腺苷荧光检测新方法及腺苷分子印迹聚合物的制备
发布时间：2018-09-08 17:45
【摘要】：本论文的研究工作主要由两部分构成:首先,针对现有腺苷检测方法大多数存在着样品前处理复杂、操作费时、仪器设备昂贵、不便于普及等不足而构建的全新的腺苷检测体系,旨在研究出一种样品使用量少、成本低、操作简单、检测快速、仪器易于普及,并且可实现生物样品中腺苷的特异及准确定量分析的检测方法,并构想与第二部分研究内容相结合以实现对腺苷分子印迹聚合物微球所富集腺苷的定量检测;其次,构建出一种简单而有效的制备核壳型腺苷分子印迹聚合物微球的方法,并使其制备出来的印迹聚合物微球不仅能对腺苷进行特异性识别,而且对于ADP-核糖基化的蛋白质也可以表现出良好的选择识别性。以下内容为该论文的主要研究工作:(1)将TPE所具有的聚集诱导发光特性与核酸适配体(ABA)对目标物质的特异识别性能相结合构建出一种基于TPE和ABA的腺苷检测新方法,并确定该检测体系中TPE和ABA的最佳浓度配比为10:0.1,且在最佳浓度配比条件下腺苷的最低检测浓度为10 pM,通过对腺苷及其三种类似物(胞腺、尿腺、鸟腺)进行荧光检测考察发现该检测方法对腺苷有良好的选择性,同时对尿样中腺苷的分析检测进行了精密度和回收率实验,回收率为86.8%~90.0%,证明该检测方法在尿样中具有良好的精密度,且准确可靠。(2)采用传统自由基沉淀聚合法和可逆加成-断裂链转移聚合反应(RAFT)相结合的方法,制备出表面带有RAFT链转移基团的聚苯乙烯微球(CP)。在一定的反应条件(反应温度:70℃,聚合时间:3.5 h)下制备出三组平行样品,并对三组平行样品的产率和外貌形态进行比较,实验结果说明在相同条件下利用RAFTPP法合成CP微球的产率、粒径及其外貌形态都较为稳定。(3)利用CP微球表面RAFT链转移基团可以再次发生活性聚合的特点,以腺苷为模板分子进行第二部聚合反应,使得微球的外表面形成一层分子印迹薄壳层,从而制备出表面具有亲水性能的核壳型腺苷分子印迹聚合物(MIP)微球,并对不同温度和时间条件下制备的MIP微球的产率,粒径及分散性进行比较分析,确定了最佳反应温度为70℃和反应时间为60 min。(4)利用扫描电镜、水中悬浮实验、腺苷重吸附实验以及对不同蛋白的吸附实验对CP、MIP、NIP分别进行表征。实验结果表明利用RAFTPP法制备的MIP在水中有较好的分散稳定性,而MIP分子印迹外壳的亲水性能有助于改善MIP在水溶液体系中对生物样品的分子识别性能;三种微球对腺苷的重吸附性相差并不明显,并且MIP和NIP对gapA蛋白都有大量吸附且吸附效果也无明显差别,其主要原因可能是在制备MIP过程中腺苷印迹不成功,其表面的腺苷印迹孔穴数量太少或是孔穴形状有问题从而无法识别腺苷分子。
[Abstract]:The research work of this thesis consists of two parts: firstly, most of the existing adenosine detection methods have complex sample pretreatment, time-consuming operation and expensive equipment. A new adenosine detection system, which is not easy to be popularized and so on, is designed to develop a new system for the detection of adenosine, which is characterized by low sample usage, low cost, simple operation, rapid detection, and easy popularization of instruments. It can also be used to detect adenosine in biological samples by specific and accurate quantitative analysis, and to combine with the second part of the study to realize the quantitative detection of adenosine enriched by adenosine molecularly imprinted polymer microspheres. A simple and effective method for the preparation of core-shell molecularly imprinted polymer microspheres was developed, and the imprinted polymer microspheres were not only able to recognize adenosine specifically. Moreover, ADP- ribosylation proteins can also exhibit good selectivity and recognition. The following are the main works of this thesis: (1) A new method of adenosine detection based on TPE and ABA was constructed by combining the agglomeration induced luminescence of TPE with the specific recognition of target substance by aptamer (ABA). The optimum concentration ratio of TPE and ABA was 10: 0.1, and the lowest concentration of adenosine was 10 pM, through the detection of adenosine and its three analogues (cytosolic gland, urinary gland). The fluorescence detection of bird gland showed that the method had good selectivity for adenosine, and the precision and recovery rate of analysis of adenosine in urine samples were tested. The recovery rate was 86.8% and 90.0%, which proved that the method was accurate and reliable in urine samples. (2) the traditional radical precipitation polymerization method and reversible addition-break chain transfer polymerization (RAFT) method were used. Preparation of Polystyrene Microspheres (CP). With RAFT chain transfer Group on the Surface Three groups of parallel samples were prepared under certain reaction conditions (reaction temperature: 70 鈩，
本文编号：2231236
[Abstract]:The research work of this thesis consists of two parts: firstly, most of the existing adenosine detection methods have complex sample pretreatment, time-consuming operation and expensive equipment. A new adenosine detection system, which is not easy to be popularized and so on, is designed to develop a new system for the detection of adenosine, which is characterized by low sample usage, low cost, simple operation, rapid detection, and easy popularization of instruments. It can also be used to detect adenosine in biological samples by specific and accurate quantitative analysis, and to combine with the second part of the study to realize the quantitative detection of adenosine enriched by adenosine molecularly imprinted polymer microspheres. A simple and effective method for the preparation of core-shell molecularly imprinted polymer microspheres was developed, and the imprinted polymer microspheres were not only able to recognize adenosine specifically. Moreover, ADP- ribosylation proteins can also exhibit good selectivity and recognition. The following are the main works of this thesis: (1) A new method of adenosine detection based on TPE and ABA was constructed by combining the agglomeration induced luminescence of TPE with the specific recognition of target substance by aptamer (ABA). The optimum concentration ratio of TPE and ABA was 10: 0.1, and the lowest concentration of adenosine was 10 pM, through the detection of adenosine and its three analogues (cytosolic gland, urinary gland). The fluorescence detection of bird gland showed that the method had good selectivity for adenosine, and the precision and recovery rate of analysis of adenosine in urine samples were tested. The recovery rate was 86.8% and 90.0%, which proved that the method was accurate and reliable in urine samples. (2) the traditional radical precipitation polymerization method and reversible addition-break chain transfer polymerization (RAFT) method were used. Preparation of Polystyrene Microspheres (CP). With RAFT chain transfer Group on the Surface Three groups of parallel samples were prepared under certain reaction conditions (reaction temperature: 70 鈩，
本文编号：2231236
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