서강대학교

초록/요약

Single cell-based assay has been used for detection the effect of drugs, toxicants agents, and other cell functional characterization. However, there is an increasing demand for more rapid and simple procedures for studying the cell viability and the effect of drugs on cell behavior at the single cell level. The focus of this dissertation is three-fold: development of various homogenous SERS-active surfaces with high sensitivity; applications of SERS-active surfaces as cell-based chips for study the cell viability, the effect of anticancer drugs on the viability of cells, monitor the behaviors of proteins and other macromolecules inside a cell during the key cellular processes such as cell differentiation, division and apoptosis as well as differentiation between different cell lines in addition to monitor the neural cell differentiation based on SERS technique; and attempting to overcome some of the difficulties encountered in electrical methods by combination between electrical and SERS techniques as spectroelectrochemical (SEC) technique. The first part of this dissertation including the development of highly sensitive SERS-active surfaces to enhance the Raman signals of living cells and its applications for real time monitoring the effect of the chemotherapeutic agents on cancer cells as wall as monitoring the neural Stem cell differentiation. First, we developed a highly sensitive SERS-active surface based on the fabrication of gold (Au) nanoflowers array modified ITO (indium tin oxide) substrates. Au nanoflower array modified ITO substrate was developed based on electrochemical deposition of Au from Au3+ solution in presence of polyethylene glycol (PEG) as a surfactant onto an ITO surface. This Au nanoflowers/ITO substrate shows two surface plasmon absorption peaks, a strong broad peak in the NIR region (700 nm) that could be due to longitudinal oscillation of the conduction band electrons, and a weak narrow wavelength band at around 540 nm that contributed by transverse electronic oscillation. The longitudinal absorption band has been reported to be a much higher sensitivity to the local dielectric environment than the absorption band of spherical nanoparticles (NPs). On account of the enhanced surface electric field depend on the surface plasmon excitation, Au nanoflowers could be absorb and scatter electromagnetic radiation strongly. Moreover, the longitudinal absorption of Au nanflowers/ITO substrate (700 nm) could be in resonance with the excitation wavelength from a diode laser (785 nm) that may be cause another additional enhancement factor. Au nanoflowers/ITO substrate was demonstrated to generate a higher enhancement of Raman signals compared to that of Au NPs/ITO substrate. Also, the focus of the NIR laser that fluorescence free and can also penetrate much deeper into the sample, on Au nanoflower arrays can induce strong surface plasmon effects and cause highly enhanced Raman scattering, which enables intensive SERS-based study the biochemical composition and/or its variance of living cells without potential cell damage. Therefore, Au nanoflower modified ITO substrate was directly applied for detection of biochemical composition changes of living HepG2 cell that exposed to different kinds of anticancer drugs. The different anticancer agents caused different effects on the cells. These different effects of the three kinds of anticancer drugs were successfully detected based on analysis each peak of the SERS signals. These anticancer drugs which used in this study were found to have significant effects on the nucleus of HepG2 cells, especially for disintegration of DNA structure or prevention of its synthesis. These negative effects on cellular chromosomes led to decreased cell viability and/or proliferation, which were confirmed by cyclic voltammetry (CV). Compared to bare ITO electrode, electrochemical signals from Au nanoflower modified ITO electrode also showed significantly increased signals, confirming its unlimited ability for enhancement of both optical and electrochemical signals from cells. Our study based on the SERS and electrochemical techniques using Au nanoflower modified ITO substrate can be readily applied for sensitive in vitro drug screening with multiple detection and high sensitivity. Moreover, this Au nanoflower array modified ITO substrate was applied as SERS-active surface for monitoring the differentiation of mouse neural embryonic stem (ES) cells, human adult neural stem (HB1F3) cells and pheochromocytoma (PC12) cells. The results show that each cell line demonstrate different behavior during their differentiation, moreover, differentiation of neural ES cell could be analysis with time since it is stepwise process. However, the results demonstrate also that the DNA content seems to decrease during the differentiation of stem cells. On the other hand, proteins percentages were increased. The increase of proteins can be reflecting local variations in protein structure and a maturational shift. Therefore, the SERS analysis technique offers more rapid sample analysis without burden-some staining, and halves the number of cell lines that must be prepared and maintained. In this part of this thesis, a highly sensitive SERS-active surface as cell-based chip has been developed to induce a highly enhanced Raman scattering, which enables intensive SERS-based study the biochemical composition and/or its variance of living cells as wall as monitoring the neural stem cell differentiation without potential cell damage. The second part in this work focuses on exploring the application of homogenous SERS-active surface as a cell analyzer. In the first part we have developed Au nanoflower array on an ITO substrate as SERS-active site that not uniform enough that could be affected on the distribution of enhanced factor. Therefore, a geometrically well-organized SERS-active substrate with control of both nanostructures size and shape has been highly desirable. In this part, we developed a uniformly distributed Au nanodot SERS-active substrate, which provide low signal variances in the SERS signals with high intensity and reproducibility. We found that the controlled nanostructure array provided broader distribution of hot spots and higher signal-to-noise ratio than the current SERS techniques. The homogeneous nanostructures arrays composed of 60 nm Au nanodots was prepared by thermal evaporation of pure Au metal onto ITO surface through a nanoporous Al mask. This SERS-active surface was applied to differentiate different kinds of cancer cell lines (HepG2, MCF-7 and HeLa cells), normal and cancerous breast cells (HMEC and MCF-7 cells) and live/dead cells under physiological-like conditions. All SERS signals from different kinds of cell lines and live/dead cells showed clear differences, and variance of cells cycles such as mitotic event or cell resting cycle in same cell line was also detectable in same conditions. Therefore, the developed SERS-active substrate was so sensitive and reproducible to provide a powerful tool for an effective single cell analyzer, protein expression levels and the distribution of proteins inside a cell. The third part of this thesis highlights an attempt to understand the origin of electrical properties of cells and the effect of applied direct voltage on the chemical composition of the bulk cells as well as the single cell based on the combination between SERS and electrochemical techniques. In this part of the thesis, we designed the SERS-LSV as SEC substrate based on the fabrication of Au nanodots array on ITO substrate. Although, nanoporous Al mask could offer a highly uniform distributed Au nanodot array, however, the preparation processes of nanoporous Al mask needed long time. Hence, we developed polystyrene mask onto ITO substrate that was prepared in one step, through this Polystyrene (PS) mask Au nanodots array was developed based on Au thermal evaporation method. A sterile cell-chamber of size 10 mm x10 mm was developed to allow the measurements of SERS spectra of living cells in like physiological conditions. We applied SEC technique to study the biochemical changes of PC12 cells under oxidation or reduction potential. Also, we applied this technique for dopamine (DA) solution to validate the hypothesis that if DA play a role in electrochemical behavior of PC12 cells or not. Our results demonstrate that not only DA peaks were involved within the redox processes of PC12 cells, but there are number of other components which play a role in the electrochemical behavior of PC12 cells. Finally, we fabricated Au nanodots inside microgap between Au microelectrode, and then single PC12 cell was immobilized over the microgap through PDMS (polydimethylsiloxane) microchannel to get the SERS and the electrical properties from the single cell to reduce the effect of bulk cells.