基于微流控芯片电泳的生物分子间相互作用研究*

用合适的手段表征生物分子的相互作用对于深刻理解生命过程的本质以及进行医药开发都具有重要意义。将微流控芯片和毛细管电泳相结合的微流控芯片电泳技术具有快速、高效、高通量、样品用量少和易于整合等诸多优势。本文对近年来进行生物分子间相互作用结合常数测定以及结合动力学研究的微流控芯片电泳分离模式、分析方法和芯片检测方法分别做了介绍;简单对比了微流控芯片技术和微阵列生物芯片生物分子间相互作用研究技术;最后分析了微流控芯片技术目前的不足,并对其未来的发展进行了展望。     

毛细管区带电泳检测九州虫草核苷类成分

1　引　　言核苷是参与遗传信息储存、传递与表达的核酸“构件”分子 ,亦是生物氧化、能量代谢中的能源物质和抗病毒、抗肿瘤药物的中间体。虫草素和腺苷作为虫草属重要的核苷活性成分 ,具有显著的药理作用。高效毛细管电泳应用于中药活性组分的分离、鉴定具有极大的优越性 ,是中药现代化的有益探索。本文采用毛细管区带电泳检测虫草素 ,首次报道了九州虫草 (Ｃｏｒｄｙｃｅｐｓｋｙｕｓｈｕｅｎｓｉｓ)子座中虫草素的存在 ,测定了虫草素、腺苷的含量 ,并初步确定了部分核苷及相关碱基的特征峰。2　实验部分2 .1　仪器与试剂　Ｐ ＡＣＥＴＭ…     

毛细管电泳药物筛选方法与技术

毛细管电泳（CE）在新药研发领域显示着重要的应用前景。CE使用水溶液介质作为实验体系,保证了药物筛选在类似于生命介质的环境中进行,优于其他传统体外仪器筛选方法。除了维持被筛选分子和作用对象的生物活性外,CE筛选过程着重突出配体与受体之间的相互作用。毛细管电泳药物筛选瞄准与药理学理论相关的重要参数,如结合常数K_b 、结合速率常数K_on 和解离速率常数K_off ,有利于模拟并预测机体内靶标与药物之间的相互作用过程。该文回顾了毛细管电泳进行药物筛选的历史,评述了毛细管电泳药物筛选方法所依据的理论和相对成熟的各种常用方法,并抽取了部分典型实例以及相关技术进行说明,对以亲和毛细管电泳、动力学毛细管电泳为手段的药物筛选方法进行了介绍,包括分子和细胞层次的药物筛选,以及针对不同类型的候选药物的研究工作都有提及。毛细管电泳与多种技术的联用,包括与质谱以及化学发光等联用发挥了更大的效能。联用方法还应用于中药有效成分的筛选。毛细管电泳在DNA编码化合物库筛选中将有良好应用前景。馏分收集的发展为筛选药物提供了广阔前景,它配合指数富集配体系统进化技术为毛细管电泳药物筛选提供了更多可能。总之,毛细管电泳多样可选的药物筛选方法和技术将为新概念的药物筛选与药物评价提供有力支撑。     

pH对氟喹诺酮药物与BSA之间相互作用影响的研究

采用毛细管区带电泳法,通过测定在不同pH值、不同牛血清白蛋白(BSA)浓度缓冲溶液的条件下药物迁移时间的变化,并分别计算出了pH为6.8、7.4和8.0时培氟沙星、左氧氟沙星、氧氟沙星、环丙沙星等四种氟喹诺酮类药物与BSA相互作用的结合常数.结果表明:pH对结合常数有较大影响,四种药物分子结合常数的最大值均出现在pH=6.8时,并随着pH的增大,结合常数值明显下降.根据实验结果,还对四种氟喹诺酮类药物与BSA之间相互作用的类型、作用位置进行了分析探讨.研究结果对于进一步阐明药用机理并迅速开发出更高效的广谱抗菌药物具有较强的理论意义.     

新多肽配体GE11与表皮生长因子受体的结合能力分析

应用亲和毛细管电泳（ACE）分析方法,对表皮生长因子受体（EGFR）和新多肽配体GE11之间的结合能力进行分析。结果表明,EGFR与多肽配体GE11之间存在特异性相互作用,考察EGFR在不同浓度GE11溶液中的迁移情况,采用非线性、双倒数、Y-倒数和X-倒数4个数据处理方法得到较好的数据拟合,并测得结合常数。该文为筛选多肽配体以及测定受体与多肽配体之间的结合常数提供了简便的方法,将有力推动肿瘤靶向药物输送的研究。     

毛细管区带电泳分离麦芽寡糖的研究

高效毛细管电泳是近年来分离科学中发展最为迅速的方法之一．它具有分离效率高、所需样品量极少、适合于生物大分子的分离测定和自动化程度高等特点．在毛细管电泳中，区带电泳作为主要的分离模式发挥了重要的作用［1］．本文中以麦芽寡糖类物质为对象，研究了离子流体动力学半径在分离中的作用和规律，提出了麦芽寡糖的迁移时间、淌度与其所含糖基数目的线性关系，并在不同的体系与操作电压下得到了验证．同时，也为利用毛细管电泳技术估算麦芽寡糖所含糖基数提供了简易的途径．1实验部分自行组装毛细管电泳电化学检测系统，高压电源0～30k…     

杯芳烃衍生聚合物用作毛细管气相色谱固定相的研究

合成了两种不同构象的杯芳烃衍生聚合物（杯芳冠醚聚硅氧烷ｃ－ｃ［４］ｃ５－ｐｓｏ和二乙氧基杯［４］芳烃聚硅氧烷ｐ－ｃ［４］－ｐｓｏ），并将其用作毛细管气相色谱固定液，使用温度高达３２０℃，对不同的极性化合物，特别是对酚类异构体有很好的分离能力。同时也研究了分离机理。     

二元络合体系的毛细管电泳相互作用分析

建立了一种利用毛细管电泳相互作用测定络合比为1：2的二元络合体系各级络合常数。依据所设计的毛细管电泳二级峰漂移模型,利用间接紫外检测方法,在运行电压为30kV、缓冲溶液组成为咪唑和醋酸（pH5．50）的条件下,测定了Mg^2＋与草酸体系的相互作用参数,求出了Mg^2＋与草酸的一级条件稳定常数的对数值为lgK1=3．40,二级条件稳定常数对数值lgK2=0．90,与文献值具有较好的一致性。     

药物与细胞膜相互作用的毛细管电泳方法研究

以细胞膜在毛细管内构成假固定相,建立一种基于毛细管电泳测定药物与细胞膜相互作用参数的方法．以西酞普兰和兔红细胞膜为相互作用模型,以不同浓度的细胞膜混悬液为电泳缓冲液,采用峰漂移法,并结合Scatchard分析,测得西酞普兰与兔红细胞膜的结合常数为0．977g^-1·L．该方法简单、快速,为研究药物与细胞膜的相互作用提供了新的技术手段,为高通量筛选药物膜通透性和活性,以及评价药物在体内吸收提供一种新的方法．     

核盘菌多糖的构象及其构象变化的研究

研究了一种由Ｓｃｌｅｒｏｔｉｎｉａｓｃｌｅｒｏｔｉｏｒｕｍｓｓ－００１产生的具有潜在抗肿瘤活性的微生物胞外多糖──核盘菌多糖的溶液行为及构象变化．通过对其在不同离子强度和不同ｐＨ值下［η］值的考察，发现其在不同离子强度下和一定的ｐＨ范围内溶液行为稳定．但当ｐＨ＞１２．３６时其［η］值急剧变小．利用透射电镜对样品在不同条件下分子形貌的研究表明，这种溶液行为的变化是由分子构象的变化产生的     

亲和毛细管电泳测定孕酮与其单克隆抗体的结合常数

采用亲和毛细管电泳的配体分离模式,以激光诱导荧光作为检测手段,测定了荧光素标记的孕酮与孕酮我隆抗体之间的结合常数,并研究了温育时间、电泳条件等因素对测定的影响。     

毛细管区带电泳法考察牛凝血酶、核酸适配体与丹参活性分子间的相互作用

采用毛细管区带电泳法(CZE)研究牛凝血酶(B-Thr)、凝血酶核酸适配体(Apt29)与丹参中3种活性物质(丹参素钠(SAS)、原儿茶酸(PA)、阿魏酸(FA))间的相互作用。电泳条件为:毛细管长度40 cm,紫外检测波长214 nm,分离电压15 kV,压力进样3.45 kPa,进样时间5 s。分别固定3种活性药物分子的浓度,改变B-Thr或Apt29的浓度,采用Scatchard方程和非特异性结合方程计算结合常数(K_b),表征药物分子和B-Thr或Apt29的相互作用大小。结果表明,PA与B-Thr结合能力最强,K_b为3.39×104L/mol,FA与B-Thr的K_b为1.05×104L/mol,SAS与B-Thr未表现出结合能力;在与Apt29的相互作用的研究中显示,SAS基本未与Apt29结合,FA与Apt29结合能力强于PA与Apt29的结合能力,FA、PA与Apt29的K_b分别为1.48×104L/mol和1.32×104L/mol。该研究报道了丹参活性分子与B-Thr、Apt29的相互作用结果,可为核酸适配体(蛋白质)与中药分子相互作用的探索提供新方法,适配体与中药分子相互作用亦可为中药分子的靶点发现、靶向转运提供借鉴。     

Application of capillary zone electrophoresis with off-line solid-phase extraction to in vitro metabolism studies of antifungals

A simple and robust solid-phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itraconazole, fluconazole, and voriconazole) and their metabolites after incubation with human liver microsomes, as well as a simplified capillary zone electrophoresis (CZE) method for their rapid analysis, have been developed to determine the stability of these compounds in in vitro samples. Three different sample pretreatment procedures using SPE with reversed-phase sorbents (100 mg C8, 100 mg C18, and 30 mg Oasis-HLB) were studied. The highest and most reproducible recoveries were obtained using a 30 mg Oasis-HLB sorbent and methanol containing 2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates to a small volume. For the CZE separation, good separations without interfering peaks due to the in vitro matrix were obtained with a simple running electrolyte using a fused-silica capillary. The best separation for all components originated by each tested drug after incubation with human liver microsomes (unmetabolized parent drug and its metabolites) was obtained using a 0.05 M phosphate running buffer (pH 2.2) without additives. The effect of the injection volume was also investigated in order to obtain the best sensitivity. Performance levels in terms of precision, linearity, limits of detection, and robustness were determined.     

Optimization of a nano‐enzymatic reactor for on‐line tryptic digestion of polypeptide conjugates by capillary electrophoresis

This work aims at studying the optimization of an on‐line capillary electrophoresis (CE)‐based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on‐line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.     

Capillary electrophoresis of polycationic poly(amidoamine) dendrimers

Generation 2 to generation 5 poly(amidoamine) (PAMAM) dendrimers having different terminal functionalities were analyzed by capillary electrophoresis (CE). Polyacrylamide gel electrophoresis was also used to assess the composition of the individual generations for comparison with the CE results. Separation of PAMAMs can be accomplished by either using uncoated silica or silanized silica capillaries, although reproducibility is poor using the uncoated silica capillary. To improve run-to-run reproducibility, silanized capillary was used and various internal standards were also tested. Relative and normalized migration times of primary amine terminated PAMAM dendrimers were then determined using 2,3-diaminopyridine (2,3-DAP) as an internal standard. Using silanized capillaries and internal standards, the relative and normalized migration times are fully reproducible and comparable between runs. Apparent dimensionless electrophoretic mobilities were determined and the results were compared to theoretical calculations. It is concluded that for PAMAMs a complex separation mechanism has to be considered in CE, where the movement of the ions is due to the electric field, but the separation is rather the consequence of the adsorption/desorption equilibria on the capillary wall ("electrokinetic capillary chromatography"). The described method may be used for quality control and may serve as an effective technique to analyze polycationic PAMAM dendrimers and their derivatives with different surface modifications.     

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.     

蛋白质样品的非竞争性毛细管电泳免疫分析激光诱导荧光检测

以牛血清白蛋白与其单克隆抗体为样品,初步研究了蛋白质的非竞争性毛细管电泳免疫分析方法。混合温育可以在２０ｍｉｎ内达到平衡,电泳分离在１２ｍｉｎ内完成。当示踪物浓度为３２０ｎｍｏｌ／Ｌ时,所得到的标准曲线的线性范围为８￣１５０ｎｍｏｌ／Ｌ,检出限为５ｎｍｏｌ／Ｌ,并考察了缓冲溶液的浓度、ｐＨ、分离电压等因素的影响。     

寡糖的毛细管电泳分析

多种寡糖经α－萘胺衍生化后，用硼砂作为电泳介质，实现了高效毛细管电泳分离。比较了毛细管区带电泳和胶束毛细管电动色谱分离寡糖α－萘胺衍生物的电泳行为，对影响分离度的诸因素进行了考察，选择了最佳分离条件。     

以羟丙基-β-环糊精为手性选择剂的毛细管电泳法分离测定日夜百服宁片剂中的伪麻黄碱

 建立了伪麻黄碱手性对映体的毛细管电泳分离分析方法 ,分离用缓冲液为 2 5mmol/L的三羟甲基氨基甲烷 磷酸缓冲液 (pH 2 6 5 ) ,含 38mmol/L的羟丙基 β 环糊精。测定了日夜百服宁片剂中右旋伪麻黄碱的含量。方法简便快速 ,具有良好的精密度、回收率和线性关系。     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.