    1.Introduction

    Enzyme,a kind of biocatalyst which has high catalytic efficiency and specificity,is essential for maintaining normal life activities.Enzymes take part in the process of physiological metabolism including cells regulating homeostasis,growth and reproduction of living organisms.So some human diseases often result from abnormal regulation of enzymes,like in flammation,tumor,cardiovascular disease,central nervous system disease,and infectious disease.Therefore,we can treat these diseases with targeted therapy by intervening in enzyme pathway and pathologic expression[1,2].It has been revealed that enzymes are potential drug targets[3–9].One situation is that the enzyme expression level is higher than the expected value,resulting in disturbed body functions.One ofthe therapeutic ideas is restricting the enzyme activity by enzyme inhibitors.So the study and development of enzyme inhibitors is one way to find new drugs.Therefore,it is important to study effective methods to screen enzyme inhibitors for the treatment of human diseases.

    Ultraviolet(UV)and fluorescence spectrophotometry,electrochemical method and high performance liquid chromatography(HPLC)are conventional methods,which are usually used as analytical tools to screen enzyme inhibitors.But these methodshave some disadvantages.UV and fluorescence spectrophotometry are only suitable for the situation in which substrates and products have significant difference in their spectrometric properties.The fluorescence signal could also be interfered by background absorption and auto- fluorescence in biological samples[10].Electrochemical method requires that substrates and products have electrochemical activity.HPLC can avoid these defects from the above methods.However,long elution time in HPLC limits high efficiency of inhibitor screening and the consumption of large amounts of organic solvent increases the cost.

    Capillary electrophoresis(CE)has been widely applied in various fields including enzyme inhibitor screening,as it has many advantages such as high separation efficiency,quick analysis,low sample consumption,low solvent volume[11,12],automation and ability to be combined with various detection techniques[13]including UV,laser induced fluorescence(LIF)[14,15],electrochemical detectors[16,17]and mass spectroscopy(MS)[18,19].

    In general,the methods for screening enzyme inhibitors by CE can be divided in two categories:(i)pre-capillary enzyme assays in which the enzymatic reaction takes place off-line and the capillary is just used as the separation channel;(ii)in-capillary enzyme assays in which enzymatic reaction takes place on-line and sampling,reaction,separation,and detection can be integrated into a single capillary.Electrophoretically mediated microanalysis(EMMA)and immobilized enzyme microreactor(IMER)are included in in-capillary enzyme assays.These methods are summarized in this review.

    This review covers the literatures and gives an overview of the recent advances in the developments and applications of these methods in the field of enzyme inhibitor screening,over the period from 2013 to late 2017,which is a continuation of previous reports[20,21]and a supplement of recent reports[22,23].

    2.Pre-capillary enzyme assays

    In pre-capillary enzyme assays,incubation reaction and sample analysis are separate.The enzymatic reaction is initiated by mixing substrate,enzyme and cofactor,and off-line incubated for a specific time.After the incubation reaction is stopped,the reaction mixture is injected into the CE system for subsequent separation and detection.This method is easy to realize because enzymatic reaction is easy to carry out outside the CE system and there is no need to specifically modify separation capillary.And incubation reaction and sample analysis can be performed without mutual interference under respectively optimized conditions.Therefore,this method has been applied to enzyme inhibitor screening,determination of enzyme activity and kinetics,and drug metabolism studies for many years.

    Iqbal et al.developed a pre-capillary enzyme assay for characterization and inhibition study of bovine carbonic anhydrase(CA)II.It was the first time to carry out CA enzyme assay by CE.The enzyme and substrate with or without inhibitors were added in a vial.After incubation at 37°C for 10 min,the reaction was terminated by freezing the reaction mixture.The reaction mixture was injected into the capillary by applying 0.5 psi for 5s,followed by the application of 15 kV voltages for separation of substrate and product.The developed method was used to determine the Michaelis-Menten kinetics of CA and inhibition constant of furosemide,a standard inhibitor of CA.The result showed it was a fast and efficient pre-capillary CA inhibitor screening method[24].Iqbal's group also applied a similar pre-capillary enzyme assay for the characterization and inhibition study ofα-glucosidase.The results obtained with the established method were in excellent agreement with data from other literatures[25].Zhang et al.combined high performance purification of HPLC with enzyme assay of CE to screen inhibitors of mammalian target of rapamycin from natural product extracts.This method facilitated in finding bioactive components among minor constituents of natural extracts[26].

    The real-time analysis of the metabolism of 6-mercaptopurine by xanthine oxidase in the absence and presence of inhibitor allopurinol was evaluated using CE by Lu et al.It is worthy for us to study metabolism of 6-mercaptopurine,which is a drug used in the treatment of acute lymphatic leukemia,Crohn's disease,and ulcerative colitis[27,28].Nehméet al.used the pre-capillary enzyme assay to evaluate tau phosphorylation by the glycogen synthase kinase 3-βfor the first time.This work showed that it is possible to follow phosphorylation of tau in vitro by CE assays in a rapid and safe manner.It also showed its potential to screen different modulators(inductors or inhibitors)of tau phosphorylation which may efficiently treat neurodegenerative diseases[29].Malina et al.carried out pre-capillary phosphofructokinase-1(PFK-1)catalytic reaction which is about the rate-limiting step of glycolytic pathway in polypropylene microcentrifuge tubes.Inhibition study of PFK-1 was performed by aurintricarboxylic acid.The separation by CE avoided the spectral interference from inhibitors[30].Nowak et al.revealed the potential of the CE-based enzyme assay in plant membrane enzyme-chlorophyllase.The reaction was conducted in a vial placed directly on the thermostated sample tray.While the incubation was happening,the separation and detection were simultaneously carried out.The product accumulation could be monitored at any time.The proposed method demonstrated a great potential of CE as a convenient analytical tool for monitoring enzyme reaction progress,determining activity of membrane enzymes and screening enzyme inhibitors[31].

    Sun et al.developed a facile chiral ligand exchange capillary electrophoresis(CLE-CE)system with Zn(II)-L-alanine as the chiral selector in the presence ofβ-cyclodextrin for enantioseparation of dansyl amino acids.It was applied to study the activity of tyrosinase and determine the inhibitory effect of kojic acid on tyrosinase.The results implied that the proposed CLE-CE system was a useful tool for studying enzymatic reaction of tyrosinase,investigating the substrate specificity,and providing a new strategy for screening tyrosinase inhibitors[32].A new CLE-CE system with L-leucine as the chiral ligand,Zn(II)as the central ion andβ-cyclodextrin as the additive was developed by Su et al.The substrates of tyrosinase,D,L-tyrosine were well separated.The method has been applied in tyrosinase inhibitor screening with chalcones as the model compounds[33].Then Su et al.constructed dual chiral selectors with Mn(II)–[1-butyl-3-methylimidazolium][L-alanine]amino acid ionic liquids(AAILs)complex and β-cyclodextrin.The CLE-CE system was further developed for enantioseparation of dansyl D,L-amino acids and applied to screen tyrosinase inhibitors[34].In addition,new kinds of AAILs with pyridinium as cations and L-lysine as anion were also developed as the available chiral ligands coordinated with Zn(II)in CLE-CE by Sun et al.This system was successfully applied to study the specificity of substrates and the kinetic constants of L-amino acid oxidase[35].

    Some pre-capillary enzymatic reactions were mainly carried out with free enzyme solutions.These methods were often hampered by the low operational stability and brought difficulties in recovery and reuse of enzymes.In addition,separation and detection of substrates and products sometimes can be interfered by free enzymes.Therefore,it was significant to explore a simple and efficient approach to improve the stability and reusability and reduce interference.Mu et al.provided a facile and efficient approach to fabricate the enzyme functionalized magnetic nanoparticles modified by a reactive polymer poly(glycidyl methacrylate)[36].The immobilized D-amino acid oxidase(DAAO)presented high enzyme activity,good reusability and satisfactory stability.A CLE-CE system with AAIL as the chiral ligand was applied to screen inhibitors of DAAO which is related to schizophrenia[37].Zhang et al.prepared an IMER in capillary which was just used for enzymatic reaction.Gold nanoparticles(AuNPs)were covalently attached to surface of the pores of the porous polymer capillary monolith via the formation of an Au–S bond.And αglucosidase was then immobilized onto AuNPs through the strong affinity of gold with amino groups of the enzyme.Substrate solution was injected by the syringe and the ef fluent was collected for the subsequent analysis by CE[38].

    Therearesomelimitationsto thepre-capillary mode.Firstly,the enzymatic reaction which is very fast must be terminated by adding quenching reagents or changing the reaction conditions before analysis by CE system.Secondly,although only nanoliter-scale sample is consumed by CE,the precapillary enzyme assay requires a large number of reactants to initiate the reaction,which would cause the waste of reagents especially for those expensive enzymes.Thirdly,enzymatic reaction and analysis by CE are separate in the pre-capillary mode which increases complexity of operation and is also time consuming.

    3.In-capillary enzyme assays

    In in-capillary enzyme assays,the capillary can be used not only as a separation channel but also as a microreactor.Injection of reactants,mixture,enzyme reaction,separation and detection of analytes can be integrated into a single capillary,which improves the analysis efficiency.The in-capillary enzyme assay realizes automatic operation,short analysis time,low consumption of sample and low cost[39].It can be divided into two categories:electrophoretically mediated microanalysis(EMMA)and immobilized enzyme microreactor(IMER).

    3.1.EMMA

    Bao and Regnier[40] firstly reported the in-capillary enzyme assay known as electrophoretically mediated microanalysis(EMMA),in which the reactants were mixed and the enzymatic reaction was triggered by utilizing the difference of electrophoretic mobility of each reactant under electric field.EMMA was generally divided into two major modes according to the pattern of sampling and mixing:the continuous engagement EMMA(long contact mode)and transient engagement EMMA(plug-plug or short contact mode).

    In the long contact mode,the capillary is initially completely filled with either substrate or enzyme and the second reactant is introduced as a plug(zonal sample introduction)[40]or in continuous flow(moving boundary sample introduction)[41].The product was continuously produced after applying the voltage which brings the electrophoretic mixing of enzyme and substrate.

    In the plug-plug mode,plugs of enzyme and substrate are consecutively introduced into the capillary.The enzymatic reaction is initiated by the application of voltage,because the difference of their electrophoretic mobility under electric field brings interpenetration among zones.It has been the most commonly used EMMA mode because of less consumption of reactants compared with long contact mode.However,in classical plug–plug mode,the buffer is required to be suitable for enzymatic reaction and separation,which limits its application when the separation buffer is incompatible with the enzymatic reaction buffer.To solve this problem,additional plugs of incubation buffer were injected between reactants and running buffer.This approach is termed as“partial filling mode”,as shown in Fig.1A[42].

    In the typical EMMA,reactants were mixed by difference of electrophoretic mobility under electric field.However,its application is limited when some enzymes are sensitive to the electric field or electrophoretic mobility of some reactants is similar.In these cases,techniques of longitudinal diffusion and transverse diffusion of laminar flow profiles(TDLFP)are applied to mix enzyme and substrate at the inlet of the capillary.The techniques have no requirements for electrophoretic mobility of reactants and reactants were mixed by simple diffusion.The schematic of the three mixing modes of reactants in the capillary is shown in Fig.2.

    EMMA is a very useful tool for the study of enzyme kinetics and inhibition,and screening enzyme inhibitors.Pochet et al.determined the inhibitory potency of argatroban toward thrombin by EMMA combined with short-end injection,partial- filling mode and rapid polarity switching technology[43].The mixing method which is performed by alternative application of positive and negative potentials to move the sample plugs back and forth in the capillary is termed rapid polarity switching[44].The schematic diagramsofshort-end injection and rapid polarityswitching technology are shown in Figs.1B and C.Nehméet al.developed the in-capillary enzyme assay,which was mixed by TDLFP to screen protein kinase inhibitors.It was the first time for those four human kinases to be assessed by CE.Compared with conventional radiometric enzyme assays,this method was eco-friendly since no radioactivity was required[45].Later,Nehméet al.developed a TDLFP-based method again in order to assess tau phosphorylation and to study the effect of modulators such as glycosaminoglycans,heparin sulfate and heparin on phosphorylation[29].Wang et al.combined EMMA and LC–MS/MS for screening of protein kinase inhibitors in natural extracts.Baicalin was successfully screened as an inhibitor of protein kinase A with the method[46].

    Fig.1.EMMA combined with partial filling mode(A),short-end injection(B),rapid polarity switching technology(C),and sandwich mode(D).

    Fig.2.Mixing of reactants in the capillary:mixing by electrophoresis(A),longitudinal diffusion(B),and TDLFP(C).

    Fig.3.The overlaid electropherograms for neuraminidase inhibitor screening by online enzymatic inhibition assay.The figure was reproduced with permission from Ref.[48].

    Fig.4.Schematic overview of EMMA.(A)Sequential injection of the incubation buffer,tyrosinase,L-DOPA,tyrosinase,incubation buffer,BGE.(B)Mixing by applying a voltage switch sequence.(C)Mixture separation and product detection.The figure was reproduced with permission from Ref.[49].

    Our group has made a great contribution to the development of EMMA in enzyme inhibitor screening.Zhao and Chen developed an EMMA method with partial filling mode for screening aromatase inhibitors in traditional Chinese medicine(TCM).A long plug of coenzyme reducedβ-nicotinamide adenine dinucleotide 2′-phosphate hydrate was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer,enzyme,and substrate solution.The reaction was initiated with a voltage of 5 kV for 40 s.Then the voltage was switched off for 20 min to increase the product amount and switched on again to separate all the analytes at the voltage of 20 kV.With the method,seven compounds were found to have potent inhibitory activity for aromatase[47].Then Zhao and Chen screened neuraminidase inhibitors by in-capillary enzyme assays again.The enzyme,substrate and inhibitors were sequentially injected,mixed by TDLFP,incubated and separated in the same capillary.To enhance the efficient mixture of reactants,+5 kPa and-5 kPa at the capillary inlet was alternately applied.To eliminate the interference from natural compounds,dual-wavelength detection was employed.The method has been applied to determine the kinetic constant of neuraminidase and four compounds have been screened as neuraminidase inhibitors,as shown in Fig.3[48].When it is hard to get the electrophoretic mobility of the reactants,sandwich mode injection was applied to further facilitate mixture of reactants.Its injection sequences are like substrate-enzyme-substrate or enzyme-substrate-enzyme(Fig.1D).As shown in Fig.4,Tang et al.integrated EMMA with sandwich mode,partial filling,and rapid polarity switching technique to screen tyrosinase inhibitors from TCM[49].Han and Chen developed a cathepsin B inhibitor screening method that integrated longitudinal diffusion and TDLFP to efficiently mix reactants.Twelve potential compounds were evaluated and dauricine showed inhibitory potential for cathepsin B.This work provides a new approach to screen cathepsin B inhibitors[50].

    Besides enzyme inhibitor screening,EMMA can also be used for the determination of enzyme activity and stereospecificity of enzymes,the understanding of enzyme-mediated metabolic reactions and other determination which is related to enzymatic reaction.Nowak et al.applied the EMMA method to realize routine,high-throughput and cost-effective assessment of the activity of plant membrane enzyme chlorophyllase[31].Harada et al.developed an in-capillary enzyme assay for the determination of enzyme activity of lignin peroxidase using micellar electrokinetic chromatography(MEKC)for separation of reaction mixture,which combined with sandwich mode and partial filling mode for injection.This method is more sensitive than conventional spectrophotometry since the background originating from the enzyme and the culture medium can be removed via MEKC separation[51].Zhu et al.developed EMMA combined with partial- filling mode to determine the stereoselective reduction of L-methionine sulfoxide diastereomers by methionine sulfoxide reductase(Msr)and validated using fluorenylmethyloxycarbonyl(Fmoc)-L-methionine sulfoxide as substrate.The assay was applied for the analysis of stereoselective reduction of Fmoc-L-methionine-(S)-sulfoxide by human MsrA and of the Fmoc-L-methionine-(R)-sulfoxide by MsrB.It was demonstrated for the first time that Fmoc-L-methionine-(R)-sulfoxide is a substrate of MsrB[52].An enzyme mediated fluorescence-amplification method by EMMA with LIF detector was developed for glucose quantification in human serum samples by Guan and Zhou.The samples spiked with a novel fluorescent reagent named 2-[6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid(APF)and the mixed enzyme solutions of glucose oxidase and horseradish peroxidase(HRP),were individually injected into the capillary with the sandwich mode[53].Then they developed a similar method again to determine the plasma total cholesterol.In this study,the diluted samples were also spiked with APF and the mixed enzyme solutions contained cholesterol oxidase and HRP[54].All recent studies related to EMMA are summarized in Table 1.

    3.2.IMER

    IMERs mean that the enzyme is immobilized in the first part of the capillary for enzymatic reaction,while the remaining part of the capillary is used for separation of analytes.Compared with the enzymatic reaction in solution,the immobilized enzyme has someadvantages.First,the immobilized enzyme is reusable which avoids the waste of enzyme and reduces the experimental cost.Second,the stability of the immobilized enzyme may be improved.Third,the efficiency of enzymatic reaction can be increased.Fourth,because the enzyme is immobilized,adsorption of the enzyme on the inner wall of capillary can be avoided,which is helpful to increase reproducibility of separation.But the IMERs are not suitable for enzymatic reaction system in which the incubation buffer and separation buffer are different.In addition,preparation of IMERs is complicated and different immobilization methods have their own defects.Generally,there are three methods for enzyme immobilization:physical adsorption,covalent attachment and encapsulation[55].Physical adsorption has advantages of low cost,easy operation,mild immobilization conditions and moderate harm for the enzyme.And the capillary can be reused by rinsing with NaCl,HCl and NaOH.Enzymes immobilized by covalent attachment have good stability,but the activity of enzymes may decrease because of chemical bonding.Encapsulation brings large enzyme adsorption and keeps high enzyme activity[56,57],but the operation is complicated and hard.

    Table 1 Summary of electrophoretically mediated microanalysis.

    Fig.5.Overlaid electropherograms for neuraminidase inhibitor screening by on-line immobilized enzyme assay detected at 320 nm with reference wavelength of 260 nm(A)and at 320 nm without reference wavelength(B).The figure was reproduced with permission from Ref.[63].

    IMERs are a good tool to study enzyme assays.Min et al.established an efficient trypsin IMER at the inlet of the capillary by glutaraldehyde cross-linking technology.The method was successfully used to study the enzyme kinetics of trypsin and on-line screen trypsin inhibitors from 19 kinds of natural extracts[58].Jiang et al.created a tyrosinase IMER based on layer-by-layer assembly for inhibitor screening.Tyrosinase was immobilized on the surface of fused silica capillary via ionic binding technique with cationic polyelectrolyte hexadimethrine bromide(HDB).Then,HDB solution with the same plug length as the tyrosinase was introduced again into the capillary to cover the immobilized tyrosinase by forming HDB-Tyrosinase-HDB sandwich-like structure.The inhibition kinetics of the immobilized tyrosinase was studied by the method[59].Camara et al.developed a multilayer IMER by layer-by-layer electrostatic assembly which can easily enhance theglucose 6-phosphate dehydrogenase(G6PDH)loading capacity.After poly(diallyldimethylammonium chloride)(PDDA)was modified on the inner surface of the capillary to create a positively charged coating,G6PDH enzyme solution was injected into the capillary to make negatively charged G6PDH electrostatically absorb on the PDDA layer.A multilayer IMER was produced by repeating the above procedures to coat PDDA and G6PDH.With the method,on-line inhibition of several green tea catechins on the G6PDH enzyme was investigated[60].Schejbal et al.developed a novel IMER based on magnetic microparticles for study of pharmacologically important cytochrome P450 2C9(CYP2C9).The CYP2C9 was attached to magnetic SiMAG-carboxyl microparticles using the carbodiimide method.The formation of an IMER at the inlet end of the capillary was ensured by two permanent magnets fixed in a cassette from the CE apparatus in the repulsive arrangement.The IMER was applied for on-line kinetics and inhibition study of CYP2C9 with diclofenac as a model substrate and sulfaphenazole as a model inhibitor[61].

    Fig.6.Procedure for immobilizing tyrosinase on capillary inner wall(A)and schematic of tyrosinase IMER assay(B).The figure was reproduced with permission from Ref.[64].

    A dual-enzyme co-IMER was constructed by Lin et al.The adenosine deaminase and xanthine oxidase were firstly immobilized on AuNPs,and the functionalized AuNPs were then assembled on the inner wall of the inlet end which was treated with polyethyleneimine.The developed method could be used for on-line simultaneous screening of multiple enzyme inhibitors.This co-immobilized enzyme technology contributes to highthroughput screening of enzyme inhibitors and provides a new strategy for discovering multitargeted enzyme inhibitors as drug lead compounds[62].

    Our group has been studying the CE-based IMERs for many years and has made some great achievements.Zhao and Chen fabricated a simple and effective neuraminidase IMER by glutaraldehyde cross-linking technology for screening neuraminidase inhibitors which are effective for in fluenza type A and B viruses in humans,avians and animals.With short-end injection mode,the substrate and product were separated by CE within 2 min.As shown in Fig.5,six compounds were found as potent inhibitors[63].Cheng and Chen prepared a tyrosinase IMER with the same method for screening tyrosinase inhibitors,as shown in Fig.6.Four compounds including quercetin,kaempferol,bavachinin,and bakuchiol from 15 compounds of TCM were found to have inhibitory potentials.Meanwhile,molecular docking study was carried out to further demonstrate their inhibitory potential on the basis of molecular interaction between the enzyme and inhibitors.As shown in Fig.7,it was obvious that bakuchiol fitted well into the binding pocket and several key interactions were observed[64].Cheng and Chen developed a trypsin IMER at the inlet of capillary treated with polydopamine(PDA).Based on strong noncovalent adsorption of PDA and skillful use of PDA for many years in our group,the trypsin IMER was successfully established for the first time and had good stability.Baicalin was screened as a trypsin inhibitor and molecular docking study well supported the experimental result[65].

    Enzymatic reactions in IMERs are used not only for enzyme inhibitor screening but also for other applications.An IMER using graphene oxide as support was developed based on layer-by-layer electrostatic assembly by Yin et al.Angiotensin and bovine serum albumin(BSA)were successfully digested by the CE-based IMER[66].Liu et al.fabricated a novel CE-based IMER by particlepacking technique.The IMER was accomplished by utilizing largepore beads as the enzyme supports and perfusive silica single particles as the frits.The IMER was successfully applied for accurate analysis of trypsin inhibition and on-line digestion of standard proteins(myoglobin and BSA)[67].All recent studies about IMER mentioned above are summarized in Table 2.

    Fig.7.Binding mode of bakuchiol(in stick model)and the active site cavity of tyrosinase(PDB ID:2Y9X).The hydrogen-bonding interaction is shown in the green dashed lines.The interactions between Cu and residues are shown in red dashed lines.The figure was reproduced with permission from Ref.[64].

    Table 2 Summary of immobilized enzyme microreactors.

    4.Conclusions

    As can be concluded from the literature collected in the present review article,over past years great advances have been made in the CE-based enzyme inhibitor screening.In pre-capillary assays,the enzymatic reaction conditions and separation conditions can be optimized separately,which is universal for most enzymes.In the mode of in-capillary assays,the sampling,mixture,reaction,separation and detection are integrated into one single capillary,which simplifies the operation,reduces reagent consumption,shortens analysis time,facilitates the automatization and miniaturization of enzymatic assays and realizes high-throughput enzyme inhibitor screening.With the rapid development of CE-based enzyme inhibitor screening,the CE will be more widely applied to enzyme assays for study of enzyme kinetics,understanding of enzyme-mediated metabolic reactions,study of rapid peptide mapping in proteomic and so on.

    Conflicts of interest

    The authors declare that there are no conflicts of interest.

    Acknowledgments

    We are grateful for the financial support from the National Natural Science Foundation of China(Grant nos.81573384 and 21375101).

    人人妻人人澡人人爽人人夜夜| 成人手机av| 性少妇av在线| 中文字幕av电影在线播放| 曰老女人黄片| 99国产极品粉嫩在线观看| 久久国产精品男人的天堂亚洲| 欧美午夜高清在线| 在线天堂中文资源库| 一个人免费在线观看的高清视频 | 国产有黄有色有爽视频| 午夜福利在线观看吧| 国产精品 国内视频| 最近最新中文字幕大全免费视频| 成年美女黄网站色视频大全免费| 91国产中文字幕| 成人三级做爰电影| a级毛片黄视频| 国产野战对白在线观看| 欧美日韩亚洲综合一区二区三区_| 国产精品 欧美亚洲| 亚洲av成人一区二区三| 人人妻,人人澡人人爽秒播| 十八禁网站网址无遮挡| 久久久国产成人免费| 亚洲情色 制服丝袜| 国产精品1区2区在线观看. | 男人爽女人下面视频在线观看| 久久久国产欧美日韩av| 国内毛片毛片毛片毛片毛片| 亚洲国产av影院在线观看| 亚洲国产精品一区三区| 热re99久久精品国产66热6| 国产欧美日韩一区二区三 | 免费一级毛片在线播放高清视频 | 午夜福利,免费看| 日韩人妻精品一区2区三区| 可以免费在线观看a视频的电影网站| 亚洲国产欧美网| 悠悠久久av| 亚洲久久久国产精品| 超色免费av| 男女高潮啪啪啪动态图| 午夜两性在线视频| 日韩,欧美,国产一区二区三区| 大香蕉久久成人网| 日日爽夜夜爽网站| 久久精品国产a三级三级三级| 日韩三级视频一区二区三区| 人人妻人人澡人人看| 日日夜夜操网爽| 婷婷成人精品国产| 国产亚洲欧美在线一区二区| 亚洲伊人久久精品综合| 色播在线永久视频| 国产亚洲精品第一综合不卡| 国产有黄有色有爽视频| 可以免费在线观看a视频的电影网站| 美国免费a级毛片| 人人妻,人人澡人人爽秒播| 91成年电影在线观看| 99热国产这里只有精品6| 日本av手机在线免费观看| 国产精品一区二区精品视频观看| 考比视频在线观看| 久久天躁狠狠躁夜夜2o2o| av网站在线播放免费| 咕卡用的链子| 国产精品麻豆人妻色哟哟久久| 国产精品av久久久久免费| 菩萨蛮人人尽说江南好唐韦庄| 黄色毛片三级朝国网站| 97在线人人人人妻| 国产精品久久久久久人妻精品电影 | 欧美日韩中文字幕国产精品一区二区三区 | 久久人妻福利社区极品人妻图片| 中文精品一卡2卡3卡4更新| 欧美人与性动交α欧美软件| 少妇被粗大的猛进出69影院| 国产精品偷伦视频观看了| 黑人欧美特级aaaaaa片| 建设人人有责人人尽责人人享有的| 久久天堂一区二区三区四区| 亚洲熟女精品中文字幕| 99国产精品99久久久久| 午夜免费成人在线视频| 永久免费av网站大全| 国产亚洲欧美精品永久| 欧美激情久久久久久爽电影 | 久久人妻熟女aⅴ| 亚洲成国产人片在线观看| 精品久久久精品久久久| 国产野战对白在线观看| 亚洲欧美一区二区三区黑人| 欧美一级毛片孕妇| 亚洲中文字幕日韩| 啦啦啦 在线观看视频| 美女主播在线视频| 亚洲专区字幕在线| 国产不卡av网站在线观看| 日韩大码丰满熟妇| 亚洲欧美日韩高清在线视频 | 日本黄色日本黄色录像| 精品久久久久久电影网| 亚洲va日本ⅴa欧美va伊人久久 | 国产成人免费观看mmmm| 精品福利观看| www.999成人在线观看| 亚洲天堂av无毛| av在线播放精品| 久久人人97超碰香蕉20202| 亚洲人成77777在线视频| 十分钟在线观看高清视频www| 一本久久精品| 热99久久久久精品小说推荐| 国产欧美日韩综合在线一区二区| 无限看片的www在线观看| 国产真人三级小视频在线观看| 纵有疾风起免费观看全集完整版| 淫妇啪啪啪对白视频 | 午夜福利在线观看吧| 亚洲国产精品一区三区| 97在线人人人人妻| 汤姆久久久久久久影院中文字幕| 免费一级毛片在线播放高清视频 | 久久国产精品大桥未久av| 爱豆传媒免费全集在线观看| 麻豆乱淫一区二区| 亚洲精品美女久久久久99蜜臀| 久久香蕉激情| 亚洲va日本ⅴa欧美va伊人久久 | 国产男人的电影天堂91| 欧美亚洲日本最大视频资源| 久久国产精品大桥未久av| 精品久久蜜臀av无| 久久久久久免费高清国产稀缺| 国产在线视频一区二区| 久久久久久亚洲精品国产蜜桃av| 一边摸一边抽搐一进一出视频| av视频免费观看在线观看| 欧美精品av麻豆av| 12—13女人毛片做爰片一| 大香蕉久久网| 欧美日韩亚洲国产一区二区在线观看 | 一级,二级,三级黄色视频| 久久久久久久久免费视频了| 国产野战对白在线观看| 国产黄频视频在线观看| 一本综合久久免费| 午夜福利,免费看| 女警被强在线播放| 亚洲va日本ⅴa欧美va伊人久久 | 久久久国产一区二区| 久久人人爽人人片av| 十八禁网站免费在线| 菩萨蛮人人尽说江南好唐韦庄| 岛国在线观看网站| 18禁国产床啪视频网站| 视频区图区小说| 欧美人与性动交α欧美软件| 在线精品无人区一区二区三| 99久久精品国产亚洲精品| 欧美国产精品va在线观看不卡| 久久毛片免费看一区二区三区| 久久香蕉激情| 9热在线视频观看99| 亚洲第一青青草原| e午夜精品久久久久久久| 成年人午夜在线观看视频| 久久天堂一区二区三区四区| 久久久久久亚洲精品国产蜜桃av| 久久久久国产精品人妻一区二区| 90打野战视频偷拍视频| 91大片在线观看| 欧美日韩黄片免| 国产男女超爽视频在线观看| 久久久久精品国产欧美久久久 | 亚洲一区二区三区欧美精品| 啦啦啦视频在线资源免费观看| 国产成人系列免费观看| 久久久久国内视频| 亚洲中文日韩欧美视频| 人妻 亚洲 视频| 亚洲熟女精品中文字幕| 又黄又粗又硬又大视频| 久久久久精品人妻al黑| 老司机福利观看| 一区二区三区激情视频| 91av网站免费观看| 一级毛片女人18水好多| 美女午夜性视频免费| 嫁个100分男人电影在线观看| 99国产精品99久久久久| 国产不卡av网站在线观看| 国产欧美日韩综合在线一区二区| 亚洲专区国产一区二区| 欧美日韩亚洲综合一区二区三区_| www.熟女人妻精品国产| 精品亚洲乱码少妇综合久久| 一区二区三区精品91| netflix在线观看网站| 久久精品熟女亚洲av麻豆精品| 嫁个100分男人电影在线观看| 大片免费播放器 马上看| 国产精品香港三级国产av潘金莲| 热99re8久久精品国产| 曰老女人黄片| 国产男女超爽视频在线观看| 国产成人av激情在线播放| 极品少妇高潮喷水抽搐| 1024视频免费在线观看| av电影中文网址| 国产精品国产av在线观看| 在线观看免费午夜福利视频| 一级黄色大片毛片| 中文字幕人妻丝袜制服| 国产精品一区二区免费欧美 | 正在播放国产对白刺激| av线在线观看网站| 久久性视频一级片| 丝袜脚勾引网站| 日韩,欧美,国产一区二区三区| 91麻豆av在线| av在线app专区| 最近最新免费中文字幕在线| 在线观看舔阴道视频| 欧美老熟妇乱子伦牲交| 一级片'在线观看视频| 欧美亚洲日本最大视频资源| 男女边摸边吃奶| 国产成人a∨麻豆精品| 人妻 亚洲 视频| 精品一区二区三区四区五区乱码| 一区二区三区激情视频| 亚洲av欧美aⅴ国产| 日日爽夜夜爽网站| 欧美黄色片欧美黄色片| 欧美日韩亚洲国产一区二区在线观看 | 一区二区av电影网| 亚洲成人免费电影在线观看| 欧美精品一区二区大全| 人妻人人澡人人爽人人| 国产精品.久久久| av一本久久久久| 99国产极品粉嫩在线观看| 国产成人影院久久av| 亚洲精品国产av成人精品| 精品乱码久久久久久99久播| 欧美日韩亚洲国产一区二区在线观看 | 亚洲国产av新网站| 一级黄色大片毛片| 纵有疾风起免费观看全集完整版| 日韩免费高清中文字幕av| 美女高潮喷水抽搐中文字幕| 国产成人啪精品午夜网站| 久久久久久久国产电影| 最新的欧美精品一区二区| 午夜福利影视在线免费观看| 在线天堂中文资源库| 欧美国产精品一级二级三级| 国产精品久久久av美女十八| 精品一区二区三区四区五区乱码| 欧美日韩亚洲高清精品| 成人手机av| 亚洲国产精品成人久久小说| 叶爱在线成人免费视频播放| 免费一级毛片在线播放高清视频 | 电影成人av| 91九色精品人成在线观看| 精品乱码久久久久久99久播| 欧美另类一区| 91成人精品电影| 亚洲色图 男人天堂 中文字幕| 女性被躁到高潮视频| av有码第一页| 免费在线观看日本一区| 美女高潮喷水抽搐中文字幕| 欧美精品av麻豆av| 80岁老熟妇乱子伦牲交| 国产男女内射视频| 日本五十路高清| 亚洲天堂av无毛| 狂野欧美激情性bbbbbb| 永久免费av网站大全| 午夜91福利影院| 国产欧美日韩精品亚洲av| 国产男女内射视频| 男人添女人高潮全过程视频| 久久久久久亚洲精品国产蜜桃av| 亚洲精品一区蜜桃| 国产精品秋霞免费鲁丝片| 亚洲熟女毛片儿| 青草久久国产| 老司机亚洲免费影院| 五月开心婷婷网| av天堂久久9| 久久久国产欧美日韩av| e午夜精品久久久久久久| 午夜视频精品福利| 制服诱惑二区| 麻豆乱淫一区二区| 久久久久国内视频| 亚洲精品第二区| 亚洲一码二码三码区别大吗| 真人做人爱边吃奶动态| 国产在线视频一区二区| 人人妻人人添人人爽欧美一区卜| 国产不卡av网站在线观看| 婷婷丁香在线五月| 在线观看一区二区三区激情| 久久久久久亚洲精品国产蜜桃av| 亚洲少妇的诱惑av| 亚洲人成电影观看| 97精品久久久久久久久久精品| 狠狠狠狠99中文字幕| 亚洲国产欧美网| 啦啦啦 在线观看视频| 久久热在线av| 欧美午夜高清在线| 中文字幕最新亚洲高清| 91麻豆精品激情在线观看国产 | 久久亚洲国产成人精品v| 国产成人啪精品午夜网站| 国产日韩欧美在线精品| 婷婷丁香在线五月| 精品国产一区二区三区四区第35| 亚洲精品乱久久久久久| 免费黄频网站在线观看国产| 一本色道久久久久久精品综合| 狠狠精品人妻久久久久久综合| 91国产中文字幕| 久久久久国产精品人妻一区二区| 麻豆av在线久日| 老司机亚洲免费影院| 久久人妻福利社区极品人妻图片| 午夜福利影视在线免费观看| 少妇裸体淫交视频免费看高清 | 91麻豆精品激情在线观看国产 | 捣出白浆h1v1| 一区二区三区四区激情视频| 亚洲成av片中文字幕在线观看| 国产片内射在线| 日韩电影二区| 老司机午夜福利在线观看视频 | 午夜免费观看性视频| 91麻豆av在线| 精品视频人人做人人爽| 国产精品 欧美亚洲| 国产淫语在线视频| √禁漫天堂资源中文www| 日本欧美视频一区| 中文字幕色久视频| 免费少妇av软件| 国产成人免费观看mmmm| 少妇精品久久久久久久| 狂野欧美激情性xxxx| av在线app专区| 老司机午夜十八禁免费视频| 国产精品影院久久| 日韩一卡2卡3卡4卡2021年| 久久久久久久国产电影| 日本wwww免费看| 中文字幕制服av| 成年人黄色毛片网站| 99热全是精品| 香蕉国产在线看| 一边摸一边抽搐一进一出视频| 精品人妻熟女毛片av久久网站| 欧美少妇被猛烈插入视频| 国产精品香港三级国产av潘金莲| 黑人猛操日本美女一级片| 一区福利在线观看| 精品一区在线观看国产| 亚洲男人天堂网一区| 国产一区二区 视频在线| 久久天堂一区二区三区四区| 人人澡人人妻人| 免费在线观看完整版高清| 久久热在线av| 99国产精品免费福利视频| 我的亚洲天堂| 国产精品.久久久| 精品高清国产在线一区| 国产一区二区三区在线臀色熟女 | 人成视频在线观看免费观看| 97人妻天天添夜夜摸| av超薄肉色丝袜交足视频| 精品福利观看| 免费人妻精品一区二区三区视频| 另类精品久久| 波多野结衣一区麻豆| 999精品在线视频| 别揉我奶头~嗯~啊~动态视频 | 国产男人的电影天堂91| 80岁老熟妇乱子伦牲交| 久久人人爽av亚洲精品天堂| 国产在线免费精品| 丰满少妇做爰视频| 亚洲欧美精品自产自拍| 老司机在亚洲福利影院| 欧美亚洲 丝袜 人妻 在线| 激情视频va一区二区三区| 国产精品成人在线| 18禁观看日本| 欧美黄色淫秽网站| 精品一区二区三卡| 欧美另类一区| 十八禁网站免费在线| 窝窝影院91人妻| 亚洲av日韩精品久久久久久密| 精品高清国产在线一区| 欧美激情高清一区二区三区| 岛国在线观看网站| 日韩大码丰满熟妇| 女人被躁到高潮嗷嗷叫费观| 99热网站在线观看| avwww免费| 久久ye,这里只有精品| 亚洲av美国av| 亚洲中文日韩欧美视频| 成年美女黄网站色视频大全免费| 国产精品一区二区精品视频观看| 青草久久国产| 丝瓜视频免费看黄片| 国产区一区二久久| 美女中出高潮动态图| 欧美中文综合在线视频| 男女床上黄色一级片免费看| 亚洲中文日韩欧美视频| 亚洲欧美一区二区三区黑人| 黑人巨大精品欧美一区二区mp4| 999久久久国产精品视频| 一区在线观看完整版| 三上悠亚av全集在线观看| 国产精品秋霞免费鲁丝片| 久久久国产欧美日韩av| 欧美另类亚洲清纯唯美| 亚洲男人天堂网一区| 日日摸夜夜添夜夜添小说| 99精国产麻豆久久婷婷| 老司机亚洲免费影院| 日韩人妻精品一区2区三区| 水蜜桃什么品种好| 久久性视频一级片| 99久久人妻综合| 精品久久蜜臀av无| 国产一卡二卡三卡精品| 一区二区三区四区激情视频| 香蕉丝袜av| 男人爽女人下面视频在线观看| 亚洲av国产av综合av卡| 亚洲精品国产av蜜桃| 色婷婷av一区二区三区视频| 亚洲精品久久成人aⅴ小说| 精品第一国产精品| 91成年电影在线观看| av不卡在线播放| 亚洲精品自拍成人| 免费在线观看影片大全网站| 国产成人免费观看mmmm| 国产av一区二区精品久久| 50天的宝宝边吃奶边哭怎么回事| 久久女婷五月综合色啪小说| 老熟妇乱子伦视频在线观看 | 老熟妇乱子伦视频在线观看 | 水蜜桃什么品种好| 国产欧美日韩一区二区精品| 日本五十路高清| 熟女少妇亚洲综合色aaa.| 黄片小视频在线播放| 狂野欧美激情性xxxx| 久久毛片免费看一区二区三区| 午夜免费鲁丝| 日韩欧美免费精品| 久久久久国产精品人妻一区二区| 在线观看免费午夜福利视频| 亚洲第一av免费看| 黄片播放在线免费| 国产精品av久久久久免费| 97人妻天天添夜夜摸| 欧美日韩视频精品一区| 亚洲少妇的诱惑av| 久久久久精品国产欧美久久久 | 制服人妻中文乱码| 欧美日本中文国产一区发布| 91精品伊人久久大香线蕉| 国产免费福利视频在线观看| 亚洲熟女毛片儿| 亚洲欧美成人综合另类久久久| 搡老岳熟女国产| 亚洲avbb在线观看| 亚洲精品美女久久av网站| 日韩免费高清中文字幕av| 中国美女看黄片| 老司机靠b影院| 亚洲免费av在线视频| 国产精品久久久人人做人人爽| 亚洲 欧美一区二区三区| 肉色欧美久久久久久久蜜桃| 黄网站色视频无遮挡免费观看| 国产成人一区二区三区免费视频网站| 男人添女人高潮全过程视频| 久久久久久久精品精品| 日韩欧美免费精品| 天堂俺去俺来也www色官网| 国产一区二区在线观看av| 国产主播在线观看一区二区| 日日摸夜夜添夜夜添小说| 亚洲国产精品一区二区三区在线| 国产无遮挡羞羞视频在线观看| 极品人妻少妇av视频| 国产av一区二区精品久久| 国产1区2区3区精品| 欧美xxⅹ黑人| 999精品在线视频| 亚洲精品乱久久久久久| 亚洲av美国av| 亚洲专区字幕在线| 两性夫妻黄色片| 一个人免费看片子| 中国国产av一级| 在线天堂中文资源库| 免费日韩欧美在线观看| 一区在线观看完整版| 国产日韩欧美在线精品| 亚洲欧美日韩高清在线视频 | 婷婷丁香在线五月| 国产在线视频一区二区| 老司机亚洲免费影院| 国产欧美日韩一区二区精品| 美女午夜性视频免费| 精品国内亚洲2022精品成人 | 免费黄频网站在线观看国产| 久久综合国产亚洲精品| 中文字幕另类日韩欧美亚洲嫩草| 妹子高潮喷水视频| 在线av久久热| 亚洲成av片中文字幕在线观看| 我要看黄色一级片免费的| 国产色视频综合| 91成人精品电影| 久久综合国产亚洲精品| 欧美xxⅹ黑人| 午夜视频精品福利| 欧美中文综合在线视频| 午夜福利免费观看在线| 国产亚洲av高清不卡| 日韩制服骚丝袜av| 久久亚洲国产成人精品v| 人妻人人澡人人爽人人| 欧美精品一区二区免费开放| 在线观看一区二区三区激情| 久久青草综合色| 狠狠婷婷综合久久久久久88av| 亚洲欧美激情在线| 一本色道久久久久久精品综合| 亚洲久久久国产精品| www日本在线高清视频| 99精品久久久久人妻精品| 国产亚洲av片在线观看秒播厂| 国产日韩一区二区三区精品不卡| 五月开心婷婷网| 欧美精品亚洲一区二区| 少妇人妻久久综合中文| 蜜桃在线观看..| 亚洲精品国产av成人精品| 香蕉国产在线看| 精品一区二区三卡| 美女福利国产在线| 午夜福利在线观看吧| 女人高潮潮喷娇喘18禁视频| 国产高清videossex| 99国产综合亚洲精品| 999久久久精品免费观看国产| 色婷婷av一区二区三区视频| 男人添女人高潮全过程视频| 制服人妻中文乱码| 国内毛片毛片毛片毛片毛片| 国产精品久久久久久精品古装| 亚洲av男天堂| 精品人妻在线不人妻| 精品第一国产精品| 天堂中文最新版在线下载| 中文字幕制服av| 国产片内射在线| 狠狠婷婷综合久久久久久88av| 12—13女人毛片做爰片一| 国产精品 欧美亚洲| 精品国产一区二区三区久久久樱花| 性色av一级| 欧美一级毛片孕妇| videosex国产| 麻豆乱淫一区二区| 91麻豆av在线| 人人妻人人澡人人爽人人夜夜| 一区二区三区激情视频| 成人三级做爰电影| 一个人免费在线观看的高清视频 | 美女大奶头黄色视频| 精品人妻1区二区| 精品国产乱子伦一区二区三区 | 亚洲国产成人一精品久久久| √禁漫天堂资源中文www| 99国产综合亚洲精品| 丝袜在线中文字幕| 高清在线国产一区| 狠狠婷婷综合久久久久久88av| 久久久久久人人人人人| 欧美精品av麻豆av| 国产免费福利视频在线观看| 嫩草影视91久久| 国产精品一区二区免费欧美 | 女人被躁到高潮嗷嗷叫费观| 精品一区在线观看国产| 日本a在线网址|