【作者】 刘斯佳；

【导师】 蒋健晖；

【作者基本信息】 湖南大学 ， 分析化学， 2013， 博士

【摘要】 生物传感是基于生物分子识别及其下游信号转换，进行目标物检测的一类分析方法，是分析化学与生命科学的交叉研究领域。比较其它分析方法，它有着不少的优势，特别是提高了分析速度和应用灵活性。随着电子、微电子和机械系统的进步，便携式、集成式传感元件的开发，生物传感方法研究有机会为医疗、环境保护、食品安全乃至公共安全等领域提供更多现场快速检测技术，为公共健康与公共安全建立第一道防线。近年来，纳米材料领域的研究不断突破，核酸识别、核酸信号放大新技术的持续发展，为构建高灵敏、高选择性、快速高效的新型生物传感器提供了更完美的设计思路，为生物传感器实现不同分析靶标的现场快速检测提供了更有力的平台。本论文则基于当前热点纳米材料-安全环保的碳、硅纳米材料发展了三个颇具医学诊断应用前景的生物传感器，其中有细胞信号通路重要蛋白酶-磷脂酶D的检测，活细胞及亚细胞器的示踪。首次报道非氧化石墨烯通过非共价生物功能化应用于生物传感器，也是首次展示利用无毒纳米材料对细胞能量工厂-线粒体的示踪研究效果能够堪比有机染料。同时涉及其中的纳米材料-非氧化石墨烯、硅量子点、碳量子点的生物功能化研究都各自有小小突破，为它们在生物传感领域的进一步发展奠定了良好的基础。本论文还基于核酸杂交技术和金属离子介导的核酸伪杂交技术分别发展了针对生物体重要的癌症标记物-小RNA（microRNA）和环境污染物-汞离子的检测技术，并且对这两种靶标都实现了高灵敏高选择性的分析。所有研究具体内容如下所述：为了进一步拓宽非氧化石墨烯在生物传感领域的应用，在第2章中发展了一种新颖的双层磷脂膜修饰的非氧化石墨烯复合纳米材料，它是通过非氧化石墨烯大面积双面芳香环层和已合成的脂质体疏水烷烃夹层利用范德华力和疏水作用力形成的，并且这种非共价组装过程不会破坏石墨烯片层的电子大共轭结构，使得该复合纳米材料保留了非氧化石墨烯卓越的电学和光学性质，而表面修饰的磷脂膜层则能赋予非氧化石墨烯良好的生物相容性、亲水性以及表面可控修饰性，为石墨烯的生物功能化提供了一个新的实用平台，该复合纳米材料由磷脂双分子层和疏水的还原石墨烯片组成，类似细胞膜的结构，通过利用荧光素标记的磷脂掺杂在复合纳米材料中，发展出了一个检测磷脂酶D活性的荧光生物传感器。该生物传感器具有高灵敏和宽检测范围的特性，其检测下限低至0.010U/L，优于以往报道的磷脂酶D分析方法，而且基于该分析模式是一步均相法，该生物传感器有极好的适应性，操作简易性，以及进一步实现高通量分析和实时监测反应动力学的可能。近年来，硅纳米材料凭借它出色的光学、电学、力学性质，以及表面可控修饰，可适应硅材料作为半导体材料的特殊应用吸引科学家们的关注，尤其是零维硅纳米材料-硅量子点，基于硅材料本身无毒、化学惰性、生物相容性极好，非常适合生物传感方法研究，特别是生物成像领域。在第3章中我们首先将电化学刻蚀法合成的硅点用双氧水氧化钝化，再用硅烷化试剂在有机相中进一步老化，转入水相后通过碳二亚胺/硫代琥珀酰亚胺法（EDC/Sulfo-NHS）进行表面水溶性改性获得最终的硅量子点，该硅量子点具有优良的水分散性，强荧光性（量子产率30%），合适的尺寸（约4nm），非常适合作为荧光成像探针。此方法合成的硅点表面保留了一定数目的伯胺基团，可以被进一步功能化。研究中我们分别选用线粒体定位试剂三苯基膦和溶酶体定位试剂吗啉通过EDC/Sulfo-NHS交联法进行功能化，并通过双光子荧光共聚焦成像证明其示踪效果，实验结果证明，通过和商品化的有机染料对比，硅量子点能准确、快速（约15min）对亚细胞器线粒体和溶酶体进行定位，且成像效果不逊于有机染料探针，此外线粒体会在细胞凋亡初期肿胀变圆，通过诱导剂引发细胞开始凋亡，实时成像图显示了我们合成的功能化硅量子点能很好的表现出线粒体形状变化，进行细胞早期凋亡的示踪，表明所得的硅量子点是有潜质的生物成像探针。不光是石墨烯，碳纳米材料本身优异的光学、电学性质和无毒、化学惰性、容易代谢等特性促使科学家们不断开发其在生物、医学领域的应用。为了拓宽荧光碳纳米材料在生物成像领域的发展空间，在第4章中我们合成了一个双层脂质体共价修饰的新型碳量子点，因为传统一步法合成出的碳点往往光学性质差强人意（量子产率通常低于3%），很多相关研究都着力于表面改性提高量子产率，例如通过氨基修饰的低聚PEG（分子量约1500）进行表面钝化处理，实现光学性能提高（量子产率约10%），而我们则尝试用相似分子量的磷脂代替氨基修饰的低聚PEG对碳点进行表面钝化，首先通过传统的硝酸回流法合成出碳点前体，再通过超滤装置筛选出所需颗粒大小（小于10nm），同时合成好双分子层脂质体。利用碳二亚胺/硫代琥珀酰亚胺法将二棕榈酰磷脂酰乙醇胺的氨基末端和碳点表面因氧化产生的羧基官能团交联获得最终的碳量子点，该碳点荧光强度比同浓度的前体碳点高3倍，尺寸约5nm，表面包裹的脂质体双分子层能赋予碳点优异的生物相容性和表面可控修饰性。我们进一步探索了碳点在HeLa（宫颈癌）细胞中的双光子荧光共聚焦成像效果，实验证明碳点在细胞各个位置都有分布，说明该方法合成的碳点非常适合生物成像领域的应用。汞是高毒的环境污染物，尤其是其具有高迁移性、持久性、甲基化作用性、生物富集性及食物链放大性等特点，即便是极微量的存在于环境中，对动植物及人类的健康也是极大的威胁。在第5章中我们设计了一个非常新颖的，基于T-Hg2+-T配位化学原理，能高灵敏性、高选择性的检测Hg2+的电化学传感器。这项技术的设计思路是邻近的聚T DNA链之间在Hg2+介导条件下产生的协同效应。先是在金电极表面，通过Au-S共价键，有序地组装上一层由8个碱基组成的全TDNA单链探针，且该探针末端标记了电活性物质二茂铁，当没有Hg2+存在时，电极表面的聚T DNA单链探针都各自柔软地趴在金盘表面，二茂铁离金表面很近，电子发生传递，可检测到二茂铁的氧化还原电流；当Hg2+存在条件下，最相邻的聚T DNA单链探针可以成对协同性地结合Hg2+，即电极表面的探针构象在Hg2+介导的T-T错配情况下可由柔软的单链结构变成相对刚性的类似双链的结构，这个构象变换导致了二茂铁远离电极表面，电子传递受阻，检测到的氧化还原电流大大减弱。通过电化学信号的变化可以判断Hg2+的存在及定量分析。这个检测机理已经得到毛细管电泳及一系列电化学方法的充分验证，并且实验结果表明这个传感器能在1nM-2μM之间高灵敏检测Hg2+，检测下限能到达0.5nM，优于本文中介绍的其它检测方法，并且这项技术表现出了优异的选择性，这是和传统的电化学方法（阳极溶出分析法ASV）检测Hg2+最大的区别，而且这个Hg2+传感器所需试剂非常少、操作步骤很简单又有着极好的再生性。如此经济高效的电化学传感器在实时现场监测环境中Hg2+将有很好的应用前景。MicroRNA（miRNA）在生物体内发挥着至关重要的调控功能，不仅对细胞的生长、分化和凋亡有着重要的影响，而且还与许多疾病有着密切的关系，研究者发现在白血病、结肠直肠癌、乳腺癌和心血管障碍等疾病中，相关的miRNA都异常表达。由于miRNA具有核酸序列短、含量少以及相互之间序列相似性等特点，分析测试有一定的挑战性。传统的检测方法有印记杂交技术、微阵列检测技术、基于RCA信号放大的检测技术、阳离子聚合物等。本论文第6章中将向大家报道一种基于双重侵入式信号放大技术（Invader assay）高灵敏高选择性检测miRNA的方法。曾有研究小组基于侵入式信号放大技术检测miRNA，但是都是将目标miRNA作为模板探针，对相似序列的miRNA不能实现很好的区分，我们则是将miRNA设计成第一重循环放大的侵入式探针，并且miRNA的存在不会立即形成侵入位点重叠结构，我们借鉴缺口连接PCR的原理，设计出miRNA的3’端距侵入位点数个核苷酸距离。当加入嗜热性Taq聚合酶，和相应的核苷三磷酸，完全匹配的miRNA成为引物开始从3’端开始聚合延伸，当聚合延伸到达信号探针和模板探针杂交区的第一对核苷酸（即侵入位点），将距信号探针侵入位点核苷酸和后一位（靠近3’端）的核苷酸之间磷酸骨架断开，信号探针5’翘起一段离开，另一段也因杂交序列变短，与模板杂交稳定性减弱并脱离模板，遂成为第二重循环放大的引物探针。第二重循环放大类似第一重循环，也是需要借助聚合的作用才能形成侵入式结构，进一步消弱了非特异性切刻作用，第二重循环的信号探针是5’标记荧光团荧光素，中间标记淬灭基团的Taqman探针，但侵入式结构形成，信号探针5’翘起并带有荧光素修饰的“翼”离开，和信号探针中间标记的淬灭基团分开，荧光信号增强，并且与靶序列miRNA的浓度成正比。实验结果证明，该方法可以一步实现两重循环放大，检测灵敏度高，下限可达10fmol/L，尤其是能区分相似的miRNA序列，实现了难得的miRNA选择性检测。更多还原

【Abstract】 Biosensor is based on the identification of biological molecules and theirdownstream signaling conversion, carried out a class of analytical methods to detectthe target, crossover study is to analyze the field of chemistry and the life sciences.Compared with other analysis methods, it has many advantages, especially toimprove the speed of analysis and application flexibility. With advances inelectronics, microelectronics and mechanical systems, portable, integrateddevelopment of the sensing element, biosensing methods have the opportunity tostudy medicine, environmental protection, food security,,and public safety and otherareas to provide more on-site rapid testing technology the establishment of the firstline of defense for the public health and public safety. In recent years, researcherscontinue to make breakthroughs in the field of nano-materials, nucleic acidrecognition, nucleic acid signal amplification continuous development of newtechnologies for building high sensitivity, high selectivity, fast and efficient novelbiosensor provides perfect design ideas, as biological sensors to achieve differenttarget site rapid testing analysis provides a more powerful platform. This paper isbased on the current hot spots of nanomaterials-environmentally safe carbon, siliconnanomaterials developed three medical diagnosis application prospects biosensors,including an important cell signaling pathway protease-phospholipase D testing, livecells and subcellular organelles tracer. It’s the first reported non-graphene oxidebiosensor applied by non-covalent biological function of the first to demonstrate theuse of non-toxic nanomaterials capablely traced of mitochondria-cellular energyfactory effectively comparable organic dyes. At the same time, which involvesnanomaterials-Non-graphene oxide, silicon quantum dots, bio-functional study ofcarbon quantum dots each have a small breakthrough, laid a better foundation fortheir further development in the field of bio-sensing. The paper is also based onnucleic acid hybridization technology and metal ion-mediated nucleic acidhybridization techniques, developed detection of important cancer markers-smallRNA (microRNA) of organisms and environmental pollutants-mercury ion, and thesetwo techniques are sensitive to target to achieve a high selectivity of the analysis.All research specific contents are as follows:In order to further broaden the application of the non-oxidized graphene biosensor field, in Chapter2We developed a novel non-bilayer lipid membranesmodified graphene oxide nanocomposites, The nanoassembly can be prepared easilythrough noncovalent hydrophobic interactions between the lipid tails and thegraphene without destroying the electronic conjugation within the graphene sheet.This imparts the nanoassembly with desired electrical and optical properties withnonoxidative graphene. The phospholipid coating offers excellent biocompatibility,facile solubilization, and controlled surface modification for graphene, making thenanoassembly a useful platform for biofunctionalization of graphene. Thenanoassembly is revealed to comprise a bilayer of phospholipids with a reducedgraphene oxide sheet hosting in the hydrophobic interior, thus affording a uniqueplanar mimic of the cellular membrane. By using a fluorescein-labeled phospholipidin this nanoassembly, a fluorescence biosensor is developed for activity assay ofphospholipase D. The developed biosensor is demonstrated to have high sensitivity,wide dynamic range, and very low detection limit of0.010U/L. Moreover, becauseof its single-step homogeneous assay format it displays excellent robustness,improved assay simplicity and throughput, as well as intrinsic ability to real-timemonitor the reaction kinetics.In recent years, silicon nanomaterials are a type of important semiconductornanomaterials with attractive properties including excellentoptical/electronic/mechanical properties, favorable biocompatibility, hugesurface-to-volume ratios, surface tailorability, improved multifunctionality, as wellas their compatibility with conventional silicon technology. Silicon materials ofvarious nanostructures have been developed, among which zero-dimensionalnanomaterials-silicon quantum dots (SiQDs) have been employed as an effectivenanomaterials for various sensing applications and fluorescent biological probes forin vitro and in vivo imaging because of their unique fluorescence properties,favorable biocompatibility, noncytotoxic property, fast response and goodreproducibility. In chapter3, we report a novel kind of SiQDs which simultaneouslypossess appropriate size (approximately4nm), excellent aqueous dispersibility, highphotoluminescent quantum yield (approximately30%), and excellent photostability.In brief, fluorescent SiQDs are facilely prepared via simple one-pot electrochemicaletching method, the as-prepared SiQDs can then be passivated by hydrogen peroxideoxidation, further aging using silane-coupling reagent in the organic phase prior tothe nanoparticles transfer to the aqueous phase, then the surface of the SiQDs aremodified with carbodiimide/sulfosuccinimidyl (EDC/Sulfo-NHS) to form final hydrophilic SiQDs. The decorated SiQDs retain a certain number of primary aminegroups, may be further functionalized with functional ligands, and they arewell-suited to fluorescence imaging. In this study, we have designed and synthesizedtwo high-friendly and brightly luminescent silicon quantum dots bioconjugates,silicon quantum dots-triphenylphosphonium (SiQDs-TPP) and silicon quantumdots-morpholine (SiQDs-MP), for subcellular mitochondrial/lysosomal imaging.Two-photon confocal imaging experiments establish that SiQDs-TPP and SiQDs-MPwere able to accurately and quickly localize to mitochondrias and lysosomesrespectively (approximately15min). Colocalization studies of the probe withcommercial colocalization reagents in cells demonstrated the specific localization ofthe probe in the mitochondrias or lysosomes with an extremely high colocalizationefficiency. Furthermore, SiQDs-TPP possesses high specificity to mitochondria,superior photostability, and appreciable tolerance to environmental change, allowingimaging and tracking of the mitochondrial morphological changes in a long period oftime, in early apoptosis, for example, swelling of mitochondria are clearly foundwhen the cells were treated with staurosporine(STS), a commonly used apoptosisinducer. The new imaging probe has a number of properties that far exceed those ofcommercial colocalization probes, including high mitochondrial/lysosomalselectivity, good fluorescence quantum yield, and, importantly, high photostability,high-friendly, all resulting in a superior figure of merit.As the classical graphene, carbon-based nanomaterial offer several favorableattributes including excellent optical/electrical properties, non-toxic, chemicallyinert, easy metabolism and promise significant advantages in several imaging andbioanalytical applications. The aim of this work is to further extend the applicationof fluorescent carbon nanomaterials in the field of biochemistry to develop anfluorescent biological probes for cell imaging. In Chapter4, we synthesized a novelkind of carbon nanodots (C-dots), which specifically involves decorating thenanoparticles surface with liposome bilayer. Current synthetic methods of C-dots aremainly deficient in accurate control of uniform dimensions and the resulting surfacechemistry, as well as in obtaining fluorescent materials with low quantum yields(usually below3%). Most of these synthesis methods need post treatment withsurface passivating agents in order to improve their water solubility andluminescence property. Recently, for example, serious efforts have been made tosyntheses of amino-functionalized fluorescent C-dots (quantum yields wasapproximately10%) by amino-modified PEG (MW=1500). On the basis of the above, we first synthesis carbon precursor through conventional nitric acid reflux method,and obtain required uniform particle size (less than10nm) by ultrafiltration, andfurther tried a similar molecular weight phospholipids instead of amino-modifiedPEG for carbon surface passivation. The final C-dots were obtained via specialcoordination crosslinked interaction between dipalmitoyl phosphatidylethanolamine(DPPE) of the as-prepared liposome decorated C-dots and carboxyl functionalgroups that present in the surface of C-dots due to oxidation using the EDC andsulfo-NHS. The as-prepared liposome decorated C-dots have a uniform dispersionwithout apparent aggregation and particle diameters of5nm, excellentbiocompatibility, controllable surface modification and their fluorescence intensitythree times than carbon precursor at same concentration. We further explored thetracking and distribution of as-prepared liposome decorated C-dots in cells, Asexpected, after incubating with a C-dots-culture solution, two-photon confocalimaging experiments establish that the fluorescence in the Hela cells retained a highintensity, and distributed in various locations. This indicating the as-preparedliposome decorated C-dots ideally suited for applications in the field of biologicalimaging.Mercury is highly toxic global environmental pollutants, especially its highmobility, persistence, the role of methylation, bioaccumulation and amplification ofthe characteristics of the food chain, even a very small amount present in theenvironment, for plants, animals and human health is also a great threat. In Chapter5, we have designed a very novel, based on T-Hg2+-T coordination chemistry, energysensitivity, high selectivity to detect Hg2+. This strategy exploited the cooperativityof proximate poly-T oligonucleotides in coordination with Hg2+. Ferrocene(Fc)-tagged poly-T oligonucleotides were immobilized on the electrode surface viaself-assembly of the terminal thiol moiety. In the presence of Hg2+, a pair of poly-Toligonucleotides could cooperatively coordinate with Hg2+, which triggered aconformational reorganization of the poly-T oligonucleotides from flexible singlestrands to relatively rigid duplexlike complexes, thus drawing the Fc tags away fromthe electrode with a substantially decreased redox current. The responsecharacteristics of the sensor were thoroughly investigated using capillaryelectrophoresis and electrochemical measurements. The results revealed that thesensor showed a sensitive response to Hg2+in a concentration range from1.0nM to2.0μM, with a detection limit of0.5nM. Also, this strategy afforded exquisiteselectivity for Hg2+against a reservoir of other environmentally related metal ions, compared to existinganodic stripping voltammetry (ASV) techniques. In addition,this sensor could be implemented using minimal reagents and working steps withexcellent reusability through mild regeneration procedure. It was expected that thiscost-effective electrochemical sensor might hold considerable potential in on-siteapplications of Hg2+detection.MicroRNA (miRNA) plays a crucial regulatory function in vivo and importantimplications on not only for cell growth, differentiation and apoptosis, but alsorelated closely with many diseases. The researchers found that miRNA are related tothe abnormal expression in leukemia, colorectal cancer, breast cancer andcardiovascular disorders diseases. Since miRNA is very short, less content andsimilarity characteristics than other sequences, analytical testing has somechallenges. The traditional method included the mark detecting hybridization,microarray technology, based RCA signal amplification detection techniques, andcationic polymers. In Chapter6of this paper, we reported double-invasive signalamplification based on highly selective sensitive miRNA detection methods andtechniques (Invader assay). Other researchers had invasive signal amplificationtechnology to detect miRNA, but their template can not achieve a good distinctionwith similar miRNA sequences. We designed the first heavy sucked miRNA invasiveprobe amplification cycle, and there is not formed immediately invade miRNA locioverlapped structure, we can learn the principles of notches ligated PCR, design ofmiRNA3’ end from the intrusion bit points nucleotides distance. When thethermophilic Taq polymerase was added, and the corresponding nucleosidetriphosphates, miRNA become fully matched primer extension to initiatepolymerization when the polymerization to the5’ end of the signal probe and probehybridizing region of the template (invasion locus), Taq polymerase5’-3’exonuclease activity began to play a role in the invasion site nucleotide probe signala (near the3’ end) of the disconnect between the nucleotide and the phosphatebackbone, the signal probe5’ tilt leave, but also because of another section of shortersequences hybridize with a template from the template and hybrid stability weakened,became the second heavy cycle amplification primers probes. The second cycle ofamplification similar to the first heavy weight cycling, also needs the role ofpolymerization to form intrusive structure, and further weaken the role ofnon-specific nicking, the second re-circulation of the probe signal is5’ fluorophorelabeled fluorescein, Taqman probes labeled quenching intermediate groups, butintrusive structure formation, the signal probe5’ cocked and with fluorescein modified left, middle, and signal probes labeled quencher separate fluorescencesignal enhancement and is proportional to the concentration of the target miRNAsequence. Experimental results show that the method can achieve double loopamplification step, high detection sensitivity, the lower limit of up to10fmol/L, inparticular, can distinguish with similar miRNA sequences, achieved a rare miRNAselective detection.更多还原