分子印迹技术结合化学发光成像和荧光光纤传感器的研究

【作者】 王丽；

【导师】 章竹君；

【作者基本信息】 陕西师范大学 ， 分析化学， 2008， 博士

【摘要】 分子印迹属超分子化学中的主客体化学范畴,是源于高分子化学、生物化学、材料科学等学科的一门交叉学科。分子印迹聚合物即是这样一种合成的人工受体:它具有形状与底物分子相匹配的空腔,而且有着特定排列的功能基团可以与底物分子产生识别作用。与常规和传统的分离和分析介质相比,基于分子识别的分子印迹聚合物的突出特点是对被分离物或分析物具有高度的选择性。同时分子印迹聚合物的物理化学稳定性好,能够耐受高温、高压、酸碱、有机溶剂等,具有构效预定性、特异识别性和广泛实用性三大特点。因而,在化学催化、材料科学、色谱分离、仿生传感等方面得到了日益广泛的应用。最常用的分子印迹聚合物的制备方法是,将模板分子、交联剂致孔剂和引发剂的混合溶液在容器中通过光照射或者加热进行引发,制备得到块状的分子印迹聚合物,经过研磨、过筛和洗涤等步骤得到一定尺寸的分子印迹聚合物的粉末颗粒,然后进行聚合物的吸附性能或者识别性能评价。这种方法制备得到的分子印迹聚合物的缺点是:其一,在研磨的过程中很多识别位点受到了损坏,不能发挥分子识别和吸附的作用;其二,许多识别位点被深深地包埋在聚合物颗粒的内部,从而造成对模板分子的洗脱难度比较大,即便是洗脱出来,在识别过程中难于高效发挥作用。而直接合成分子印迹聚合物微球,不但可以避免繁杂的后处理过程,获得粒径均一的产品,聚合物产率高,而且印迹孔穴暴露在微球表面的几率大,是一类有广阔发展前景的分子印迹材料。目前制备分子印迹聚合物微球的方法主要有:悬浮聚合法、多步溶胀悬浮聚合法、乳液聚合法、种子悬浮聚合法等。这些聚合法虽然印迹效果很好,但其制备过程非常繁琐,制备周期较长,且所得到的聚合物微球的粒径大都在微米级。而沉淀聚合是合成分子印迹聚合物微球的一种简单易行的方法,该方法一步即可完成,且不需在反应体系中加入任何稳定剂,获得的聚合物微球表面洁净,可避免由稳定剂或表面活性剂对模板分子的非选择性吸附。合成的微粒可以达到亚微米级。本论文的研究工作主要由两部分组成,第一部分为分子印迹-化学发光成像研究。化学发光成像分析是一种高通量的,可以快速检测多个样品的分析方法。在这一部分的工作中,采用沉淀聚合法合成了单分散性较好的,单一的亚微米级的几种物质的聚合物微球,并结合化学发光成像分析,建立了几种物质的高选择性、快速的检测方法,特别是应用于手性分子的快速识别和检测,取得了成功。第二部分为分子印迹-荧光光纤传感器研究。在分子印迹技术中,如何有效的洗脱模板分子是一个难题,传统的方法尽管进行了多次充分的洗涤,仍然有很大一部分模板分子留存在聚合物的空穴中,传感器的可逆性和重现性较差。本论文这一部分的工作,是用氧化反应破坏模板分子的结构,使其易于洗脱,并用于96孔板上,将其作为一次性传感器的识别元件,避免了多次测量之间的清洗困难,结合荧光光纤技术检测了复杂样品中的模板分子。具体内容如下:第一部分分子印迹-化学发光成像研究第1章分子印迹技术的研究进展本章对分子印迹技术的研究进展进行了论述,内容主要包括分子印迹的发展概况、基本原理、合成方法及其应用。第2章化学发光成像分析本章对化学发光成像分析的研究进展进行了综述,内容主要包括化学发光成像分析的发展概况、应用以及发展前景。第3章沉淀聚合法制备分子印迹聚合物结合化学发光成像分析测定反式白藜芦醇以反式白藜芦醇为模板分子,用沉淀印迹微球技术合成了粒径均一的亚微米级微球。采用SEM表征印迹微球的行貌。详细探讨了该印迹微球合成条件。该研究解决了传统的印迹聚合物呈块状的缺点,无需研磨,可以直接制备呈规则微球型的聚合物颗粒。该方法既避免了块状聚合物所需要研磨带来的结合位点的破坏以及原料利用率低等缺点,相比其他微球聚合技术又具有合成过程简单的优点,且合成的微粒直径可以达到亚微米级。结合化学发光成像分析,建立了测定葡萄酒以及虎杖提取物中白藜芦醇含量的新方法。具体过程是以聚乙烯醇(PVA)为固定剂固定分子印迹聚合物微球到96微孔板上,选择性吸附溶液中的反式白藜芦醇,以CCD摄像机记录化学发光成像信号。该方法简单、快速且具有高的选择性。第4章分子印迹-化学发光成像分离分析丹酰化-苯丙氨酸手性异构体的研究在分子印迹的手性分离应用中,大部分都是结合色谱或毛细管电泳分离方法,而色谱或毛细管电泳方法不仅仪器昂贵,且费时、费试剂。分子印迹用于传感器又有响应时间过长,分子印迹的洗脱困难等缺点。本文建立了一种MIP-化学发光成像手性识别检测丹酰化-苯丙氨酸异构体的新方法。采用微阵列检测,不仅节省时间且没有多次洗脱模板分子的困扰。印迹聚合物微球用沉淀聚合的方法制成,并将其固定于96微孔板上,用一台商品化的带有低温冷却装置的CCD成像系统检测化学发光信号。在最佳实验条件下,L-和D-型异构体的检出限分别为0.025μmol/L和0.075μmol/L(3σ)。将本方法用于L-,D-型混合样品的分析,测定结果与毛细管电泳法做了对照,结果相吻合。第5章分子印迹-化学发光成像分析检测人尿液中的双嘧达莫建立了一种选择性好的高通量检测双嘧达莫的方法。该方法结合分子印迹技术和化学发光成像技术的优点,即以具有专一性识别特性的分子印迹聚合物为识别元件,固定到96微孔板上,选择性吸附被测物后,加入化学发光试剂,以高通量的成像分析其化学发光信号。在最佳条件下,双嘧达莫的先行范围为0.02～10μg/mL。检出限为0.006μg/mL。成功的用于人尿液中双嘧达莫的分析。拓宽了化学发光成像和分子印迹技术的应用领域。本方法的最大优点就是能在30 min之内同时检测96个样品。第6章化学发光成像结合分子印迹技术测定废水中的罗丹明B本文采用一种灵敏、简单的方法实现对罗丹明B的选择性检测。采用沉淀聚合法首次成功地制备了粒径均一的罗丹明B印迹聚合物微球,优化了聚合条件。并将洗脱了模板分子的聚合物固定于96微孔板上,用一台商品化的带有低温冷却装置的CCD成像系统检测化学发光信号。在最佳实验条件下,罗丹明B在0.002～10μg/mL浓度范围内线性关系良好(r=0.9994)。沉淀既避免了块状聚合物所需要研磨带来的结合位点的破坏以及原料利用率低等缺点,相比其他微球聚合技术又具有合成过程简单的优点,且合成的微粒直径可以达到亚微米级。化学发光成像分析是一种高通量的检测手段,可以实现多个样品的同时检测。第7章化学发光成像结合分子印迹技术测定环境水中的荧蒽荧蒽属于多环芳烃(PAHs)中的一种。多环芳烃是一类广泛分布于天然环境中的有毒有机污染物,难于生物降解。是最早被发现的环境致癌物,因为PAHs在水中的低溶解度和疏水性测定水中的PAHs难度尤其大,本实验结合高灵敏度的化学发光成像方法和高选择性的分子印迹技术,富集检测环境水中的荧蒽,结果显示即便是没有任何官能团的分子也可以用分子印迹作为这一类物质的识别手段。第二部分分子印迹-荧光光纤研究第1章光纤分析本章对光纤分析的研究进展进行了综述,内容主要包括光纤传感器的发展概况、光纤传感原理、光纤化学传感器试剂相的固定方法和光纤化学传感器研究发展趋势。第2章分子印迹-荧光光纤传感器检测6-巯基嘌呤采用沉淀聚合合成粒径均一的亚微米级6-巯基嘌呤分子印迹聚合物微球。以聚乙烯醇固定合成的分子印迹聚合物微球到96孔板上,结合荧光光纤技术成功制备印迹传感器的微阵列。荧光分析法具有灵敏、快速、简便等优点,本文采用氧化的方法把原本微弱荧光的6-巯基嘌呤转变成强荧光的物质,大大提高了灵敏度。分子印迹聚合物作为选择性识别元件,可以提高方法的选择性。且本方法采用微阵列的方式,相比其它的传感器,被测物分子不用不断的从换能器上洗脱,简化了实验过程,节省了实验时间,已成功地用于人血清中6-巯基嘌呤的检测。第3章分子印迹荧光荧光光纤传感器分析检测卡马西平研究了一种结合分子印迹技术和荧光分析技术相结合的光纤微阵列传感器并将其用于血样中卡马西平的测定。利用沉淀聚合方法,以及基丙烯酸为功能单体,TRIM为交联剂合成亚微米级分子印迹聚合物微球,其对卡马西平存在特异性吸附。通过聚乙烯醇包埋聚合物微球到96孔板上,采用氧化荧光光纤传感的方法测定卡马西平的含量。虽然已经有许多分子印迹作为识别元件的传感器,但是分子印迹作为识别元件的最大的困扰之一就是洗脱困难,重现性差。本方法采用微阵列的方式,相比其它的传感器,被测物分子不用不断的从换能器上洗脱。同时分子印迹聚合物作为识别元件,极大地提高了荧光分析方法的选择性和灵敏度。第4章分子印迹荧光荧光光纤传感器分析测定叶酸含量合成了叶酸的分子印迹聚合物,并将其固定到96孔板上,构建了微阵列光纤荧光传感器。基于分子印迹微球的传感器响应时间快,尤其是采用微阵列的方式,相比其它的传感器,被测物分子不用不断的从换能器上洗脱。同时分子印迹聚合物作为识别元件,极大地提高了荧光分析方法的选择性和灵敏度,使该方法可直接用于测定奶粉中的叶酸含量。更多还原

【Abstract】 Molecular imprinting belongs to the category of host-guest chemistry in super-molecule chemistry. It is an interdiscipline subject, which origins from macromelocular chemistry, biochemistry, material chemistry, etc. Molecular imprinted polymers （MIPs） are such a synthetically man-made receptor: it owns the cavities which match up substrate molecules in space and can recognize the substrate molecules through the ordered functional groups. Compared with the routine and traditional medium used for separation and analysis, the outstanding character of MIPs based on molecular recognition is its excellent selectivity to the substrates. Further more, MIPs are highly stable and able to bear high temperature, high pressure, acid or alkaline condition, and organic solution. Due to their predetermination, specific recognition and practicability, MIPs have been largely put into use in catalysis, material chemistry, separation and biomimic sensor.A typical imprinting system consists of a print molecule, at least one type of functional monomer and crosslinker, and a porogenic solvent. To induce radical polymerization an appropriate initiator is included as well. In their most common format, MIPs are prepared in the form of a macroporous monolith that is then ground and sieved to appropriate particle sizes. The grinding and sieving process is time-consuming and yields only moderate amounts of ’useful’ product. The MIP particles are also irregularly shaped and are not ideal. Although suspension polymerization and seed polymerization can provide spherical, molecularly imprinted polymer beads, they require either use of special dispersing phases/surfactants, or complicated swelling processes. Precipitation polymerization was a more general and simple imprinting method applicable to a wide range of print molecules giving uniform MIP spheres.The research work of the dissertation is made up of two sections of development of MIP-chemiluminence （CL） imaging assay, MIP-fluorescence optical fiber sensor assay. In the first part, imprinted uniform microspheres were synthesized by precipitation polymerization against several templates. The simple and specific methods have been developed coupled of chemiluminescence （CL） imaging assay. In the second part, fiber optic fluorescence sensors have been developed by combinding MIP and fiber optic fluorescence technology. Although MIP has many advantages, a number of problems still exist. One of the main disadvantages is the difficulty in removing the entire template analyte molecule, even after extensive washing. In this part, we are aiming to resolve the problems through oxidizing the template to a compound that can be detected with higher sensitivity. The oxidization reaction could change the template molecular structure and made the compound washed off easily. As a result the cavities can be released and retain their memory for the target analyte. Details are presented in this thesis as follows:Part One MIP-CL imaging assayChapter 1 The development of the molecular imprinted technologyThis review summarizes recent research efforts on the development of molecularly templated （sometimes called molecularly imprinted） polymers. It’s contained the progress of MIPs in recent years, the principle of imprinting procedure, the method of synthesiz and MIPs’ applications.Chapter 2 CL imageing assayThe development, applicationd and the trend of the CL imageing method were reviewed in this chapter.Chapter 3 Molecular imprinted polymer-based chemiluminescence imaging for the detection of trans-resveratrolImprinted uniform microspheres were synthesized by precipitation polymerization against trans-resveratrol. The conditions of polymerization were carefully studied. Microtiter plates （96 wells） were coated with polymer microspheres imprinted with trans-resveratrol, which were fixed in place using poly（vinyl alcohol） （PVA） as glue. The amount of polymer-bound trans-resveratrol was quantified using imidazole （IMZ）-catalyzed peroxyoxalate chemiluminescence （PO-CL） reaction. The light produced was then measured with a high-resolution CCD camera. These results showed that the MIP-based CL imaging can become a useful analytical tool for quick simultaneous detection of trans-resveratrol in a large number of real samples.Chapter 4 Molecularly imprinted polymer based on chemiluminescence imaging for the chiral recognition of Dns-phenylalanineA new molecularly imprinted polymer （MIP）-chemiluminescence （CL） imaging detection approach towards chiral recognition of Dns-phenylalanne （Phe） is presented. The polymer microspheres were synthesized using precipitation polymerization with Dns-L-Phe as template. Polymer microspheres were immobilized in microtiter plates （96 wells）. The analyte was selectively adsorbed on the MIP microspheres. After washing, the bound fraction was quantified based on peroxyoxalate chemiluminescence （PO-CL） analysis. In the presence of Dns-Phe, bis（2, 4, 6-trichlorophenyl）oxalate （TCPO） reacted with hydrogen peroxide （H₂O₂） to emit chemiluminescence. The signal was detected and quantified with a highly sensitive cooled charge-coupled device （CCD）. Influencing factors were investigated and optimized in detail. Control experiments using capillary electrophoresis showed that there was no significant difference between the proposed method and the control method at a confidence level of 95%. The method can perform 96 independent measurements simultaneously in 30 min and the limits of detection （LODs） for Dns-LPhe and Dns-D-Phe were 0.025μmol/L and 0.075μmol/L （3σ）, respectively. The relative standard deviation （RSD） for 11 parallel measurements of Dns-L-Phe （0.78μmol/L） was 8%. The results show that MIP-based CL imaging can become a useful analytical technology for quick chiral recognition.Chapter 5 Chemiluminescence imaging assay dipyridamole based on molecular imprinted polymer as recognition materialA simple, sensitive and specific method has been developed for high throughput detection of dipyridamole. The proposed method is based on chemiluminescence （CL） imaging assay combined molecularly imprinted polymer （MIP） recognition. Polymer as the artificial biomimetic recognition element for dipyridamole was coated in 96- microtiter well plates with poly（vinyl alcohol） （PVA） as glue. The amount of polymer-bound dipyridamolewas determination based on dipyridamole peroxyoxalate chemiluminescence （PO-CL） reaction. The emitting light was measured with a high-resolution charge couple device （CCD） camera. Influencing factors were investigated and optimized in detail. The proposed method in this paper exhibits high selectivity and sensitivity to dipyridamole. Under the optimum conditions, the relative CL imaging intensity （AVG） is proportional to the concentration of dipyridamole ranging from 0.02 to 10μg/mL. The detection limit is 0.006μg/mL. The method can perform 96 independent measurements simultaneously in 30 min. These results show that the MIP-based CL imaging method can become a useful analytical technology for quick detection of dipyridamole in real sample.Chapter 6 Determination of rhodamine B in water by molecular imprinted polymer and CL imageingA simple, sensitive and specific method has been developed for high throughput detection of rhodamine B. Molecular imprinted polymer microspheres were prepared by precipitation polymerization. Polymer microspheres were immobilized in microtiter plates （96 wells）. The analyte was selectively adsorbed on the MIP microspheres. After washing, the bound fraction was quantified based on peroxyoxalate chemiluminescence （PO-CL） analysis. The signal was detected and quantified with a highly sensitive cooled charge-coupled device （CCD）. In addition, the developed method was satisfactorily applied to the determination of rhodamine B in water samples.Chapter 7 Determination of fluoranthene in water by molecular imprinted polymer and CL imageingFluoranthene is a representative compound among polycyclic aromatic hydrocarbons （PAHs）. PAHs is a kind toxic organic contamination distributing in environment abroad and difficult to be degraded by biology, is a kind environmental carcinogens described earliest.oweing to the lower solubility in water and hydrophobicity, the detection of these compounds was very difficult. This work detected fluoranthene in the water by conbinding the high sensitivity CL imageing assay and high selectivity MIP. The results showed that MIP could be an enrichment technology.Part Two MIP-fluorescence optical fiber sensor assayChapter 1 The development of fiber optic sensorThis charpter reviewed the development of fiber optic sensor. It is containing the progress of fiber optic sensor in recent years, the mechanisms of fiber optic sensor, the methods of fixing sensitive materials and the trend of fiber optic sensor was described.Chapter 2 The study of oxidization fluorescence sensor with molecular imprinting polymer and its application for 6-Mercaptopurine （6-MP） determinationThis paper developed high-performance optical fiber sensors based on molecular imprinting polymer （MIP） and studied the application in 6-Mercaptopurine （6-MP） pharmaceutical formulations, human serum and urine samples. The sensors were fabricated by coupling the optical fibers with the polymer coated in 96-microtiter well plates using poly（vinyl alcohol） （PVA） as glue. The polymer acted as the artificial biomimetic recognition element for 6-MP. The MIP microspheres with uniform shape were prepared using precipitation polymerization method. During the determination, 6-MP was oxidized to a strong fluorescent compound with H₂O₂ in the alkaline media, which was one of the maim advantages that made it could be washed off easily from MIP and detected sensitively with fluorescence. This molecular imprinting approach offered a relatively nice selectivity for 6-MP detection. In addition, the developed method was satisfactorily applied to the determination of 6-MP in human serum samples.Chapter 3 The study of oxidization fluorescence sensors with molecular imprinting polymer and the application for carbamazepine determinationThis paper developed high-performance optical fiber fluorescence sensors based on molecular imprinting polymer （MIP）. As a selective recognition material, the polymers were coupled with optical fiber fluorimetry detection for the efficient determination of carbamazepine （CBZ） in human serum. The sensors were fabricated by coupling the optical fiber fluorimetry with 96-microtiter well plates coated MIP. After adsorbed to MIP, the CBZ was detected by fiber fluorescence spectrophotometer after oxidation with lead dioxide in the media of sulfuric acid and methanol （V:V=3:1）, which was one of the maim advantages that made it could be washed off easily from MIP and detected sensitively with fluorescence. This molecular imprinting approach offered a relatively nice selectivity for CBZ detection. The relative standard deviation （RSD） was 5% for a same sensor （n=5） when CBZ concentration was 0.01μg/mL in serum. In addition, the developed method was satisfactorily applied to the determination of CBZ in human serum samples without any necessity for sample treatment or time-consuming extraction steps prior to the analysis.Chapter 4 The study of oxidization fluorescence sensor with molecular imprinting polymer andits application for folic acid determinationFluorescence feber optic sensor combined with MIP was fabricated. Imprinted uniform microspheres againsted folic acid were synthesized by precipitation polymerization and were fixed to microtiter plates （96 wells）. The selective of the sensor was inhenced based on the MIP recognition of template. And its application to the analysis of milk powder, value of RSD （n = 5） obtained was 3.5%.更多还原