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。     

柱前衍生化-高效液相色谱法跟踪检测酶法制备L-蛋氨酸反应过程

以邻硝基苯磺酰氯为柱前衍生试剂,RP-HPLC为分离手段,选用Lichrospher C18柱,15 mmol/L的磷酸盐溶液(pH5.0)和乙腈为流动相,梯度洗脱,检测波长230 nm,建立了柱前衍生-高效液相色谱法测定N-乙酰-DL-蛋氨酸和L-蛋氨酸的方法,所有组分于20min内洗脱结束。N-乙酰-DL-蛋氨酸和L-蛋氨酸在2.75～254μmol/L范围内线性良好,检出限分别为153 nmol/L和55 nmol/L。加标回收率分别为101.1%和99.8%,相对标准偏差为1.4%和0.89%。本方法可用于酶法或固定化酶法制备蛋氨酸反应体系的跟踪分析。     

基于毛细管电泳酶微反应器的吉非替尼对表皮生长因子受体抑制率的测定

采用共价键法将表皮生长因子受体固定在毛细管内壁,制备了一种毛细管电泳酶微反应器。结合毛细管电泳高效分离技术,以三磷酸腺苷为底物,吉非替尼为抑制剂,利用表皮生长因子受体酶微反应器在线测定了吉非替尼对皮生长因子受体的抑制性能。结果显示,吉非替尼可竞争性的抑制三磷酸腺苷与表皮生长因子受体的结合,从而影响三磷酸腺苷与表皮生长因子受体的结合量以及产物二磷酸腺苷的生成量。根据不同浓度吉非替尼下三磷酸腺苷峰面积的变化计算了吉非替尼对表皮生长因子受体的抑制率。同时绘制了抑制曲线,得到IC50值为32.44±0.82μmol/L。实验表明该酶微反应器可实际用于酪氨酸激酶抑制剂的抑制率的快速测定。     

多层开管毛细管酶反应器的制备及在蛋白质酶切中的应用

以石英毛细管作为酶固定化的载体, 在毛细管内壁上逐步合成树枝形大分子聚酰胺-胺(PAMAM), 再通过交联剂戊二醛将胰蛋白酶直接键合到该大分子的末端氨基上, 并对酶固定化条件进行了优化, 制备了多层酶反应器. 利用该酶反应器对马心细胞色素C等蛋白质进行了酶切, 并对酶切的条件进行了优化. 实验结果表明, 该固定化酶反应器具有较高的酶切效率、良好的重现性和稳定性, 可用于蛋白质组学的研究.     

纳米沸石修饰微通道反应器内固定化酶催化的水解反应动力学

通过在毛细管内层叠层组装纳米沸石并固定脂肪酶来构建纳米沸石修饰的固定化酶微反应器通道,将纳米沸石良好的生物相容性和高的酶固定能力与微反应器反应效率高、扩散传质快等优点相结合. 以对硝基苯棕榈酸酯的水解作为探针反应对该微反应器内固定化酶催化水解反应动力学进行了研究和计算,并与普通反应器内同样的反应进行比较. 通过对比米氏方程参数,证实在微反应器内酶催化水解反应效率可比普通反应器内提高3倍以上并可提高酶和反应底物的亲和能力.     

乙酰鸟氨酸脱乙酰酶固定化细胞拆分D,L-缬氨酸

报道了一种利用具有乙酰鸟氨酸脱乙酰酶活性的固定化细胞拆分D,L-缬氨酸的新方法. 该酶促反应最适条件: pH=6, 反应温度50 ℃, 底物N-乙酰-D,L-缬氨酸浓度200 mmol/L, 固定化细胞用量0.2 g/mL(或100 U/mL). 0.1 mmol/L CoCl₂条件对该酶促反应有显著的激活作用. 在以上条件下反应2～3 h, 测得产物L-缬氨酸浓度95 mmol/L. 该固定化细胞连续10次使用, 平均转化率为90.8%(以N-乙酰-L-缬氨酸计), 显示出了良好的工业化应用前景.     

氧化石墨烯基质毛细管电色谱胰蛋白酶微反应器的性能研究

基于石墨烯优良的物化性能,利用层层组装法将氧化石墨烯修饰于石英毛细管内壁,制备了氧化石墨烯基质的毛细管电色谱,通过电渗流、拉曼光谱等对其进行表征。在此基础上,基于离子键合法将胰蛋白酶固定于毛细管电色谱柱头,制备胰蛋白酶微反应器。两者结合构成毛细管电色谱胰蛋白酶微反应器。实验结果显示,氧化石墨烯作为基质既可提高样品的分离效率,还能促进胰蛋白酶的催化性能。氧化石墨烯修饰的毛细管电色谱对N-苯甲酰-L-精氨酸乙酯盐酸盐(BAEE)和N-苯甲酰-L-精氨酸(BA)混合物的分离度从裸毛细管的3.70提升至4.71,而其固定化酶活性(米氏常数K_m=1.10 mmol/L,最大反应速率V_(max)=0.32 mmol·L~(-1)·s~(-1))也明显优于裸毛细管(K_m=109.77 mmol/L,V_(max)=0.000 46 mmol·L~(-1)·s~(-1))。利用所制备的微反应器从10种中药材中筛选胰蛋白酶抑制剂活性成分的药材,结果发现三七和大黄中均存在胰蛋白酶抑制剂活性成分。     

亲和毛细管电泳研究谷氨酸底物循环双酶反应

近年来,毛细管电泳(CE)~([1])以及固定化的酶反应器~([2])在生命体系中的酶催化反应以及药物的高通量筛选等研究中得到广泛的应用.本实验制备了一种新型的双酶微反应器,研究了谷氨酸底物循环反应,对临床医学、神经生物学以及食品化学的谷氨酸测定研究具有重要意义.     

基于DNA定向固定化技术构建毛细管固定化酶微反应器的研究进展

生物酶影响着物质代谢和质能转换等生命活动,生物体内某些酶的活性变化会导致疾病的发生。发展新型的酶分析方法对深刻理解生物代谢过程、疾病诊断和药物研发等具有重要意义。毛细管电泳（CE）具有分离效率高、分析速度快、操作简单和样品消耗少以及可与多种检测手段联用等优点,在酶分析研究中越来越受到关注。CE酶分析主要包括离线和在线两种模式,其中,固定化酶微反应器与毛细管电泳联用（CE-IMER）的在线酶分析已经成为主要的酶分析方法之一。CE-IMER充分结合了固定化酶和CE的优势,将游离酶固定在毛细管内,不仅可以显著提高酶的稳定性和重复使用性,而且可以实现纳升规模溶液的自动化酶分析,进而显著降低酶分析成本。目前已有大量方法制备IMER用于CE酶分析,然而如何构建性能良好、可再生使用、酶固载量大、自动化程度高的CE-IMER一直是该领域重点研究的问题。DNA定向固定化技术（DDI）可以充分利用DNA分子的碱基互补配对（A-T,C-G）,在温和的生理条件下特异性固定生物大分子。由于短链双螺旋DNA分子具有较强的机械刚性和物理化学稳定性,通过DDI将酶固定在载体表面,有利于降低传质阻力,提高酶与底物的接触能力,进而促进酶促分析过程。该文主要综述了利用DDI构建新型IMER在CE酶分析中的应用现状,并对其未来发展进行了展望。     

固定化酶纳升微反应器用于痕量蛋白质快速肽谱分析的研究

利用毛细管作为酶固定化的载体,将酶直接键合到毛细管内壁,制成毛细管纳升反应器,结合质谱分析水解产物,获得了蛋白质的肽谱.实验发现,以毛细管为反应器后,蛋白质肽谱分析所需量大大减少,只需10^-13mol,甚至几个10^-15mol的量就可满足分析要求.     

Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca²⁺ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86 s residence time in the μIMER followed by a wash step).     

Indirect Measurement of Yoctomole Alkaline Phosphatase by Capillary Electrophoresis with Electrochemical Detection

A method for indirectly detecting yoctomole (ymol) alkaline phosphatase was developed by capillary electrophoresis with electrochemical detection. In this method, disodium phenyl phosphate was used as the enzyme substrate and the product (phenol) of its hydrolysis reaction catalyzed by alkaline phosphatase was detected at the carbon fiber electrode. The optimum conditions of detection are 1.0×10^?2 mol/L Na₂B₄O₇ (pH 9.8) for the running buffer; 1.00×10^?3 mol/L disodium phenyl phosphate for the enzyme substrate; 20.0 kV for the separation voltage; 5 kV and 10 s for the injection voltage and injection time; 1.05 V (vs. saturated calomel electrode) for the detection potential and 10 min for the incubation time, respectively. In order to enhance the ratio of signal to noise, the shape and size of the working electrode, the shape of detection end of the capillary, and the capillary/electrode alignment method were studied in detail. When a single carbon fiber microcylinder electrode of 6 μm, a capillary of 10 μm ID with the etched detection end and the in‐capillary alignment were used, a ymol mass limit of detection for alkaline phosphatase was achieved.     

Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis

A novel kind of hydrophilic monolith based immobilized enzyme reactors (IMERs) was prepared both in UV-transparent capillaries and on glass microchips by the photopolymerization of N-acryloxysuccinimide and poly(ethylene glycol)diacrylate, followed by trypsin immobilization. The performance of capillary IMERs for protein digestion was evaluated by the digestion of myoglobin with the residential time from 12s to 71 s. With μRPLC-ESI-MS/MS analysis, the obtained sequence coverages were all over 80%, comparable to that obtained by in-solution digestion for 12 h. The nonspecific absorption of BSA on monolithic support was evaluated, and no obvious protein residue was observed by a fluorescence assay. Moreover, no carry-over of the digests on the capillary IMER was found after the digestion of myoglobin (24 μg) and BSA (9 μg), which further demonstrated the good hydrophilicity of such matrix. In addition, an integrated microchip-based system involving on-line protein digestion by microchip-based IMER, peptides separation by nanoRPLC and identification by ESI-MS/MS was established, by which a mixture of standard proteins and one RPLC fraction of Escherichia coli extract were successfully identified, indicating that the hydrophilic monolith based IMER might provide a promising tool for high-throughput proteomic analysis.     

Polymethacrylate-based monolithic capillary column with weak cation exchange functionalities for capillary electrochromatography

Polymethacrylate-based monolith with weak cation exchange functionalities was prepared in capillary column (i.d. 100 μm, o.d. 375 μm) by in situ polymerization of butyl methacrylate, ethylene dimethacrylate and N-methacryloyl-L-glutamic acid in presence of porogens. The porogen mixture included N,N-dimethyl formamide and phosphate buffer. The preparation procedure of stationary phase contained the synthesis of monomer, silanization of capillary inner wall and in situ polymerization. The use of amino acid based monomer for the monolith synthesis is one of the originalities of this novel approach. N-methacryloyl-L-glutamic acid has two carboxyl functionalities. The separation of the solutes were performed at different acetonitrile/phosphate buffer and acetonitrile/sodium hydroxide contents. The applied voltage for the alkyl benzenes was changed between +5 and +30 kV. CEC separations of alkyl benzenes, acidic, basic, phenolic and some polycylic aromatic compounds were succesfully performed under capillary-electrochromatography mode with cathodic electroosmotic flow.     

Rapid analysis of unsaturated acidic xylooligosaccharides from kraft pulps using capillary zone electrophoresis

Several acidic xylooligosaccharides containing unsaturated “hexenuronic acid” units, i.e. 4-deoxy-L -threo-hex-4-enopyranosy-lurinic acid (4-ΔU) units, were separated as their alditol derivatives by capillary zone electrophoriesis in 438 mM borate buffer (pH 10.3) and were detected selectively at the μM level on-column UV detection at 232 nm. These acidic oligosaccharides were obtained from birch and pine kraft pulps on enzymatic hydrolysis with endoxylanases and subsequent treatment with other Trichoderma reesei enzymes. Under the conditions empolyed, acidic 4-ΔU-containing xylooligosaccharides with a molecular size renging from trisaccharides up to nonasaccharides could be separated. Oligosaccharides with higher molecular mass were detected first. Two 4-ΔU-xylotetraose isomers, with the 4-ΔU-group linked to different xylose units in the iligosaccharide backbone, could be resolved from each other with a resolution of about 1. By using a disaccharide (4-ΔU α-(1 → 4) linked to N-acetyl glucosamine) as a model compound the minimum detectable concentration was determined as 10 μM.     

A technique for capillary joining in capillary electrophoresis

Summary Aspects of cracking and joining capillaries have been investigated. Capillary coupling was achieved using various methods. The most successful used hydrofluoric acid-etched capillaries to form male and female ends which were then joined together. This type of joint was used to connect sections of capillary of similar and different internal diameters with minimal loss in resolution, peak width and number of theoretical plates. (Uridine and hypoxanthine was used as a test mixture). For hypoxanthine on a 50 m/50 m etched joined capillary 10 cm from the detector window the number of theoretical plates was 96.6% of that for hypoxanthine on an unbroken capillary. Following the relative success of capillary joining, a coupled capillary flowcell (50 m/200 m) was produced and evaluated.     

Simultaneous analysis of metformin and cyanoguanidine by capillary zone electrophoresis and its application in a stability study

A capillary zone electrophoresis method was established for stability study of metformin (MET). MET and cyanoguanidine (CGN; a major degradation product) were well separated (with a resolution of 38.9) in 40 mM citrate buffer (pH 6.7) using a fused‐silica capillary with an effective length of 60 cm and an inner diameter of 50 μm, injection at 50 mbar for 5 s at 30°C with an applied voltage of 15 kV and diode array detection at 214 nm. Method validation showed good linearity (r² > 0.99), precision (%RSDs < 1.98%), and accuracy (%recovery between 98.3 and 100.9%). Limits of detection and quantification were <30 and 100 μg/mL, respectively. The method was robust upon alteration of pH and voltage (%RSDs < 1.99%). Stability profiles of metformin from 11 stress conditions and the degradation kinetics could be established, using the simple capillary zone electrophoresis system. A mechanism for the degradation of MET was also proposed. MET was stable in neutral hydrolysis, but degraded under alkaline hydrolysis and oxidation. Under both conditions, CGN was quantified as the degradation product. An assay of MET in raw material and tablets showed that content of the drugs in all samples met the requirements of pharmacopeias and CGN was not detected.     

An electroosmotic pump for packed capillary liquid chromatography

An electroosmotic pump (EOP) capable of generating pressure above 3 MPa and μl/min flow rate with reverse phase mobile phases of HPLC was constructed and evaluated. The pump consisted of three parallel connected fused silica capillary columns (25 cm×320 μm I.D.) packed with 2 μm silica materials, hollow electrodes, a high voltage DC power supply, and a liquid pressure transducer. The EOP was applied in a capillary liquid chromatographic system for mobile phase delivery instead of a mechanical pump. Standard samples containing thiourea, naphthalene, anthracene, phenanthrene and acetonitrile were separated on a 15 cm×320 μm I.D. 5 μm Chromasil C₁₈ packed capillary column with acetonitrile/water as mobile phase.