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。
The rapid development of life science, clinical medicine, environmental science and material engineering has driven a higher demanding on analytical chemistry and analytical apparatuses. High-sensitivity, high throughput, multi-informatization and visualization are the most important trends in regards to these fields. Electrochemiluminescence (ECL) imaging is such an effective method conforming to the growing tendency. As a new imaging platform, ECL imaging has attracted considerable attention in the fabrication of ECL biosensors with portability, miniaturization, and high throughput. Research in this area is still at an early stage and has a broad prospect of development.
Fingerprints have long been used as the gold standard for personal identification in forensic investigations, and continue to be the most reliable tool in crime cases and law enforcement. Although innumerable methods have been exploited for fingerprint detection, it still remains challenging to acquire fingerprint information in a technically simple, rapid, and easy handling way. Additionally, research interest has been recently directed to detect the secretion chemicals in fingerprints. Taking into account the advantages of ECL imaging, e.g., high sensitivity, controlled reaction, fast imaging and good compatibility with bioassay, herein we present its application in the development and component recognition of latent fingerprint. The aim of this thesis is to establish a prospective study that integrates imaging analysis, bioanalysis and fingerprint identification, in order to provide new methods and ideas for the application of ECL imaging in life science and forensic science.
Firstly, a home-designed ECL imaging system was established. It consists of an electrochemical workstation and an imaging device. The electrochemical workstation was used to control the electrochemical reaction and detect the signal. And the imaging device was used to record the images and input them into a computer for post-processing. The imaging device equipped with a high-resolution CCD camera, an appropriate Macro Zoom lens and a three-dimensional translational stage is the key component of the whole system.
ECL imaging for the development of latent fingerprints was carried out by using the established imaging system. Selective control of ECL generation at the electrode surface is shown to be an effective means of visualizing latent fingerprints. Two operating schemes designated negative and positive imaging modes was reported. The negative imaging mode was performed by directly exposing the electrodes bearing sebaceous fingerprints to the reaction solution containing Ru(bpy)32+and TPrA. Since the fingerprint pattern functions as an insulating mask or template, ECL is only generated from the bare electrode surface when biasing a suitable voltage. The ending outcome is that the furrow areas are lighted up by ECL and contrasted distinctively with the dark ridge details, yielding a negative image of the fingerprint. Factors, including the applied potential and the concentration of ECL luminophore, were investigated to achieve a satisfactory visualization enhancement. The negative mode is suitable for identifying the second and even the third level detail in latent fingerprints, as well as enhancing the visualization of aged latent fingerprints. In addition, the imaging of latent fingerprints in the negative mode also works in other ECL reaction systems, such as the well-known one involving luminol-hydrogen peroxide pair, as well as on other conducting substrates, such as stainless steel plates, with a satisfactory visual contrast. The proposed ECL imaging approach could be utilized to enhance sebaceous fingerprints collected from various object surfaces including coin, desk, disk and computer screen, proving the practical significance of the approach.
In the positive imaging mode, an ECL-generating luminophore, ruthenium bis(2,2'-bipyridine)(2,2'-bipyridine-4,4'-dicarboxylic acid) N-hydroxysuccinimide ester (Ru(bpy)2(dcbpy)NHS), the NHS group in which is reactive toward the amino acids via the formation of amide bonds, was synthesized to functionalize the fingerprint ridges. After the covalent interaction, the ridge details were tagged by the luminophores, which could then react with dissolved co-reactant, namely DBAE, to generate ECL, eventually producing a positive image of the fingerprint. The covalent bonding of Ru(bpy)2(dcbpy)NHS with amino acids present in the ridge details were investigated by MALDI-TOF measurements. Factors, including the applied potential, the concentration of ECL luminophore and the incubation time, were investigated to achieve. Similar enhancement effect was also observed for eccrine fingerprints.
In combination with the enzyme immunassay, the ECL detection of the target components in latent fingerprints was presented with high specificity and sensitivity. To exemplify the fingerprint visualization approach, hIgG was chosen as a test protein. The artificial simulated fingerprint was incubated with goat anti-human1gG and HRP-conjugated rabbit anti-goat IgG for a certain time successively. After the immune reactions, HRP was immobilized onto the fingerprint ridges, which can then catalyze the oxidation of luminol by electrochemically generated H2O2to yield an ECL image of the fingerprint. A satisfactory visualization enhancement was determined by optimizing operating conditions in regards to the incubation time and temperature of the immunolabeling, and the concentration of immuno reagents. BSA inked fingerprints were used to investigate the specificity of the ECL enzyme immunoassay. Results confirmed that the antibodies specifically target the hlgG antigen in the fingerprint residue. Furthermore, the ECL visualization of a blood fingermprint was realized through the immunodetection of hlgG.
Finally, the ECL imaging strategy was further improved by introducing the biotin-streptavidin which have been known as a sufficient signal amplification system for the detection of Dermcidin, EGF and lysozyme in eccrine fingerprints. This technique provides an effective method for fingermark detection that enables both identification of an individual and recognition of the secretions in the human perspiration. It has promise for developing a low cost and portable method to detect more other species that are practically and potentially useful for forensic work or diagnostic assay, such as metabolites of illicit drugs, chemical products of explosives, and biomarkers for in vitro diagnosis.
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