基于DNA适体的生物传感信号增强方法的设计与应用
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摘要
传感系统中最重要的两个部分是识别单元和信号转换单元。在过去的几十年中,各种各样的分子识别单元被开发了出来,例如酶,凝集素,分子印迹技术,抗体以及适体(适配体)。适配体是单链的核酸分子,对特定的目标物具有高度的亲和力。其作为一种多功能的分子感受器已经被广泛的研究和应用。随着对传感系统灵敏度要求的增加,多种通过信号增强或扩增的手段来提高化学/生物传感的灵敏度的方法已经被设计出来。本研究致力于在DNA适配体为基的生物传感中信号增强方法的设计与应用。本论文的主要内容包括以下五个方面：
1.因其独特的光学性能,尤其是其高摩尔吸光系数(是普通的有机发光团的3-5个数量级),纳米金在本工作中作为信号增强单元,同时又是信号转换单元,来构建铅离子(Pb2+)检测的生物传感系统。本论文的第一项工作是用两条富含G碱基的序列(TBA (5'-GGT TGG TGT GGT TGG-3')和PW17(5'-GGG TAG GGC GGG TTG GG-3'))作为Pb2+的识别原件来做传感系统。其原理是无修饰的纳米金能够识别非折叠结构的DNA和折叠结构的DNA,比如G四联体。圆二色光谱表征了这些TBA-Pb2+和PW17-Pb2+所形成的G四联体。紫外吸收差谱证明了在Pb2+响应上序列的特异性。透射电子显微镜和紫外光谱表征了柠檬酸钠还原法合成的纳米金。本研究优化了实验条件,包括盐浓度,单链DNA浓度和加盐之后聚集时间。本工作构建的对Pb2+检测的传感系统能达到30nM的检测下限。结果表明,PW17的体系在铅检测中展现出了更加优异的性能。在相同的DNA浓度下,PW17体系对Pb2+浓度的变化有更大的响应和较小的误差线。
2.为了探明TBA体系和PW17体系在铅检测性能上的不同,我们研究了G四联体与无修饰纳米金之间的相互作用。研究发现G四联体溶液具有保护纳米金的作用,表明仍有DNA吸附在了纳米金表面。这一现象不同于以往的结论,即G四联体不能吸附在纳米金表面并保护纳米金。我们通过跟踪纳米金的表面等离子体共振吸收(A650/A520)对时间的变化曲线研究了纳米金对G四联体溶液中DNA的吸附行为。这项研究包含了三个体系：TBA,PW17和PSO。结果表面,在Pb2+稳定的G四联体溶液中通过5小时的作用大约有93%的DNA被吸附在了纳米金上。这一结果可能是两种原因导致的：(1)G四联体能吸附在纳米金表面；(2)G四联体解缠了进而吸附在了纳米金表面。为了探明原因,我们采用了电感耦合等离子体发射光谱分析了溶液中Pb2+浓度。结果发现G四联体与纳米金作用后吸附在纳米金表面,但Pb2+存留在了水中。这表明G四联体可能被解缠了从而释放出了Pb2+。PW17-Pb2+在纳米金上的吸附速度比TBA-Pb2+更慢,说明PW17-Pb2+形成的G四联体更稳定。这也就解释了两者在铅检测中的差异。在对PSO-K+的体系中,我们观察到了相似的吸附现象,说明G四联体在纳米金存在下的解缠可能是一个普遍的现象。这种相互作用表明长时间的相互作用使得纳米金无法区分非折叠的和折叠的DNA。
3.将巯基修饰的DNA化学键合在纳米金的表面可以避免2中所叙述的问题。在这一部分中,我们用巯基修饰的裂开型适配体设计了一种新型的DNA-纳米金传感系统。在这里ATP被选用为模型分子。ATP的适配体被切为两段,每段在5’端或3’端修饰了巯基。这样,每种纳米金上修饰了同一序列但适配体片段的方向不同,即有的是5’端键接在纳米金表面,有的是3’端键接在纳米金表面。所以这样得到的每种纳米金都是双修饰的。由于在目标分子的存在下,裂开的适配体片段能和目标分子重新组合成完整的适配体二级结构,因此其与纳米金的复合物能产生目标分子诱导的组装,进而由于纳米金颜色的距离依赖性使其颜色发生由红到紫的变化。在新设计的双修饰DNA-纳米金体系中,我们发现此体系对ATP产生的表面等离子体共振吸收变化是传统的单修饰体系的两倍。本工作通过跟踪纳米金的表面等离子体共振吸收研究了不同修饰方式,镁离子浓度和适配体链段在纳米金表面的密度对该体系在目标分子存在下的组装动力学。在最优实验条件下,对ATP的检测可达到24μtM的检测下限,这个结果优于传统组装方法对ATP的检测。
4.然而,双修饰的纳米金-DNA体系对目标分子的响应只是产生两倍于常规的修饰方法的信号增强。因此,我们设计了基于链替换反应的催化循环来放大信号。该体系包括了一个由两步链替换反应组成的熵驱动的催化循环。一共有五条链参与了这个循环,分别表示为"Substrate-1","Fuel-1","Catalyst-1","C1" and "C2"。"Catalyst-1"是ATP的适配体,其在这个催化循环中起催化剂的作用促成"Substrate-Fuel-1"双链的形成。催化循环过程中的各组分通过聚丙烯酰胺凝胶电泳得以证明。由于ATP的引入导致“Catalyst-1"这条链与其相互作用形成了G四联体的结构,从而使其失去了催化活性并抑制‘"Substrate-Fuel-1"双链的形成。很显然这种目标分子抑制的催化循环可用来进行ATP的检测。当核酸链"Substrate-1"和‘"Fuel-1"标记了FAM荧光基团和DABCYL淬灭集团后,催化反应的进程因"Substrate-Fuel-1"双链形成导致的荧光共振能量转移而反映出来。该荧光基团标记的催化体系对ATP的检测显示出"switch on"的响应。在优化了实验条件后,包括催化剂浓度,镁离子浓度,孵化温度等,该体系可以对ATP达到50nM的检测下限和上至1400nM的线性范围。这种目标分子抑制的催化循环提供了一利无酶的传感手段并有前景应用于其他基于适配体的信号放大的传感系统。
5.上述目标分子抑制的催化循环反应体系中存在这么几个问题：检测过程是多步操作并且十分费时(总过程8小时)。因此我们在这部分工作中设计了目标分子触发的催化循环体系。相比于上述的目标分子抑制的催化体系,该触发型体系除了包含基于链替换反应的催化循环,还包含一步用于释放催化剂链的目标分子诱导的链替换反应。对ATP的检测是通过在"Substrate-2"和“C4”链上分别标记FAM和DABCYL来实现的。ATP的加入引发了核酸链"Catalyst-2"从双链"Catalyst-2-ATP aptamer"上的释放,进而催化了下面的催化循环。这样原本以双链状态结合的"Substrate-2"和“C4”就因"Substrate-2-Fuel-2"的形成而分开,并伴随着荧光的出现。用聚丙烯酰胺凝胶电泳表征反应过程的各组分证明了该催化特性。催化过程通过跟踪催化体系的荧光变化得以表征。优化了实验条件后,该目标分子触发的催化体系提供了更为灵敏的检测性能(对ATP的检测下限为20nM)。最重要的是,该反应速度很快,整个检测操作过程小于一个小时。
With the increasing requirements on the sensitivity of biosensing system, various strategies have been devised to boost detection sensitivity of chemosensing and biosensing processes via the enhancement/amplification of sensing responses. The recognition element and the signal transducer are two core sections in a biosensing system. Various recognition elements have been developed in the past several decades, such as enzymes, lectins, antibodies, molecular imprinting and aptamers. Aptamer, which is single-stranded oligonucleotides with high affinity to a special target, is one of the emerging classes of versatile receptors. This study aims to the design and application of signal enhancement strategies in DNA aptamer-based biosensing. The main content is as follows:
1. Because of its unique optical properties especially high extinction coefficient (3-5orders of magnitude higher than that of organic chromophores), gold nanoparticle (AuNP) was employed as the signal enhancement element and transducer to construct biosensing system for lead (II) detection. The sensing systems with two guanine-rich sequences (TBA (5'-GGT TGG TGT GGT TGG-3') and PW17(5'-GGG TAG GGC GGG TTG GG-3')) respectively as recognition elements were developed base on the principle that ummodified AuNP can distinguish unfolded ssDNA from folded ssDNA, such as G-quadruplex. The formation of G-quadruplexes by TBA-Pb2+and PW17-Pb2+were characterized by circular dichroism. The specificity of the sequence to Pb2+was analyzed by difference absorption spectrum. The AuNP of13nm synthesized using citrate reduction method was characterized by TEM and UV-vis spectroscopy. Experimental conditions, such as NaCl concentrations, ssDNA concentrations, aggregation time after the addition of salt were optimized. Results showed that a limit of detection of30nM can be easily obtained for Pb+detection. The PW17system was found to possess a much better performance for Pb2+detection than TBA system. In the same [Pb2+] range and ssDNA concentration, PW17system shows a larger LSPR response than TBA system with a relative smaller standard deviation.
2. To investigate the difference of TBA system and PW17system in the performance of Pb2+sensing, the interaction between DNA G-quadruplexes and13nm gold nanoparticles (AuNPs) was studied. The adsorption of DNAs in G-quadruplex solutions onto AuNPs was observed in DNA-AuNP-based sensing system. The adsorption behavior was studied through monitoring of the localized surface plasmon resonance (LSPR) absorbance of13nm AuNPs at520and650nm (A650/A520) in the solutions of three widely studied guanine-rich sequences, TBA, PW17, and PSO (5'-GGG TTA GGG TTA GGG TTA GGG-3'). It was found that the degree of the adsorption of DNAs in Pb2+stabilized G-quadruplex solutions is up to93%after more than5h of incubation. Two interpretations, the adsorption of G-quadruplexes and the unfolding of G-quadruplexes in the presence of AuNPs, were proposed for these observations. To explore the possible explanation, the lead concentrations in the solutions containing G-quadruplex and AuNP were analyzed by inductively coupled plasma atomic emission spectrometer. The results showed that Pb2+had been released from the G-quadruplexes, which means the G-quadruplexes may be unfolded in the presence of AuNPs. The adsorption rate in PW17-Pb2+system was lower than that in TB A-Pb2+system, demonstrating that the G-quadruplex formed from PW17and Pb2+is more stable in the presence of AuNPs. This result can interpret the difference in their performance in Pb2+sensing. Similar results were also observed in PSO-K+system, which indicate that the potential unfolding of G-quadruplexes in the presence of AuNPs is a general phenomenon in DNA AuNP-based sensing system. This interaction between G-quadruplexes and AuNP demonstrated that long time incubation between DNAs and AuNPs would possibly make it unable to distinguish G-quadruplex from ssDNA.
3. To avoid the adverse effect of unmodified AuNPs on the formation of folded structure of ssDNA, the thiolated-aptamer conjugated AuNPs sensing system was adopted for biosensors design. The newly designed AuNP functionalized with split aptamer was developed for the detection of adenosine triphosphate (ATP). The ATP aptamer was split into two parts with their5'prime or3'prime modified with thiol. Both the5'SH and3'SH modified strands for each split aptamer fragment were functionalized onto the same AuNP to construct double-functionalized AuNP-DNA conjugates. Thus, the split aptamer can be reassembled into intact folded structure in the presence of ATP molecule with two potential assembly types, which induces the assembly of AuNP-DNA conjugates. In this double-functionalized system, the traditional assembly type might facilitate another assembly type, which was found to give two-fold increase in LSPR response of AuNPs in the presence of ATP than the traditional assembly type, and improved the sensitivity for ATP detection. Time courses of the assemble processes with different assembly types, Mg2+concentrations, and aptamer fragments densities on AuNP were followed using the absorption ratio at650nm and520nm. A limit of detection of24μM with highly selectivity was determined which has greatly surpassed the traditional assembly type in ATP sensing.
4. However, the double-functionalized AuNP-DNA system could only provide two-fold increase in LSPR response of AuNPs to target molecules than the traditional one. Therefore, a strand displacement reaction (SDR)-based catalytic cycle was employed to amplify signals. This system involves an entropy-driven catalytic cycle of two strand displacement reactions with five oligonucleotides, denoted as "Substrate-1","Fuel-1","Catalyst-1","Cl" and "C2", respectively. The "Catalyst-1" is an ATP aptamer catalyzing the SDRs to form the "Substrate-Fuel-1" duplexes. All the intermediates in the SDR processes have been identified by PAGE (polyacrylamide gel electrophoresis) analysis. Introduction of ATP into the SDR system will induce the "Catalyst-1" to form G-quadruplex conformation so as to inhibit the catalytic activity and cut down the formation of the "Substrate-Fuel-1" duplexes. Obviously, this target-inhibited catalytic cycle can be applied to an ATP sensing system. When the "Substrate-1" and "Fuel-1" oligonucleotides were labeled with a carboxyfluorescein (FAM) fluorophore and a4-([4-(dimethylamino)phenyl] azo)benzoic acid (DABCYL) quencher, this SDR catalytic system exhibits a "switch-on" response for ATP. Conditions for detecting ATP, such as the loading of the "catalyst", buffer concentration of Mg+and incubation temperature, have been optimized to afford a detection limit of50nM and a linear response up to1400nM of ATP. This target inhibited catalytic cycle provides an enzyme-free biosensing strategy with higher sensitivity than many aptamer-based biosensing systems and even some enzyme-based amplification systems.
5. The problems, which exist in the above target inhibited catalytic cycle-based sensing system, are the multi-step operations and time-consuming detection process (8h). Therefore, a target triggered catalytic cycle was designed for biosensing. Besides the SDR-based catalytic cycle, this new system also contains a target-induced strand displacement process which releases the catalyst strand for the cycle from the ATP aptamer-"Catalyst-2" duplex. The sensing of ATP was achieved by labeling "Substrate-2" and "C4" strands with FAM and DABCYL respectively. The addition of ATP triggered the release of "Catalyst-2" so as to catalyze the SDR-based catalytic cycle. Then, the bounded "Substrate-2" and "C4" strands in "S-C-2" complex were separated in the presence of ATP and resuming the fluorescence. The intermediates in the system were analyzed by PAGE and the time course of the catalysis process was followed in the FAM and DABCYL labeled system. After optimizing the Mg2+concentration, this target triggered catalytic-based sensing system provided a more sensitive response (Limit of detection is20nM) to ATP than the target inhibited one. It also provides a faster detection process (less than1hour's detection process) which overcomes the disadvantage in the target inhibited catalytic cycle-based system.
1.因其独特的光学性能,尤其是其高摩尔吸光系数(是普通的有机发光团的3-5个数量级),纳米金在本工作中作为信号增强单元,同时又是信号转换单元,来构建铅离子(Pb2+)检测的生物传感系统。本论文的第一项工作是用两条富含G碱基的序列(TBA (5'-GGT TGG TGT GGT TGG-3')和PW17(5'-GGG TAG GGC GGG TTG GG-3'))作为Pb2+的识别原件来做传感系统。其原理是无修饰的纳米金能够识别非折叠结构的DNA和折叠结构的DNA,比如G四联体。圆二色光谱表征了这些TBA-Pb2+和PW17-Pb2+所形成的G四联体。紫外吸收差谱证明了在Pb2+响应上序列的特异性。透射电子显微镜和紫外光谱表征了柠檬酸钠还原法合成的纳米金。本研究优化了实验条件,包括盐浓度,单链DNA浓度和加盐之后聚集时间。本工作构建的对Pb2+检测的传感系统能达到30nM的检测下限。结果表明,PW17的体系在铅检测中展现出了更加优异的性能。在相同的DNA浓度下,PW17体系对Pb2+浓度的变化有更大的响应和较小的误差线。
2.为了探明TBA体系和PW17体系在铅检测性能上的不同,我们研究了G四联体与无修饰纳米金之间的相互作用。研究发现G四联体溶液具有保护纳米金的作用,表明仍有DNA吸附在了纳米金表面。这一现象不同于以往的结论,即G四联体不能吸附在纳米金表面并保护纳米金。我们通过跟踪纳米金的表面等离子体共振吸收(A650/A520)对时间的变化曲线研究了纳米金对G四联体溶液中DNA的吸附行为。这项研究包含了三个体系：TBA,PW17和PSO。结果表面,在Pb2+稳定的G四联体溶液中通过5小时的作用大约有93%的DNA被吸附在了纳米金上。这一结果可能是两种原因导致的：(1)G四联体能吸附在纳米金表面；(2)G四联体解缠了进而吸附在了纳米金表面。为了探明原因,我们采用了电感耦合等离子体发射光谱分析了溶液中Pb2+浓度。结果发现G四联体与纳米金作用后吸附在纳米金表面,但Pb2+存留在了水中。这表明G四联体可能被解缠了从而释放出了Pb2+。PW17-Pb2+在纳米金上的吸附速度比TBA-Pb2+更慢,说明PW17-Pb2+形成的G四联体更稳定。这也就解释了两者在铅检测中的差异。在对PSO-K+的体系中,我们观察到了相似的吸附现象,说明G四联体在纳米金存在下的解缠可能是一个普遍的现象。这种相互作用表明长时间的相互作用使得纳米金无法区分非折叠的和折叠的DNA。
3.将巯基修饰的DNA化学键合在纳米金的表面可以避免2中所叙述的问题。在这一部分中,我们用巯基修饰的裂开型适配体设计了一种新型的DNA-纳米金传感系统。在这里ATP被选用为模型分子。ATP的适配体被切为两段,每段在5’端或3’端修饰了巯基。这样,每种纳米金上修饰了同一序列但适配体片段的方向不同,即有的是5’端键接在纳米金表面,有的是3’端键接在纳米金表面。所以这样得到的每种纳米金都是双修饰的。由于在目标分子的存在下,裂开的适配体片段能和目标分子重新组合成完整的适配体二级结构,因此其与纳米金的复合物能产生目标分子诱导的组装,进而由于纳米金颜色的距离依赖性使其颜色发生由红到紫的变化。在新设计的双修饰DNA-纳米金体系中,我们发现此体系对ATP产生的表面等离子体共振吸收变化是传统的单修饰体系的两倍。本工作通过跟踪纳米金的表面等离子体共振吸收研究了不同修饰方式,镁离子浓度和适配体链段在纳米金表面的密度对该体系在目标分子存在下的组装动力学。在最优实验条件下,对ATP的检测可达到24μtM的检测下限,这个结果优于传统组装方法对ATP的检测。
4.然而,双修饰的纳米金-DNA体系对目标分子的响应只是产生两倍于常规的修饰方法的信号增强。因此,我们设计了基于链替换反应的催化循环来放大信号。该体系包括了一个由两步链替换反应组成的熵驱动的催化循环。一共有五条链参与了这个循环,分别表示为"Substrate-1","Fuel-1","Catalyst-1","C1" and "C2"。"Catalyst-1"是ATP的适配体,其在这个催化循环中起催化剂的作用促成"Substrate-Fuel-1"双链的形成。催化循环过程中的各组分通过聚丙烯酰胺凝胶电泳得以证明。由于ATP的引入导致“Catalyst-1"这条链与其相互作用形成了G四联体的结构,从而使其失去了催化活性并抑制‘"Substrate-Fuel-1"双链的形成。很显然这种目标分子抑制的催化循环可用来进行ATP的检测。当核酸链"Substrate-1"和‘"Fuel-1"标记了FAM荧光基团和DABCYL淬灭集团后,催化反应的进程因"Substrate-Fuel-1"双链形成导致的荧光共振能量转移而反映出来。该荧光基团标记的催化体系对ATP的检测显示出"switch on"的响应。在优化了实验条件后,包括催化剂浓度,镁离子浓度,孵化温度等,该体系可以对ATP达到50nM的检测下限和上至1400nM的线性范围。这种目标分子抑制的催化循环提供了一利无酶的传感手段并有前景应用于其他基于适配体的信号放大的传感系统。
5.上述目标分子抑制的催化循环反应体系中存在这么几个问题：检测过程是多步操作并且十分费时(总过程8小时)。因此我们在这部分工作中设计了目标分子触发的催化循环体系。相比于上述的目标分子抑制的催化体系,该触发型体系除了包含基于链替换反应的催化循环,还包含一步用于释放催化剂链的目标分子诱导的链替换反应。对ATP的检测是通过在"Substrate-2"和“C4”链上分别标记FAM和DABCYL来实现的。ATP的加入引发了核酸链"Catalyst-2"从双链"Catalyst-2-ATP aptamer"上的释放,进而催化了下面的催化循环。这样原本以双链状态结合的"Substrate-2"和“C4”就因"Substrate-2-Fuel-2"的形成而分开,并伴随着荧光的出现。用聚丙烯酰胺凝胶电泳表征反应过程的各组分证明了该催化特性。催化过程通过跟踪催化体系的荧光变化得以表征。优化了实验条件后,该目标分子触发的催化体系提供了更为灵敏的检测性能(对ATP的检测下限为20nM)。最重要的是,该反应速度很快,整个检测操作过程小于一个小时。
With the increasing requirements on the sensitivity of biosensing system, various strategies have been devised to boost detection sensitivity of chemosensing and biosensing processes via the enhancement/amplification of sensing responses. The recognition element and the signal transducer are two core sections in a biosensing system. Various recognition elements have been developed in the past several decades, such as enzymes, lectins, antibodies, molecular imprinting and aptamers. Aptamer, which is single-stranded oligonucleotides with high affinity to a special target, is one of the emerging classes of versatile receptors. This study aims to the design and application of signal enhancement strategies in DNA aptamer-based biosensing. The main content is as follows:
1. Because of its unique optical properties especially high extinction coefficient (3-5orders of magnitude higher than that of organic chromophores), gold nanoparticle (AuNP) was employed as the signal enhancement element and transducer to construct biosensing system for lead (II) detection. The sensing systems with two guanine-rich sequences (TBA (5'-GGT TGG TGT GGT TGG-3') and PW17(5'-GGG TAG GGC GGG TTG GG-3')) respectively as recognition elements were developed base on the principle that ummodified AuNP can distinguish unfolded ssDNA from folded ssDNA, such as G-quadruplex. The formation of G-quadruplexes by TBA-Pb2+and PW17-Pb2+were characterized by circular dichroism. The specificity of the sequence to Pb2+was analyzed by difference absorption spectrum. The AuNP of13nm synthesized using citrate reduction method was characterized by TEM and UV-vis spectroscopy. Experimental conditions, such as NaCl concentrations, ssDNA concentrations, aggregation time after the addition of salt were optimized. Results showed that a limit of detection of30nM can be easily obtained for Pb+detection. The PW17system was found to possess a much better performance for Pb2+detection than TBA system. In the same [Pb2+] range and ssDNA concentration, PW17system shows a larger LSPR response than TBA system with a relative smaller standard deviation.
2. To investigate the difference of TBA system and PW17system in the performance of Pb2+sensing, the interaction between DNA G-quadruplexes and13nm gold nanoparticles (AuNPs) was studied. The adsorption of DNAs in G-quadruplex solutions onto AuNPs was observed in DNA-AuNP-based sensing system. The adsorption behavior was studied through monitoring of the localized surface plasmon resonance (LSPR) absorbance of13nm AuNPs at520and650nm (A650/A520) in the solutions of three widely studied guanine-rich sequences, TBA, PW17, and PSO (5'-GGG TTA GGG TTA GGG TTA GGG-3'). It was found that the degree of the adsorption of DNAs in Pb2+stabilized G-quadruplex solutions is up to93%after more than5h of incubation. Two interpretations, the adsorption of G-quadruplexes and the unfolding of G-quadruplexes in the presence of AuNPs, were proposed for these observations. To explore the possible explanation, the lead concentrations in the solutions containing G-quadruplex and AuNP were analyzed by inductively coupled plasma atomic emission spectrometer. The results showed that Pb2+had been released from the G-quadruplexes, which means the G-quadruplexes may be unfolded in the presence of AuNPs. The adsorption rate in PW17-Pb2+system was lower than that in TB A-Pb2+system, demonstrating that the G-quadruplex formed from PW17and Pb2+is more stable in the presence of AuNPs. This result can interpret the difference in their performance in Pb2+sensing. Similar results were also observed in PSO-K+system, which indicate that the potential unfolding of G-quadruplexes in the presence of AuNPs is a general phenomenon in DNA AuNP-based sensing system. This interaction between G-quadruplexes and AuNP demonstrated that long time incubation between DNAs and AuNPs would possibly make it unable to distinguish G-quadruplex from ssDNA.
3. To avoid the adverse effect of unmodified AuNPs on the formation of folded structure of ssDNA, the thiolated-aptamer conjugated AuNPs sensing system was adopted for biosensors design. The newly designed AuNP functionalized with split aptamer was developed for the detection of adenosine triphosphate (ATP). The ATP aptamer was split into two parts with their5'prime or3'prime modified with thiol. Both the5'SH and3'SH modified strands for each split aptamer fragment were functionalized onto the same AuNP to construct double-functionalized AuNP-DNA conjugates. Thus, the split aptamer can be reassembled into intact folded structure in the presence of ATP molecule with two potential assembly types, which induces the assembly of AuNP-DNA conjugates. In this double-functionalized system, the traditional assembly type might facilitate another assembly type, which was found to give two-fold increase in LSPR response of AuNPs in the presence of ATP than the traditional assembly type, and improved the sensitivity for ATP detection. Time courses of the assemble processes with different assembly types, Mg2+concentrations, and aptamer fragments densities on AuNP were followed using the absorption ratio at650nm and520nm. A limit of detection of24μM with highly selectivity was determined which has greatly surpassed the traditional assembly type in ATP sensing.
4. However, the double-functionalized AuNP-DNA system could only provide two-fold increase in LSPR response of AuNPs to target molecules than the traditional one. Therefore, a strand displacement reaction (SDR)-based catalytic cycle was employed to amplify signals. This system involves an entropy-driven catalytic cycle of two strand displacement reactions with five oligonucleotides, denoted as "Substrate-1","Fuel-1","Catalyst-1","Cl" and "C2", respectively. The "Catalyst-1" is an ATP aptamer catalyzing the SDRs to form the "Substrate-Fuel-1" duplexes. All the intermediates in the SDR processes have been identified by PAGE (polyacrylamide gel electrophoresis) analysis. Introduction of ATP into the SDR system will induce the "Catalyst-1" to form G-quadruplex conformation so as to inhibit the catalytic activity and cut down the formation of the "Substrate-Fuel-1" duplexes. Obviously, this target-inhibited catalytic cycle can be applied to an ATP sensing system. When the "Substrate-1" and "Fuel-1" oligonucleotides were labeled with a carboxyfluorescein (FAM) fluorophore and a4-([4-(dimethylamino)phenyl] azo)benzoic acid (DABCYL) quencher, this SDR catalytic system exhibits a "switch-on" response for ATP. Conditions for detecting ATP, such as the loading of the "catalyst", buffer concentration of Mg+and incubation temperature, have been optimized to afford a detection limit of50nM and a linear response up to1400nM of ATP. This target inhibited catalytic cycle provides an enzyme-free biosensing strategy with higher sensitivity than many aptamer-based biosensing systems and even some enzyme-based amplification systems.
5. The problems, which exist in the above target inhibited catalytic cycle-based sensing system, are the multi-step operations and time-consuming detection process (8h). Therefore, a target triggered catalytic cycle was designed for biosensing. Besides the SDR-based catalytic cycle, this new system also contains a target-induced strand displacement process which releases the catalyst strand for the cycle from the ATP aptamer-"Catalyst-2" duplex. The sensing of ATP was achieved by labeling "Substrate-2" and "C4" strands with FAM and DABCYL respectively. The addition of ATP triggered the release of "Catalyst-2" so as to catalyze the SDR-based catalytic cycle. Then, the bounded "Substrate-2" and "C4" strands in "S-C-2" complex were separated in the presence of ATP and resuming the fluorescence. The intermediates in the system were analyzed by PAGE and the time course of the catalysis process was followed in the FAM and DABCYL labeled system. After optimizing the Mg2+concentration, this target triggered catalytic-based sensing system provided a more sensitive response (Limit of detection is20nM) to ATP than the target inhibited one. It also provides a faster detection process (less than1hour's detection process) which overcomes the disadvantage in the target inhibited catalytic cycle-based system.
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