基于量子点和石墨烯的化学生物传感新方法及环境应用研究

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英文题名：Research on Novel Chem-/Bio-sensors Based on Quantum Dots and Graphenes and Their Applications in Environment 作者：刘猛 论文级别：博士 学科专业名称：环境工程 中文关键词：量子点 ; 石墨烯 ; 传感器 ; 模拟酶 ; 脱氧核酶 ; 适配子 英文关键词：Quantum Dot ; Graphene ; Sensor ; Petoxidase Mimetic ; DNAzyme ; Aptamer 学位年度：2012 导师：全燮 学科代码：083002 学位授予单位：大连理工大学 论文提交日期：2012-05-01

摘要

实现水体中污染物的实时、快速、高灵敏和特异性检测,对于监控水污染控制措施的实施、保障水安全等具有重要意义。传统的分析检测技术,如仪器分析等,大多存在仪器昂贵、耗时、操作复杂、专业性较强等问题,已经难以满足实际需求。纳米材料与技术独特的优势和性能为实现水中污染物的检测提供了新的契机,其中量子点材料和石墨烯材料由于具有优异的光学、电学和结构性质,已经被广泛用于构建各种功能性纳米传感器件,但是受传感机制自身缺陷的影响,这些传感检测方法仍然存在灵敏度低、选择性差等问题。本论文针对上述问题,探索了纳米传感检测新方法和新原理,设计了几种基于量子点和石墨烯的新型化学与生物传感器,用于快速、实时、高灵敏和高选择性检测环境中的重金属离子、有毒化合物及重要生物分子等,主要开展了以下几方面的研究：
     (1)采用光辐射法直接制备了高量子产率(60%)的水溶性CdTe量子点,在此基础上,发展了一种新型的层层自组装工艺实现了CdTe量子点在导电玻璃基底的有效固定。固定后的量子点对重金属铜离子表现出较好的选择性和灵敏性,对于低浓度范围的铜离子(0.02-1.0μM),该自组装体系具有较好的可重复使用性能,使用四次后,其荧光强度仍可以恢复到95%以上,在很大程度上克服了传统溶液相量子点在离子检测过程中存在的无法回收利用等缺点；将离子交换反应引入传感设计,构建了基于无机发光材料—dTe纳米棒和有机荧光染料—钙黄绿素蓝的超灵敏荧光比色探针,对铜离子的检出限达到0.13nM,灵敏度是传统量子点荧光探针的60倍,该方法也为构建其它超灵敏荧光比色探针提供了新的思路。
     (2)利用石墨烯材料独特的二维结构和优异的荧光猝灭能力,’发展了基于石墨烯/CdTe量子点的新型荧光免疫传感器用于甲胎蛋白的高灵敏检测,检测限可以达到0.15ng/mL,远低于其它量子点荧光免疫传感器；发展了一种基于胶体石墨烯的新型均相荧光免疫传感器用于水体中MC-LR的快速和高灵敏检测,检测时间小于35min,检出限可以达到0.14μg/L；发展了一种基于滚环扩增(RCA)机理和石墨烯基分子信标的适配子荧光传感器用于环境样品中土霉素的高灵敏和高选择性检测,检出限可以达到0.87nM。此外,石墨烯的引入将传统的基于能量共振转移机理构建的荧光免疫传感系统中供体-受体的有效作用距离从100A提高到223A,为其它高效荧光免疫传感器的构建奠定了基础。
     (3)利用石墨烯对单链DNA和双链DNA不同的结合力,设计了基于石墨烯／T4DNA连接酶的荧光探针,用于快速和高灵敏检测单核苷酸多态性,该探针可以在40分钟内有效检出2.6%的突变DNA,与传统方法相比,具有灵敏度高、响应快等优点,该设计也为实现DNA甲基化,DNA损伤和酶活的检测开辟了新途径。
     (4)通过控制活性氧自由基引发的DNA断裂反应,有效调节石墨烯和DNA之间作用力大小,为构建新型的石墨烯/DNA传感器提供了新的思路。在此基础上,构建了基于石墨烯和脱氧核酶的荧光传感器,用于高灵敏和高选择性检测铜离子,检测限可以达到0.365nM,远低于传统的基于脱氧核酶构建的铜离子荧光传感器；结合核酸结合染料GelRed的发光性质,发展了一种基于石墨烯和脱氧核酶的完全无标记的铜离子荧光传感器,有效避免了传统方法中存在的操作复杂、酶的识别和催化能力低等问题。
     (5)通过控制胶体石墨烯所处环境盐度(NaC、MgCl2)大小,有效调节胶体石墨烯表面电荷分布,从而诱导胶体石墨烯自组装形成“堆叠态”的石墨烯,表现为胶体石墨烯的平均粒径随着体系中盐浓度的增大而增加。而且该构型的石墨烯与传统的胶体石墨烯相比,可以进一步与多种DNA结构自组装形成石墨烯/DNA复合物,在此基础上,构建了基于该石墨烯/DNA复合物的新型荧光传感器,在体外实现了对有毒物质,如铜离子和黄酮类有机物的快速和实时筛选,避免了传统电化学传感器中存在的操作复杂、离线检测等问题,大大拓展了石墨烯在环境检测领域中的应用。
     (6)利用石墨烯材料独特的电子性质以及贵金属材料的催化性能,构建了基于石墨烯材料和贵金属金纳米颗粒的高效类过氧化氢酶,其对过氧化氢酶底物TMB的最大催化氧化速率是商品化的辣根过氧化氢酶的1.3倍。在此基础上,构建了基于纳米人工模拟酶和核酸适配子的新型比色传感器,用于快速和高灵敏检测DNA分子、丙肝病毒、胰岛素分子和酶活等,该方法也为其它无标记适配了比色传感器的设计提供了新的思路。
     综上所述,本论文针对我国水质监测的需求和发展趋势,利用量子点材料和石墨烯材料独特的性质以及分子生物学,生物化学、免疫学等领域相关技术,建立了几种针对水体中典型性污染物的新型化学与生物传感器,实现了纳米传感器的性能调控,揭示了纳米传感检测机理,并阐明了功能化纳米材料的界面反应过程与传感机制之间的内在关系,有利于推动功能性纳米材料在环境监测领域的应用。
It is of great significance to develop new techniques and methods in realizing real-time, rapid, highly sensitive and selective analysis of pollutants in environment, which will play an important role in monitoring the implementation of water pollution control measures and further guaranteeing the drinking water security. Generally, the traditional methods, such as instrumental analysis, are expensive, time-consuming, complicated and necessary for technical skills, which limit their further applications. The development of nanomaterials and nanotechnology with their unique advantages and performance opens new opportunities for the detection of pollutants in water. Among these, quantum dots and graphene-based materials have been widely used to develop the functional nano-devices due to their unique optical, electrical and structural properties. However, these sensing methods still suffer from low sensitivity and poor selectivity originated from the defects in the sensing mechanism. In order to solve the above issues, we have explored the novel methods and principles in nanosensors, and designed several new chem-/bio-sensors sensors based on quantum dots and graphenes for rapid, real-time, highly sensitive and selective detection of environmental heavy metal ions, toxic compounds and biologically important molecules. Several works are as follows:
     (1) A simple photoillumination procedure was developed to produce thiol-capped CdTe quantum dots with high quantum yield (60%). Based on it, a novel layer-by-layer self-assembly technology was developed to immobilize the quantum dots on ITO substrate. It was found that the solid-phase of quantum dot allowed sensitive and selective determination of Cu(Ⅱ) ions. Furthermore, we realized the "on-off-on" detection of Cu(Ⅱ) ions at low concentration levels (0.02-1.0μM). It was observed that the quenched fluorescence could recover to more than95%after four cycles, which overcame the shortcomings of the non-renewable of colloidal quantum dots in the ion detection, thus possessing important application values compared with that in traditional solution assays; A novel ratiometric fluorescent sensor was developed based on inorganic luminescent CdTe nanorods and organic calcein blue through the cation-exchange reaction, which realized the ultrasensitive detection of Cu(Ⅱ) ions with a detection limit of0.13nM. The sensitivity of the ratiometric sensor was enormously enhanced60-fold in comparison with the traditional quantum dot-based sensor. Furthermore, this method can be applied to design other fluorescent sensors for a wide range of metal ions.
     (2) A novel graphene/CdTe quantum dot-based fluoroimmunoassay was developed by taking advantage of the unique two-dimensional structure and the excellent fluorescence quenching efficiency of grapheme, which realized the highly sensitive detection of the biomarker-a fetoprotein. The detection limit was determined to be0.15ng/mL, which was much lower than that of previously reported quantum dot-based fluoroimmunoassay; A novel graphene-based homogenous fluorescence-based immunoassay was developed for rapid and sensitive detection of MC-LR in water samples. The whole assay time was less than35min, and the detection limit was calculated to be0.14μg/L; A novel fluorescent aptasensor was developed for sensitive and sensitive detection of OTC in environmental samples based on RCA technique and graphene-based molecular beacon. The detection limit was calculated to be0.87nM for OTC assay. Moreover, the donor-acceptor distance was estimated to be223A, which significantly broke the distance limit (100A) in traditional fluoroimmunoassay based on energy transfer mechanism. Therefore, graphene can be used as novel acceptor in the design of fluorescent biosensors with high performance.
     (3) A graphene/T4DNA ligase-based fluorescent sensor was designed by taking advantage of the different adsorption affinity of graphene for single-stranded DNA and double-stranded DNA, which realized the rapid and highly sensitive detection of the single nucleotide polymorphism (SNP) compared with traditional methods. The results indicated that it was possible to accurately determine SNP with frequency as low as2.6%within40min. Furthermore, the presented method opened new opportunities for the development of other biosensing platforms for DNA methylation, DNA damage and multiple nucleases activity assay.
     (4) A promising and controllable internal method was developed to regulate the interaction between graphene sheets and DNA based on oxidative DNA damage, which also provided novel basis for the design of other extended graphene/DNA-based sensing platforms. Furthermore, we designed a novel graphene/DNAzyme-based catalytic beacon for Cu(Ⅱ), which allowed the highly sensitive and selective detection of Cu(Ⅱ) in aqueous solution. The detection limit was determined to be0.365nM, which was much lower than that of previously reported DNAzyme-based fluorescent sensors. Base on it, a promising graphene/DNAzyme-based label-free fluorescent sensor for Cu(Ⅱ) detection was developed by means of the unique luminescent property of extrinsic fluorophore GelRed, which avoided the complicated operation and weak performance of DNAzyme in traditional DNAzyme-based sensors.
     (5) Fine control over the surface charge density of colloidal graphene is possible through the control of salt concentration (NaCl or MgCl2) in the solution, thus resulting in the spontaneously assembled stacked-graphene, the average size distribution of which could be increased with the increase of salt concentration. Furthermore, this type of graphene-derived material possessed great capabilities in the capturing of various DNAs independent of their structure compared with the traditional colloidal graphene. Based on it, we designed a novel graphene/DNA-based fluorescent sensor for rapid and real-time screening genotoxic chemicals, such as Cu(II) ions and organic flavonoids, not only avoiding the complex operation and off-line detection in traditional electrochemistry-based sensors, but also improving the signal transduction, thus broadening the graphene-based applications in environemtal monitoring.
     (6) A highly active graphene/Au-NPs-based peroxidase mimetic was constructed in combination with the unique electronic property of graphene and the intrinsic catalytic activity of Au-NPs. It was observed that the calculated values of reaction rate for our catalyst with TMB as substrate was about1.3times higher than those for commercial HRP. Based on it, a novel peroxidase mimetic/aptamer-based colorimetric sensor was developed for rapid and highly sensitive detection of nucleic acids, hepatitis C virus, insulin and enzyme activity. Furthermore, this convenient approach can be applicable to the other extended label-free aptamer-based sensors for colorimetric detection of a broad range of analytes.
     In combination with the needs and development of water quality monitoring, the unique properties of quantum dots and graphenes, and various technologies in molecular biology, biochemistry and immunology, we have designed several novel nanosensors for typical pollutions in water environment, realized the regulation of the sensing performance, revealed the detection mechanism, and illustrated the intrinsic relationship between the interfacial reaction and sensing mechanism in functional nanomaterials, which greatly promoted the application of functional nanomaterials in environmental monitoring.

引文

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