Fabrication and Characteristics of Dual-Faced Multifunctional Nanobeads for Single Particle Tracking and Raman Detection

• Main Authors: Hsieh, Hsin-Yi, 謝馨儀
• Other Authors: Tseng, Fan-Gang
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/89762158256873797456

博士 === 國立清華大學 === 奈米工程與微系統研究所 === 101 === The purpose of this study is to develop a batch manufacturing approach to massively produce surface-enhanced Raman scattering (SERS) fluorescent nanoparticles, mushroom-like Au semishell fluorescent nanoparticle (AuFNMs), for a potential application of targeting cancer cells, delivering drug, and subsequently observing cell behavior via SERS detection of intracellular biomolecule. This nanoparticle system was established on commercial available polystyrene beads with full range of sizes (from tens of nanometer to micrometers in diameter) and many fluorescent specifications. Based on the chemical property of polystyrene, the fluorescent nanoparticle can be treated with surface roughness on the upper hemisphere and carboxyl groups on the entire surface through plasma ion bombardment and chemical oxidation. Followed by the electron-beam deposition of gold film on the upper hemisphere, the nanoparticle can perform as a SERS-active fluorescent bead with high enhancement factor. After the deposition of metal, thiol (-SH) molecules and the amine (-NH2) of protein molecules could be modified simultaneously and selectively onto the top gold surfaces and bottom carboxyl groups through Au-S and peptide bonds, respectively. In the experiments, the technique of densely packed nanoparticle array was employed to obtain a monolayer distribution of polystyrene beads on a 4-inch glass wafer. A 250-μL droplet containing 1-10% polystyrene bead, ranging from 220 nm to 920 nm in diameter, was sufficient to cover the entire surface of 4-inch wafer by a three-step spin-coating, 400rpm/10s, 800rpm/120s, and 3000rpm/2-5s. The following plasma treatment process was processed under inert argon plasma and/or vigorous oxygen plasma for the investigation and comparison of surface roughness on polystyrene nanoparticles with different intrinsic carboxyl density (0-14.7 carboxyl groups/nm2). In the meanwhile X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were utilized to analyze the change of surface chemistry and the correlation coefficient between carboxyl group density and surface roughness, respectively. The results suggest that bare polystyrene bead surface can be oxidized with carboxyl groups under oxygen plasma treatment, and the higher bonding energy of the carboxyl groups play the role as nanomasks decorating on the surface of polystyrene. As a result, the upper hemisphere of the polystyrene beads were treated with corrugated surface due to the selective etching under vertical plasma ion bombardment. Besides, in order to optimize the SERS enhancement factor on the nanoparticles, various gold-coated and plasma-treated polystyrene bead arrays with 100 μM Rhodamine 6G solution were observed their Raman scattering intensities under 632 nm laser excitation. Raman intensity enhancement on a 20-nm gold coated nanocorrugated polystyrene bead array is summarized by three factors: (1) the effect of plasmonic coupling among neighboring particles, (2) the nanocorrugation-contributed roughness, and (3) the pitch size of nanocorrugations, through the analysis of SEM images, AFM height images, and LSPR signals. Among these factors, the pitch size of nanocorrugations (ranging from ~6 nm to ~12 nm on the surface of polystyrene beads) dominates the SERS enhancement, and the average enhancement factor can reach up to 10E6. To equip the SERS fluorescent nanoparticles (AuFNMs) with more applications, an anti-CD44 antibody was selectively modified onto the carboxyl hemisphere for the recognition of overexpressive CD44 transmembrane glycoprotein on most cancer cells, such as HeLa and MCF-7 cells. A cleavable disulfide linker of Sulfo-NHS-SS-with biotin was chosen for the modification either on the gold film or on the primary amine for releasing drug in the cell intracellular environment, via the reduction of a disulfide bond (-SS-) to two thiols. In the experiments, streptavidin-linked QDots was modified onto the disulfide linker of Sulfo-NHS-SS-biotin for the compatibility testing of multiple modifications. Moreover, the cancer cell targeting ability was compared among AuFNMs with a variety of modification order, and the cleavage of disulfide bond was examined in the extracellular environment. The AuFNMs suspension, which was verified with >99% purity and uniformed particle size in a concentration of ~10E10 numbers/mL in 2-mL DI water, can be employed to target cell-surface overexpressive glycoproteins (CD44) on cancer cells and release the loads via cleaving the disulfide bonds in cytoplasm after endocytosis of 30 minutes. A 12-fold cancer targeting ability of our AuFNMs was achieved on HeLa cells when compared to a normal cell of chondrocyte. For the applications of 3D confocal particle tracking and Raman mapping, the ~200 nm AuFNMs demonstrate excellent long-lasting single-particle fluorescence and superior biomolecule sensing ability. This technique provides a potential platform for the research of cell endocytosis pathway and the cancer cell theranostics.