【作者】 徐宁；

【导师】 吴梅笙；

【作者基本信息】 南京农业大学 ， 工程硕士（专业学位）， 2019， 硕士

【摘要】 随着我们进入二十一世纪,医疗技术的进步有助于发现更多的遗传疾病。DNA传感对于遗传疾病的快速筛查和检测变得越来越重要。与传统的杂交测定相比,DNA生物传感器以更快,更简单和更便捷的方式为获得人、病毒和细菌核酸中的序列特异性信息提供了相当大的前景。早在1927年,格式试剂被发现在无水乙醚溶液中能够产生发光,1929年Harvey课题组报道了有关鲁米诺在碱性溶液中电解时,在阴阳两极都有发光的现象,直到20世纪60年代才开始了对电致化学发光进行了详细的研究,并且随着电子技术以及高灵敏的光电传感器的出现,使得电致化学发光技术得到迅猛的发展,目前,电致化学发光技术已经成为生命分析化学领域的主要研究手段之一。电致化学发光是化学发光和电化学相结合的产物,它是通过在电极上施加一定的电位,使得物质之间或物质与体系中其他的组分之间产生电子转移形成激发态,由激发态返回到基态所产生的发光现象。生物传感器(biosensor),是一种对生物物质敏感并将其浓度转换为电信号进行检测的仪器。是由固定化的生物敏感材料作识别元件(包括酶、抗体、抗原、微生物、细胞、组织、核酸等生物活性物质)、适当的理化换能器(如氧电极、光敏管、场效应管、压电晶体等等)及信号放大装置构成的分析工具或系统。DNA杂交生物传感器的开发有助于病原体检测和分子诊断能力的提升。如果我们能够更加快速、有效、便捷、准确的检测出疾病中的相关致病因素,那么将对现在的医疗检测做出巨大的帮助,有利的推进医疗检查技术的进步。因此,本文旨在研究简单、快捷、低成本且准确性高的检测装置。根据以上的研究现状和现有理论依据,本篇论文的具体工作相关如下:1.利用两层聚二甲基硅氧烷(PDMS)和氧化铟锡(ITO)电极进行层层构建,组装了简便、便携的闭合式双极电极-电化学发光检测体系。在这两层PDMS表面都具有两个小孔,作为储液池和放置外界驱动电极的位点。这两层上的小孔尺寸并不相同。通过在驱动电极上施加一定的外加电压,使得ITO 双极电极(BPE)的阴极可以电沉积一层金颗粒。颗粒的致密性和尺寸可以通过氯金酸的浓度进行调节。镀了金颗粒的一端作为阴极,它可以极大的催化阴极的电化学反应,如:亚甲基蓝和双氧水,从而促进阳极的电致化学发光(ECL)信号。随后,我们用扫描电镜对金纳米颗粒的形貌进行了表征,并通过光学成像考察了金膜的颜色变化;研究了不同氯金酸浓度所沉积的金颗粒的对钌联吡啶ECL信号的影响,为后续的生物分子的组装和生物传感器的构建奠定基础。2.在上述构建好的双极电极芯片的镀金端通过Au-S键进行DNA的层层组装,并通过杂交链反应实现高效的信号放大构建生物传感器。通过在金颗粒表面组装一层capture DNA,它可以通过碱基互补配对进行特异性的捕获target DNA;随后通过两个发夹结构的DNA(H1和H2)与Target DNA相互作用,将发卡结构的H1和H2依次打开,循环往复,使得大量的H1和H2连接到电极表面,从而增加了阴极的电阻,导致阳极处的ECL信号猝灭。该方法不仅可以检测target DNA的浓度,还可以检测双氧水的浓度。这主要是由于双氧水在双极电极的阴极可以催化阳极的ECL信号。因此,我们构建了一个既可以检测DNA含量又可以检测双氧水浓度的生物传感器。探究镀金浓度、发光物质浓度、DNA孵育时间等因素对发光信号的影响。同时还进行了特异性实验,结果显示:该实验具有较高的选择性和准确性。该传感器对目标DNA检测的线性范围是0.1 nM～0.5 μM,相关系数为0.9905。此外,它还可以用于H2O2的检测,线性范围为:0～5 mM,相关系数为0.9923。更多还原

【Abstract】 As we enter the 21stcentury,advances in medical technology can help us to discover more genetic diseases.Sensitive DNA sensing approach is becoming increasingly important for rapid screening and detection of genetic diseases.Compared to traditional hybridization assays,DNA biosensors offer considerable promise for obtaining sequence-specific information in human,viral and bacterial nucleic acids in a faster,simpler and less expensive manner.As early as 1927,Grignard reagents were found to produce luminescence in an anhydrous ether solution.In 1929,the Harvey team reported on the luminescence of luminol at the cathode and anode in an alkaline solution.Until the 1960s,large number of studies has been focused on the investigation of the detail mechanism of electrochemiluminescence(ECL).With the development of electronic technology and highly sensitive photoelectric sensors,ECL technology has developed rapidly.Currently,ECL technology has become one of the main research methods in the field of life analysis chemistry.ECL is a product of the combination of chemiluminescence and electrochemistry.It exerts a certain potential on the electrode to cause electron transfer between substances or other components in the system to form an excited state.The phenomenon of luminescence produced by the return of the excited state to the ground state.A biosensor is a device that is sensitive to biological substances and converts its concentration into electrical signals for detection.It is an analytical tool or system consisting of immobilized bio-sensitive materials as identification components,appropriate physical and chemical transducers,and signal amplification devices.The development of DNA hybrid biosensors contributes to the improvement of pathogen detection and molecular diagnostic capabilities.If we can detect the relevant pathogenic factors in the disease more quickly,effectively,conveniently and accurately,it will greatly help the current medical testing and promote the advancement of medical examination technology.Therefore,this paper aims to study detection devices that are simple,fast,low-cost,recyclable,and highly accurate.Based on the above research status and existing theoretical basis,the specific work of this paper is related to the following:1.A simple and portable closed bipolar electrode-electrochemiluminescence(BPE-ECL)detection system was constructed based on the combination of two layers of PDMS slices and an indium tin oxide(ITO)electrode.Two holes are punched on each PDMS slices with different diameters which serve as reservoirs and the anchors for the placing of driving electrodes.After applying a suitable voltage on the driving electrodes,AuCl4-would be reduced on one pole of ITO BPE and water was oxidized on the opposite pole of ITO BPE.Therefore,we can observe the deposition of Au layer on the cathode of ITO electrode.The density and the morphology are closely related to the concentration of AuCl4-.They are analyzed by the optical images and Scanning Electron Microscope(SEM)images.The prepared Au-ITO BPE is then characterized by the ECL approach by adding different types of electroactive species on the Au layer(cathode of BPE)and ECL reagent on ITO electrode(anode of BPE),such as,methylene blue(MB)and hydrogen peroxide(H2O2).Results have indicated that both of them can be used to trigger the ECL signal in closed-BPE device.Additionally,the concentration of AuCl4-has a great impact on the ECL signal of Ru(bpy)32+/DBAE,which laid a foundation for the subsequent assembly of biomolecules and the construction of biosensors.2.A sensitive biosensor is then constructed on the above device by self-assembly through the Au-S bond between Au layer and the thiol group at the terminal of DNA.Meanwhile,a high-efficiency signal amplification effect of hybridization chain reaction is integrated in the above sensing interface.A layer of capture DNA is first modified on Au layer which can be used to hybridize with target DNA.The residual sequence of target DNA could trigger the decomposition of hairpin DNA(H1 and H2),leading to the improving length of DNA sequence.Due to the large amount of H1 and H2 is captured on electrode surface,the resistance is greatly enhanced,leading to the inhibition of ECL signal at the anode of BPE.Due to the reduction of H2O2 at the anode of BPE can trigger the ECL signal at the anode,the designed biosensor could not only be used to detect the concentration of target DNA,but also be applied for the measurement of H2O2.In order achieve sensitive detection,we have explored the effects of Au plating concentration,the concentration of ECL reagent,DNA incubation time and other factors on the ECL signal.At the same time,specific experiments were carried out,and the results show that the experiment has high selectivity and accuracy.The linear range of the sensor for target DNA detection is 0.1 nM to 0.5 μM with a correlation coefficient of 0.9905.In addition,it can also be used for the detection of H2O2 with a linear range of 0～5 mM and a correlation coefficient of 0.9923.The results show that the proposed biosensor can be used for DNA and H2O2 detection.更多还原