【作者】 何鹏；

【导师】 张书圣；

【作者基本信息】 青岛科技大学 ， 应用化学， 2014， 博士

【摘要】 本文采用表面等离子共振及表面增强拉曼光谱分析检测方法,基于核酸适体与靶分子的高亲和力和特异性识别作用,结合纳米颗粒生物条码技术及DNA聚合酶、核酸内切酶等工具酶的自身特性,构建了基于双适体识别滚环复制放大的放大方法、纳米颗粒增强-链替换循环放大方法、靶向激活双重循环放大方法及级联循环放大方法,实现了蛋白质、基因及肿瘤细胞等肿瘤标志物的高灵敏及高选择性检测。主要研究内容如下：1、提出了新型的表面等离子共振适体传感器并将其用于蛋白质的高灵敏检测。首次将滚环复制放大与表面等离子共振技术相结合,以纳米金生物条码探针作为信号标记,通过滚环复制反应,将信号探针与带有大量重复序列的滚环复制的长链相结合,使检测信号显著增强,以凝血酶为靶蛋白质,检测限可达0.78aM。采用发夹结构的适体捕获探针及双适体识别体系,提高了检测的选择性,并采用加标回收法将该方法用于实际血清样品的检测,进一步验证了检测体系的实用性。该方法操作简单,线性范围宽,灵敏度高,选择性好,适用于复杂生物样品中蛋白质的选择性分析,为生物学研究、基因检测以及临床诊断提供了有效的分析手段。2、构建了一种基于纳米颗粒增强及多级信号放大的SPR检测DNA及肿瘤细胞的新方法。通过靶DNA引发链置换循环放大和滚环复制放大形成双重信号放大反应,并结合磁珠作为DNA载体的性质,以磁珠作为信号放大的“中转站”,磁珠生物条码探针不仅作为反应单元,发生链置换循环放大反应,还作为信号放大的载体,增大了滚环复制产物的负载量,形成多级信号放大体系,以金纳米粒子作为信号探针,采用表面等离子共振技术实现了靶DNA的高灵敏检测,检测限可达1fM。并进一步基于细胞适体对细胞表面蛋白的特异性识别及高亲和力,考察了该反应体系对肿瘤细胞检测的适用性,通过细胞适体负载的磁珠进行磁性分离,降低了体系的背景噪音,对淋巴瘤Ramos细胞的检测限可达76个细胞,实现了肿瘤细胞的高灵敏检测并具有良好的选择性,适用于复杂生物样品的检测。3、基于适体与靶分子的结构互变性质,构建了靶向激活双重循环放大的SPR检测系统用于溶菌酶的检测。将靶分子与适体的特异性识别作用转化成双重循环放大反应的切入点,从而引发靶分子的聚合替换源头放大和DNA链的聚合剪切放大反应,提高了反应的放大效率及选择性,以溶菌酶作为目标分析物,生物功能化的量子点探针为信号标记,利用表面等离子共振检测技术实现了溶菌酶的快速、灵敏检测,检测限可达76fM。并成功应用于人血清实际样品中溶菌酶的分析。该方法适用范围广、选择性强、操作便捷,在蛋白质分析及临床诊断方面具有广阔的应用前景。4、构建了新型的DNA级联循环放大分子器件,并将其应用于DNA和蛋白质的SERS分析检测中。以罗丹明染料分子标记的纳米金生物条码作为信号探针,金芯片作为检测基底,以溶菌酶作为目标蛋白质,通过溶菌酶与核酸适体的靶向识别,将与适体互补的靶DNA释放出来从而引发上游的内切酶循环放大,同时上游的产物又可以作为下游循环体系的DNA触发器,引发链置换和发夹探针辅助的指数放大反应,在检测基底上捕获大量的纳米金拉曼信号探针,使拉曼信号显著增强,从而实现了DNA和溶菌酶的高灵敏检测。DNA和溶菌酶的检测限分别为72aM和1fM。并进一步将该检测方法用于实际样品的分析,展现了优异的选择性和良好的分析性能,为该方法在临床及复杂生物样品的分析应用方面提供了有力的保障。更多还原

【Abstract】 Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. In this thesis, a series of novel biosensing assay systems were developed for ultrasensitive detection of cancer biomarker based on aptamer-target recognition, bio-bar-code nanoprobe enhancement and enzyme assisted multiple cycle amplification strategy. The proposed methods were also implemented in the practical determination of realistic biological samples. These results demonstrated the high potential of the developed sensor devices as platforms for clinical cancer diagnosis and prognosis. The detailed content was described as follows:1. A novel approach for thrombin detection was developed by the combination of rolling circle amplification (RCA) reaction with surface plasmon resonance (SPR) array. An aptamer-based sandwich assay was performed on gold substrate. The primary aptamer could specifically capture thrombin and the hairpin structure of aptamer capture probe could be used to effectively reduce the nonspecific adsorption. The second aptamer served as a template for the ligation of a padlock probe, and the circularized probe could in turn be used as a template for RCA to produce thousands of repeated oligonucleotide sequences. Then, the secondary aptamer domain of the products bound to the immobilized protein in a classic sandwich assay format to localize the long single-strand DNA directly to the thrombin. Thus, a large amount of Au NP labeled bio-bar-coded probes could be linearly and periodically assembled to the RCA products for amplification of recognition event. The enhancement of the SPR signals was achieved by increasing surface mass and refractive index from clustered AuNP conjugates on the SPR chip. The detection limit of0.78aM could be achieved. Based on hairpin aptamer capture probe and dual-aptamer recognition system, the excellent selectivity was also obtained. The high sensitivity and selectivity of RCA-based SPR aptasensor would make it a potential tool for proteomics research and clinical diagnostics.2. A cascade signal amplification strategy was proposed for detection of specific DNA sequences and Ramos cells by combining aptamer-based target-triggering strand displacement amplification (SDA) with rolling circle amplification (RCA) technique, magnetic nanoparticles (MNPs) bio-bar-code probe and surface plasmon resonance detection. MNPs acted as both DNA molecular carriers and the amplification intermediary for DNA amplification. Bio-bar-code MNPs were functionalized with the primer DNA of the SDA reaction and the primer DNA of the RCA reaction. The RCA product containing tandem-repeat sequences could serve as excellent template for periodic assembly of AuNPs which presented per cell recognition event to numerous AuNP tags for SPR signal response. Both the two-stage amplification reaction and the MNPs bio-bar-code technique showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. The detection of specific DNA sequences and Ramos cells as low as1fM and76cells were achieved, respectively. The proposed cascade signal amplification strategy would become a powerful tool for early diagnosis of disease.3. A simple and sensitive method for the detection of lysozyme was developed based on aptamer-based target-triggering triple-amplification system. Via combining an aptamer with a common hairpin DNA probe, the homogeneous parallel amplifica-tion reactions were performed, target molecule displacement polymerization amplification (TDPA) and strand nicking-polymerization amplification (SNPA). On the basis of TDPA and SNPA signal amplification, the SPR signal was dramatically enhanced. This detection method exhibited excellent specificity and high sensitivity with a detection limit of76fM and a detection range of more than2orders of magnitude. Moreover, this detection method exhibited impressive simplicity, convenience, and rapid without multiple separation and washing steps. Furthermore, this method might be extended to sensitive detection of a variety of biomolecules whose aptamers undergo similar conformational changes.4. An ultrasensitive surface enhanced Raman scattering (SERS) method has been designed to selectively and sensitively detect the specific DNA sequences and lysozyme. The gold chip as detection substrate, the aptamer-based target-triggering cascade multiple cycle amplification and gold nanoparticles (AuNPs) bio-barcode Raman probe enhancement on the gold substrate were employed to enhance the SERS signals. The cascade amplification process consisted of nicking enzyme signaling amplification (NESA), strand displacement amplification (SDA), and circular hairpin assisted exponential amplification reaction (HA-EXPAR). With the involvement of an aptamer-based probe, two amplification reaction templates and a Raman probe, the whole circle amplification process was triggered by the target recognition of lysozyme. The products of upstream cycle (NESA) could acted as the "DNA trigger" of the downstream cycle (SDA and circular HA-EXPAR) to generate further signal amplification, resulting in the immobility of abundant AuNPs Raman probes on the gold substrate."Hot spots" were produced between the Raman probe and the gold film, leading to significant SERS enhance-ment. This detection method exhibited excellent specificity and sensitivity towards specific DNA sequences and lysozyme with the detection limit of72aM and1fM, respectively. Moreover, The practical determination of lysozyme in human serum demonstrated the feasibility of this SERS approach in the analysis of a variety of biological specimens.更多还原