金属纳米粒子与DNAzyme在电化学与比色传感中的研究与应用
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摘要
纳米技术的出现为纳米材料的发展和应用开辟了新的思路。在现代科学中,纳米技术结合化学、生物、物理和医学发展超灵敏检测和成像方法在分析化学或生物科学中越来越重要,已经成为一个非常有前景的前沿领域。由于粒径小(通常在1-100nm范围),金属纳米粒子具有独特的不同于其他材料的化学、物理和电子性质,可用于构建和完善新颖的传感设备,特别是在电化学传感器与生物传感器等方面。金属纳米粒子用于电化学传感器主要利用其促进电子传递与催化性能以及大的比表面积；且由于具有大的表面积和小的质量,金属纳米粒子也可用作载体固定其他分子,如DNA、酶、小分子等。除了利用其催化与固定能力外,利用金属纳米粒子自身光学性质的报道也有很多。金属纳米粒子的运用能提高传感器的灵敏度,降低检测限,以及完成一些其他材料不能完成的工作。另外,纳米粒子之间的距离不同,其溶液颜色可能也会不同,因此他们在比色分析中具有非常广阔的应用前景。
另外,DNAzyme(脱氧核酶,DNA enzyme, deoxyribozyme)是具有催化活性的DNA。科学家筛选出了多种具有不同催化活性的DNAzyme,具有切割核酸、链接核酸、激酶、过氧化物酶等活性。尤其是具有过氧化物酶活性的DNAzyme,其组成是核酸分子,比同功能蛋白质酶稳定,具有催化过氧化物的能力,从而可以替代蛋白质酶而运用到化学生物传感中实现放大检测,提高检测灵敏度。另外,DNAzyme的活性可以通过DNA的杂交／解链进行可逆调节,这也是比蛋白酶先进的地方。DNAzyme在过氧化物协助下能催化某些底物产生颜色,也可用于比色分析。但是,现有DNAzyme的活性却不及蛋白酶,因此通过简单的方法提高DNAzyme活性,将会是一项非常有意义且负挑战性的工作。
基于以上考虑,综合文献报道,本论文利用金属纳米粒子与具有过氧化物酶活性的DNAzyme,发展了一系列的电化学与比色化学生物检测体系,研究了提高与抑制DNAzyme活性的方法。另外,利用金属特异结合多肽与金纳米粒子(AuNPs),设计了一组比色逻辑门。具体细节如下：1.基于金属纳米粒子的小分子电化学传感器研究
利用层层自组装技术,第2章构建了一种由磷钼酸(H3PMO12O40, PMO12)与铂纳米粒子掺杂的壳聚糖(Pt-CHIT)多层复合膜((PMo12/Pt-CHIT)n)。带负电的磷钼酸纳米簇与带正电的壳聚糖分子能按照秩序,通过静电吸附作用一层层的交替构建到修饰有多壁碳纳米管的石墨电极表面,从而形成稳定的超薄多层膜。虽然壳聚糖的引入会部分阻碍电子的传递,但是铂纳米粒子的掺杂能促进电子传递,从而消除此不利因素。循环伏安法用来证实多层膜的一步一步的构建,并用来考察此多层膜的电化学行为。作为一个碘酸根(103-)传感器,(PMo12/Pt-CHIT)4/CPG电极展现出了良好的性能,如灵敏度高、线性范围宽、检测限低以及响应快速。
基于AuNPs的放大作用,第3章开发了一个灵敏且无标记的电化学适体传感器用来检测小分子。此适体传感器包含3个DNA部分：组装在金电极表面的ADNA,修饰到AuNPs表面的RDNA,以及连接ADNA与RDNA的TRDNA。带正电的六氨合钌分子([Ru(NH3)6]3+, RuHex)按一定比例通过静电作用吸附到带负电的DNA上,通过计时库仑法可以获取其电化学信号。本章中,三磷酸腺苷(ATP)与抗ATP适体被当作模板分析物与模板分子识别部件。ATP的引入可以触发TRDNA结构转变成复杂的适体-ATP超分子,导致结合有RDNA的AuNPs从电极表面离去,使得电极表面的电活性物质减少。AuNPs的引入能明显提高灵敏度。在优化的条件下,此适体传感器对ATP的线性范围为1nM-10gM,最低可检测浓度低至亚nM级别(0.2nM)。这些结果表明,该基于AuNPs放大的方法对分析ATP是可行的,且提供了一个潜在的通用的构造其他小分子适体传感器的方法。
2.基于DNAzyme的甲基转移酶与限制性内切酶活性的比色分析以及DNAzyme的活性改造的研究
甲基转移酶(MTase)催化的DNA甲基化对调控基因表达而言是一个显著的表观遗传过程。研究甲基转移酶活性的传统方法需要费劲且昂贵的DNA标记。第4章中,我们提出了一个简单的、比色的而且不用标记的基于甲基化响应的DNAzyme (MR-DNAzyme)方法来分析甲基转移酶活性。这个新的分析策略依靠具有类辣根过氧化物酶活性的核酶(DNAzyme)和甲基化响应的DNA序列(methylation-responsive sequence, MRS)其中DNA可以通过甲基转移酶／核酸内切酶的偶联反应被甲基化后切断。甲基化诱导的MRS的断裂能激活DNAzyme, DNAzyme可以催化底物产生颜色信号,从而放大检测DNA甲基化行为。以Dam MTase和DpnI限制性内切酶为例,我们开发了两种基于MR-DNAzyme策略的比色检测方法。第一种方法是利用一条精心设计的发卡DNAzyme杂交探针来简便的turn-on检测Dam MTase活性,此方法拥有很宽的线性范围(6-100U/mL)和低的检测限(6U/mL)。此外,这个方法还能很容易的拓展到检测限制性内切酶的活性及筛选酶的抑制剂。第二个方法利用基于DNAzyme的甲基化触发的DNA机器,通过两步信号串联放大,实现了超高灵敏的检测Dam MTase活性(检测限低至0.25U/mL)。
DNAzyme活性不及其对应的蛋白质酶活性,因此,提高DNAzyme活性对扩大其应用范围提高检测灵敏度有重要的促进意义。第5章提出了一种全新的DNAzyme活性调节方式——邻位增强效应。在DNAzyme序列3’末端有腺嘌呤A的存在下能使酶活性提高近5倍,并且该效应具有末端选择性和碱基选择性(仅3’端的A)；此外还具有拓扑构象选择性,倾向于G四面体(G4)平行结构。A与G43’末端G平面以及hemin的相对位置也是重要的影响因素。我们推测了该现象可能的机理以及应用该效应检测了核酸酶活性。邻位增强效应相对于其它DNAzyme活性调节方法实现更加简单,仅在序列上延伸一个碱基即可实现,不引入外加成分或因素,因此在分析领域有广泛的应用前景,同时也有助于加深我们对DNAzyme催化机理的理解。
抑制DNAzyme活性一般的方法是杂交部分DNAzyme序列。第6章提出了
个更为简单可行的抑制DNAzyme活性的方法——邻位抑制效应。我们发现,如果DNAzyme序列3’端第一个碱基为嘌呤碱基,只需要把此碱基杂交即可几乎完全抑制DNAzyme活性。该方法不需要杂交DNAzyme序列中的任何一个碱基即可实现对DNAzyme活性的抑制,且抑制效果比杂交部分DNAzyme序列的抑制效果更佳。
3.基于双功能多肽与未修饰AuNPs的Zn2+快速比色检测及比色逻辑门研究
第7章开发了一种新型而简单的基于双功能化的多肽探针及未修饰AuNPs的方法来比色检测锌离子。此双功能多肽包含锌离子特异性结合位点和可导致AuNPs变色的精氨酸残基。多肽的巯基能与金作用,使精氨酸接近AuNPs表面,引起AuNPs不稳定而聚集；而多肽上巯基与Zn2+结合后,则不能与AuNPs结合,使多肽不能组装到AuNPs表面,精氨酸失去使AuNPs变色的能力。该Zn2+比色检测方法检测过程迅速(不到半分钟),灵敏度高,检测限低(10nM)且无需标记,已成功应用于营养品及药物中的Zn2+分析。
利用第7章中提到的原理,我们在第8章中设计了一组基于多肽与AuNPs的比色逻辑门。以Zn2+与糜蛋白酶为输入信号,AuNPs溶液的颜色为输出信号,比较容易地实现了“是”(“非”)门、“或”门和“与”门。
Nanotechnology has given a new insight into the development and application of nanomaterials. In the modern science, combined with chemistry, biology, physics and medicine science, nanotechnology has become one of the most exciting forefront fields in developing the ultra-sensitive detection and imaging analysis. Owing to their small size (normally in the range of1-100nm), metal nanoparticles exhibit unique chemical, physical and electronic properties that are different from those of bulk materials, and can be used to construct novel and improved sensing devices; especially, electrochemical sensors and biosensors. We take advantage of the promotion of electronic transfer, catalytic performance and large specific surface area of the metal nanoparticles for electrochemical sensors; and with the large surface and the small mass, they can also be used as carriers fixed on other molecules, such as DNA, enzyme, and small molecule. In addition to the catalytic and fixed functions, some reports have focused on the optical properties of the metal nanoparticles. Using the metal nanoparticles can improve the sensitivity, lower the detection limit of the sensor, and perform the work that other materials can't finish. In addition, when the distance between the nanoparticles is different, the color of the solution may also be different, thus it owns broad application prospects in colorimetric analysis.
DNAzymes are catalytic DNA. To date, various DNAzymes have emerged through in vitro selection. Some DNAzymes are able to catalyze the incision or ligation of nucleic acids, and others have the catalytic activity of kinase or peroxidase. One interesting example of DNAzymes is the horseradish peroxidase mimicking DNAzyme, which has the catalytic activity of horseradish peroxidase and is more stable than horseradish peroxidase. Thus, this kind of DNAzyme can be used to replace protein enzymes in chemo/biosensing for signal amplification and sensitive detection. Furthermore, the catalytic activity of DNAzyme can be regulated through DNA, which is superior to protein enzymes. Cooperating with peroxide, DNAzyme can catalyze some substrates to generate color change for colorimetric assay. However, the catalytic activity of DNAzyme is inferior to that of protein enzymes; therefore, increasing the activity of DNAzyme through straightforward methods will be a significant and challenging work.
Based on the above consideration and literatures reported previously, takeing the advantage of AuNPs and mimicking-HRP DNAzyme, we have developed a series of electrochemical and colorimetric chemo/biosensors. We also have studied the way to improve and inhibit the activity of DNAzyme. Additionally, we have developed logic gates based on specific metal ions binding peptides and AuNPs. The details are summarized as follows:
1. Electrochemical sensor based on the metal nanoparticles
A strategy to construct H3PMo12O40(PMo12) and platinum nanoparticles-chitosan (Pt-CHIT) multilayer film ((PMo12/Pt-CHIT)n) has been developed in chapter2. Negatively charged PMo12and positively charged CHIT have been employed to fabricate stable ultrathin multilayer film on the multiwalled carbon nanotubes coated pyrolytic graphite (CPG) electrode using layer-by-layer self-assembly technique. The doping of Pt nanoparticles minimized the disadvantage of CHIT. Cyclic voltammetry was used to confirm the consecutive growth of the multilayer film and to investigate the electrochemical behavior of the resulting (PMo12/Pt-CHIT)4/CPG detailedly. As an IO3-sensor, the (PMo12/Pt-CHIT)4/CPG exhibits excellent characteristics, such as high sensitivity, wide linear range, lower detection limit and fast response time.
A sensitive, label-free electrochemical aptasensor for small molecule detection has been developed in chapter3based on gold nanoparticles (AuNPs) amplification. This aptasensor was fabricated as a tertiary hybrid DNA-AuNPs system, which involved the anchored DNA (ADNA) immobilized on gold electrode, reporter DNA (RDNA) tethered with AuNPs and target-responsive DNA (TRDNA) linking ADNA and RDNA. Electrochemical signal is derived from chronocoulometric interrogation of [Ru(NH3)6]3+(RuHex) that quantitatively binds to surface-confined DNA via electrostatic interaction. Using adenosine triphosphate (ATP) as a model analyte and ATP-binding aptamer as a model molecular recognization element, the introduction of ATP triggers the structure switching of the TRDNA to form aptamer-ATP complex, which results in the dissociation of the RDNA capped AuNPs (RDNA-AuNPs) and the release of abundant RuHex molecules trapped by RDNA-AuNPs. The incorporation of AuNPs in this strategy significantly enhances the sensitivity because of the amplification of electrochemical signal by the RDNA-AuNPs/RuHex system. Under optimized conditions, a wide linear dynamic range of4orders of magnitude (1nM-10μM) was reached with the minimum detectable concentration at sub-nanomolar level (0.2nM). These results demonstrate that our nanoparticles-based amplification strategy is feasible for ATP assay and presents a potential universal method for other small molecule aptasensors.
2. Colorimetric detection of the activities of ethyltransferase and restriction endonuclease based on DNAzyme and the reform of the DNAzyme activity
DNA methylation catalyzed by methyltransferase (MTase) is a significant epigenetic process for modulating gene expression. Traditional methods to study MTase activity required laborious and costly DNA labeling process. In chapter4, we report a simple, colorimetric, and label-free methylation-responsive DNAzyme (MR-DNAzyme) strategy for MTase activity analysis. This new strategy relies on horseradish peroxidase (HRP) mimicking DNAzyme and the methylation-responsive sequence (MRS) of DNA which can be methylated and cleaved by MTase/endonuclease coupling reaction. Methylation-induced scission of MRS would activate the DNAzyme that can catalyze the generation of a color signal for the amplified detection of methylation events. Taking Dam MTase and DpnI endonuclease as examples, we have developed two colorimetric methods based on MR-DNAzyme strategy. The first method is to utilize an engineered hairpin-DNAzyme hybrid probe for facile turn-on detection of Dam MTase activity, with a wide linear range (6-100U/mL) and a low detection limit (6U/mL). Furthermore, this method could be easily expanded to profile the activity and inhibition of restriction endonuclease. The second method involves a methylation-triggered DNAzyme-based DNA machine, which achieves the ultra-high sensitive detection of Dam MTase activity (detection limit=0.25U/mL) by two-step signal amplification cascade.
The activity of DNAzyme is much poorer than that of protein enzymes. Thus, it is significant to improve the activity of DNAzyme for expanding its application and improving the sensitivity. In chapter5, a new method to improve the activity of DNAzyme is introduced, called neighboring improved effect. We have found that if the DNAzyem sequence with an A base at3'terminal, its activity can be improved about5fold. This effect has terminal and base selectivity (A base at3'terminal). In addition, it also has the topology configuration selectivity that trends forming a G4parallel structure. The position of base A between hemin and G4(3'terminal) is a factor to affect the DNAzyme activity. In this chapter, we try to give a possible mechnism of the catalytic reaction and employ it to assay the activity of nuclease. Compared with other DNAzyme activity regulation method, neighboring improved effect is simple for that it can be realized only by adding an A base to the3'terminal of DNAzyme sequence. This method doesn't need to introduce extra components or factors, and can be widely applied in analytical field. It also can help us to deepen our understanding of the DNAzyme catalytic mechanism.
General method for the inhibition of the DNAzyme activity is based on the binding or release of partial DNAzyme sequence. In chapter6, a more simple and feasible method for inhibition of the DNAzyme activity is developed, called neighboring inhibited effect. We found that, if the first base at3'terminal of DNAzyme sequence is purine base, DNAzyme activity can be almost fully inhibited just by hybriding this purine base. The inhibition effect of this strategy is superior to general methods, and it doesn't need to hybrid any base of DNAzyme sequence.
3. Rapid colorimetric Zn2+assay and colorimetric logic gates based on bifunctional peptide and unmodified AuNPs
In chapter7, a new and simple mechanism for the label-free and rapid colorimetric Zn2+assay is developed based on bifunctional peptide probe and unmodified AuNPs. This bifunctional peptide probe contains specific Zn2+binding site and arginine that can lead to the color change of AuNPs. The combination of hydrosulfuryl in peptide with Au in AuNPs can make the arginine residual close to AuNPs surface, and the AuNPs will be unstable and aggregated. Once the-SHs in the side chain of peptide are all coordinated with Zn2+, the peptide probe can't combine to the surface of AuNPs, therefore, the arginine residual cannot cause the aggregation of AuNPs. This colorimetric method for the detection of Zn2+exhibits excellent characteristics, such as rapid detection process (less than0.5min), high sensitivity, lower detection limit (10nM), and label-free. This strategy has been successfully used in Zn2+analysis of foods and drugs.
Using the principle in chapter7, we have designed a series of colorimetric logic gates based on peptide and AuNPs in chapter8. Employing Zn2+and chymotrypsin as input, the color of the solution of AuNPs as output, it is easy to achieve the "YES"("NO"),"OR" and "AND" logic gates.
另外,DNAzyme(脱氧核酶,DNA enzyme, deoxyribozyme)是具有催化活性的DNA。科学家筛选出了多种具有不同催化活性的DNAzyme,具有切割核酸、链接核酸、激酶、过氧化物酶等活性。尤其是具有过氧化物酶活性的DNAzyme,其组成是核酸分子,比同功能蛋白质酶稳定,具有催化过氧化物的能力,从而可以替代蛋白质酶而运用到化学生物传感中实现放大检测,提高检测灵敏度。另外,DNAzyme的活性可以通过DNA的杂交／解链进行可逆调节,这也是比蛋白酶先进的地方。DNAzyme在过氧化物协助下能催化某些底物产生颜色,也可用于比色分析。但是,现有DNAzyme的活性却不及蛋白酶,因此通过简单的方法提高DNAzyme活性,将会是一项非常有意义且负挑战性的工作。
基于以上考虑,综合文献报道,本论文利用金属纳米粒子与具有过氧化物酶活性的DNAzyme,发展了一系列的电化学与比色化学生物检测体系,研究了提高与抑制DNAzyme活性的方法。另外,利用金属特异结合多肽与金纳米粒子(AuNPs),设计了一组比色逻辑门。具体细节如下：1.基于金属纳米粒子的小分子电化学传感器研究
利用层层自组装技术,第2章构建了一种由磷钼酸(H3PMO12O40, PMO12)与铂纳米粒子掺杂的壳聚糖(Pt-CHIT)多层复合膜((PMo12/Pt-CHIT)n)。带负电的磷钼酸纳米簇与带正电的壳聚糖分子能按照秩序,通过静电吸附作用一层层的交替构建到修饰有多壁碳纳米管的石墨电极表面,从而形成稳定的超薄多层膜。虽然壳聚糖的引入会部分阻碍电子的传递,但是铂纳米粒子的掺杂能促进电子传递,从而消除此不利因素。循环伏安法用来证实多层膜的一步一步的构建,并用来考察此多层膜的电化学行为。作为一个碘酸根(103-)传感器,(PMo12/Pt-CHIT)4/CPG电极展现出了良好的性能,如灵敏度高、线性范围宽、检测限低以及响应快速。
基于AuNPs的放大作用,第3章开发了一个灵敏且无标记的电化学适体传感器用来检测小分子。此适体传感器包含3个DNA部分：组装在金电极表面的ADNA,修饰到AuNPs表面的RDNA,以及连接ADNA与RDNA的TRDNA。带正电的六氨合钌分子([Ru(NH3)6]3+, RuHex)按一定比例通过静电作用吸附到带负电的DNA上,通过计时库仑法可以获取其电化学信号。本章中,三磷酸腺苷(ATP)与抗ATP适体被当作模板分析物与模板分子识别部件。ATP的引入可以触发TRDNA结构转变成复杂的适体-ATP超分子,导致结合有RDNA的AuNPs从电极表面离去,使得电极表面的电活性物质减少。AuNPs的引入能明显提高灵敏度。在优化的条件下,此适体传感器对ATP的线性范围为1nM-10gM,最低可检测浓度低至亚nM级别(0.2nM)。这些结果表明,该基于AuNPs放大的方法对分析ATP是可行的,且提供了一个潜在的通用的构造其他小分子适体传感器的方法。
2.基于DNAzyme的甲基转移酶与限制性内切酶活性的比色分析以及DNAzyme的活性改造的研究
甲基转移酶(MTase)催化的DNA甲基化对调控基因表达而言是一个显著的表观遗传过程。研究甲基转移酶活性的传统方法需要费劲且昂贵的DNA标记。第4章中,我们提出了一个简单的、比色的而且不用标记的基于甲基化响应的DNAzyme (MR-DNAzyme)方法来分析甲基转移酶活性。这个新的分析策略依靠具有类辣根过氧化物酶活性的核酶(DNAzyme)和甲基化响应的DNA序列(methylation-responsive sequence, MRS)其中DNA可以通过甲基转移酶／核酸内切酶的偶联反应被甲基化后切断。甲基化诱导的MRS的断裂能激活DNAzyme, DNAzyme可以催化底物产生颜色信号,从而放大检测DNA甲基化行为。以Dam MTase和DpnI限制性内切酶为例,我们开发了两种基于MR-DNAzyme策略的比色检测方法。第一种方法是利用一条精心设计的发卡DNAzyme杂交探针来简便的turn-on检测Dam MTase活性,此方法拥有很宽的线性范围(6-100U/mL)和低的检测限(6U/mL)。此外,这个方法还能很容易的拓展到检测限制性内切酶的活性及筛选酶的抑制剂。第二个方法利用基于DNAzyme的甲基化触发的DNA机器,通过两步信号串联放大,实现了超高灵敏的检测Dam MTase活性(检测限低至0.25U/mL)。
DNAzyme活性不及其对应的蛋白质酶活性,因此,提高DNAzyme活性对扩大其应用范围提高检测灵敏度有重要的促进意义。第5章提出了一种全新的DNAzyme活性调节方式——邻位增强效应。在DNAzyme序列3’末端有腺嘌呤A的存在下能使酶活性提高近5倍,并且该效应具有末端选择性和碱基选择性(仅3’端的A)；此外还具有拓扑构象选择性,倾向于G四面体(G4)平行结构。A与G43’末端G平面以及hemin的相对位置也是重要的影响因素。我们推测了该现象可能的机理以及应用该效应检测了核酸酶活性。邻位增强效应相对于其它DNAzyme活性调节方法实现更加简单,仅在序列上延伸一个碱基即可实现,不引入外加成分或因素,因此在分析领域有广泛的应用前景,同时也有助于加深我们对DNAzyme催化机理的理解。
抑制DNAzyme活性一般的方法是杂交部分DNAzyme序列。第6章提出了
个更为简单可行的抑制DNAzyme活性的方法——邻位抑制效应。我们发现,如果DNAzyme序列3’端第一个碱基为嘌呤碱基,只需要把此碱基杂交即可几乎完全抑制DNAzyme活性。该方法不需要杂交DNAzyme序列中的任何一个碱基即可实现对DNAzyme活性的抑制,且抑制效果比杂交部分DNAzyme序列的抑制效果更佳。
3.基于双功能多肽与未修饰AuNPs的Zn2+快速比色检测及比色逻辑门研究
第7章开发了一种新型而简单的基于双功能化的多肽探针及未修饰AuNPs的方法来比色检测锌离子。此双功能多肽包含锌离子特异性结合位点和可导致AuNPs变色的精氨酸残基。多肽的巯基能与金作用,使精氨酸接近AuNPs表面,引起AuNPs不稳定而聚集；而多肽上巯基与Zn2+结合后,则不能与AuNPs结合,使多肽不能组装到AuNPs表面,精氨酸失去使AuNPs变色的能力。该Zn2+比色检测方法检测过程迅速(不到半分钟),灵敏度高,检测限低(10nM)且无需标记,已成功应用于营养品及药物中的Zn2+分析。
利用第7章中提到的原理,我们在第8章中设计了一组基于多肽与AuNPs的比色逻辑门。以Zn2+与糜蛋白酶为输入信号,AuNPs溶液的颜色为输出信号,比较容易地实现了“是”(“非”)门、“或”门和“与”门。
Nanotechnology has given a new insight into the development and application of nanomaterials. In the modern science, combined with chemistry, biology, physics and medicine science, nanotechnology has become one of the most exciting forefront fields in developing the ultra-sensitive detection and imaging analysis. Owing to their small size (normally in the range of1-100nm), metal nanoparticles exhibit unique chemical, physical and electronic properties that are different from those of bulk materials, and can be used to construct novel and improved sensing devices; especially, electrochemical sensors and biosensors. We take advantage of the promotion of electronic transfer, catalytic performance and large specific surface area of the metal nanoparticles for electrochemical sensors; and with the large surface and the small mass, they can also be used as carriers fixed on other molecules, such as DNA, enzyme, and small molecule. In addition to the catalytic and fixed functions, some reports have focused on the optical properties of the metal nanoparticles. Using the metal nanoparticles can improve the sensitivity, lower the detection limit of the sensor, and perform the work that other materials can't finish. In addition, when the distance between the nanoparticles is different, the color of the solution may also be different, thus it owns broad application prospects in colorimetric analysis.
DNAzymes are catalytic DNA. To date, various DNAzymes have emerged through in vitro selection. Some DNAzymes are able to catalyze the incision or ligation of nucleic acids, and others have the catalytic activity of kinase or peroxidase. One interesting example of DNAzymes is the horseradish peroxidase mimicking DNAzyme, which has the catalytic activity of horseradish peroxidase and is more stable than horseradish peroxidase. Thus, this kind of DNAzyme can be used to replace protein enzymes in chemo/biosensing for signal amplification and sensitive detection. Furthermore, the catalytic activity of DNAzyme can be regulated through DNA, which is superior to protein enzymes. Cooperating with peroxide, DNAzyme can catalyze some substrates to generate color change for colorimetric assay. However, the catalytic activity of DNAzyme is inferior to that of protein enzymes; therefore, increasing the activity of DNAzyme through straightforward methods will be a significant and challenging work.
Based on the above consideration and literatures reported previously, takeing the advantage of AuNPs and mimicking-HRP DNAzyme, we have developed a series of electrochemical and colorimetric chemo/biosensors. We also have studied the way to improve and inhibit the activity of DNAzyme. Additionally, we have developed logic gates based on specific metal ions binding peptides and AuNPs. The details are summarized as follows:
1. Electrochemical sensor based on the metal nanoparticles
A strategy to construct H3PMo12O40(PMo12) and platinum nanoparticles-chitosan (Pt-CHIT) multilayer film ((PMo12/Pt-CHIT)n) has been developed in chapter2. Negatively charged PMo12and positively charged CHIT have been employed to fabricate stable ultrathin multilayer film on the multiwalled carbon nanotubes coated pyrolytic graphite (CPG) electrode using layer-by-layer self-assembly technique. The doping of Pt nanoparticles minimized the disadvantage of CHIT. Cyclic voltammetry was used to confirm the consecutive growth of the multilayer film and to investigate the electrochemical behavior of the resulting (PMo12/Pt-CHIT)4/CPG detailedly. As an IO3-sensor, the (PMo12/Pt-CHIT)4/CPG exhibits excellent characteristics, such as high sensitivity, wide linear range, lower detection limit and fast response time.
A sensitive, label-free electrochemical aptasensor for small molecule detection has been developed in chapter3based on gold nanoparticles (AuNPs) amplification. This aptasensor was fabricated as a tertiary hybrid DNA-AuNPs system, which involved the anchored DNA (ADNA) immobilized on gold electrode, reporter DNA (RDNA) tethered with AuNPs and target-responsive DNA (TRDNA) linking ADNA and RDNA. Electrochemical signal is derived from chronocoulometric interrogation of [Ru(NH3)6]3+(RuHex) that quantitatively binds to surface-confined DNA via electrostatic interaction. Using adenosine triphosphate (ATP) as a model analyte and ATP-binding aptamer as a model molecular recognization element, the introduction of ATP triggers the structure switching of the TRDNA to form aptamer-ATP complex, which results in the dissociation of the RDNA capped AuNPs (RDNA-AuNPs) and the release of abundant RuHex molecules trapped by RDNA-AuNPs. The incorporation of AuNPs in this strategy significantly enhances the sensitivity because of the amplification of electrochemical signal by the RDNA-AuNPs/RuHex system. Under optimized conditions, a wide linear dynamic range of4orders of magnitude (1nM-10μM) was reached with the minimum detectable concentration at sub-nanomolar level (0.2nM). These results demonstrate that our nanoparticles-based amplification strategy is feasible for ATP assay and presents a potential universal method for other small molecule aptasensors.
2. Colorimetric detection of the activities of ethyltransferase and restriction endonuclease based on DNAzyme and the reform of the DNAzyme activity
DNA methylation catalyzed by methyltransferase (MTase) is a significant epigenetic process for modulating gene expression. Traditional methods to study MTase activity required laborious and costly DNA labeling process. In chapter4, we report a simple, colorimetric, and label-free methylation-responsive DNAzyme (MR-DNAzyme) strategy for MTase activity analysis. This new strategy relies on horseradish peroxidase (HRP) mimicking DNAzyme and the methylation-responsive sequence (MRS) of DNA which can be methylated and cleaved by MTase/endonuclease coupling reaction. Methylation-induced scission of MRS would activate the DNAzyme that can catalyze the generation of a color signal for the amplified detection of methylation events. Taking Dam MTase and DpnI endonuclease as examples, we have developed two colorimetric methods based on MR-DNAzyme strategy. The first method is to utilize an engineered hairpin-DNAzyme hybrid probe for facile turn-on detection of Dam MTase activity, with a wide linear range (6-100U/mL) and a low detection limit (6U/mL). Furthermore, this method could be easily expanded to profile the activity and inhibition of restriction endonuclease. The second method involves a methylation-triggered DNAzyme-based DNA machine, which achieves the ultra-high sensitive detection of Dam MTase activity (detection limit=0.25U/mL) by two-step signal amplification cascade.
The activity of DNAzyme is much poorer than that of protein enzymes. Thus, it is significant to improve the activity of DNAzyme for expanding its application and improving the sensitivity. In chapter5, a new method to improve the activity of DNAzyme is introduced, called neighboring improved effect. We have found that if the DNAzyem sequence with an A base at3'terminal, its activity can be improved about5fold. This effect has terminal and base selectivity (A base at3'terminal). In addition, it also has the topology configuration selectivity that trends forming a G4parallel structure. The position of base A between hemin and G4(3'terminal) is a factor to affect the DNAzyme activity. In this chapter, we try to give a possible mechnism of the catalytic reaction and employ it to assay the activity of nuclease. Compared with other DNAzyme activity regulation method, neighboring improved effect is simple for that it can be realized only by adding an A base to the3'terminal of DNAzyme sequence. This method doesn't need to introduce extra components or factors, and can be widely applied in analytical field. It also can help us to deepen our understanding of the DNAzyme catalytic mechanism.
General method for the inhibition of the DNAzyme activity is based on the binding or release of partial DNAzyme sequence. In chapter6, a more simple and feasible method for inhibition of the DNAzyme activity is developed, called neighboring inhibited effect. We found that, if the first base at3'terminal of DNAzyme sequence is purine base, DNAzyme activity can be almost fully inhibited just by hybriding this purine base. The inhibition effect of this strategy is superior to general methods, and it doesn't need to hybrid any base of DNAzyme sequence.
3. Rapid colorimetric Zn2+assay and colorimetric logic gates based on bifunctional peptide and unmodified AuNPs
In chapter7, a new and simple mechanism for the label-free and rapid colorimetric Zn2+assay is developed based on bifunctional peptide probe and unmodified AuNPs. This bifunctional peptide probe contains specific Zn2+binding site and arginine that can lead to the color change of AuNPs. The combination of hydrosulfuryl in peptide with Au in AuNPs can make the arginine residual close to AuNPs surface, and the AuNPs will be unstable and aggregated. Once the-SHs in the side chain of peptide are all coordinated with Zn2+, the peptide probe can't combine to the surface of AuNPs, therefore, the arginine residual cannot cause the aggregation of AuNPs. This colorimetric method for the detection of Zn2+exhibits excellent characteristics, such as rapid detection process (less than0.5min), high sensitivity, lower detection limit (10nM), and label-free. This strategy has been successfully used in Zn2+analysis of foods and drugs.
Using the principle in chapter7, we have designed a series of colorimetric logic gates based on peptide and AuNPs in chapter8. Employing Zn2+and chymotrypsin as input, the color of the solution of AuNPs as output, it is easy to achieve the "YES"("NO"),"OR" and "AND" logic gates.
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