發(fā)布時間：2018-06-18 17:07

  本文選題：電化學發(fā)光成像 + 生物分析�。� 參考：《浙江大學》2014年博士論文

【摘要】：生命科學、臨床醫(yī)學、環(huán)境科學及材料科學等領域的迅速發(fā)展對分析化學及分析儀器提出了越來越高的要求。高靈敏度、高通量、多信息化和可視化是當前分析儀器和分析科學發(fā)展的一大重要趨勢。電化學發(fā)光成像就是順應這一時代背景發(fā)展起來的一種有效的分析手段。作為一種全新的成像技術,電化學發(fā)光成像目前在研制便攜式、微型化、高通量電化學發(fā)光生物傳感器方面逐漸引起人們的重視。然而該領域的研究尚處于起步階段,仍然存在著廣闊的研究空間。 指紋鑒定是進行個人識別的最可靠的方法之一,在法庭科學中用來有效地查證、揭露和證實犯罪。盡管指紋顯現(xiàn)方法繁多,但是目前仍然需要建立一種簡單、快速、適用范圍廣以及造價低廉的新方法。另外,指紋的成分分析近年來引起人們越來越濃厚的研究興趣。本項研究基于電化學發(fā)光成像高靈敏、反應可控、快速成像以及對于生物分析兼容性的特點,將其用于潛在指紋的顯現(xiàn)與成分識別,旨在發(fā)展一種集成像分析、生物分析、指紋識別于一體的前瞻研究,為電化學發(fā)光成像技術在生命科學和法庭科學中的應用提供新的方法和思路。進行的具體工作如下： 首先自行組建了一套電化學發(fā)光成像系統(tǒng)。該系統(tǒng)由電化學工作站和成像裝置組成。其中,電化學工作站用于電化學反應的控制和電化學信號的接收；成像裝置用于發(fā)光圖像的采集,并將圖像輸入計算機進行后期處理。成像裝置是該系統(tǒng)的核心部件,主要包括一個高分辨率數(shù)字冷卻CCD相機、一個高通透微距鏡頭以及一個升降樣品臺。 利用該成像系統(tǒng),展開了潛在指紋的電化學發(fā)光成像研究。通過在空間上選擇性地控制電極表面的電化學發(fā)光反應,可以實現(xiàn)潛在指紋的反相成像和正相成像兩種顯現(xiàn)模式。在反相模式中,指紋覆蓋的電極表面會由于沉積的有機脂肪酸等惰性物質而對電子轉移造成抑制作用,因此Ru(bpy)32+/TPrA體系產(chǎn)生的電化學發(fā)光圖像可以間接反襯出指紋的嵴線紋路。考察了反應電位、發(fā)光體Ru(bpy)32+濃度對顯現(xiàn)效果的影響。反相模式可實現(xiàn)潛在指紋中不同二級結構特征和三級結構特征的檢測成像,并適用于陳舊指紋的成像分析。在此基礎上,進一步研究和完善了不同承載客體及其他電化學發(fā)光體系。在不銹鋼片上,成功實現(xiàn)了潛在指紋的顯現(xiàn)。并用采集膠帶將日常物面(如硬幣、桌面、光盤和電腦屏等)上的殘留指紋轉移到不銹鋼導電基底上,證明了該方法的實踐價值。利用魯米諾/K2S208(或H202)進一步拓展了電化學發(fā)光反應體系。 合成了具有生物反應活性的二(2,2’-聯(lián)吡啶)(2,2’-聯(lián)吡啶-4,4’-二甲酸)釕的N-羥基琥珀酰亞胺酯(Ru(bpy)2(dcbpy)NHS),實現(xiàn)了潛在指紋的正相模式顯現(xiàn)。在正相模式中,電化學發(fā)光活性分子Ru(bpy)2(dcbpy)NHS可以通過分子上的N-羥基琥珀酰亞胺酯與指紋氨基酸中的氨基發(fā)生共價結合,從而標記到指紋上；繼而與共反應劑DBAE在一定電位下產(chǎn)生電化學發(fā)光反應,顯現(xiàn)出指紋的形貌。利用MALDI-TOF MS驗證了Ru(bpy)2(dcbpy)NHS與指紋氨基酸的共價連接,考察了反應電位、發(fā)光體濃度及反應時間對顯現(xiàn)效果的影響。正相模式可以對痕量汗?jié)撝讣y進行有效顯現(xiàn)。 結合酶聯(lián)免疫分析技術,實現(xiàn)了潛在指紋中目標成分的特異性高靈敏電化學發(fā)光檢測。首先以人IgG人工模擬指紋為檢測對象,驗證了方法的可行性。將指紋依次與羊抗人IgG、HRP標記兔抗羊IgG孵育一定時間,通過抗體與目標物的特異性免疫反應使HRP標記到指紋上。然后利用電化學反應使電解質溶液中的溶解O2還原生成H202,在指紋HRP的催化作用下,H202與底物溶液中的魯米諾產(chǎn)生波長425nm的化學發(fā)光,進而顯現(xiàn)出指紋紋路。考察了免疫反應溫度、反應時間及抗體濃度對顯現(xiàn)效果的影響。用BSA模擬指紋作為對照,結果表明抗體對人IgG模擬指紋的標記是特異性的。進一步地,將該方法用于真實血指紋中人IgG的成功檢測。 最后,在上述方法的基礎之上,進一步引入生物素-鏈霉親和素放大系統(tǒng),實現(xiàn)了汗?jié)撝讣y中人汗腺抗菌肽Dermcidin、人表皮生長因子和溶菌酶等目標代謝物的電化學發(fā)光檢測。該方法在進行潛在指紋顯現(xiàn)的同時,可以實現(xiàn)人汗腺代謝物的特異性識別,有望發(fā)展成為一種簡單、便攜、通用的指紋檢測技術,用于興奮劑檢測、病患臨床診斷以及爆炸物檢測等領域,具有重要的醫(yī)學診斷和安全保障價值。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：O657.1;D918.91

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