摘要
近年来,纳米材料由于其独特的光学和电化学性能,已成为当前研究的焦点,纳米材料也在生物传感器的研究中得到越来越广泛的应用。纳米材料与生物传感器的结合,涉及到生物技术、信息技术、纳米科学等多个学科,因此纳米材料在生物传感器中的研究为许多基础研究提供了许多创新性的研究思路,同时对临床检测、医学诊断、环境监测等领域有着重要影响,也得到了前所未有的发展机遇。本文利用纳米材料氧化石墨烯和金纳米颗粒,开发了一系列成本低、操作简单、灵敏度高的新型纳米生物传感技术,成功实现了对核酸、蛋白质的高灵敏检测和抗癌药物的筛选。此外,针对当前癌症标志物端粒酶检测方法中存在的灵敏度较低、检测时间较长,容易出现假阴性信号等问题,结合本实验室已发展的方法和技术,开发了一些新颖的生物传感策略用于端粒酶的灵敏检测。其主要内容如下：
(1)在第2章中发展了一种新型的基于核酸外切酶Ⅲ辅助目标循环放大的生物传感策略用于目标DNA高灵敏的检测。在这种方法中,我们设计了一条标记有荧光基团的发夹状信号探针,其3’末端突出了7个碱基,此时核酸外切酶Ⅲ不能水解信号探针,信号探针吸附在氧化石墨烯表面上,荧光被氧化石墨烯猝灭。当存在目标DNA时,目标DNA的5’末端与信号探针的3’末端杂交,形成双链结构的平齐末端,从氧化石墨烯表面上解离下来,荧光得到恢复,而核酸外切酶Ⅲ会从3’末端开始水解信号探针,而目标DNA的3’末端突出了6个碱基,目标DNA不会被核酸外切酶Ⅲ水解,被释放出来进入下一个循环反应,继续与发夹状信号探针杂交,然后信号再被核酸外切酶Ⅲ水解,这样目标DNA就可以得到循环利用。该方法实现了对目标DNA的高灵敏、高特异性检测,响应动态范围为1pM到50nM,检测下限可达1pM,比传统的DNA检测方法要低3个数量级,同时该方法具有较好的碱基错配识别能力。
(2)在第3章中建立了一个基于氧化石墨烯的通用的生物传感平台用于抗癌药物的筛选。该方法以人类端粒DNA的重复序列为模板设计了一条标记有荧光的信号探针,信号探针会吸附在氧化石墨烯表面,从而猝灭荧光。当目标药物出现时,信号探针上的碱基与中药单体之间通过π-π共轭作用和氢键作用形成分子内反平行的G-四股螺旋复合结构,从而使信号探针从氧化石墨烯表面上解离下来,荧光信号得到恢复,进而达到对抗癌抗肿瘤药物筛选的目的。该方法对三类传统中药单体进行了筛选,结果发现黄酮类中药单体能与信号探针形成G-四股螺旋复合结构,是一种潜在的抗癌药物,同时该方法在发现新的抗癌药物方面具有潜在的应用价值。
(3)为了进一步拓宽氧化石墨烯的应用领域,在第4章中,利用氧化石墨烯优越的负载能力,构建了一个通用的生物传感平台用于蛋白水解酶的检测,并用于活细胞内蛋白水解酶的成像分析。这种生物传感技术利用共价交联的方法,将多肽信号探针固定到氧化石墨烯的表面,达到降低背景荧光的目的。当目标蛋白水解酶存在时,蛋白水解酶特异性地水解多肽信号探针,荧光基团从氧化石墨烯表面解离下,远离氧化石墨烯,从而产生荧光信号。在这种方法中,氧化石墨烯一方面作为荧光猝灭剂,用来猝灭信号探针的荧光,另一方面,氧化石墨烯又作为多肽信号探针的优良载体,将多肽信号探针运送到肿瘤细胞的内部。当肿瘤细胞被诱导产生目标蛋白水解酶时,蛋白水解酶特异性地水解固定在氧化石墨烯表面的多肽信号探针,从而产生荧光成像信号。该方法实现了对目标蛋白水解酶caspase-3的灵敏、快速检测,其线性范围在7.25ng/mL到362ng/mL之间,同时也具有较好的选择性,此外还可实现对多元目标蛋白水解酶的同时检测。
(4)在第5章中,利用金纳米颗粒具有优越的荧光猝灭能力,建立了一种新颖的生物传感技术用于肿瘤细胞中端粒酶活性的检测。该方法先将信号和巯基捕获探针退火杂交形成双链DNA,再将双链DNA自组装到金纳米颗粒表面,得到DNA-纳米金探针。巯基捕获探针的3’端包含端粒酶扩增的引物序列,在目标端粒酶存在的条件下,引物进行扩增,扩增产物与捕获探针上的互补序列形成分子内发夹结构,将信号探针置换下来,从而产生荧光信号。该方法实现了对目标端粒酶的灵敏检测,动态响应范围为100到30000个HeLa细胞,并且当HeLa细胞在0到1600个范围内时,荧光强度与HeLa细胞的个数呈现良好的线性关系。此外该传感技术还可用于二价铅离子高灵敏、高特异性的检测,检测下限可达2nM。
(5)在第6章中,制备了一种基于构象变换的电化学DNA生物传感器用于肿瘤细胞中端粒酶活性的检测。该方法将3’端包含了端粒酶扩增引物序列的捕获探针组装到金电极表面,再将二茂铁标记的信号探针与捕获探针的5’端杂交。在目标端粒酶的作用下,引物进行扩增,扩增产物使得捕获探针由线型转换成发夹结构,通过捕获探针的构象变换,二茂铁标记的信号探针远离电极表面,从而使得电化学信号减弱。这种方法成功地实现了对肿瘤细胞中端粒酶活性的灵敏检测,动态响应范围为100到60000个HeLa细胞,并且在该范围内峰电流信号与HeLa细胞个数的对数呈现良好的线性关系,同时该方法具有较高选择性,线性范围宽,高特异性等优点。
(6)在第7章中,开发了一种基于T7核酸外切酶辅助目标循环的放大方法用于肿瘤细胞中端粒酶活性的高灵敏检测。在该方法中,我们设计了一条5’标记有荧光素(FAM)和中间标记有四甲基罗丹明(TAMRA)的Taqman探针,由于T7核酸外切酶对单双链DNA具有较好的区分能力,几乎不水解单链状态的DNA,因此,在没有目标端粒酶时,单链状态的Taqman探针不被水解,体系的背景信号也很低。此外,T7核酸外切酶是从双链DNA的5’端开始水解DNA,所以引物3’端的扩增产物不会被水解,从而得到进一步的循环利用,大大提高了目标检测的灵敏度。该方法成功地实现了对端粒酶活性的灵敏检测,检测范围为5到1000个HeLa细胞,并且在5到100个HeLa细胞范围内呈现良好的线性关系,同时还具有高选择性,高特异性的优点,为肿瘤细胞中端粒酶活性的快速灵敏检测提供了一种新的思路。
In recent years, nanomaterials have been regarded as the research focus, and applied in biosensors more and more widespreadly, due to their excellent optical and electrochemical properties. The combination of nanomaterials and biosensors is concerned with the fields of biotechnology, information technology, nanoscience and so on. Therefore, the study of nanomaterials in biosensors provides a lot of innovative ideas for basic research, and plays a most important role in clinical testing, medical diagnosis, and environmental monitoring. Nanomaterials are also facing an unprecedented opportunity for development. In this paper, a seris of novel nano-biotechology as a simple and low-cost platform for sensitive detection of DNA and proteins, screening antitumor drugs. Because reported methods for detecting telomerase activity were time-consuming, low-sensitive and false negative, a seris of novel biosensing strategys were developed for sensitive detection of telomerase activity. The detailed contents are described as follows:
(1) In chapter2, we developed a novel biosensing strategy for high-sensitive detection of DNA based on Exo Ⅲ-assisted target recycling amplification. In this assay, we designed a fluorescein amidite (FAM)-labeled hairpin signal probe which with extensions7bases at its3'terminus. In the absence of target DNA, signal probe first adsorbed onto the surface of graphene oxide through π-stacking interaction between the ring structrue in the nucleobases and the hexagonal cells of GO, and the fluorescence of the dye was quenched. While the target DNA was introduced, signal probe hybridezed with target DNA to form double-stranded DNA structure, which led to the releasing of signal probe from the surface of graphene oxide and the fluorescence intensity recovered. The duplex DNA was digested by Exo Ⅲ, a sequence-independent nuclease that catalyzes the removal of mononucleaotides from blunt or recessed3'-hydroxyl termini of duplex DNA and is not active on single-stranded DNA and3'-protruding termini with extensions4bases or longer. This digestion not only make the dye far from the graphene oxide, but also released the target DNA, which then hybridize with fresh signal probes and restart the digestion process. The results revealed that this strategy offered a sensitive and selective method for the detection of target DNA over the concentration range of1pM to50nM with the detection limit of1pM. The sensitivity of the assay is3orders magnitude better than previously reported methods.
(2) In chapter3, we constructed a novel biosensing platform for screening antitumor drugs based on graphene oxide sheets. In this method, we chose human telomeric DNA as the template to design a fluorescein amidite (FAM)-labeled signal probe which adsorbed onto the surface of graphene oxide sheets, and the fluorescence of the FAM was quenched. When the quadruplex-binding ligands were introduced, the signal probe folded to form intramolecular antiparallel G-quadruplex structure through the π-π conjugated interactions and hydrogen bindings between ligands and bases of signal probe. It led to the releasing of FAM-labeled signal probe from the surface of graphene oxide and the fluorescence intensity recovered. Three series of Chinese medicine monomers were investigated by the proposed method, and the flavonoids were demonstrated to be the potential quadruplex-binding ligands. Furthermore, the strategy could find wide applications in the discovery of new antitumor drugs.
(3) In order to further extend the application range of graphene oxide, we exploited a universal biosensing platform for protease detection and intracellular imaging in live cells based on graphene oxide-peptide conjugate in chapter4. In this strategy, peptide probe was conjugated to the surface of graphene oxide by covalently crosslinking method to reduce the background signal. In the presence of target protease, peptide probe was cleaved by target protease specificly, which led to the releasing of FAM-labeled peptide probe from the surface of graphene oxide and the fluorescence intensity enhanced substantially. On one hand, graphene oxide was a fluorescence quencher for fluorophores adjacent to its surface. On the other hand, graphene oxide is intrinsically a nanocarrier for delivering peptide cargos inside live cells. After being transported into cells followed by cleavage of the peptide by intracellular proteases, the GO-peptide conjugate provided greatly enhanced fluorescence imaging. The results demonstrated that this strategy could afford a simple, sensitive and selective biosensor for the detection of caspase-3in the dynamic range of7.25-362ng/mL. In addition, it could be extended to multiplex in vitro assays or live-cell imaging of multiple proteases by use of the conjugate of GO to different peptide substrates with multicolor fluorophore tags.
(4) In chapter5, we presented a novel biosensing technology for the detection of telomerase activity in cancer cells by using gold nanoparticles as a fluorescence quencher. Firstly, thiolated capture probe was mixed with signal probe and slowly cooled to room temperature. Then the DNA duplexes were added to gold nanoparticles (GNPs) to form the DNA-GNPs composite probes by self-assembly technology. The TS primer contained at the thiolated capture probe elonged in the presence of target telomerase. While the telomerase extension products could folded into intramolecular hairpin structure with the complementary sequences at the capture probe and released signal probe to enhance the fluorescence intensity. The results indicated that the method could be used for sensitive determination of telomerase in a concentration range from100to30000HeLa cells with the linear range of0to1600HeLa cells. Moreover, it could be extended to detection of Pb2+sensitively and selectively with the detection limit of2nM.
(5) In chapter6, we developed a novel eletrochemical DNA sensor for sensitive and selective detection of telomerase based on target-induced structure-switching DNA. In this assay, capture probe with the sequences of TS primer at its3'terminus was firstly immobilized on the gold electrode via self-assembly of the terminal thiol moiety and then hybridized with a ferrocene-tagged signal probe, leading to a high redox current. In the presence of telomerase, the TS primer elonged and the telomerase extension products could fold into intramolecular hairpin structure with the complementary sequences at the capture probe, resulting in the release of the ferrocence-tagged signal probe far from the electrode with a substantially decreased redox current. The results show that the eletrochemical DNA sensor displayed a quantitative analysis of telomerase with the linear range of100to60000HeLa cells besides desirable specificity and sensitivity.
(6) In chapter7, we exploited a high-sensitive fluorescence strategy for telomerase detection in cancer cells base on T7Exonuclease-assisted target recycling amplification. In this assay, we designed a Taqman probe modified with6-carboxy-fluorescein (FAM) as a fluorophore at its5'terminus and tetramethyl-6-carboxyrhodamine (TAMRA) as a quencher at the neighboring three-nucleotide position. The T7Exonuclease, a sequence-independent nuclease that catalyzes the removal of5'mononucleotides from5'termini of double-stranded DNA, couldnot digest the Taqman probe, hence this probe gives very weak fluorescence signal from FAM due to fluorescence resonant energy transfer from FAM to TAMRA. The telomerase can bind to the substrate sequence (TS) and enzymatically elongate it with TTAGGG repeats in the presence of dNTPs. Once the telomerase extension products hybridize with the Taqman probes, the T7Exonuclease will digest product-bound Taqman probes. This digestion not only makes the quencher-fluorophore pair of Taqman probe separate from each other, leading to significantly enhanced fluorescence, but also releases the intact product strands, which then hybridize with fresh Taqman probes and re-start the digestion process. Therefore, the fluorescence signal is amplified repeatedly through recycle of the telomerase extension products. The results revealed that it offered a sensitive and selective biosensing strategy for the detection of telomerase over the concentration range of5to1000HeLa cells with the detection limit of5HeLa cells. This strategy holds great promise as a simple, sensitive method for telomerase detection in proteomics and clinical diagnostics.
引文
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