【作者】 王薇；

【导师】 王升富；

【作者基本信息】 湖北大学 ， 材料制备与测试技术， 2020， 博士

【摘要】 金属纳米簇（metal nanoclusters,MNCs）是由几个到几十个金属原子组成的分子级聚集体。复杂多变的微观结构给这种新型纳米材料带来丰富、未知的物理和化学性质,激起越来越多研究者们浓厚的研究兴趣。然而金属纳米簇较大的比表面积和高度配位不饱和性使其在制备和保存方面遇到了较大的困难。目前,急需解决的问题包括如何高效、简易且可控地制备出分析检测所需的金属纳米簇;如何进一步提高以生物分子为模板制备的金属纳米簇的荧光强度;如何在不影响金属纳米簇荧光性能的前提下对其进行纯化等。基于先进表征技术的材料结构信息的精准获取,能够深入探索MNCs结构与各种特性间的关联性,尤其是光学、催化、电子和磁性等,以提高结构对性能调整的高度可控性,并以此为理论依据,寻求高效、简易、可控的制备方法,为MNCs在后续生物分析应用的研究中提供牢固的基础。因此,本文主要围绕以下几个方面开展工作:研究金、银纳米簇聚集态变化对其性能的影响;寻找简单的纯化MNCs的方法;探讨制备方法、检测模型的通用性;基于MNCs电化学和光学性能构建电化学和荧光传感器,并应用于生物分析检测;采用多种表征手段和技术对MNCs材料的结构、性能以及物质之间的相互作用等进行了表征和研究。具体的研究工作如下:1、以石墨烯为模板的金纳米簇制备、表征及其对特定序列DNA的电化学分析应用:构建了一种新型的基于功能化金纳米簇@石墨烯（AuNCs@GR）纳米复合物和核酸外切酶III（Exo III）辅助级联目标物循环放大策略的超灵敏电化学生物传感器。以GR作为一种通用的模板分子,利用超声法快速、简便地制备了AuNCs@GR和PtNCs@GR。检测过程中,由微量的目标DNA引发的Exo III辅助级联目标物循环放大过程富集了二级目标物。借助二级目标物组装到电极表面的AuNCs@GR-DNA-碱性磷酸酶（ALP）纳米复合物能使溶液中Ag⁺发生催化沉积反应,从而以Ag的电化学信号检测目标DNA的含量,且还可借助AuNCs和GR对电子传递的促进作用,进一步提高检测的灵敏度。同时,可将该检测原理拓展到PtNCs体系。由于PtNCs本身具备类过氧化物酶活性,因此构建的电化学生物传感器更为简便。利用PtNCs@GR-DNA对TMB-H₂O₂体系的催化作用,由此产生的电信号变化同样可以对溶液中的目标DNA进行超灵敏检测。这种基于MNCs@GR纳米复合物构建的电化学生物传感器结合了多重放大策略,提供了一种通用的DNA检测方法。2、以牛血清白蛋白为模板的金纳米簇制备、表征及其在荧光分析检测中的应用:选用常见的生物大分子牛血清白蛋白（BSA）为模板制备了金纳米簇（AuNCs@BSA）。首先研究了不同酸度对其性能的影响。由于AuNCs@BSA对H⁺较为敏感,在酸性条件下生成聚集沉淀,荧光强度降低。当恢复碱性条件,其荧光强度几乎完全恢复。因此可借助该特性对AuNCs@BSA进行纯化,该方法简单、易行。同时,由此构建了AuNCs@BSA-琼脂糖凝胶pH荧光传感器。其次,选择了两种常见的、经济的咪唑鎓盐型离子液体（ILs）,即1-乙基-3-甲基咪唑四氟硼酸盐（[C₂mim][BF₄]）和1-丁基-3-甲基咪唑四氟硼酸盐（[C₄mim][BF₄]）作为反应介质,成功制备得到AuNCs@BSA/[C₄mim],其稳定性和荧光性能均得到明显加强。为了验证这种提高策略的通用性,进一步制备得到银纳米簇@谷胱甘肽（GSH）/ILs和铜纳米簇@BSA/ILs,其荧光性能和稳定性均不同程度地提高。其中AgNCs@GSH/ILs发生聚集现象,荧光性能增强更为明显。初步研究了H⁺、ILs与模板分子之间的作用以及ILs对MNCs性能增强的机理。3、以二氢硫辛酸为模板的银纳米簇制备、表征及其对细菌代谢过程监测的荧光分析应用:制备了以水溶性二氢硫辛酸（DHLA）为模板的银纳米簇（AgNCs@DHLA）。这种药物小分子对H⁺更为敏感,AgNCs@DHLA荧光强度在更为宽泛的pH范围具备良好的可逆性。因此,可通过pH诱导的沉淀反应,简便、有效地纯化AgNCs@DHLA。同时,基于AgNCs@DHLA-琼脂糖水凝胶,构建了“turn off/on”pH荧光传感器,pH响应范围为8.0～4.0。由于该传感器具有较低的细胞毒性和较强的稳定性,因而可成功应用于细菌环境的pH监测,从而可对细菌的代谢过程进行研究。4、以万古霉素为模板的金纳米簇制备、表征及其对含磷酸盐代谢物的荧光分析应用:利用万古霉素（Van）作为模板分子和还原剂,制备得到金纳米簇（AuNCs@Van）。发现AuNCs@Van在芬顿试剂(Fe²⁺/H₂O₂)中荧光猝灭效果明显,并细致研究了其猝灭机理。由于Fe²⁺/H₂O₂体系发生芬顿反应生成了Fe³⁺,Fe³⁺与AuNCs表面的Van发生配位反应,因而荧光得以猝灭。进一步研究发现在抗氧化剂和含磷代谢物体系中,AuNCs@Van-Fe²⁺/H₂O₂复合物的荧光强度保持良好。由此构建了基于AuNCs@Van-Fe²⁺/H₂O₂复合物的荧光传感器,用于检测三磷酸腺苷的含量。该传感器能准确测定人血样品中代谢物的总含磷量,且检测时间不超过1分钟。将AuNCs@Van应用于抗菌实验,发现其对革兰氏阴性菌和革兰氏阳性菌具有广谱抗菌效果。由于AuNCs@Van对人肝癌细胞（HepG2）表现出较低的细胞毒性,说明其有望成为具有潜力的抗菌药物。更多还原

【Abstract】 Metal nanoclusters（MNCs）are fluorescent,water-soluble molecular-level aggregates consisting of several to dozens of metal atoms.More and more researchers focus on the diverse and undiscovered physical and chemical properties of the new nanomaterials due to the complex and mutative microstructure.But a large specific surface area and a high degree nonsaturation of coordination bring the difficulty to their synthesis and preservation.At present,the urgent problems to be solved include how to prepare MNCs efficiently,easily and controllablely;how to improve the fluorescence intensity of MNCs stabilized by biological template molecules further;and how to purify MNCs without affecting their fluorescence properties.It is very important to acquire structural information accurately with advanced characterization techniques and explore the correlation between MNCs structures and various properties,especially particular optical,catalytic,electronic and magnetic properties deeply.And more efficient,simple and controllable preparation methods can be proposed based on the theories,which promote applications of MNCs in biological analysis widely.Therefore,this paper mainly focuses on the following aspects:studying the effects of aggregation to the properties of MNCs;finding simple purification methods of MNCs;exploring the generality of preparation methods and detection models;constructing electrochemical and fluorescence sensors based on electrical and optical properties of MNCs to apply in bioanalytical detection;characterizing and studying the properties of MNCs and the ineraction between all kinds of substances by various characterization methods and techniques.The specific research work is as follows:1、The preparation,characterization of gold nanoclusters@graphene and its application in the ultrasensitive electrochemical DNA biosensor based on functionalized nanohybrids:A novel and ultrasensitive electrochemical biosensor was constructed for DNA detection based on functionalized gold nanoclusters@graphene nanohybrids（AuNCs@GR nanobybrids）and exonuclease III（Exo III）-aided cascade target recycling.By utilizing the capacity of GR as universal template,different metal nanoclusters including AuNCs@GR nanobybrids and PtNCs@GR nanohybrids were synthesized through convenient ultrasonic method.Exo III-aided cascade recycling was initiated by target DNA,generating the final cleavage product（S2）,which acted as a linkage between capture probe and the functionalized MNCs@GR conjugates in the construction of the biosensor.The AuNCs@GR-DNA-enzyme conjugates acted as interfaces of enzyme-catalyzed silver deposition reaction,achieving DNA detection ranging from 0.02 f M to 20 p M with a detection limit of 0.057 f M.In addition,the electrochemical biosensor based on PtNCs@GR nanohybrids also realized DNA detection by catalyzing the 3,3’,5,5’-tetramethyl-benzidine-hydrogen peroxide（TMB-H₂O₂）system to produce electrochemical signal.This metal nanoclusters@GR-based multiple-amplified electrochemical biosensor provided a universal method for DNA detection.2、The preparation,characterization of gold nanoclusters@bovine serum albumin and its application in fluorescence analysis and detection:A common biological template molecule,bovine serum albumin（BSA）was selected to prepare gold nanoclusters（AuNCs@BSA）.First,we studied the effect of H⁺on the properties of AuNCs@BSA.Since AuNCs@BSA was sensitive to H⁺,it could be purified by pH-induced precipitation under acidic conditions.The approach is simple and feasible,and the fluorescence of AuNCs could be almost completely recovered upon the re-dispersion of nanoparticles into basic media.A fluorescence sensor pH AuNCs@BSA-agarose gel was constructed based on this property.Secondly,two common and economical short-chain imidazolium ionic liquids（ILs）,1-ethyl-3-methylimidazolium tetrafluoroborate([C₂mim][BF₄]and 1-butyl-3-methylimidazolium tetrafluoroborate（[C₄mim][BF₄]）,were selected as the reaction media.AuNCs@BSA/[C₄mim]was obtained.Both its fluorescence property and stability were significantly enhanced.Furthermore,silver nanoclusters@glutathione/ILs and Copper nanoclusters@BSA/ILs were prepared to verify the generality of this properties enhancement.The interactions between ILs and template molecules and the properties enhancement mechanism of MNCs were studied preliminarily.3、The preparation,characterization of silver nanoclusters@dihydrolipoic acid and its applications in fluorescent pH sensors for bacterial monitoring:A convenient approach to purify dihydrolipoicacid（DHLA）-capped Ag nanoclusters（AgNCs@DHLA）by pH-induced precipitation under acidic conditions was reported.The fluorescence of AgNCs could be completely recovered by re-dispersing the precipitate into a basic solution using DHLA and Na BH₄ as stabilizing ligands and etching reagent.AgNCs@DHLA-agarose hydrogels were prepared to monitor pH with a wide range from 8.0 to 4.0,which showed low cytotoxicity and long stability.Accordingly,a fluorescent pH sensor for bacterial monitoring was employed based on the“off/on”signal switch of the AgNCs@DHLA-agarose hydrogels.4、The preparation,characterization of gold nanoclusters@vancomycin and its application in the fluorescence analysis and detection of phosphate-containing metabolites:A water-soluble,monodispersed gold nanoclusters（AuNCs）were prepared using vancomycin（Van）as a temple and reducing agent.AuNCs@Van exhibited a high tendency to undergo fluorescence quenching in the presence of Fe²⁺/H₂O₂.The fluorescence quenching mechanism was investigated in detail.The luminescence of AuNCs@Van was quenched when chelating with iron ions,which were the main product of the Fenton reaction.In successive experiments,the luminescence of AuNCs@Van could be switched on in the presence of phosphate-containing molecules and some antioxidants,which suggested the possibility of using the AuNCs@Van-Fe²⁺/H₂O₂ complexes as selective luminescent switches for phosphate-containing metabolites and antioxidants,particularly adenosine triphosphate.The total assay duration could be completed within 1 min.The prepared AuNCs@Van had low toxicity to the HepG2 cell in the tested concentration range.In addition,for the first time,AuNCs@Van was used in an antimicrobial application,and AuNCs@Van had good antimicrobial activities towards both Gram-negative and Gram-positive bacteria.更多还原