【摘要】：本論文的研究工作主要由兩部分構(gòu)成:首先,針對現(xiàn)有腺苷檢測方法大多數(shù)存在著樣品前處理復(fù)雜、操作費(fèi)時、儀器設(shè)備昂貴、不便于普及等不足而構(gòu)建的全新的腺苷檢測體系,旨在研究出一種樣品使用量少、成本低、操作簡單、檢測快速、儀器易于普及,并且可實(shí)現(xiàn)生物樣品中腺苷的特異及準(zhǔn)確定量分析的檢測方法,并構(gòu)想與第二部分研究內(nèi)容相結(jié)合以實(shí)現(xiàn)對腺苷分子印跡聚合物微球所富集腺苷的定量檢測;其次,構(gòu)建出一種簡單而有效的制備核殼型腺苷分子印跡聚合物微球的方法,并使其制備出來的印跡聚合物微球不僅能對腺苷進(jìn)行特異性識別,而且對于ADP-核糖基化的蛋白質(zhì)也可以表現(xiàn)出良好的選擇識別性。以下內(nèi)容為該論文的主要研究工作:(1)將TPE所具有的聚集誘導(dǎo)發(fā)光特性與核酸適配體(ABA)對目標(biāo)物質(zhì)的特異識別性能相結(jié)合構(gòu)建出一種基于TPE和ABA的腺苷檢測新方法,并確定該檢測體系中TPE和ABA的最佳濃度配比為10:0.1,且在最佳濃度配比條件下腺苷的最低檢測濃度為10 pM,通過對腺苷及其三種類似物(胞腺、尿腺、鳥腺)進(jìn)行熒光檢測考察發(fā)現(xiàn)該檢測方法對腺苷有良好的選擇性,同時對尿樣中腺苷的分析檢測進(jìn)行了精密度和回收率實(shí)驗(yàn),回收率為86.8%~90.0%,證明該檢測方法在尿樣中具有良好的精密度,且準(zhǔn)確可靠。(2)采用傳統(tǒng)自由基沉淀聚合法和可逆加成-斷裂鏈轉(zhuǎn)移聚合反應(yīng)(RAFT)相結(jié)合的方法,制備出表面帶有RAFT鏈轉(zhuǎn)移基團(tuán)的聚苯乙烯微球(CP)。在一定的反應(yīng)條件(反應(yīng)溫度:70℃,聚合時間:3.5 h)下制備出三組平行樣品,并對三組平行樣品的產(chǎn)率和外貌形態(tài)進(jìn)行比較,實(shí)驗(yàn)結(jié)果說明在相同條件下利用RAFTPP法合成CP微球的產(chǎn)率、粒徑及其外貌形態(tài)都較為穩(wěn)定。(3)利用CP微球表面RAFT鏈轉(zhuǎn)移基團(tuán)可以再次發(fā)生活性聚合的特點(diǎn),以腺苷為模板分子進(jìn)行第二部聚合反應(yīng),使得微球的外表面形成一層分子印跡薄殼層,從而制備出表面具有親水性能的核殼型腺苷分子印跡聚合物(MIP)微球,并對不同溫度和時間條件下制備的MIP微球的產(chǎn)率,粒徑及分散性進(jìn)行比較分析,確定了最佳反應(yīng)溫度為70℃和反應(yīng)時間為60 min。(4)利用掃描電鏡、水中懸浮實(shí)驗(yàn)、腺苷重吸附實(shí)驗(yàn)以及對不同蛋白的吸附實(shí)驗(yàn)對CP、MIP、NIP分別進(jìn)行表征。實(shí)驗(yàn)結(jié)果表明利用RAFTPP法制備的MIP在水中有較好的分散穩(wěn)定性,而MIP分子印跡外殼的親水性能有助于改善MIP在水溶液體系中對生物樣品的分子識別性能;三種微球?qū)ο佘盏闹匚叫韵嗖畈⒉幻黠@,并且MIP和NIP對gapA蛋白都有大量吸附且吸附效果也無明顯差別,其主要原因可能是在制備MIP過程中腺苷印跡不成功,其表面的腺苷印跡孔穴數(shù)量太少或是孔穴形狀有問題從而無法識別腺苷分子。
[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，

本文編號：2231236