基于表面增强拉曼光谱技术的鼻咽癌与胃癌检测方法研究

发布时间：2018-03-28 22:06

本文选题：表面增强拉曼光谱　切入点：细胞　出处：《福建师范大学》2011年硕士论文

【摘要】：表面增强拉曼光谱(SERS)在生物医学领域具有非常广阔的应用前景,本论文跟踪国际热点研究问题,结合我国研究现状和临床需求,基于SERS技术进行鼻咽癌与胃癌检测方法研究,主要研究成果如下： 1)分别利用细胞内吞法、细胞自还原法以及细胞电穿孔法三种金属纳米粒子传输法进行细胞SERS光谱研究并对三种方法的优缺点进行比较。重点讨论利用电穿孔方法实现金属纳米粒子到细胞的快速输送；首次利用电穿孔技术将银纳米粒子快速导入鼻咽癌C666细胞系、皮肤癌A431细胞系及白血病细胞CA46细胞系,并成功测得细胞内SERS信号。结合电穿孔导入技术及全细胞SERS检测方法,不仅可以显著缩短细胞SERS光谱的准备和测试时间,而且很大程度上提高了细胞SERS光谱的重复性和稳定性。细胞电穿孔-SERS这一新方法将在生物医学应用中(如细胞内生化物质的实时检测、癌细胞筛查等)展现出极大的应用潜力。 2)利用金纳米粒子为增强基底获取人正常鼻咽组织与鼻咽癌组织：SERS光谱与图像,并进行组织SERS光谱归属分析;探讨在组织层面上观察到的SERS光谱闪烁现象,研究发现组织SERS波动现象与激发光功率密度高度相关。利用SERS成像技术研究正常与癌变鼻咽组织在生化成分上的差异；在此基础上进一步构建EB病毒SERS免疫探针,证实该免疫SERS探针可实现高效的EB病毒免疫检测。 3)首次提出利用血浆SERS光谱检测实现非标记鼻咽癌的无损诊断分析；利用表面增强拉曼光谱技术进行人体血浆生化分析,通过谱峰指认进一步探讨鼻咽癌患者与健康人血浆SERS光谱的差异。人体血浆SERS光谱结合主成分分析和线性判别统计分析可区分正常人与鼻咽癌患者血浆,诊断特异性与灵敏度分别达到100%与90.7%(针对43例鼻咽癌患者和33例正常人血浆样品分析结果).通过对比研究发现鼻咽癌患者血浆与正常健康人血浆之间存在某些特定的生化成分差异。研究结果表明,血浆表面增强拉曼光谱分析很有希望发展为一种无损检测与筛查鼻咽癌的临床诊断工具。 4)首次利用四种不同偏振态激光激发血浆表面增强拉曼光谱并结合多变量统计分析方法进行胃癌的无损检测研究。与使用非偏振激光和线偏振激光相比,利用圆偏振态激发光能够大大提高诊断的特异性与灵敏度。利用左旋圆偏振激光进行胃癌患者血浆SERS光谱无损诊断分析,最佳诊断效果可以实现灵敏度为100%,特异性为97%(针对32例胃癌患者和33例正常人血浆样品分析结果)；而利用非偏振激光激发,其诊断灵敏度与特异性大概都只有72%.诊断效果的大幅度提高可能是由于左旋圆偏振激光比较适合于探测与胃癌有关的血浆手性分子。 5)首次将膜电泳提纯血浆蛋白技术与表面增强拉曼光谱相结合应用于无损癌症检测分析。与未经过任何处理的血浆直接与银胶混合后进行SERS检测相比,这种膜电泳与SERS相结合方法可大大减少由于癌症患者血浆中含有的外源性物质以及除白蛋白和球蛋白之外的其它血浆成分所引起的光谱干扰变化。将血浆膜电泳蛋白SERS光谱进行主成分分析,统计结果显示无论是胃癌样本还是鼻咽癌样本都可准确地与健康样本组区分开,诊断的灵敏度和特异性都达到100%(针对31例胃癌患者、43例鼻咽癌患者以及33例正常人血浆样品分析结果)。我们发明的这种新方法可能会对所有类型的癌症表现出敏感性,并可能成为一种非侵入性的癌症检测通用筛查方法。
[Abstract]:Surface - enhanced Raman spectroscopy ( SERS ) has a very wide application prospect in the field of biomedical field . This paper tracks international hotspot research , combines the current situation and clinical demand of our country , and studies the detection methods of nasopharyngeal carcinoma and gastric cancer based on SERS technique . The main research results are as follows :

1 ) The cell SERS spectra of three kinds of metal nanoparticles were studied by means of endocytosis , self - reduction and electroporation . The advantages and disadvantages of the three methods were compared .
In the first time , silver nanoparticles were rapidly introduced into NPC C666 cell line , skin cancer A431 cell line and leukemia cell CA46 cell line by electroporation technique , and SERS signal was successfully measured . Combined with electroporation and full cell SERS assay , not only the preparation and testing time of SERS spectra were significantly shortened , but also the repeatability and stability of SERS spectra were greatly improved .

2 ) using gold nanoparticles as the enhancement substrate to obtain the human normal nasopharyngeal tissue and the nasopharyngeal carcinoma tissue : SERS spectra and images , and carrying out SERS spectrum attribution analysis ; and exploring the SERS spectrum flicker phenomenon observed at the tissue level , and researching the difference of the tissue SERS fluctuation phenomenon and the excitation light power density .
On the basis of this , the EB virus SERS immunoprobe was further constructed , and it was confirmed that the immune SERS probe could realize high - efficiency EB virus immunodetection .

3 ) A nondestructive diagnostic analysis of non - labeled nasopharyngeal carcinoma ( NPC ) using plasma SERS ( SERS ) was proposed for the first time .
The plasma SERS spectra of patients with nasopharyngeal carcinoma ( NPC ) and normal healthy human plasma were analyzed by means of surface enhanced Raman spectroscopy ( SERS ) . The results showed that the plasma surface enhanced Raman spectrum analysis was promising to develop a clinical diagnostic tool for non - destructive testing and screening of nasopharyngeal carcinoma .

4 ) Using four different polarization states laser to excite plasma surface enhanced Raman spectrum and combined with multivariate statistical analysis method to study the non - destructive examination of gastric cancer . Compared with non - polarized laser and linear polarized laser , using circular polarized laser to excite light can greatly improve the specificity and sensitivity of diagnosis .
With non - polarized laser excitation , the diagnostic sensitivity and specificity can be only 72 % . The significant increase in diagnostic effectiveness may be due to the comparison of the left - handed circular polarized laser to the detection of the plasma chiral molecules associated with gastric cancer .

The plasma membrane electrophoretic protein SERS ( SERS ) spectra were analyzed by SERS spectra . The results showed that the sensitivity and specificity of plasma membrane electrophoresis were 100 % ( for 31 patients with gastric cancer , 43 patients with nasopharyngeal carcinoma and 33 normal human plasma samples ) . This new method of our invention may show sensitivity to all types of cancer and may be a non - invasive cancer detection universal screening method .

【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63;R735.2;O657.37

【引证文献】