【作者】 张丹；

【导师】 周国宝；

【作者基本信息】 浙江师范大学 ， 分析化学， 2024， 硕士

【摘要】 本论文主要围绕银纳米簇多信号通路生物传感器的制备和核酸检测应用展开,通过经典的杂交链式反应（hybridization chain reaction,HCR）等温扩增技术放大传感器各通路信号,并利用多信号通路提升稳定性的特点,实现核酸灵敏、准确检测。此外,研究过程中创新的提出了多种封闭探针,用于封闭杂交链式反应过程中过量的茎环探针,不仅能降低背景信号,还能简化核酸检测过程,进一步提升所设计生物传感器的检测性能。论文从以下两个方面展开研究:1.通过设计双功能封闭探针辅助杂交链反应（HCR）,提出了一种新的双荧光通路比率银纳米簇（AgNCs）传感方法。发夹探针HP1在3’端和5’端各含有1个特殊的DNA片段（5’-CACCGCT-3’和5’-ATTTGCCTTGGGGACGGATA-3’）,2个DNA片段合在一起为一条红色银纳米簇（r-AgNCs）成核序列（r NS,5’-CACCGCTATTTGCCTTTTGGGGAGGATA-3’）。我们发现HP1中用于参与HCR反应的粘性末端（5’-TGCCC-3’）的存在会显著封闭r NS合成r-AgNCs的荧光。加入目标DNA后,引发两个发夹探针HP1和HP2发生HCR反应,从而形成具有多支r NS的长DNA双链。由于HP1中的粘性末端参与了长双链的形成,其对r-AgNCs荧光的影响得以被消除,在670 nm处能观察到r-AgNCs显著增强的荧光。更为重要的是,设计的双功能封闭探针不仅能进一步降低r-AgNCs红色荧光,还能作为绿色银纳米簇（g-AgNCs）成核序列,实验中该绿色荧光可用于内参提升核酸检测灵敏度。因此,封闭探针一方面可以提高所构建生物传感器的信噪比,另一方面,封闭探针还有助于构建具有自校准能力的HCR-AgNCs比率传感策略,提升检测结果重现性。与传统的HCR-AgNCs传感器相比,基于双功能封闭探针辅助HCR的比率分析法具有更高的可靠性,对构建能在各种领域应用的生物传感器具有重要的实际意义。2.为了进一步提高所构建传感器的综合性能,在下一个工作中,从多方面改进传感器的设计与构建。首先,在前述HCR信号放大的基础上,提出了三维杂交链式反应（3D-HCR）策略用来放大检测信号,进一步提升传感器的核酸检测灵敏度。在实验过程中,只需通过控制一维杂交链式反应（1D-HCR）所生成长双链上手臂链的方向（相邻手臂链方向呈90o）,3D-HCR便可通过1D-HCR产物手臂链平行杂交自组装而成。相比传统3D-HCR,我们所设计的3D-HCR方法更加简便高效。其次,利用DNA作为模版合成的银纳米簇不仅能产生荧光,还能抑制脲酶和尿素存在下发生的显色反应,因此构建出荧光和显色双信号通路传感器。为了更简便高效的实现双信号检测,本实验合成了聚多巴胺球/磁性四氧化三铁/DNA探针（PDA@Fe₃O₄/DNA）复合材料,该复合材料的核心作用是将溶液中的3DHCR/AgNCs产物分离,而分离的桥梁是目标核酸引发的1D-HCR。一方面溶液中的银纳米簇减少使荧光强度降低,另一方面复合材料上的银纳米簇可以抑制脲酶活性,使得尿素不会被氧化并发生相应的显色反应,从而实现双信号通路核酸检测,提高其检测稳定性和可靠性。由于过量的未反应完的茎环探针会与复合材料界面探针杂交,实验中还巧妙的设计了茎环封闭探针用于封闭过量的未反应的茎环探针,避免其干扰3D-HCR产物捕获。此外,本实验合成的PDA@Fe₃O₄/DNA具有很好的分散性,有利于复合材料界面进行杂交反应,因而进一步提升其检测性能。综上所述,所设计双信号通路银纳米簇传感器能实现快捷、稳定和灵敏检测核酸,具有较好的实际应用前景。更多还原

【Abstract】 This thesis mainly focuses on the preparation of silver nanocluster multi-signal pathway biosensor and the application of nucleic acid detection.The classical hybrid chain reaction（hybridization chain reaction,HCR）isothermal amplification technique is used to amplify the signals of each pathway of the biosensor,and using the characteristics of multi-signal pathway to improve stability to achieve sensitive and accurate detection of nucleic acid.In addition,a variety of blocked probes were creatively proposed in the research process,which can be used to block the excess stem-ring probe in the process of hybrid chain reaction,which can not only reduce the background signal,but also simplify the nucleic acid detection process.Further improve the detection performance of the designed biosensor.This paper carries out research from the following two aspects:1.we proposed a ratiometric silver nanoclusters（Ag NCs）fluorescent assay by designing a bifunctional-blocker-aided hybridization chain reaction（HCR）.Hairpin probe 1（HP1）containing two special DNA fragments（5’-CACCGC-3’and 5’-ATTTGCC TTTTGGGGACGGATA-3’）at two terminals creates a red-emitting Ag NCs nucleation sequence（r NS,5’-CACCGCTATTTGCCTTTTGGGGACGGA TA-3’）.We found that the presence of a toehold fragment（5’-TGCCC-3’）in HP1 could silence the r NS.Upon the addition of a target nucleic acid,HCR of HP1 and hairpin probe 2（HP2）could be initiated,resulting in the formation of long chain of DNA duplexes with multibranched r NS.As the toehold fragment in HP1 participated in generating duplexes,a strong emission of r NS-templated Ag NCs was observed at 670 nm.More significantly,a bifunctional blocker was introduced not only to reduce the background red-emitting fluorescence but also to play as an internal green-emitting Ag NCs nucleation sequence.On the one hand,the blocker could increase the signal-to-noise ratio of the constructed biosensor,and on the other hand,the blocker also helped to prepare ratiometric HCR-Ag NCs assay with self-calibrating ability to strengthen its reproducibility.Compared with the traditional HCR-Ag NCs sensors,the developed ratiometric assay based on the bifunctional-blocker-aided HCR has higher reliability,which is important for the fabrication of biosensors in various fields for practical biosensing applications.2.In order to further improve the sensitivity of nucleic acid detection,we introduced three-dimensional HCR（hybridization chain reaction）strategy on the basis of DNA-Ag NCs,combined with functional PDA@Fe₃O₄ to capture DNA and further magnetic separation of HCR products,in order to achieve the construction of biosensors.In this work,firstly,the base sequence of multidirectional HCR reaction in space was designed to synthesize the three-dimensional HCR product,and there were many protruding chains on the main chain of the product,and then Ag NCs was synthesized on the protruding chain of the three-dimensional HCR product,so as to obtain the fluorescence signal and amplify the fluorescence signal by three-dimensional HCR.In addition,poly-dopamine（PDA）was synthesized by polymerization of dopamine（DA）in alkaline environment,and in situ ferric oxide（Fe₃O₄）nanoparticles were loaded on its surface to synthesize PDA@Fe₃O₄complexes.Then,amino-functionalized PDA@Fe₃O₄ complexes were obtained by using the interaction between PDA and modified amino DNA,so that DNA was successfully linked to PDA@Fe₃O₄ by amino groups for subsequent DNA capture.In the presence of the target DNA,two single strands not involved in hybridization of the one-dimensional HCR products initiated by the target DNA can form a triple sandwich structure by complementary pairing with the extended strands of three-dimensional HCR and the DNA on the amino functional PDA@Fe₃O₄,respectively.After magnetic separation,the fluorescence of the supernatant decreased,and the silver atoms in the precipitate could be oxidized by hydrogen peroxide（H₂O₂）to inactivate urease,which further led to the yellow color of urea solution containing phenol red,thus realizing the sensitive detection of double signals of DNA.更多还原