基于纳米金和酶切信号放大的生物传感分析方法及应用研究
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摘要
随着分析科学的不断发展,生命科学领域中的各种分析和检测过程越来越多地需要要借助于生物传感技术获取所需的信息。生物传感器具有选择性好、灵敏度高、分析速度快、成本低、能在复杂体系中进行在线连续监测等优点,在化学、生物医学、环境监测、食品、医药和军事等领域有重要的应用价值。理想的生物传感器对目标物应该具有非常高的检测灵敏度(低检测限),高特异性(低干扰)宽的动力学检测范围,快速响应时间,以及通用性等特点。
功能核酸是基于体外筛选或指数富集配体系统进化法(systematic evolution of ligands by exponential enrichment, SELEX),筛选得到的与生物小分子、蛋白质分子、细胞、金属离子等高亲和力和高特异性结合的DNA或RNA片段。功能核酸通常包括两大类具有特殊功能的核酸分子：一类具有类似于蛋白酶的催化活性,称为DNA酶(DNAzymes),另一类能够像抗体一样特异性结合目标分子,称为核酸适配体(Aptamer)。功能核酸的发现突破了传统意义上关于核酸只是遗传信息存储和转运载体的认识,为构建用于各种分析对象的生物传感体系提供了全新的设计思路和平台。
灵敏度是衡量生物传感器性能的最重要参数之一,而单纯利用功能核酸结合目标物前后构象变化引起信号的改变,在进一步提高灵敏度方面已经十分有限。近年来,设计新的信号放大方法用于目标物的高灵敏检测已经引起了研究人员的广泛关注。提高传感器灵敏度的方法通常有两种,一种是降低检测背景,另外种是采用信号放大技术。其中,由于信号放大技术在提高灵敏度方面的显著效果,近年来受到了研究者更为广泛的关注
基于以上考虑,综合文献报道,本文主要利用信号放大技术在提高生物传感器的检测灵敏度方面做了一些工作,主要内容如下：
(1)基于纳米金信号放大技术的生物传感器的研究。利用纳米金比表面积大、具有良好生物相容性以及表面核酸高负载量的特点,在第2章提出了一种基于
‘T-Hg2+-T”特异性强结合作用和纳米金信号放大的新型非标记Hg2+电化学传感器。在Hg2+存在的条件下,连接DNA与电极表面的捕获探针通过“T-Hg2+-T”相互杂交,然后引入纳米金功能化的信号DNA与连接DNA另外一端序列杂交。以亚甲基蓝作为电活性物质,由于纳米金功能化信号DNA的引入能够放大检测信号,因此当进行DPV检测时,可得到明显增强的电化学信号。该方法可获得0.5nM的检测限,且选择性好、操作简单,并可用于实际样品的检测。在第3章,我们基于利用距离决定电磁场(EM)增强作用的指数减弱,即拉曼标记物离纳米粒子表面越近,Raman信号越强的原理,发展了一种基于树枝状纳米分子结信号放大技术的新型SERS传感器。以基于GR-5DNA酶的Pb2+SERS传感器和基于核酸适配体的腺苷SERS传感器作为分析模型,验证了通过层层自组装形成纳米分子实现拉曼信号放大的可行性,以及该设计策略的通用性。为了构建Pb2+SERS传感器,首先将巯基标记的GR-5DNA酶组装在金电极表面,当Pb2+存在时,能够催化镶嵌在该DNA酶底物链中RNA碱基的水解,使其被剪切为两部分。金电极表面剩余的核酸片段部分可以和纳米金标记的报告探针链杂交,通过层层自组装,纳米金之间形成了纳米结,拉曼信号得到显著增强,对Pb2+检测限可达0.1nM。为了检测腺苷,采用目标导致链置换构建核酸适配体SERS传感器。核酸适配体首先与金电极表面的捕获探针互补杂交被固定在电极表面,当腺苷存在时,与核酸适配体发生特异性结合,导致其构型发生变化,从电极表面脱落。然后,捕获探针可以和纳米金标记的报告探针链杂交,同样通过层层自组装形成纳米结,从而导致拉曼信号的显著增强。该传感器具有较好的选择性,检测下限为50nM。
(2)基于酶切循环信号放大技术的生物传感器的研究。基于核酸切口酶能够识别特异性的双链序列而只酶切其中一条单链从而可以实现循环信号放大的特点,在第4章利用分子信标低背景、高灵敏的优点提出了一种新型、能够实现目标诱导酶切循环信号放大过程的“Y型”探针用于高灵敏检测目标DNA,该探针同时具有较强的单碱基错配识别能力。当目标DNA存在时,可以和辅助探针协同打开分子信标的发夹型结构,三者相互杂交形成“Y型”结构,从而形成了切口酶Nt.BbvCI的识别序列。分子信标被核酸切口酶识别切断后,从传感系统上游离释放出来。释放出的辅助探针和目标DNA可以继续和另外一个分子信标进行杂交,同时诱发第二次酶切循环。经多次酶切循环反应后,实现了真正的目标DNA诱发酶切循环信号放大,与第一代基于中间标记的直线型信号探针构建的“Y型”探针相比更加灵敏,检测下限为5pM。在第5章,我们采用将分子识别和信号报告基团进行分离的设计策略,避免了对DNA酶进行修饰,并采用酶切循环信号放大技术构建了一种新型荧光催化信标,显著提高了对目标物的检测灵敏度。以L-组氨酸作为分析模型,采用DNA酶链和底物链以聚T连接的一体化DNA酶作为识别基团,L-组氨酸导致底物链酶切后的产物序列能够与作为信号报告基团的分子信标杂交,进一步采用核酸切口酶实现酶切循环信号放大,实现了对目标物的高灵敏和特异性检测,检测限为200nM。由于DNA酶具有可以循环剪切的性质,也可以用于酶切信号放大技术。在第6章,我们利用连接反应触发DNA酶和催化分子信标信号放大技术,提出了一种零背景、高灵敏的小分子检测方法。以ATP和NAD+作为分析模型,利用DNA连接酶对生物小分子的高特异性依赖,以连接反应触发劈开DNA酶的激活,然后与设计为分子信标的底物链杂交,在Zn2+存在的条件下进行DNA酶的酶切循环反应,实现了对生物小分子高灵敏、高特异性的荧光检测,其灵敏度和特异性均优于以前所报道的传感器。
(3)基于纳米金和酶切循环双信号放大的生物传感器的研究。基于核酸外切酶和纳米金双重信号放大策略,在第7章提出了一种非标型、高灵敏、高特异性电化学DNA传感器。该传感器首先在均相中进行发夹型探针和目标DNA之间的杂交及酶切反应,避免了电极表面进行酶切所遇到的酶效率降低等缺点,而且该发夹型探针能够提供较好的碱基错配识别能力；然后将酶切产物序列与电极表面的发夹型捕获探针杂交,进一步提高了碱基错配识别能力；最后利用纳米金功能化的报告探针进行信号放大,以亚甲基蓝为电活性物质,实现了对目标DNA的高灵敏和高特异性电化学检测,检测限为0.6pM。
With the continuous development of the analytical science, various analysis and testing process in life sciences are increasingly needed the help of biosensing technology to obtain the required information. Due to many advantages such as good selectivity, high sensitivity, fast response, low cost and continuous on-line detection in complex system, biosensors have valuable applications in chemistry, biomedicine, environmental protection, food industry, medicine and military affairs. Ideally, biosensors for the target detection should have high detection sensitivity (low detection limit), high specificity (low interference), wide dynamic range, fast response time, and universal characteristics.
Functional nucleic acids are short synthetic DNA and RNA sequences which have been isolated via a combinatorial biology technique known as in vitro selection, or a process also known as systematic evolution of ligands by exponential enrichment (SELEX). The functional nucleic acids can specifically bind a broad of analytes including small biomolecules, proteins, cancer cells as well as metal ions with high affinity. Typically, functional nucleic acids include two types of nucleic acid molecules, one type of them have been shown to perform catalytic reactions (called DNAzymes, or deoxyribozymes) like protein enzymes, and the other can bind to a specific target molecule (called aptamers) like antibodies. In the traditional understanding, the nucleic acids are only the storage and transfer carrier of genetic information. However, the discovery of functional nucleic acids has resulted in a breakthrough in such traditional sense, and provided an interesting alternative to biosensing system.
One of the most important performances of biosensor is sensitivity, and it is very limited in further improving the sensitivity by simply using the conformational changes of the functional nucleic acid before and after bind to the target. In recent years, the design of new signal amplification method for ultrasensitive detection of target has attracted attentions of researchers. Two different strategies have been widely employed to improve the sensitivity of a sensor:lower the detection background and signal amplified detection. Recently, signal amplification technology has attracted more concerns due to the significant effect on improving sensitivity.
Based on the above considerations and the reported literatures, in this doctoral thesis, several bioassay systems have been developed focused on new methods for enhancing the detection sensitivity of biosensors by using signal amplification technology. The details are summarized as follows:
(1) Study on the biosensors based on gold nanoparticle signal amplification technology. Due to the large specific surface area, good biocompatibility and surface characteristics of nucleic acids of high load, a novel electrochemical label-free biosensor for Hg2+has been developed in chapter2based on the "T-Hg2+-T" specific interaction and the gold nanoparticle signal amplification technology. In the presence of Hg2+, the linker DNA hybridized with the electrode surface-tethered capture probe via the "T-Hg2+-T" specific interaction, and the gold nanoparticles functionalized reporter DNA subsequently hybridized with the linker. Methylene blue was selected as electrically active substances, which can specifically bind with guanine bases to form methylene blue-guanine complexes, resulting in a significant increase in the electrochemical signal. The proposed biosensor exhibits high sensitivity, high specificity, and a detection limit of0.5nM could be achieved for Hg2+, which makes the biosensor favorable for Hg2+assays in practical samples of very low concentration. Based on the distance dependent exponentially decreased enhancement of the EM (electromagnetic) field, in chapter3, the use of a nanoscale DNA-Au dendrimer as a signal amplifier was proposed for the design of functional DNA-based ultra-sensitive SERS biosensors. As a proof-of-concept, a Pb2+-dependent DNAzyme and the anti-adenosine aptamer was chosen to develop novel SERS biosensors to verify the feasibility of our DNA-Au dendrimer amplification strategy. For the detection of Pb2+the DNAzyme strand was immobilized on the electrode surface first, in the presence of Pb2+, the DNAzyme was activated and it cleaved the substrate section into two parts. The remaining oligonucleotide moiety on the electrode surface hybridized with the reporter DNA1and DNA2to form nanoscale junctions through layer by layer assembling. The DNA-Au dendrimer could then be formed to give a strong SERS signal, and a detection limit of0.1nM could be achieved for Pb2+. For the detection of adenosine, the SERS aptamer sensor was designed based on target-induced strand displacement. A thiolated DNA sequence was first immobilized on gold electrode surface as a capture probe, and then hybridized with the anti-adenosine aptamer. The introduction of target adenosine could trigger the release of the aptamer from the duplex. The free capture probe could then hybridize with the reporter DNA2and DNA1, thereby forming the DNA-Au dendrimer to trigger a remarkable SERS signal enhancement. A low detection limit of50nM was obtained with good selectivity.
(2) Study on the biosensors based on enzymatic recycling signal amplification technology. Based on ssDNA-cleaved property of nicking endonuclease, and take advantage of the unique features of molecular beacons (MBs) such as high sensitivity and low fluorescent background, in chapter4, a new MB-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs was developed. In the presence of a target, the assistant probe, together with the target, can hybridize with the MB and open its hairpin structure to form a "Y" junction structure, as well as form the doublestranded recognition sequence for nicking endonuclease Nt.BbvCI. Once the MB is cleaved, it is dissociated from the sensing system, and fluorescence is restored. The released hybrid of the assistant probe with the target can then hybridize with another MB and trigger the second cycle of cleavage. Eventually, each hybrid of the assistant probe with the target can undergo many cycles to trigger the cleavage of many MBs, providing a true and efficient target-triggered enzymatic recycling amplification signal. Moreover, compared with the first-generation junction sensing system, our proposed sensing sytem also afforded a faster and more sensitive response, and a detection limit of5pM was obtained. In chapter5, by separating the molecular recognition module from the signal reporter to avoid DNAzyme modifications, and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification, we constructed a new fluorescent cascadic catalytic beacon sensing platform. An L-histidine-dependent DNAzyme was chosen as a model to construct the cascadic catalytic beacon. The unmodified unimolecular DNAzyme was linked by a polyT sequence which serves as a molecular recognition module for L-histidine, and a molecular beacon was selected as the signal reporter. The introduction of target resulted in the cleavage of DNAzyme, and the cleaved partial substrate is released from the DNAzyme and hybridized with the MB, the further using of nicking endonuclease resulted in signal amplification detection for the target. Under the optimized experimental conditions, the proposed sensing strategy provides a low detection limit of200nM with high selectivity. Due to the enzymatic recycling cleavage property, the DNAzyme also can be used to develop signal amplification technology. In chapter6, we proposed a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based signal amplification to develop a ligation-triggered DNAzyme cascade for small molecules detection. ATP and NAD+were chosen as analytical models to verify the feasibility of the CAMB sensing system. As two kinds of DNA ligases which specifically employ ATP and NAD+as cofactors, respectively, we employed the ligation to trigger DNAzyme cascade and realize zero-background signal. Then the activated DNAzyme could hybridize with substrate sequence which was designed as MB to form the CAMB, which was employed for amplified signal detection. The cycling and regenerating of DNAzyme could lead to a true enzymatic multiple turnovers of catalytic beacons. This combination of and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of100pM and50pM for ATP and NAD+, respectively, much lower than those of previously reported biosensors. Moreover, the developed DNAzyme cascades show significantly high selectivity towards the target cofactor (ATP or NAD+), and the target biological small molecule can be distinguished from its analogues.
(3) Study on the biosensors based on dual signal amplification strategy that combines the gold nanoparticle with enzymatic recycling signal amplification technologies. In chapter7, a label-free electrochemical DNA biosensor with high sensitivity and selectivity has been reported by employing both gold nanoparticle and enzymatic recycling signal amplification technology. In the design, take advantage of the improved mismatch distinguishing ability of MB, the hairpin probe first hybridizes with target DNA to trigger the enzymatic recycling reaction in the presence of exonuclease Ⅲ, which could avoid the low enzymatic reaction efficiency, effectively. Then, the cleaved sequences hybridize with the hairpin capture probe, which can further improve the mismatch distinguishing ability of the sensing system. Finally, the gold nanoparticles functionalized reporter DNA hybridized with the opened capture probe to amplify the electrochemical signal. Methylene blue was selected as electrically active substances as the sensor in chapter2, and the results demonstrated that the presented dual signal amplification strategy was highly sensitive and specific with a detection limit of0.6pM for target DNA.
功能核酸是基于体外筛选或指数富集配体系统进化法(systematic evolution of ligands by exponential enrichment, SELEX),筛选得到的与生物小分子、蛋白质分子、细胞、金属离子等高亲和力和高特异性结合的DNA或RNA片段。功能核酸通常包括两大类具有特殊功能的核酸分子：一类具有类似于蛋白酶的催化活性,称为DNA酶(DNAzymes),另一类能够像抗体一样特异性结合目标分子,称为核酸适配体(Aptamer)。功能核酸的发现突破了传统意义上关于核酸只是遗传信息存储和转运载体的认识,为构建用于各种分析对象的生物传感体系提供了全新的设计思路和平台。
灵敏度是衡量生物传感器性能的最重要参数之一,而单纯利用功能核酸结合目标物前后构象变化引起信号的改变,在进一步提高灵敏度方面已经十分有限。近年来,设计新的信号放大方法用于目标物的高灵敏检测已经引起了研究人员的广泛关注。提高传感器灵敏度的方法通常有两种,一种是降低检测背景,另外种是采用信号放大技术。其中,由于信号放大技术在提高灵敏度方面的显著效果,近年来受到了研究者更为广泛的关注
基于以上考虑,综合文献报道,本文主要利用信号放大技术在提高生物传感器的检测灵敏度方面做了一些工作,主要内容如下：
(1)基于纳米金信号放大技术的生物传感器的研究。利用纳米金比表面积大、具有良好生物相容性以及表面核酸高负载量的特点,在第2章提出了一种基于
‘T-Hg2+-T”特异性强结合作用和纳米金信号放大的新型非标记Hg2+电化学传感器。在Hg2+存在的条件下,连接DNA与电极表面的捕获探针通过“T-Hg2+-T”相互杂交,然后引入纳米金功能化的信号DNA与连接DNA另外一端序列杂交。以亚甲基蓝作为电活性物质,由于纳米金功能化信号DNA的引入能够放大检测信号,因此当进行DPV检测时,可得到明显增强的电化学信号。该方法可获得0.5nM的检测限,且选择性好、操作简单,并可用于实际样品的检测。在第3章,我们基于利用距离决定电磁场(EM)增强作用的指数减弱,即拉曼标记物离纳米粒子表面越近,Raman信号越强的原理,发展了一种基于树枝状纳米分子结信号放大技术的新型SERS传感器。以基于GR-5DNA酶的Pb2+SERS传感器和基于核酸适配体的腺苷SERS传感器作为分析模型,验证了通过层层自组装形成纳米分子实现拉曼信号放大的可行性,以及该设计策略的通用性。为了构建Pb2+SERS传感器,首先将巯基标记的GR-5DNA酶组装在金电极表面,当Pb2+存在时,能够催化镶嵌在该DNA酶底物链中RNA碱基的水解,使其被剪切为两部分。金电极表面剩余的核酸片段部分可以和纳米金标记的报告探针链杂交,通过层层自组装,纳米金之间形成了纳米结,拉曼信号得到显著增强,对Pb2+检测限可达0.1nM。为了检测腺苷,采用目标导致链置换构建核酸适配体SERS传感器。核酸适配体首先与金电极表面的捕获探针互补杂交被固定在电极表面,当腺苷存在时,与核酸适配体发生特异性结合,导致其构型发生变化,从电极表面脱落。然后,捕获探针可以和纳米金标记的报告探针链杂交,同样通过层层自组装形成纳米结,从而导致拉曼信号的显著增强。该传感器具有较好的选择性,检测下限为50nM。
(2)基于酶切循环信号放大技术的生物传感器的研究。基于核酸切口酶能够识别特异性的双链序列而只酶切其中一条单链从而可以实现循环信号放大的特点,在第4章利用分子信标低背景、高灵敏的优点提出了一种新型、能够实现目标诱导酶切循环信号放大过程的“Y型”探针用于高灵敏检测目标DNA,该探针同时具有较强的单碱基错配识别能力。当目标DNA存在时,可以和辅助探针协同打开分子信标的发夹型结构,三者相互杂交形成“Y型”结构,从而形成了切口酶Nt.BbvCI的识别序列。分子信标被核酸切口酶识别切断后,从传感系统上游离释放出来。释放出的辅助探针和目标DNA可以继续和另外一个分子信标进行杂交,同时诱发第二次酶切循环。经多次酶切循环反应后,实现了真正的目标DNA诱发酶切循环信号放大,与第一代基于中间标记的直线型信号探针构建的“Y型”探针相比更加灵敏,检测下限为5pM。在第5章,我们采用将分子识别和信号报告基团进行分离的设计策略,避免了对DNA酶进行修饰,并采用酶切循环信号放大技术构建了一种新型荧光催化信标,显著提高了对目标物的检测灵敏度。以L-组氨酸作为分析模型,采用DNA酶链和底物链以聚T连接的一体化DNA酶作为识别基团,L-组氨酸导致底物链酶切后的产物序列能够与作为信号报告基团的分子信标杂交,进一步采用核酸切口酶实现酶切循环信号放大,实现了对目标物的高灵敏和特异性检测,检测限为200nM。由于DNA酶具有可以循环剪切的性质,也可以用于酶切信号放大技术。在第6章,我们利用连接反应触发DNA酶和催化分子信标信号放大技术,提出了一种零背景、高灵敏的小分子检测方法。以ATP和NAD+作为分析模型,利用DNA连接酶对生物小分子的高特异性依赖,以连接反应触发劈开DNA酶的激活,然后与设计为分子信标的底物链杂交,在Zn2+存在的条件下进行DNA酶的酶切循环反应,实现了对生物小分子高灵敏、高特异性的荧光检测,其灵敏度和特异性均优于以前所报道的传感器。
(3)基于纳米金和酶切循环双信号放大的生物传感器的研究。基于核酸外切酶和纳米金双重信号放大策略,在第7章提出了一种非标型、高灵敏、高特异性电化学DNA传感器。该传感器首先在均相中进行发夹型探针和目标DNA之间的杂交及酶切反应,避免了电极表面进行酶切所遇到的酶效率降低等缺点,而且该发夹型探针能够提供较好的碱基错配识别能力；然后将酶切产物序列与电极表面的发夹型捕获探针杂交,进一步提高了碱基错配识别能力；最后利用纳米金功能化的报告探针进行信号放大,以亚甲基蓝为电活性物质,实现了对目标DNA的高灵敏和高特异性电化学检测,检测限为0.6pM。
With the continuous development of the analytical science, various analysis and testing process in life sciences are increasingly needed the help of biosensing technology to obtain the required information. Due to many advantages such as good selectivity, high sensitivity, fast response, low cost and continuous on-line detection in complex system, biosensors have valuable applications in chemistry, biomedicine, environmental protection, food industry, medicine and military affairs. Ideally, biosensors for the target detection should have high detection sensitivity (low detection limit), high specificity (low interference), wide dynamic range, fast response time, and universal characteristics.
Functional nucleic acids are short synthetic DNA and RNA sequences which have been isolated via a combinatorial biology technique known as in vitro selection, or a process also known as systematic evolution of ligands by exponential enrichment (SELEX). The functional nucleic acids can specifically bind a broad of analytes including small biomolecules, proteins, cancer cells as well as metal ions with high affinity. Typically, functional nucleic acids include two types of nucleic acid molecules, one type of them have been shown to perform catalytic reactions (called DNAzymes, or deoxyribozymes) like protein enzymes, and the other can bind to a specific target molecule (called aptamers) like antibodies. In the traditional understanding, the nucleic acids are only the storage and transfer carrier of genetic information. However, the discovery of functional nucleic acids has resulted in a breakthrough in such traditional sense, and provided an interesting alternative to biosensing system.
One of the most important performances of biosensor is sensitivity, and it is very limited in further improving the sensitivity by simply using the conformational changes of the functional nucleic acid before and after bind to the target. In recent years, the design of new signal amplification method for ultrasensitive detection of target has attracted attentions of researchers. Two different strategies have been widely employed to improve the sensitivity of a sensor:lower the detection background and signal amplified detection. Recently, signal amplification technology has attracted more concerns due to the significant effect on improving sensitivity.
Based on the above considerations and the reported literatures, in this doctoral thesis, several bioassay systems have been developed focused on new methods for enhancing the detection sensitivity of biosensors by using signal amplification technology. The details are summarized as follows:
(1) Study on the biosensors based on gold nanoparticle signal amplification technology. Due to the large specific surface area, good biocompatibility and surface characteristics of nucleic acids of high load, a novel electrochemical label-free biosensor for Hg2+has been developed in chapter2based on the "T-Hg2+-T" specific interaction and the gold nanoparticle signal amplification technology. In the presence of Hg2+, the linker DNA hybridized with the electrode surface-tethered capture probe via the "T-Hg2+-T" specific interaction, and the gold nanoparticles functionalized reporter DNA subsequently hybridized with the linker. Methylene blue was selected as electrically active substances, which can specifically bind with guanine bases to form methylene blue-guanine complexes, resulting in a significant increase in the electrochemical signal. The proposed biosensor exhibits high sensitivity, high specificity, and a detection limit of0.5nM could be achieved for Hg2+, which makes the biosensor favorable for Hg2+assays in practical samples of very low concentration. Based on the distance dependent exponentially decreased enhancement of the EM (electromagnetic) field, in chapter3, the use of a nanoscale DNA-Au dendrimer as a signal amplifier was proposed for the design of functional DNA-based ultra-sensitive SERS biosensors. As a proof-of-concept, a Pb2+-dependent DNAzyme and the anti-adenosine aptamer was chosen to develop novel SERS biosensors to verify the feasibility of our DNA-Au dendrimer amplification strategy. For the detection of Pb2+the DNAzyme strand was immobilized on the electrode surface first, in the presence of Pb2+, the DNAzyme was activated and it cleaved the substrate section into two parts. The remaining oligonucleotide moiety on the electrode surface hybridized with the reporter DNA1and DNA2to form nanoscale junctions through layer by layer assembling. The DNA-Au dendrimer could then be formed to give a strong SERS signal, and a detection limit of0.1nM could be achieved for Pb2+. For the detection of adenosine, the SERS aptamer sensor was designed based on target-induced strand displacement. A thiolated DNA sequence was first immobilized on gold electrode surface as a capture probe, and then hybridized with the anti-adenosine aptamer. The introduction of target adenosine could trigger the release of the aptamer from the duplex. The free capture probe could then hybridize with the reporter DNA2and DNA1, thereby forming the DNA-Au dendrimer to trigger a remarkable SERS signal enhancement. A low detection limit of50nM was obtained with good selectivity.
(2) Study on the biosensors based on enzymatic recycling signal amplification technology. Based on ssDNA-cleaved property of nicking endonuclease, and take advantage of the unique features of molecular beacons (MBs) such as high sensitivity and low fluorescent background, in chapter4, a new MB-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs was developed. In the presence of a target, the assistant probe, together with the target, can hybridize with the MB and open its hairpin structure to form a "Y" junction structure, as well as form the doublestranded recognition sequence for nicking endonuclease Nt.BbvCI. Once the MB is cleaved, it is dissociated from the sensing system, and fluorescence is restored. The released hybrid of the assistant probe with the target can then hybridize with another MB and trigger the second cycle of cleavage. Eventually, each hybrid of the assistant probe with the target can undergo many cycles to trigger the cleavage of many MBs, providing a true and efficient target-triggered enzymatic recycling amplification signal. Moreover, compared with the first-generation junction sensing system, our proposed sensing sytem also afforded a faster and more sensitive response, and a detection limit of5pM was obtained. In chapter5, by separating the molecular recognition module from the signal reporter to avoid DNAzyme modifications, and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification, we constructed a new fluorescent cascadic catalytic beacon sensing platform. An L-histidine-dependent DNAzyme was chosen as a model to construct the cascadic catalytic beacon. The unmodified unimolecular DNAzyme was linked by a polyT sequence which serves as a molecular recognition module for L-histidine, and a molecular beacon was selected as the signal reporter. The introduction of target resulted in the cleavage of DNAzyme, and the cleaved partial substrate is released from the DNAzyme and hybridized with the MB, the further using of nicking endonuclease resulted in signal amplification detection for the target. Under the optimized experimental conditions, the proposed sensing strategy provides a low detection limit of200nM with high selectivity. Due to the enzymatic recycling cleavage property, the DNAzyme also can be used to develop signal amplification technology. In chapter6, we proposed a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based signal amplification to develop a ligation-triggered DNAzyme cascade for small molecules detection. ATP and NAD+were chosen as analytical models to verify the feasibility of the CAMB sensing system. As two kinds of DNA ligases which specifically employ ATP and NAD+as cofactors, respectively, we employed the ligation to trigger DNAzyme cascade and realize zero-background signal. Then the activated DNAzyme could hybridize with substrate sequence which was designed as MB to form the CAMB, which was employed for amplified signal detection. The cycling and regenerating of DNAzyme could lead to a true enzymatic multiple turnovers of catalytic beacons. This combination of and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of100pM and50pM for ATP and NAD+, respectively, much lower than those of previously reported biosensors. Moreover, the developed DNAzyme cascades show significantly high selectivity towards the target cofactor (ATP or NAD+), and the target biological small molecule can be distinguished from its analogues.
(3) Study on the biosensors based on dual signal amplification strategy that combines the gold nanoparticle with enzymatic recycling signal amplification technologies. In chapter7, a label-free electrochemical DNA biosensor with high sensitivity and selectivity has been reported by employing both gold nanoparticle and enzymatic recycling signal amplification technology. In the design, take advantage of the improved mismatch distinguishing ability of MB, the hairpin probe first hybridizes with target DNA to trigger the enzymatic recycling reaction in the presence of exonuclease Ⅲ, which could avoid the low enzymatic reaction efficiency, effectively. Then, the cleaved sequences hybridize with the hairpin capture probe, which can further improve the mismatch distinguishing ability of the sensing system. Finally, the gold nanoparticles functionalized reporter DNA hybridized with the opened capture probe to amplify the electrochemical signal. Methylene blue was selected as electrically active substances as the sensor in chapter2, and the results demonstrated that the presented dual signal amplification strategy was highly sensitive and specific with a detection limit of0.6pM for target DNA.
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