摘要
随着相关学科的迅速发展以及相关新技术的引入,荧光分析法的灵敏度、准确度和选择性日益提高,方法的应用范围遍及众多领域。目前,荧光分析法已经发展成为一种十分重要且有效的光谱化学分析手段,本论文利用荧光分析法构建传感器用于与人类健康和安全密切相关的金属离子及小分子的识别研究。自从2004年,Ono,A.和Togashi,H.研究证实Hg2+能特异性识别胸腺嘧啶(T)以来,基于T-Hg2+-T协同作用特异性检测Hg2+的研究陆续报道,本文也追随这一研究热点,构建了简单、快速、灵敏的DNA荧光传感器识别Hg2+。荧光金属簇由于量子限制效应而具有荧光,其已经作为一类新型的超小尺寸的荧光探针应用于环境、生物分析等方面。基于本实验室新开发的银纳米簇,本文还构建了银纳米簇荧光传感器识别Cr(Ⅵ)、叶酸和苦味酸爆炸物,拓展了银纳米簇的应用领域。
本文的主要研究内容及结论如下：
1.构建DNA荧光传感器用于Hg2+的识别研究
(1)基于poly(dT)DNA双链和氧化石墨烯的传感器的构建
互补的T15与FAM-A15杂交形成DNA双链,由于DNA双链与GO作用弱,FAM-A15不能靠近GO表面而保持强的荧光信号。若体系中存在Hg2+,T15与Hg2+形成稳定的T15-(Hg2+)n-T15复合物,FAM-A15以单链形式存在,与GO之间存在强的π-π堆积相互作用,FAM接近GO表面,其荧光被GO猝灭,体系显示弱的荧光信号。在一定Hg2+浓度范围内,体系荧光的猝灭程度与Hg2+浓度成正比。在最佳实验条件下,基于FAM荧光的猝灭反应,可以高灵敏度、高选择性地测定Hg2+,检测限低至0.5nM。其他可能共存的金属离子,对Hg2+的测定没有影响。将该方法用于江水样品中Hg2+的测定及回收实验,获得了满意的效果。
(2)基于乙炔基信号放大和T-Hg2+-T复合物的传感器的构建
FAM标记的poly d(T) DNA序列(记作：FAM-Tn, n为T的数目)与Hg2+反应,FAM的荧光猝灭：FAM-T1的荧光猝灭效率很低；FAM-Tn (n>1)的荧光猝灭效率较高,并随着T数目的增加,最大荧光猝灭效率降低。若FAM-T3的另一端再修饰乙炔基,Hg2+加入后,则荧光团的最大荧光猝灭效率提高,可高达95%。本研究以两端分别标记有FAM和乙炔基的HC=C-(5')TTT(3')-FAM为探针,借助乙炔基的信号放大作用,构建了灵敏度高、选择性好、快速识别Hg2+的荧光传感器,并详细探讨了乙炔基放大猝灭荧光的原理。此外,在Hg2+猝灭体系荧光的基础上,构建了半胱氨酸传感器。实验结果表明,该研究体系对半胱氨酸也显示高的灵敏度和选择性。
2.构建银纳米簇荧光传感器用于金属离子及小分子的识别研究
(1)Cr(Ⅵ)传感器的构建以及银纳米簇与不同类金属离子的作用机理初探
以聚乙烯亚胺为模板合成的银纳米簇具有较强的荧光信号,Cr(Ⅵ)能高效猝灭该银纳米簇的荧光,由此建立了识别Cr(Ⅵ)的荧光新方法。在最佳实验条件下,Cr(Ⅵ)浓度在0.1nM-1.5μM范围内与荧光猝灭效率具有良好的线性响应,方法灵敏度高(0.04nM)、速度快。基于不同金属离子对银纳米簇荧光响应的差异,利用紫外.可见吸收光谱,再结合各离子的特征电子构型、对应氢氧化物的溶解性能、碱性条件下的电极电势等相关特性探讨了银纳米簇与不同金属离子之间的作用机理,认为金属离子与银纳米簇之间可通过金属离子与模板之间的氢键作用或络合作用间接靠近银纳米簇,以及金属离子以亲金属相互作用或沉淀包覆的形式直接与银纳米簇作用两种方式相互作用,电荷转移是荧光猝灭的主要因素。
(2)叶酸传感器的构建以及银纳米簇与叶酸的作用机理初探
叶酸能有效猝灭聚乙烯亚胺为模板的银纳米簇的荧光,基于此构建了高灵敏识别叶酸的新型荧光传感器。对多种可能与叶酸共存的物质进行分析,结果显示该体系对叶酸识别具有高的选择性；广泛用于固体奶粉、液态奶、面粉、发酵面团、尿液、药片等实际样品中叶酸含量的测定,结果令人满意。利用紫外可见吸收光谱、高分辨率透射电镜、红外光谱、荧光寿命图谱以及荧光光谱等多种表征手段探讨了叶酸猝灭银纳米簇荧光的机理,提出了以聚乙烯亚胺作为桥梁,从叶酸到银纳米簇的两步电子转移荧光猝灭机理。
(3)苦味酸爆炸物传感器的构建以及银纳米簇与苦味酸的作用机理初探
基于苦味酸对银纳米簇的荧光猝灭反应,建立了荧光检测苦味酸的新方法。在最佳实验条件,银纳米簇荧光与苦味酸浓度呈现好的线性响应特征：线性响应范围1nM-2.25μM,检出限为0.7nM,优于检测苦味酸的其他方法。研究表明该体系能很好地区分苦味酸与2,4,6-三硝基甲苯等硝基甲苯类、硝基苯类爆炸物。苦味酸通过与银纳米簇模板-聚乙烯亚胺之间的静电作用靠近银纳米簇,发生从银纳米簇到缺电子的苦味酸苯环之间的电荷转移,从而使银纳米簇的荧光发生猝灭。
With the rapid development of some subjects and the introduction of new techniques, the sensitivity, accuracy and selectivity of fluorescence analysis increasingly improve and its applications range many fields. To date, fluorescence analysis has become an important and effective spectral chemical method. This work constructed sensors based on fluorescence analysis for sensing metal ions and small moleculars closely related to human health and safety. In2004, Ono A. and Togashi H. authenticated that Hg2+can specifically recognize thymine (T), and since then, Hg2+was measured based on T-Hg2+-T complex in lots of reports. In this paper, this research focus was followed and developed simple, rapid, sensitive sensors based on T-Hg2+-T based Hg2+were developed. Metal clusters are fluorescent owing to its quantum confinement effect and it has been used as a new type of ultra small size of the fluorescent probe in environmental and biological analysis. On the base of the new type of silver nanoclusters synthesized in our laboratory, fluorescent silver nanoclusters sensors were constructed for sensing Cr (VI), folic acid, and picric acid explosive, which contributed to researchers' understand the characteristics of silver nanoclusters and expands the application field of silver nanoclusters.
The main contents and conclusions of this paper are listed as follows:
1. Constructing fluorescent DNA sensor for identification of Hg2+
(1) Construction of fluorescent DNA sensor based on duplexes of poly(dT) and graphene oxide (GO)
T15hybridize to FAM-A15, forming double stranded DNA, and FAM fluoresces strongly due to the weak interaction between double stranded DNA and GO. In the presence of Hg2+, the stable T15-(Hg2+)n-T15complexes are formed and the single stranded FAM-A15interacts strongly with GO via π-π stacking, so the fluorescence of FAM is quenched by GO. Under the optimum experimental conditions, the fluorescence quenching efficiencies are proportional to the concentrations of Hg2+and a detection limit of0.5nM was obtained. Other potential coexisting metal ions had no significant effects on the determination of Hg2+. The proposed method was applied for the determination of Hg2+in river water samples and recovery experiment, the satisfactory results were achieved.
(2) Construction of fluorescent DNA sensor based on fluorescence quenching effect of T-Hg2+-T complexes and ethynyl enhanced fluorescence quenching
Hg2+can quench the fluorescence of FAM labeled polyd (T) sequences (denoted as: FAM-Tn, n for the number of T). The fluorescence quenching efficiencies were low for FAM-T1, while higher for FAM-Tn (n>1) and with the increasing number of T in sequence, the fluorescence quenching efficiencies decreased. If FAM and ethynyl were labeled on the two termini of T3, respectively, the fluorescence quenching efficiencies of FAM can be improved. In this study, HC=C-(5')TTT(3')-FAM was used as probe and highly sensitive, highly selective, and rapid Hg2+sensor was constructed by aid of signal amplification from ethynyl. In this paper, the principle of signal amplification was discussed in detail and on the basis of the fluorescence-quenching system by Hg2+, the cysteine sensor was further constructed. The experimental results indicate that the proposed method also shows high sensitivity and selectivity for cysteine.
2. Constructing fluorescent silver nanoclusters sensors for recognition of metal ions and small molecules
(1) Construction of fluorescent silver nanoclusters sensor for Cr(Ⅵ) and the investi-gation of mechanism for silver nanoclusters interacting with different metal ions
Polyethyleneimine-capped silver nanoclusters, which were easy to be synthesized and have excellent performance, were used as the probe for rapid, sensitive detection of Cr (Ⅵ). Under the optimized experimental conditions, the linear response range of0.1nM-1.5μM (R2=0.9991) and the detection limit of0.04nM were obtained. On the basis of the different response of silver nanoclusters to several metal ions, the mechanism for silver nanoclusters interacting with different metal ions were studied in detail based on UV-Vis spectroscopy, the characteristic electron configuration of ion, the solubility of corresponding hydroxide, and the electrode potential. Possible modes of action were concluded that targets were brought into close proximity with silver nanoclusters by hydrogen bonding or target complexing with template, or by metallophilic interaction or direct complexation between target and silver nanoclusters. And fluorescence quenching mechanism based on charge transfer was proposed.
(2) Construction of fluorescent silver nanoclusters sensor for folic acid and the investigation of mechanism for silver nanoclusters interacting with folic acid
Folic acid can efficiently quench the fluorescence of silver nanoclusters, based on which, the experimental conditions were optimized and the fluorescent silver nanoclusters sensor for folic acid recognition with high sensitivity was constructed. Those potential coexisting species were measured and the results show that the sensing system has high selectivity for detection of folic acid. The proposed method was well used for sensing folic acid in various real samples, such as milk powder, liquid milk, flour, ferment dough, urine, tablets, and so on. Sevral characterizations such as UV-Vis absorption spectroscopy, infrared spectra, high resolution transmission electron microscopy, fluorescence decay profiles, fluorescence spectra, and so on, were adopted to study the fluorescence quenching mechanism in detail. A two-step electron-transfer process, in which the electron is transferred from FA to AgNCs through PEI molecule functioned as a bridge, was proposed.
(3) Construction of fluorescent silver nanoclusters sensor for picric acid explosive and the investigation of mechanism for silver nanoclusters interacting with picric acid
The fluorescent silver nanoclusters sensor with high sensitivity for identification of picric acid was founded based on fluorescence quenching reaction between silver nanoclusters and picric acid. Under the optimized experimental conditions, the proposed sening syetem shows excellent linear response characteristics, of which, the linear response range was1nM-2.25μM, and the detection limit was0.7nM. The performance was better than that of other TNP detection methods reported in last few years. The as-developed TNP analysis technology can discriminate between TNT and TNP. Picric acid and silver nanoclusters were brought into close proximity through the electrostatic interaction between picric acid and PEI and the charge transfer from silver nanoclusters to TNP would result in the fluorescence quenching.
引文
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