Immobilized capillary enzyme reactor based on layer-by-layer assembling acetylcholinesterase for inhibitor screening by CE

A method for creating an immobilized capillary acetylcholinesterase (AChE) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. The unique capillary AChE reactor was easily prepared by the instrument in three steps: first, a 0.5 cm long plug of a solution of the cationic polyelectrolyte polydiallyldimethylammonium (PDDA) was injected into the capillary to produce a positively charged coating on the surface of the capillary; subsequently, the enzyme solution with the same plug length was injected into the capillary and incubated for 10 min to immobilize the enzyme on the capillary wall via electrostatic interaction; third, PDDA solution with the same plug length was injected again into the capillary to cover the immobilized enzyme by forming PDDA-AChE-PDDA sandwich-like structure. The enzyme reactor can be easily renewed after removing the immobilized enzyme by flushing the column with 1 M NaCl solution. Activity of the immobilized enzyme can be assayed simply by carrying out an electrophoretic separation, i.e., the substrate solution was injected and incubated for a short time, followed by applying a voltage to separate the product from the unreacted substrate. The measured peak area of the product then represented the enzyme activity. For enzyme inhibitor screening, the mixture solution of the substrate and the inhibitor was injected and assayed the reduction of the enzyme activity. The immobilized enzyme could withstand 100 consecutive assays by only losing 10% activity. The reproducibility in terms of time-to-time, day-to-day, and batch-to-batch was measured with RSD% less than 4.7%. Furthermore, the screening system was validated by a known inhibitor. Finally, screening a small compound library containing four known AChE inhibitors and 42 natural extracts was demonstrated, and species with inhibition activity can be straightforwardly identified with the system.     

On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts by capillary electrophoresis

In this study we developed a simple capillary electrophoresis (CE) method with an on-line acetylcholinesterase (AChE) microreactor at the inlet of capillary for inhibitor screening. The fused-silica capillary surface was modified with a polycationic polyethylenimine coating. Solutions of the enzyme and chitosan were then injected to immobilize the enzyme in approximately 2.9?cm of the capillary inlet (total length of capillary 60.2?cm) by electrostatic interaction and the film overlay technique. Separation of enzyme reaction product (thiocholine, ThCh) and unreacted substrate (acetylthiocholine, AThCh) was achieved within 3.0?min. The conditions affecting the efficiency of reaction of the enzyme were optimized by measuring the peak area of ThCh. Under the optimum conditions, using Huperzine-A as model inhibitor, K (i) and IC (50) were 0.551?μmol?L(-1) and 1.52?μmol?L(-1), respectively, for immobilized AChE. Finally, screening of a small compound library containing two known AChE inhibitors and 30 natural extracts was conducted, and species with inhibition activity were directly identified. Compared with previous publications on screening for AChE inhibitors in natural products based on CE methods, the method developed in this work has the advantages of lower cost per analysis, less leakage, and better bioaffinity for the immobilized enzyme because of the unique properties of sodium alginate and chitosan.     

Improved peak capacity for CE separations of enzyme inhibitors with activity‐based detection using magnetic bead microreactors

A technique for separating and detecting enzyme inhibitors was developed using CE with an enzyme microreactor. The on‐column enzyme microreactor was constructed using NdFeB magnet(s) to immobilize alkaline phosphatase‐coated superparamagnetic beads (2.8 μm diameter) inside a capillary before the detection window. Enzyme inhibition assays were performed by injecting a plug of inhibitor into a capillary filled with the substrate, AttoPhos. Product generated in the enzyme microreactor was detected by LIF. Inhibitor zones electrophoresed through the capillary, passed through the enzyme microreactor, and were observed as negative peaks due to decreased product formation. The goal of this study was to improve peak capacities for inhibitor separations relative to previous studies, which combined continuous engagement electrophoretically mediated microanalysis and transient engagement electrophoretically mediated microanalysis to study enzyme inhibition. The effects of electric field strength, bead injection time and inhibitor concentrations on peak capacity and peak width were investigated. Peak capacities were increased to ≥20 under optimal conditions of electric field strength and bead injection time for inhibition assays with arsenate and theophylline. Five reversible inhibitors of alkaline phosphatase (theophylline, vanadate, arsenate, L ‐tryptophan and tungstate) were separated and detected to demonstrate the ability of this technique to analyze complex inhibitor mixtures.     

Preparation of a dual‐enzyme co‐immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis

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.     

Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking

A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross‐linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short‐end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis–Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half‐maximal inhibitory concentration of kojic acid was 5.55 μM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study.     

Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in‐capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β‐nicotinamide adenine dinucleotide 2′‐phosphate hydrate dissolved in the reaction buffer 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 applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β‐nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver–Burk equation, the Michaelis–Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.     

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis–Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half‐maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.     

毛细管电泳法测定天麻素对乙酰胆碱酯酶活性的抑制

阿尔茨海默症（AD）是引起中老年人痴呆最常见的疾病。目前治疗AD的药物主要为乙酰胆碱酯酶抑制剂（AChEI）。建立快速地从天然产物中筛选AChEI的方法,将对临床治疗AD产生积极的作用。该研究建立了一种简单可靠的AChEI筛选新方法。通过海美溴铵在毛细管内壁形成一段正电荷涂层,再经过离子吸附作用制备1.5 cm长的乙酰胆碱酯酶（AChE）反应器。底物碘化乙酰硫代胆碱在0.015 MPa压力下进样10 s,在微酶反应器中停留1 min后采用毛细管电泳（CE）法对底物和酶解产物进行分离。天麻素是天麻的重要药效成分之一,对AChE具有抑制作用。该研究以天麻素为例,根据加入药物前后酶解产物峰面积的差异,完成天麻素对AChE活性的抑制能力的测定。结果表明,随着天麻素浓度的增加,产物峰面积逐渐减小,对AChE的活性抑制变大。该方法所建微酶反应器产物峰面积的RSD值小于5.3%,可连续使用300次。当天麻素浓度为5.24 μmol/L时,对AChE活性抑制率达到64.8%。根据加入不同浓度天麻素时的抑制率,测定出天麻素的IC₅₀值为（2.26±0.14）μmol/L（R²=0.9983）。与传统紫外分光光度法所得结果（2.09±0.18）μmol/L吻合较好。固定化酶微反应器的活性变差时,可以洗脱掉固定在柱上的AChE,重复酶的固定化步骤即可完成再生。该方法简单、高效,运行成本低,柱上固定的AChE酶反应器稳定性较好,可重复使用,极大地提高了工作效率,未来有望应用于各类AChEI的高通量筛选,对AD药物的研发具有积极作用。     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

Dual‐enzyme,co‐immobilized capillary microreactor combined with substrate recycling for high‐sensitive glutamate determination based on CE

A new method for high‐sensitive determination of glutamate was developed and evaluated based on CE by using dual‐enzyme co‐immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co‐immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet‐visible spectroscopy, and fluorescence spectroscopy. The GDH‐GPT co‐immobilized capillary microreactor showed great stability and reproducibility. The apparent K_m for glutamate with GDH‐GPT coupled reaction was determined to be 0.61±0.06 mM but 2.56±0.24 mM when only GDH was immobilized. Glutamate determination was based on on‐column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co‐immobilized GDH‐GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7‐fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 μM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples.     

Development of an enzymatic microreactor based on microencapsulated laccase with off-line capillary electrophoresis for measurement of oxidation reactions

Microencapsulation is used here as a new technique to immobilize enzymes in a microreactor coupled off-line to capillary electrophoresis (CE), allowing the determination of enzymatic reaction products. The redox enzyme laccase was encapsulated using the method of interfacial cross-linking of poly(ethyleneimine) (PEI). The 50 μm diameter capsules were slurry packed from a suspension into a capillary-sized reactor made easily and quickly from a short length of 530 μm diameter fused-silica tubing. The volume of the bed of laccase microcapsules in the microreactor was in the order of 1.1 μL through which 50 μL of the substrate o-phenylenediamine (OPD) was flowed. The oxidation product 2,3-diaminophenazine (DAP) and the remaining OPD were quantified by CE in a pH 2.5 phosphate buffer. Peak migration time reproducibility was in the order of 0.4% RSD and peak area reproducibility was less than 1.7% RSD within the same day. Using the OPD peak area calibration curve, a conversion efficiency of 48% was achieved for a 2-min oxidation reaction in the microreactor.     

Evaluation of xanthine oxidase inhibitory activity of flavonoids by an online capillary electrophoresis-based immobilized enzyme microreactor

Xanthine oxidase (XOD) is a key enzyme in the human body to produce uric acid, and its inhibitor can be used for the treatment of hyperuricemia and gout. In this study, an online CE-based XOD immobilized enzyme microreactor (IMER) was developed for the enzyme kinetics assays and inhibitor screening. After 30 consecutive runs, the XOD activity remained about 95.6% of the initial immobilized activity. The Michaelis–Menten constant (K_m) of the immobilized XOD was determined as 0.39 mM using xanthine as substrate. The half-maximal inhibitory concentration and inhibition constant of the known inhibitor 4-aminopyrazolo[3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2 μM, respectively. Then, the developed method was applied to evaluate the XOD inhibitory activity of 10 flavonoids, which indicated that dihydroquercetin, quercetin, biochanin A, and epicatechin had significant inhibitory effect on XOD. In addition, molecular docking results verified that the binding energy of the flavonoids with enzyme were in line with their inhibitory activity determined by XOD–IMER. Therefore, the developed XOD–IMER is a potential tool for the primary screening of XOD inhibitors from natural products.     

Screening of acetylcholinesterase inhibitors in natural extracts by CE with electrophoretically mediated microanalysis technique

An electrophoretically mediated microanalysis (EMMA) method for screening acetylcholinesterase (AChE) inhibitors in natural extracts is described. In this method, solutions of AChE and the mixture of the substrate and the natural extract were successively injected into the capillary, and mixed electrophoretically by applying a voltage for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at UV 230 nm represents the enzyme activity. Since the extract is mixed with the substrate, there is no need to separate the components before testing the inhibition. The inhibitory activity of the natural extract as a whole can be easily found if the peak area of the product is reduced. This makes the present method suitable for screening inhibitors in complex mixtures, such as natural extracts. Compared to the commonly used spectrometric method for screening of AChE inhibitors, the major advantage of the present method is the elimination of Ellman reagent, which is essential for the spectrometric method. This not only simplifies the experimental procedure but also minimizes false-positive results. Moreover, it is an obvious advantage of combining the separation power with the on-column enzyme assay for further investigating which compound(s) is/are responsible for the inhibition. The method was validated using a commercially available AChE inhibitor tacrine and a small chemical library containing four AChE inhibitors and 32 natural extracts. Inhibitors in natural extracts were identified with the present method.     

"Inject-mix-react-separate-and-quantitate" (IMReSQ) method for screening enzyme inhibitors

Many regulatory enzymes are considered attractive therapeutic targets, and their inhibitors are potential drug candidates. Screening combinatorial libraries for enzyme inhibitors is pivotal to identifying hit compounds for the development of drugs targeting regulatory enzymes. Here, we introduce the first inhibitor screening method that consumes only nanoliters of the reactant solutions and is applicable to regulatory enzymes. The method is termed inject-mix-react-separate-and-quantitate (IMReSQ) and includes five steps. First, nanoliter volumes of substrate, candidate inhibitor, and enzyme solutions are injected by pressure into a capillary as separate plugs. Second, the plugs are mixed inside this capillary microreactor by transverse diffusion of laminar flow profiles. Third, the reaction mixture is incubated to form the enzymatic product. Fourth, the product is separated from the substrate inside the capillary by electrophoresis. Fifth, the amounts of the product and substrate are quantitated. In this proof-of-principle work, we applied IMReSQ to study inhibition of recently cloned protein farnesyltransferase from parasite Entamoeba histolytica. This enzyme is a potential therapeutic target for antiparasitic drugs. We identified three previously unknown inhibitors of this enzyme and proved that IMReSQ could be used for quantitatively ranking the potencies of inhibitors.     

Rapid on‐line system for preliminary screening of lipase inhibitors from natural products by integrating capillary electrophoresis with immobilized enzyme microreactor

An on‐line system for preliminary screening lipase inhibitors from natural products with an immobilized lipase microreactor coupled to capillary electrophoresis was established. In this research, the lipase was anchored on the amino activated capillary inner wall using glutaraldehyde as a homobifunctional linker through Schiff base reaction. The immobilized lipase activity was evaluated by measuring the peak area of the hydrolyzate of p‐nitrophenyl acetate. In order to maintain the enzymatic activity of immobilized lipase, the acetonitrile content and the pH of the reaction solution were also optimized. Under the optimized reaction conditions, the Michaelis–Menten constant of the immobilized lipase and the half maximal inhibitory concentration for orlistat were studied, which were consistent with previous literature data. Furthermore, the developed method was applied to screen lipase inhibition activity from ten natural products. As a result, we found that six natural products have inhibitory effect on the activity of lipase, among which the inhibitory effect of Rhizoma atractylodis extract has never been reported before.     

Ultramicro enzyme assays in a capillary electrophoretic system.

This paper describes an ultramicro method for achieving enzyme assays. Enzyme saturating concentrations of substrate, coenzyme when appropriate, and running buffer were mixed and used to fill a deactivated fused-silica capillary in a capillary zone electrophoresis apparatus. The enzyme glucose-6-phosphate dehydrogenase was injected by either electrophoresis or siphoning and mixed with the reagents in the capillary by electrophoretic mixing. Enzyme activity was assayed by electrophoresing the product, reduced nicotinamide adenine dinucleotide phosphate, to the detector where it was detected at 340 nm. Under constant potential, the transport velocity of enzyme and the product was generally different. This caused product to be separated from the enzyme after it was formed. Because product formation was much faster than the rate of enzyme-product separation, product accumulated. The amount of accumulated product was inversely related to operating potential. In the extreme case, the operating potential was zero. Zero potential assays were generally carried out by electrophoresing the enzyme partially through the capillary and then switching to zero potential. This capillary was left at zero potential for several minutes to allow additional product to accumulate. After this additional amplification step, potential was again applied and the product transported to the detector. Product formed under constant potential appears as a broad peak with a flat plateau. When the voltage is switched to zero at intermediate migration distance, a peak will be observed on top of this plateau. Either the eight of the plateau or the area of the peak may be used to determine enzyme concentration. The lower limit of detection was 4.6.10(-17) mol of glucose-6-phosphate dehydrogenase.     

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

CE was used for the first time to study the two‐substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine‐5′‐triphosphate were in‐capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine‐5′‐triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero‐potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine‐5′‐diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose‐1,6‐bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in‐capillary mixed using an electrophoretically mediated microanalysis approach.     

Screening neuraminidase inhibitors from glycosaminoglycan and natural extract by capillary electrophoresis

An electrophoretically mediated microanalysis (EMMA) method for screening neuraminidase inhibitors in depolymerized glycosaminoglycan and natural extracts is described. In the present method, enzyme and substrate were individually introduced into the capillary as distinct plugs, and then mixed for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at 214 nm represents the enzyme activity. The electrophoretic conditions for the enzyme reaction and separation of substrate and product were optimized in this study. Under the optimal conditions, the Michaelis–Menten constant and the inhibitive mechanism of zanamivir were studied, which agreed with the literature data. Furthermore, the inhibitory ratios of enzymatic activity of depolymerized glycosaminoglycan and traditional Chinese drugs were determined. The EMMA method has superiority over traditional assay methods, in not only minimizing the false-positive results but also in simplifying the experimental procedure. Therefore, it could be employed to screen inhibitors from natural sources.     

Development of a novel electrochemical biosensor based on catalytic properties of adenosine deaminase immobilized on graphene oxide/carboxymethyl chitosan/multi-wall carbon nanotube platform

A new type of biosensor was designed based on Adenosine deaminase (ADA) immobilized on graphene oxide (GO)/carboxymethyl chitosan (CMC)/multi-wall carbon nanotube (MWCNT) platform nanostructure, fabricated and successfully applied (utilized) in Adenosine detection. Square wave voltammetry was used to study the biosensor catalytic activity. Morphological analysis of the nanostructure was performed by AFM and SEM methods. The results provided here proved that utilizing GO/CMC/MWCNT leads to effective immobilization of ADA which was confirmed by the long term stability of the biosensor during examined intervals. The immobilized ADA activity was examined and the kinetic parameters (K _m and V _max) were found to be 47.5 μM and 5.8 μM min^?1, respectively. Furthermore, benznidazole was introduced as a potent ADA inhibitor using virtual screening. Outstanding inhibition characteristics of benznidazole was observed against ADA. ADA inhibition by benznidazole was non-competitive with the inhibition constant of 0.42 μM. Such an interesting template with an easy preparation process with low cost can provide a novel matrix for developing biosensors and biocatalysts based on enzyme immobilization.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。