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发展适用于泪液及血清生物医学分析的高灵敏拉曼光谱技术 ; Development of highly sensitive Raman spectroscopy for biomedical analysis of human tears and sera

Pei Hu

拉曼光谱技术具有无标记、活体检测等优势,因而在生物医学研究中广受关注,并具有广泛的应用前景。拉曼光谱面临的一个主要挑战在于其检测灵敏度低,特别是对于生物分子的检测。为此,需要各种能够有效提高拉曼光谱技术检测灵敏度的方法。在本论文中,我们系统地比较了两种具有较高检测灵敏度的拉曼光谱技术检测方法即滴涂沉积拉曼光谱(DropcoatingdepositionRamanspectroscopy,DCDRS)技术和表面增强拉曼光谱(Surface-enhancedRamanspectroscopy,SERS)技术在对微升级的泪液样本或血清样本检测中的应用,获得了如下主要结果: 1)泪液或血清样品DCDR... Full description

Main Author: Hu, Pei
University Notes: Dissertation 2014
Published: 2014
Links: Volltext (dspace.xmu.edu.cn)
Keywords: Hochschulschrift
Language: Chinese
Physical Description: 1 Online-Ressource
PPN (Catalogue-ID): 858818922
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520 |a 拉曼光谱技术具有无标记、活体检测等优势,因而在生物医学研究中广受关注,并具有广泛的应用前景。拉曼光谱面临的一个主要挑战在于其检测灵敏度低,特别是对于生物分子的检测。为此,需要各种能够有效提高拉曼光谱技术检测灵敏度的方法。在本论文中,我们系统地比较了两种具有较高检测灵敏度的拉曼光谱技术检测方法即滴涂沉积拉曼光谱(DropcoatingdepositionRamanspectroscopy,DCDRS)技术和表面增强拉曼光谱(Surface-enhancedRamanspectroscopy,SERS)技术在对微升级的泪液样本或血清样本检测中的应用,获得了如下主要结果: 1)泪液或血清样品DCDRS分析检测中的方法学研究 对微量泪液或血清样品在干燥过程中的实验条件进行了探索及优化,主要包括检测衬底的选择、样品干燥条件的控制、样品检测区域的选择、拉曼光谱检测条件的优化等,分别确定了适合泪液及血清DCDRS检测的实验条件,并获得了高灵敏、高重现性的拉曼谱图。泪液或血清中除水分外,存在的主要成份是蛋白质如溶菌酶、血清蛋白、乳铁传递蛋白等。研究发现泪液的DCDRS谱峰主要由这些高含量的蛋白质贡献,而血清的DCDRS信号则主要由具有共振拉曼效应的类胡萝卜素所提供。 从临床应用的角度出发,分别对15例健康个体和15例结膜炎患者泪液、20例健康个体和20例肺癌患者血清的DCDRS光谱进行主成分分析,获得了较好的区分效果,诊断符合率分别为100%、95%,这使临床结膜炎、肺癌的快速、客观诊断成为可能。 2)基于DCDRS技术对泪液成份的定量分析 建立了基于DCDRS的单组分溶菌酶溶液的内标法定量及双组份溶菌酶–血清蛋白、溶菌酶–乳铁传递蛋白混合溶液中溶菌酶相对含量的定量分析模型,从定量的角度对15例健康个体、15例细菌性结膜炎患者、7例真菌性结膜炎患者、6例病毒性结膜炎患者及3例干眼症患者泪液进行了溶菌酶相对含量的分析,并得出健康个体泪液和细菌性结膜炎患者等泪液的溶菌酶含量间存在差异。 通过DCDRS技术对泪液中低浓度绿脓菌素进行检测,检测限可低达5.0×10-4mg/mL,这对于绿脓菌感染症的诊断具有重要意义。 3)基于SERS技术对泪液或血清的检测和分析 利用SERS对泪液或血清的临床样本进行分析,发现泪液和血清的SERS谱峰极为相似,其贡献主要来自于嘌呤代谢的产物尿酸和次黄嘌呤等,首次对泪液的SERS谱峰进行了可靠指认。 分别对20例健康个体和20例结膜炎患者泪液及22例健康个体和22例肺癌患者血清进行了SERS检测及主成分分析,分别获得了90%和90.5%的诊断符合率,进而可以辅助临床疾病的快速诊断。 ; Raman spectroscopy has received increasing interest in biomedical applications due to its label-free and in vivo detection capabilities. The main challenge for a wider application lies in its low detection sensitivity, especially for biomolecules. Therefore, there is a great demand for effective methods to improve the detection sensitivity of Raman spectroscopy. In this thesis, we systematically compared two Raman techniques with improved sensitivity for the detection of microliter quantities of whole human tears and blood sera: drop coating deposition Raman spectroscopy (DCDRS) and surface-enhanced Raman spectroscopy (SERS). The main results of the thesis are as follows: 1) Methodological studies in the DCDRS detection of tears and sera The experimental conditions for the drying process of micro quantites of tears and sera were explored and optimized, including selection of the substrates, control of the drying conditions, selection of the detection regions and the acquisition conditions for Raman detection. The optimized experimental conditions were determined for both tears and sera, and highly sensitive and reproducible DCDR spectra were obtained. In addition to water, tears or sera mainly consist of proteins such as lysozyme, albumin, lactoferrin, and etc. It is found that those high abundant proteins dominate the tear DCDR spectra; whereas, the low abundant carotenoids, with resonant Raman effect, provide the main signal for the blood sera DCDR spectra. From the aspect of clinical applications, principle component analysis was applied to 15 healthy and 15 conjunctivitis tear samples, and 20 healthy and 20 lung cancer serum samples to discriminate the conjunctivitis tears or lung cancer sera from the healthy ones. A good discriminative effect was achieved with diagnostic accuracies of 100% and 95% for the tear and serum samples, respectively. This greatly enables the rapid and objective diagnosis of clinical conjunctivitis and lung cancer. 2) Quantitative analysis of tear components with DCDRS A quantitative analytical model was established for the quantification of the relative content of lysozyme in the single-component system of a lysozyme solution with the internal standard method and binary-component systems of Lys–BSA or Lys–Lac with DCDRS. From a quantitative standpoint, quantitative analysis of lysozyme was made for clinical tear samples from 15 healthy, 15 bacterial conjunctivitis, 7 fungal conjunctivitis, 6 viral conjunctivitis and 3 dry eye patients. It shows that there exist difference in the relative content of lysozyme in the tears of healthy individuals and patients with some eye diseases. Pyocyanine with a low concentration in tears was also detected with the detection limit as low as 5.010-4 mg/mL, which is of great significance for the diagnosis of Pseudomonas aeruginosa infection. 3) Detection of tear or serum with SERS Clinical blood serum samples were analyzed with SERS. It turns out that tears show a similar SERS feature to blood sera and the main contribution comes from uric acid and hypoxanthine, the products from purine metabolism. This is the first time that SERS peaks of tears have been reliably assigned. SERS measurements and principle component analysis were carried out on 20 healthy and 20 conjunctivitis tear samples, and 22 healthy individuals and 22 lung cancer blood serum samples. The diagnostic accuracies for the tear and serum samples are 90% and 90.5%, respectively, which , in turn, can assist clinical rapid diagnosis of diseases. ; 学位:理学硕士 ; 院系专业:化学化工学院_分析化学 ; 学号:20520111151564 
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