分子手性调控核酸自组装及其生物传感应用

发布时间：2018-06-20 12:01

本文选题：分子手性 + 核酸自组装　；参考：《华中科技大学》2016年博士论文

【摘要】：手性是自然界的基本属性之一,构成生命体的生物大分子大多是以手性小分子为基元构成的。因此,生命体内的绝大多数生理和生物行为都与分子手性息息相关。为了探索生命体内手性选择性的奥秘,揭示自然界中手性现象的本质,本论文将生物大分子的自身属性——手性与自然界的普遍现象——核酸自组装有机结合,研究分子手性对核酸自组装的调控作用,并应用于生物传感器中,探索分子手性对特定目标分子检测性能的调控(包括检测限、灵敏度和线性范围等),为开发新型纳米生物材料和器件开启新的方向,并为临床疾病的早期诊断提供新的思路。本论文的主要内容包括四个方面：(1)以巯基和亚甲基蓝修饰的DNA为模型分子,探讨其在N-异丁酰基-D(L)-半胱氨酸(NIBC)修饰的金表面的组装行为,由于L-NIBC修饰表面对DNA有更强的吸附作用导致亚甲基蓝离金表面距离更近,所以其在L-表面的峰电流比对应的D-表面的峰电流大。进一步地,在手性表面引入与模型分子互补的核酸分子,通过方波伏安法(SWV)、石英晶体微天平(QCM)和电化学阻抗谱(EIS)等手段研究了表面手性对DNA杂交效率的调控作用。结果表明D-表面的杂交效率比L-表面的杂交效率高,并实现了分子手性对灵敏度的调控。此外,将该体系引入到复杂基质中,以满足复杂生物样品测试的需求,为临床诊断、基因治疗及相关领域研究提供帮助,并有助于更深层次理解自然界中手性现象的本质。(2)将具有信号放大机制的“三明治”结构DNA与手性分子有机结合,以电化学生物传感器为平台,探索分子手性对“传统三明治”和“超级三明治”结构DNA检测效率的调控作用。结果表明,D-NIBC构筑的“超级三明治”结构DNA的检测效率比L-NIBC构筑的“超级三明治”结构DNA的检测效率更强,检测限相差2个数量级。该体系通过“超级三明治”结构DNA不仅放大了分子手性对杂交效率的调控能力,而且不同手性分子修饰的“超级三明治”结构对“传统三明治”结构检测核酸时的信号放大能力差异显著,即使在复杂生物体系中检测效率的差异仍然存在。证明了手性有望成为核酸杂交调控的新手段,并为进一步揭示生命体手性选择性的奥秘开启新的方向。(3)研究了分子手性诱导的DNA探针用于超灵敏单核苷核苷酸多态性(SNP)分析。在检测完全互补靶分子时,L-色氨酸孵育的传感器比对应的D-色氨酸孵育传感器的检测效率低,且二者检测效率的差异较大：而在检测单碱基错配靶分子时二者检测效率的差异不显著。利用手性传感器检测效率差异的不同,实现了高灵敏的SNP分析,SNP检测的区分因子中值为7.21。此外，，我们还通过“超级三明治”结构进一步提升了特异性,区分因子的中值增加到38.71,“超级三明治”结构不仅改善了灵敏度,还提高了特异性。手性是生命体系中最重要的生化属性之一,因此该成果将有助于发展新的生物器件,并对复杂体系中的临床诊断提供新的方法和思路。(4)利用碱基错配的原理设计了不同亲和力的双链探针,并调控了ATP适配体传感器的线性范围。通过共组装不同亲和力的双标记双链探针,成功实现了线性范围的同步拓宽,线性范围对应靶分子浓度跨度从81倍增加到6400倍：通过组装无标记的高亲和力探针和双标记的低亲和力探针,实现了线性范围的同步缩窄,线性范围对应靶分子浓度跨度缩小至10倍;通过组装单标记的高亲和力探针和双标记的低亲和力探针,实现了线性范围的异向调控,同时拓宽了线性范围并提高了灵敏度。适配体传感器线性范围的可控调节将有助于克服生物分子识别在生物燃料电池及生物电子逻辑门中的局限性,更有助于拓展生物传感器在临床诊断中的应用范围。
[Abstract]:Chiral is one of the basic properties of nature. Most of the biological macromolecules are composed of chiral small molecules. Therefore, most of the physiological and biological behaviors in life are closely related to molecular chirality. In order to explore the mysteries of chiral selectivity in life, the essence of chiral phenomena in nature is revealed. In this paper, the self-assembly of chiral macromolecules - chiral and natural phenomena - nucleic acid self-assembly is organically combined, and the regulation of molecular chirality on nucleic acid self-assembly is studied and used in biosensors to explore the regulation of molecular chirality on the detection performance of specific target molecules (including detection limit, sensitivity and linear range, etc.) In order to open new directions for the development of new nano biomaterials and devices and provide new ideas for the early diagnosis of clinical diseases, the main contents of this paper include four aspects: (1) the assembly behavior of DNA modified gold surface modified by N- isobutylyl -D (L) - cysteine (NIBC) was studied by using mercapto and methylene blue modified as model molecules. As the L-NIBC modified surface has a stronger adsorption to DNA, the methylene blue is closer to the gold surface, so the peak current on the L- surface is larger than that of the corresponding D- surface. Further, the nucleic acid molecules complementary to the model molecules on the chiral surface are introduced by Fang Bo volt ampere (SWV), quartz crystal microbalance (QCM) and electrochemistry. The effect of surface chirality on the efficiency of DNA hybridization was studied by means of impedance spectroscopy (EIS). The results showed that the hybridization efficiency of the D- surface was higher than that of the L- surface, and the sensitivity was regulated by the molecular chirality. In addition, the system was introduced into the complex matrix to meet the needs of the complex biological sample test and the clinical diagnosis. The study of treatment and related fields helps to further understand the nature of chiral phenomena in nature. (2) the combination of "sandwich" structure DNA with signal amplification mechanism is organically combined with chiral molecules, and an electrochemical biosensor is used as a platform to explore the hand nature of "traditional sandwiches" and "super sandwiches". The results show that the detection efficiency of "super sandwich" structure DNA constructed by D-NIBC is more efficient than that of "super sandwich" structure DNA, which is constructed by L-NIBC, and the difference of detection limit is 2 orders of magnitude. The system not only amplifies the modulation of molecular chirality to the hybridization efficiency through the "super sandwich" structure DNA. Control ability, and the different chiral molecular modified "super sandwich" structure has significant difference in the signal amplification ability of the "traditional sandwich" structure detection nucleic acid. Even in the complex biological system, the difference of detection efficiency still exists. It is proved that chiral is expected to be a new part of nucleic acid hybridization regulation, and to further reveal the life. The new direction of the chiral selectivity of the body is opened. (3) the molecular chiral DNA probe is used to analyze the ultra sensitive mononucleotide polymorphism (SNP) analysis. The detection efficiency of the L- tryptophan incubating sensor is lower than that of the corresponding D- tryptophan incubating sensor when detecting the fully complementary target molecules, and the difference of the detection efficiency is larger than that of the corresponding D- tryptophan incubating sensor. There is no significant difference in the detection efficiency between the two of the single base mismatch targets. The high sensitive SNP analysis is achieved by using the difference of the detection efficiency of the chiral sensor. The median of the SNP detection is 7.21.. We also increase the specificity of the "super sandwich" structure, and increase the median of the differentiation factor. Adding to 38.71, the "super sandwich" structure not only improves the sensitivity, but also improves the specificity. Chirality is one of the most important biochemical properties in the life system. Therefore, the results will help to develop new biological devices and provide new methods and ideas for the clinical diagnosis of complex systems. (4) the principle of using base mismatch is not designed. The same affinity double stranded probe regulates the linear range of the ATP aptamer sensor. By CO assembling the dual labelled double stranded probes with different affinity, the linear range is successfully synchronized. The linear range increases to 6400 times from 81 times to the target molecular concentration: by assembling unlabeled high affinity probes and double markers The low affinity probe realizes synchronous narrowing in linear range, and the linear range decreases to 10 times of the target molecular concentration span. By assembling a single labeled high affinity probe and a double labeled low affinity probe, the linear range is controlled, and the linear range is widened and the sensitivity is increased. The linear norm of the aptamer sensor is linear. The controllable regulation will help to overcome the limitations of biomolecular recognition in biofuel batteries and bio electronic logic gates, and will help to expand the application of biosensors in clinical diagnosis.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：O629.74;TP212.3

【相似文献】