【作者】 李莉；

【导师】 陈坚；

【作者基本信息】 新疆医科大学 ， 药理学， 2007， 博士

【摘要】 异丙酚（2,6-二异丙基苯酚）是超短效静脉麻醉药,广泛用于诱导麻醉、维持麻醉及镇静。异丙酚在人体中代谢速度很快,需要连续输注,而药物代谢的个体差异较大,所以需要实时在线对异丙酚监测及信息反馈。异丙酚的血药浓度检测,常用高效液相色谱（紫外、荧光、质谱及电化学检测）或气相色谱-质谱联用等方法。生物样品的前处理主要采用液—液萃取或固相萃取法。但均为离线的取样分析,不利于样品的实时分析。因此,发展建立在线分析异丙酚的技术和方法,对于临床用药的安全有效具有重要意义。光纤化学传感器是一种连续、可逆、选择性感受某一化学量的装置,具有高灵敏、重量小、体积轻、高通量等特点。光辐射由光纤直接导入样品检测,避免了取样后再送到实验室检测的繁琐,尤其适合用于在线检测生物样品中的药物浓度变化。荧光光谱分析具有灵敏度高、选择性强、方法简便等优点,适用于药物监测领域。随着光纤传感技术的发展,荧光分析也在药物监测领域表现出其优越性。光纤传感技术与荧光光谱分析结合,可以提高其对分析物响应的灵敏性。分子印迹技术的出现为提高光纤传感技术的选择性提供了重要的潜在力量。分子印迹技术是近年迅速发展起来的制备对目标分子具有预定选择性识别聚合物的技术。此技术将待分析目标分子（又称印迹分子）作为模板,与功能化单体通过共价键或非共价键结合,再与过量交联剂一起发生聚合反应,形成聚合物后再将印迹分子从聚合物中抽提出来。这样在聚合物中便留下了与模板分子大小和形状相匹配的立体孔穴,同时孔穴中包含了精确排列的与模板分子官能团互补的由功能单体提供的功能基团,这样的空穴将对模板分子具有选择识别特性。分子印迹聚合物对目标分子的选择识别特性可以与天然生物分子识别系统如抗体-抗原、酶-底物的专一性相媲美。与生物抗体相比,印迹聚合物还具有针对特殊物质制备的预定性、可以反复使用的可逆性、耐酸、碱、及有机溶剂等优点。在分离科学的应用中,如对映体和异构体的拆分、色谱分析、固相萃取、传感器等领域,分子印迹聚合物的特异选择性得到积极地应用。本文的目的是利用光纤传感技术、分子印迹技术和荧光分析这几种先进研究的结合点,消除生物样品中内源成分和相似物的干扰,建立一种在线分析麻醉异丙酚药物浓度的方法。本文以异丙酚为模板分子,甲基丙烯酸作为的功能单体,二甲基丙烯酸乙二醇酯做交联剂,成功地合成了异丙酚分子印迹聚合物及其聚合物膜,用于对异丙酚高度选择性的识别。对功能单体、异丙酚和交联剂三者的比例等反应条件进行优化。通过扫描电镜、透射电镜、红外光谱、高效液相等技术对异丙酚分子印迹聚合组成结构、结合性能等进行表征。平衡结合实验显示,分子印迹聚合物对异丙酚吸附量明显大于非印迹聚合物。Scatchard分析表明,印迹聚合物主要存在两类不同的结合位点,对异丙酚有特异选择性。分子印迹聚合物高亲和力的结合位点的离解常数为Kd₁=0.4882mol/L,最大表观结合常数Q_max1=23.47μmol/g;低亲和力的结合位点的离解常数为Kd₂=5.731mol/L,最大表观结合常数Q_max2=153.90μmol/g。异丙酚在聚合物上达到结合平衡只需2min,洗脱时间也是2min,响应时间低于异丙酚的代谢速度,非常适合应用于异丙酚的快速分离检测。在考察异丙酚的紫外、荧光光谱性质,以及不同pH值介质对异丙酚和血浆荧光性质影响的基础上,建立了荧光分光光度法测定异丙酚静注乳剂含量的方法,较常用的方法比较,此法操作简便快速,更适用于异丙酚静注乳剂的质量监控。将光纤传感技术与荧光光谱分析方法结合,建立了“光纤荧光传感在线检测异丙酚”的检测系统。由氙灯光源发出的光经凸透镜聚焦、干涉滤光片选择特定波长进入光纤,由光纤传输到达装有样品的流通池,被测物质受激发出荧光,经探测光纤收集,传输至光电倍增管,将光学信号转换成电信号并放大。样品由蠕动泵通过载流代入流通池,由光纤检测。在光纤荧光传感检测系统的基础上,建立了光纤荧光传感在线检测异丙酚的方法。在111.1μg/mL浓度范围内,荧光强度与异丙酚浓度成线性关系。方法回收率为101.1～119.4%,日内日间精密度分别为4.8%,6.6%。在生物样品的监测中,存在着样品中复杂成分对检测的干扰,被测组分与干扰组分间光谱信号重叠、背景吸收等各种影响因素,致使缺乏对分析物高选择性的识别能力,因此样品的纯化前处理在快速、可靠的在线分析中起着至关重要的作用。传统的样品前处理方法常常耗时,效率低,对样品的分离缺乏选择性。用于生物样品的在线分离材料需要对分析物有特异识别能力。本文探索了分子印迹聚合物在辅助光纤传感技术在药物分析在线监测中的应用的可能性,采用本文合成的异丙酚分子印迹聚合物做固相萃取剂,不仅可以将干扰物分离,且可起到浓缩分析物的作用,显示分子印迹聚合物在线处理适用于光纤传感在线检测。更多还原

【Abstract】 Propofol（2,6-diisopropylphenol）is a short-acting intravenous administered anaesthetic largely used for inauction and maintenance of anaesthesia.The rapid metabolisation of propofol in the body makes it necessary to have a continuous infusion.However, metabolic rates vary between individuals,making on-line monitoring of propofol desirable.Various methods have been reported for the quantitation ofpropofol in plasma or brood, high pressure liquid chromatography（HPLC）with ultraviolet（UV）,fluorescence or MS electrochemical detection,gas chromatography（CJC）with atomic emission or mass spectrometric detection;Sample preparation prior to analysis by HPLC or GC requires either liquid-liquid or solid-phase extraction（SPE）.But all of the assays are off-line analysis,and have disadvantages of sampling analysis.Therefore it is of significant importance to develop and establish an on-line monitoring technique or method for propofol for clinical safe validity control.A fiber-optic sensor is a device that is able to indicate continuously and reversibly the concentration of an analyte.The optical fiber sensors have certain advantages that include high sensitivity,lightweight,small size,immunity to electromagnetic interference,large bandwidth.The fiber-optic sensors break through the conventional spectral analysis pattern;they enable optical spectroscopy to be performed on sites inaccessible to conventional spectroscopy.The analysis of the sample can be carried out by guiding light through the fiber optic to the sample,thus avoiding the need to put the sample in a spectrometer and the disadvantages of sampling analysis.It is especially applicable for on-line monitoring of therapeutic drugs in biological samples.Fluorescence analysis method has an advantage of high sensitivity,strong selectivity and simpleness and is fit for drag analysis.With the rapid development of optical-fiber technology,fluorescence analysis technology shows more and more superiority in monitoring of therapeutic drugs. The fiber-optic techniques combining the fluorescence analysis are applied to improve the sensitivity of response for analytes.And its great potentialities on improving selectivity can be fully brought out by means of the molecular imprinting technique.Molecular imprinting is an emerging technique for preparing the polymeric materials possessing highly selective and affinitive properties.The technique involves complexation in a solution of a target molecule（template）with functional monomers through either covalent or noncovalent bonds followed by a polymerization reaction with an excess of cross-linkers.Removal of the templates leaves behind specific recognition sites that are complementary to the template in terms of its shape,size,and functionality in the polymer network.These cavities have highly selective molecular recognition to capture the target analyte.Recognition ability of some MIP to target molecule is comparable with crude molecular recognition system,such as antibody to antigen, enzyme to matrix.MIPs have several advantages over classical antibodies.For example they can be tailor-made for specific substances,the imprint molecule can be recovered after polymerization,and they are resistant to pH changes and organic solvents.In analytical separation science,The selectivity of MIPs has been exploited in several applications,such as chromatography,enantiomeric separations,solid-phase extraction and sensors.Our aim is to develop a quick and reliable method for on-line determination of propofol that would be free of interferences,either from other similar compounds or from blood components and approach for process monitoring of therapeutic drugs by means of hyphenating the Fiber-optic Sensor,the Molecular Imprinting Technique and Fluorescence analysis.In this thesis,Molecular imprinted polymer（MIP）against propofol was synthesized by using propofol as template molecule,methacrylic acid（MAA）as functional monomer and ethyleneglycoldimethacrylate（EDMA）as cross-linker and it had selective adsorptivity to propofol.Reaction condition such as ratio of monomer,propofol and cross linker was investigated.FT-IR,scan electron micrography and HPLC were used to characterize the MIP structure and surface features.According to batch binding experiments,propofol imprinted polymer exhibited higher affinity to propofol compared to nonimprinted polymer.Scatchard analysis indicated that there were two classes of recognition sites in propofol imprinted polymer and they had uniform affinity to propofol.Its dissociation constant K_d1and maximum apparent binding constant Q_max,1 of binding sites with high affinity was 0.4882 mol/L,23.47μmol/g respectively,while K_d2 and Q_max,2 of binding sites with low affinity was 5.731 mol/L,153.90μmol/g.Propofol bound to this polymer in 2 min and could be removed in 2 min,and the response rate is below the rate of metabolism of the anaesthetic.These results suggest suitability for use in an on-line propofol detection.The character of fluorescence and UV of propofol had been investigated,including influence of pH on fluorescence intensity of propofol and human plasma.Based on these, we developed fluorescence spectrometry method for the determination of concentration of propofol emulsion.The method is more simple and quick compared with the conventional methods,and what’s more,it is suitable for quality control for emulsion.An Optical Fiber analysis instruments system based on Fluorescence signal for propofol determination had been developed.In the system,light from Xenon lamp passes through a collimated lens and the interference filter to achieve the correct light intensity and wavelength,then the light is guided through the fiber to the analytes,which can emit fluorescence in the flow-through cell.The fluorescence is guided back to the photomultiplier tube,converted to an electrical signal,amplified.The carrier steam is propelled with the pump;the sample is introduced into the carrier steam by means of the valve（Ⅴ）and the analyte quantity information is measured in the flow-through cell using the fiber-optic sensor.Based on the instruments system,an optical-fiber fluorescence measurement method which is used in on-line determination of propofol concentration had been established.The standard curve of propofol was linear with the concentration in the range 0.1～15μg/mL.The average recovery was 111.1%.The RSD of within-day and between-day were 4.8%and 6.6%.A quick and reliable method for on-line determination of propofol would be free of interferences,either from other similar compounds or from blood components,so the isolation or separation of lead compounds from natural resources followed by the bioassay guidance has played an important role.The final determination will be hindered while the analyte is retained together with other matrix compounds.However, conventional methods are often time-consuming and inefficient for thos procedure as a result of the poor affinity and selectivity of the analyte analysis.Materials that display molecular recognition capabilities are essential for biological samples.We investigated the potential of molecular imprinted materials used in the optic-fiber analytical system. The MIP obtained previous was developed to be the solid-phase extraction material, which allowed not only the other compounds present in the sample matrix to be removed but also the analyte to be preconcentrated.The results suggest its suitability use in an on-line propofol detection.更多还原