Font Size: a A A

Raman Scattering Techniques Applied On Single Cell Detection

Posted on:2014-01-10Degree:DoctorType:Dissertation
Country:ChinaCandidate:L SuFull Text:PDF
GTID:1221330398455397Subject:Analytical Chemistry
Abstract/Summary:PDF Full Text Request
Cell is the fundamental unite of life activity and structure in the living body. Early in1925, Wilson, the famous biologist, had proposed:"All of the key problems in the life have to resort to cell." Growth and development of living body rely on proliferation, differentiation and apoptosis of cell. Almost all diseases are caused by the pathologic change of cell. Oral squamous cell carcinoma (OSCC) is a kind of oral cancer with low survival rate, high metastatic and recurrence rate. OSCC ranks the sixth in the world cancer incidence. Up to now, no treatment has shown efficacy. Metastasis is an important reason which leads to the death of OSCC patient. Early detection and blocking of tumor genesis, progenesis, invasion and metastasis is critical for the prevention and treatment of OSCC. Conventional methods of cell biology include Western blot, enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (IC), etc. Most of the techniques require numerous preparation steps, such as cell fixation, extraction or staining. They have many disadvantages such as invasive, time consuming, as well as false positive/negative, and can not get specific structure information of biological composition in cells by real-time and in situ.Raman spectroscopy, which is based on inelastic scattering of laser photons by molecular vibrations of biopolymers, is a quantitative and non-destructive optical technique that is capable of acquiring "fingerprint" information derived from the biochemical composition of cells at molecular leval. Moreover, the development of surface enhanced Raman spectroscopy (SERS) greatly improves the sensitivity of detection and analysis. Compared with infrared and fluorescent spectroscopy, Raman spectroscopy has advantages of low aqueous interference, unease to quench, can be excited with red laser, and narrow spectral band width, which make it very suitable for the research in biological system, especially for the study of single living cell.Based on all of this, the paper set to study. The main contents are listed as follow: 1. Investaged normal and malignant tissues/cells by confocal Raman spectroscopy. After acquiring Raman spectroscopy of normal and malignant tissues/cells, spectral difference was obtained, from which the biological composition changes were seeked out, and finally principal component analysis (PCA) was employed to classify the Raman data into normal and malignant groups. The research has laid a foundation for the clinical application of Raman spectroscopy.2. Monitored nitric oxide stimulus response of single cancer cell by Raman spectroscopy. Sodium nitroprusside (SNP) was chosed as a NO source. The amount of NO could be controlled by adjusting the SNP concentration in culture solution. TCA8113cells were firstly treated with the indicated concentration of SNP for certain time intervals in incubator, and then Raman spectroscopy was obtained from the cells. Content and structure changes of biological components in cells were observed after NO stimulas. Raman spectral analysis was compared with flow cytometric analysis in the study of NO stimlulus reponse. In addition, kinetic study was carried out to analyze the NO stimulus process. Research results indicated that Raman spectroscopy is a reliable, fast, label-free, and sensitive detection method, and it could potentially be used in rapid screening of novel drugs.3. Synthesis of a novel membrane-targeted SERS nanoprobe and its application in cell imaging were presented. At first, highly stable gold nanoparticle with diameter of40nm was synthesized, and then it was linked with EGFR antibody by electrostatic adsorption. After incubating the SERS nanoprobe with TCA8113cells, gold nanoparticles were specifically targeted on the cell membrane with the aid of EGFR antibody. Raman mapping technique was employed to characterize the cells, and the results demonstrated the SERS nanoprobe is of high sensitivity and stability. It will be significant for the establishment of multi-targeted, highly sensitive, in situ and in vivo SERS nanoprobe.4. Synthesis of a novel nuclear-targeted SERS nanoprobe and its application in cell imaging were presented. At first, highly stable gold nanoparticle with diameter of20nm was synthesized, and then it was conjugated with SV-40large T nuclear localization signal (NLS) peptide by chemical coupled reaction. After incubating the SERS nanoprobe with TCA8113cells, gold nanoparticles were specifically targeted on the cell nuclear with the aid of NLS peptide. Raman mapping technique was employed to characterize the nuclear-targeted process, and the results demonstrated the SERS nanoprobe is of high sensitivity and stability. In addition, simultaneously targeted detection of different subcellular structure was realized for the first time by incubating both the membrane-and the nuclear-targeted SERS nanoprobe with TCA8113cells. It will provide an effective analysis method for the research of cell signal transduction and anticancer mechanism of novel drugs.
Keywords/Search Tags:oral squamous cell carcinoma (OSCC), Raman spectroscopy, surfaceenhanced Raman spectroscopy (SERS), single cell, multi-targeted imaging
PDF Full Text Request
Related items