电化学发光成像技术在潜在指纹显现与成分识别中的应用

发布时间：2018-06-18 17:07

本文选题：电化学发光成像 + 生物分析　；参考：《浙江大学》2014年博士论文

【摘要】：生命科学、临床医学、环境科学及材料科学等领域的迅速发展对分析化学及分析仪器提出了越来越高的要求。高灵敏度、高通量、多信息化和可视化是当前分析仪器和分析科学发展的一大重要趋势。电化学发光成像就是顺应这一时代背景发展起来的一种有效的分析手段。作为一种全新的成像技术,电化学发光成像目前在研制便携式、微型化、高通量电化学发光生物传感器方面逐渐引起人们的重视。然而该领域的研究尚处于起步阶段,仍然存在着广阔的研究空间。指纹鉴定是进行个人识别的最可靠的方法之一,在法庭科学中用来有效地查证、揭露和证实犯罪。尽管指纹显现方法繁多,但是目前仍然需要建立一种简单、快速、适用范围广以及造价低廉的新方法。另外,指纹的成分分析近年来引起人们越来越浓厚的研究兴趣。本项研究基于电化学发光成像高灵敏、反应可控、快速成像以及对于生物分析兼容性的特点,将其用于潜在指纹的显现与成分识别,旨在发展一种集成像分析、生物分析、指纹识别于一体的前瞻研究,为电化学发光成像技术在生命科学和法庭科学中的应用提供新的方法和思路。进行的具体工作如下：首先自行组建了一套电化学发光成像系统。该系统由电化学工作站和成像装置组成。其中,电化学工作站用于电化学反应的控制和电化学信号的接收；成像装置用于发光图像的采集,并将图像输入计算机进行后期处理。成像装置是该系统的核心部件,主要包括一个高分辨率数字冷却CCD相机、一个高通透微距镜头以及一个升降样品台。利用该成像系统,展开了潜在指纹的电化学发光成像研究。通过在空间上选择性地控制电极表面的电化学发光反应,可以实现潜在指纹的反相成像和正相成像两种显现模式。在反相模式中,指纹覆盖的电极表面会由于沉积的有机脂肪酸等惰性物质而对电子转移造成抑制作用,因此Ru(bpy)32+/TPrA体系产生的电化学发光图像可以间接反衬出指纹的嵴线纹路。考察了反应电位、发光体Ru(bpy)32+浓度对显现效果的影响。反相模式可实现潜在指纹中不同二级结构特征和三级结构特征的检测成像,并适用于陈旧指纹的成像分析。在此基础上,进一步研究和完善了不同承载客体及其他电化学发光体系。在不锈钢片上,成功实现了潜在指纹的显现。并用采集胶带将日常物面(如硬币、桌面、光盘和电脑屏等)上的残留指纹转移到不锈钢导电基底上,证明了该方法的实践价值。利用鲁米诺/K2S208(或H202)进一步拓展了电化学发光反应体系。合成了具有生物反应活性的二(2,2’-联吡啶)(2,2’-联吡啶-4,4’-二甲酸)钌的N-羟基琥珀酰亚胺酯(Ru(bpy)2(dcbpy)NHS),实现了潜在指纹的正相模式显现。在正相模式中,电化学发光活性分子Ru(bpy)2(dcbpy)NHS可以通过分子上的N-羟基琥珀酰亚胺酯与指纹氨基酸中的氨基发生共价结合,从而标记到指纹上；继而与共反应剂DBAE在一定电位下产生电化学发光反应,显现出指纹的形貌。利用MALDI-TOF MS验证了Ru(bpy)2(dcbpy)NHS与指纹氨基酸的共价连接,考察了反应电位、发光体浓度及反应时间对显现效果的影响。正相模式可以对痕量汗潜指纹进行有效显现。结合酶联免疫分析技术,实现了潜在指纹中目标成分的特异性高灵敏电化学发光检测。首先以人IgG人工模拟指纹为检测对象,验证了方法的可行性。将指纹依次与羊抗人IgG、HRP标记兔抗羊IgG孵育一定时间,通过抗体与目标物的特异性免疫反应使HRP标记到指纹上。然后利用电化学反应使电解质溶液中的溶解O2还原生成H202,在指纹HRP的催化作用下,H202与底物溶液中的鲁米诺产生波长425nm的化学发光,进而显现出指纹纹路。考察了免疫反应温度、反应时间及抗体浓度对显现效果的影响。用BSA模拟指纹作为对照,结果表明抗体对人IgG模拟指纹的标记是特异性的。进一步地,将该方法用于真实血指纹中人IgG的成功检测。最后,在上述方法的基础之上,进一步引入生物素-链霉亲和素放大系统,实现了汗潜指纹中人汗腺抗菌肽Dermcidin、人表皮生长因子和溶菌酶等目标代谢物的电化学发光检测。该方法在进行潜在指纹显现的同时,可以实现人汗腺代谢物的特异性识别,有望发展成为一种简单、便携、通用的指纹检测技术,用于兴奋剂检测、病患临床诊断以及爆炸物检测等领域,具有重要的医学诊断和安全保障价值。
[Abstract]:The rapid development in the fields of life science, clinical medicine, environmental science and material science has put forward more and more demands on Analytical Chemistry and analytical instruments. High sensitivity, high throughput, multi information and visualization are the major trend of the development of analytical instruments and analytical sciences. Electrochemiluminescence imaging is in conformity with the back of this era. As a new imaging technique, electrochemiluminescence imaging has attracted more and more attention in the development of portable, miniaturized and high-throughput electrochemiluminescence biosensors. However, the research in this field is still in its infancy, and there is still a wide space of research.
Fingerprint identification is one of the most reliable methods for personal identification. It is used in forensic science to effectively verify, expose and confirm the crime. Although there are many methods of fingerprint appearance, it is still necessary to establish a new method that is simple, fast, wide applicable and low cost. In addition, the analysis of fingerprint composition has caused people in recent years. This study is based on the characteristics of electrochemiluminescence imaging, such as high sensitivity, reaction control, rapid imaging, and the characteristics of bioanalytical compatibility. It is used to identify potential fingerprints and identify components. It aims to develop a prospective study of integrated image analysis, bioanalysis and fingerprint identification, for electrochemiluminescence. The application of imaging technology in life science and forensic science provides new methods and ideas.
A set of electrochemiluminescence imaging system is set up first. The system consists of an electrochemical workstation and an imaging device. The electrochemical workstation is used for the control of the electrochemical reaction and the reception of the electrochemical signal; the imaging device is used for the collection of the luminescent images, and the image input computer is later processed. The imaging device is the system The core components of the system include a high resolution digital cooling CCD camera, a high pass macro lens and a lift sample stage.
By using the imaging system, the electrochemiluminescence imaging of potential fingerprints is developed. By selectively controlling the electrochemiluminescence reaction on the surface of the electrode, two modes of latent fingerprint imaging and positive phase imaging can be realized. In the reverse phase mode, the surface of the electrode covered by the fingerprint is due to the deposition of organic fatty acids. The electrochemiluminescence images produced by the Ru (bpy) 32+/TPrA system can indirectly reflect the ridge lines of the fingerprint. The effects of the reaction potential and the concentration of Ru (bpy) 32+ on the apparent effect are investigated. The reverse phase pattern can realize the two stages of the structure and the three structure of the potential fingerprints. Characteristic detection imaging, which is applied to the imaging analysis of old fingerprints, and on this basis, further studies and perfects different bearing objects and other electrochemical luminescence systems. On stainless steel sheets, potential fingerprints are successfully realized. And the residual finger of daily surface (such as coins, desktops, CD and computer screens) is used to collect tape. The transfer of grain to the conductive substrate of stainless steel proves the practical value of this method. Luminol /K2S208 (or H202) is used to further expand the electrochemiluminescence reaction system.
N- hydroxy succinimide (Ru (bpy) 2 (dcbpy) NHS) of two (2,2 '- bipyridine - bipyridine - -4,4' - two formic acid) Ru (Ru (bpy) 2 (dcbpy) NHS) has been synthesized. In the positive phase, the electrochemiluminescence active fraction Ru (bpy) 2 (dcbpy) NHS can pass through the molecular hydroxy succinimide The ester was covalently bound to the amino acid in the fingerprint amino acid, which was marked on the fingerprint, and then the co reactants DBAE produced the electrochemiluminescence reaction at a certain potential and showed the morphology of the fingerprint. The covalent connection between Ru (bpy) 2 (dcbpy) NHS and the fingerprint amino acid was verified by MALDI-TOF MS, and the reaction potential, the concentration of luminescent body and the reaction were investigated. The effect of time on the effect of appearance is positive.
Combined with enzyme linked immunosorbent assay (ELISA), the specific high sensitive electrochemiluminescence detection of target components in potential fingerprints was realized. First, human IgG artificial fingerprint was used as a test object to verify the feasibility of the method. The fingerprints were incubated with the Sheep anti human IgG, HRP labeled Rabbit anti sheep IgG for a fixed time, and the specific immunity of the antibody and target was avoided. The pestilence reacts the HRP to the fingerprints. Then the dissolved O2 in the electrolyte solution is reduced to H202 by the electrochemical reaction. Under the catalysis of the fingerprint HRP, the chemiluminescence of the H202 and the Lumino wavelength 425nm in the substrate solution is produced, and then the fingerprint pattern is displayed. The impact of the results. Using the BSA simulated fingerprint as a contrast, the results showed that the antibody against human IgG simulated fingerprints was specific. Further, the method was used for the successful detection of human IgG in real blood fingerprints.
Finally, on the basis of the above method, the biotin streptomycin amplification system was further introduced to detect the electrochemical luminescence of the target metabolites of human sweat gland antibacterial peptide Dermcidin, human epidermal growth factor and lysozyme in sweat latent fingerprints. The method can realize human sweat gland metabolites while carrying out potential fingerprints. Specific recognition is expected to develop into a simple, portable, universal fingerprint detection technology, which is used in the field of doping, clinical diagnosis and explosive detection, and has important value in medical diagnosis and safety.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：O657.1;D918.91

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