Studies On Novel Design Methods Of SERS-based Biosensor And On New-Type Synthesis Of SERS Tags

Surface-enhanced Raman scattering (SERS) is a highly sensitive and selective tool for the identification of biological or chemical analytes based on Raman scattering. Its narrow, well-resolved bands, low SERS intensity of water and high stability make SERS widely used in material science, surface science, biological characterization and diagnostics. This dissertation presented here is focused on the fabrication and application of novel biosensors based on SERS, the novel synthesis methods of biocompatible core-shell SERS tags and probing the validity of nanometal surface energy transfer. The details are summarized as follows:1.Preparation of aptameric biosensors for small molecules based on SERSIn chapter 2, two novel strategies have been developed in the preparation of aptameric biosensors for cocaine and adenosine based on SERS. The combination of aptamer and SERS enriches the application of SERS in biosensor.(1) A proof-of-principle of a reagentless aptameric sensor based on SERS with"signal-on"architecture using a model target of cocaine was first reported. This novel aptameric sensor was based on the conformational change of the surface-tethered aptamer on binding target that draws a certain Raman reporter in close proximity to the SERS substrate, thereby increasing the Raman scattering signal due to the local enhancement effect of SERS. To improve the response performance, the sensor is fabricated by a cocaine-templated mixed self-assembly immobilization strategy. Under optimized assay conditions, one can determine cocaine at a concentration of 1μM, which compares favorably with analogous aptameric sensors based on electrochemical and fluorescence techniques. The sensor can be readily regenerated through washing with a buffer.(2) To increase the sensitivity of an aptameric sensor based on SERS, structure-switching signaling aptamer was introduced to the fabrication process. First, Au@Ag colloid film on a polished gold disc is prepared as enhanced substrate and modified with thiolated capture DNA. The formation of an aptamer/DNA duplex of expanded anti-adenosine aptamer and tetramethylrhodamine–labeled DNA (denoted TMR-DNA) is then developed, in which TMR-DNA could also hybridize completely with capture DNA. The introduction of adenosine thus triggers structure switching of the aptamer from aptamer/DNA duplex to aptamer/target complex. As a result, the released TMR-DNA is captured onto the SERS substrate, resulting in an increase of SERS signal. Under optimized assay conditions, a wide linear dynamic range (2.0×10-8 M- 2×10-6 M) was reached with low detection limit (1.0×10-8 M). Moreover, high selectivity, stability and facile regeneration are achieved.These results suggest that the SERS-based transducer might create a new dimension for future development of novel aptameric sensors for sensitive determination in biochemical and biomedical studies;2.Research on homogeneous SERS immunoassay based on aggregation of nanoparticle SERS probes. In chapter 3, a one-step homogenous sensitive immunoassay using SERS has beendeveloped. This strategy is based on the aggregation of Raman reporter-labeled immunogold nanoparticles induced by the immunoreaction with corresponding antigens. The aggregation of gold nanoparticles results in a SERS signal increase of Raman reporter. Therefore, human IgG could be directly determined by measuring the Raman signal of reporter. Using human IgG as a model protein, a wide linear dynamic range (0.1μg/mL to 15μg/mL) was reached with low detection limit (0.1μg/mL) under optimized assay conditions. The successful test suggests that the application of the proposed method holds promising potential for simple, fast detection of proteins in the fields of molecular biology and clinical diagnostics.3.The synthesis of core-shell nanoparticle-based SERS tagsIn chapter 4, two novel synthesis methods have been developed in the preparation of Au@SiO2 core-shell nanoparticle-based SERS tags and gold-aggregated, dye-embedded, polymer-protected nanoparticles.(1) An improved synthesis method of Au@SiO2 SERS tags has been described in this chapter. The common properties of the core-shell nanoparticle-based SERS tags including Au@SiO2 are strong SERS signal and extraordinary stability. The most exciting advantage of the method is its simplicity and time saving procedure as compared to other preparing methods reported before. Besides, the proposed methodology eliminates the use of vitrophilic pretreatment during the preparation, which is necessary in other proposed methodologies.(2)Gold-aggregated, dye-embedded, polymer-protected nanoparticles (GDPNs) are unusual aggregates that involve charge-driven aggregation of gold nanoparticles/poly(L-lysine) functionalized with Raman dye and a poly(L-lysine) (PLL) outer layer. Unlike most of traditional Raman tags, aggregative gold nanoparticles are used in GDPNs as the active substrate for increasing the signal of SERS. Furthermore, the outer PLL layer could lock in Au nanoparticles modified with rhodamine B isothiocyanate (RBITC) and lock out external dye molecules. Specifically, PLL as the outer layer exposes an amino-group rich surface to the surrounding environment that should facilitate the conjugation of GDPNs with biomolecules (antibody/antigen, nucleic acid). These advantages suggest the potential of using GDPNs as Raman tags for multiplex and ultrasensitive detection of biomolecules.4.The application of SERS for probing the validity of nanometal surface energy transfer (NSET) spectroscopic rulerIn chapter 5, the fluorescent behavior of rhodamine 6G at discrete distance from Ag@Au core-shell nanoparticle surface which follows d-4 distance dependence was investigated by SERS. This result indicates the necessity of theoretical studies in SERS research.