【作者】 李文华；

【导师】 聂舟；

【作者基本信息】 湖南大学 ， 分析化学， 2013， 硕士

【摘要】 金属纳米团簇是一种由几到几十个金属原子构成，尺寸小于2nm的金属团簇纳米材料。最近几年，DNA为模板合成的荧光金属银纳米簇（简称DNA-AgNCs）在生物化学传感中引起广泛的兴趣。DNA-AgNCs由几个到几十个银原子组成，具有很多优良的荧光性能，包括荧光光谱可调、荧光产率高、抗光漂白性强、生物毒性低和生物相容性好等。基于这些优点，DNA-AgNCs已经被广泛应用于生物小分子、金属离子、DNA、RNA和蛋白质等的检测，以及生物标记和成像中。相对于传统的有机荧光染料和荧光纳米颗粒，DNA-AgNCs及其复合纳米簇的合成和性质的调控更为简便，因此在生物化学传感领域具有十分广阔的应用前景。本文基于DNA稳定的荧光银纳米簇（DNA-AgNCs）以及铜/银纳米簇（DNA-Cu/AgNCs）在生物传感领域开展了几个研究工作。主要研究工作如下：1．基于巯基化合物对DNA-Cu/AgNCs荧光淬灭作用，构建了一种新型的乙酰胆碱酯酶活性以及其抑制剂的生物传感方法。在这种方法中，乙酰胆碱酯酶可以催化乙酰硫代胆碱水解为带有一个巯基基团的硫代胆碱，水解产生的硫代胆碱可以淬灭DNA-Cu/AgNCs的荧光，通过这种间接荧光淬灭作用可以对乙酰胆碱酯酶的活性以及其抑制剂进行检测和筛选。其荧光淬灭机理为巯基与银原子有较强的共价作用（Ag-S键），因而硫代胆碱可以与DNA-Cu/AgNCs中的银原子作用，减弱了DNA模板分子与银原子之间相互作用力，从而使得DNA对荧光纳米簇的保护作用减弱，使纳米簇的荧光发生淬灭。DNA-Cu/AgNCs荧光强度的变化对乙酰胆碱酯酶活性具有很好的响应，相较于很多其他已有的乙酰胆碱酯酶活性检测方法，本方法具有相对较低的检测下限，可以检测到低至0.05mU/mL的乙酰胆碱酯酶活性，其检测的线性范围为0.05mU/mL-2.0mU/mL。该方法还对乙酰胆碱酯酶的两种典型抑制剂（他克林和甲胺磷）进行了检测，利用该方法测得的他克林的半抑制剂浓度（IC50）为16.9nM，跟文献报道的相近。测得的甲胺磷的半抑制剂浓度为0.075mg/L，其检测下限可达0.0001mg/L，远低于食物中允许甲胺磷的含量。此外，该方法成功应用于实际样品中农药残留的检测，通过甲胺磷添加实验得到了与标准样品中近似结果。该方法所用的DNA-Cu/AgNCs的合成简单，原材料不需要进行耗时长和昂贵的化学标记和修饰，是一种操作简单、成本低廉、无需标记和灵敏性高的乙酰胆碱酯酶活性检测方法。2．基于末端脱氧核苷酸转移酶（Terminal deoxynucleotidyl transferase，TdT）对DNA的3’羟基端的链延伸作用，构建了一种新型的DNA-AgNCs合成方法，并将其与核酸适体相结合应用于蛋白质的检测中。在该方法中，首先选择一条单链DNA作为末端脱氧核苷酸转移酶的底物，该DNA底物不能作为模板用于DNA-AgNCs的合成。由于富含胞嘧啶的单链DNA可以用于具有荧光的DNA-AgNCs的合成，那么利用末端脱氧核苷酸转移酶在该DNA引物的3’-OH端延伸出一段含有多个胞嘧啶的DNA片段，然后利用该富含胞嘧啶的DNA片段作为DNA-AgNCs的合成模板就可以合成出具有荧光信号的DNA-AgNCs。此外，为了证明该方法在生物传感中的应用潜力，我们将该合成DNA-AgNCs的方法与核酸适体相结合，初步应用于蛋白质的分析检测中。在该蛋白质检测方法中，我们选择凝血酶作为分析检测的对象。更多还原

【Abstract】 Metal nanoclusters (NCs) are smaller than2nm which contain from two to tenmetal atoms. In recent years, DNA templated silver nanoclusters (DNA-AgNCs) haveattracted extensive attention in biosensors and chemical sensors. DNA-AgNCs consistof a few silver atoms and possess many excellent fluorescent properties includingtunable fluorescence emission, good quantum yield, high photostability, low biologicaltoxicity and excellent biocompatibility. Based on all these advantages, DNA-AgNCshave been applied to detect biomolecules, metal ions, DNA, RNA and proteins, andfurthermore biolabeling and bioimaging. Compared to traditional organic fluorescnetdyes and fluorescnet nanoparticles, the synthesis and property of tunable DNA-AgNCsare simple and convenient. Therefore, DNA-AgNCs possess extensive potential in theapplication of biosensors and chemical sensors. Based on DNA-AgNCs andDNA-Cu/AgNCs, the following biosensor research was carried out:1．A novel acetylcholinesterase (AChE) activity sensor was constructed based onthe fluorescence quenching of DNA-Cu/AgNCs. In this assay, AChE catalyzes thehydrolysis of acetlythiocholie (ATCh) to form thiocholine which contains sulfhydryland induces the fluorescence quenching of DNA-Cu/AgNCs. Therefore, the activity ofAChE can be monitored through this indirect mode. The fluorescence quenchingmechanism of AChE activity detection is that sulfhydryl of thiocholine has strongaffinity with silver atom, and thus the produced thiocholine can associate with thesilver atoms of DNA-Cu/AgNCs leading to fluorescence quenching ofDNA-Cu/AgNCs. The fluorescence intensity change of DNA-Cu/AgNCs shows goodresponse to the activity of AChE. Compared to many previous methods, this methodhas a lower detection limit of0.05mU/mL, and the linearity range is from0.05mU/mL to2.0mU/mL. Two typical AChE inhibitors (tacrine and methamidophos)were detected using this AChE assay described above. The IC50value of tacrine wasestimated to be16.9nM which is close to those by other reported methods. Thecorresponding IC50value of methamidophos was obtained to be about0.075mg/L, andits low detection limit was0.0001mg/L, which is much lower than the safetymethamidophos content in food. Furthermore, this method was also applied to detectspiked methamidophos in agricultural products successfully and the results weresimilar with that obtained in pure water. In this assay, the synthesis of DNA-Cu/AgNCs is simple, and the DNA templates used do not require thelabor-intensive and expensive modification with any label. This sensor is simple,cost-effective, label-free and with high sensitivity for the detection of AChE activityand its inhibitor.2．A novel DNA-AgNCs synthesis strategy was developed based on terminaldeoxynucleotidyl transferase (TdT) which can catalyze the sequential addition ofdNTPs to the3’ hydroxyl terminus of DNA. This assay was successfully applied to thedetection of protein combined with protein aptamer. In this assay, TdT was employedto catalyze the sequential addition of natural deoxycytidine triphosphate (dCTP) at the3′-OH of a single-stranded DNA primer which cannot be used as template forDNA-AgNCs synthesis. Due to the fact that cytosine-rich DNA can be used astemplate for DNA-AgNCs synthesis, then the cytosine-rich sequential addition DNAcatalyzed by TdT can be employed as template for the flurescent DNA-AgNCssynthesis. Furthermore, to demonstrate that this DNA-AgNCs synthesis assay haspotential for protein detection, a novel approach for protein detection based on theassay described above and protein aptamer was developed. In this protein detectionapproach, thrombin was chosen as the model analyte of interest.更多还原