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Surface-enhanced Raman scattering (SERS) is an extremely sensitive analytical tool with the detection limit ranging down to a single molecule. Moreover, SERS is a minimally invasive analytical technique that involves little or no sample preparation and has high molecular specificity. Interestingly, in recent years, the miniaturization of Raman instrumentation down to smartphone size has proved its suitability for point-of-care. Owing to these attractive features, SERS has become a promising analytical technique for rapid and accurate disease detection in recent years. Excellent uniformity and sensitivity of SERS substrates are the two key requirements to get reproducible and reliable signals from a probe. The assemblage of nanoparticles into ordered 2D and 3D arrays and thereby engineering SERS hotspots is the main strategy in designing highly uniform and ultrasensitive SERS substrates. Ultra-uniform SERS substrates can be produced using top-down nanofabrication technologies such as high-resolution electron-beam lithography. However, such top-down methods involve highly expensive and sophisticated techniques that need a tightly controlled environment. Moreover, the yield from such methods is extremely low that these methods are not suitable for mass production of low-cost SERS substrates for routine analysis. As a result, cost-effective and facile fabrication of highly sensitive and uniform SERS substrates is at the center of the practical aspect of SERS-related researches. In this thesis, three different approaches are presented to fabricate highly uniform and sensitive SES substrates and applied for cancer and bacterial detections.
In the first approach, methods to improve the reliability and sensitivity of SERS-based sandwich immunoassay have been described. AuNF was used to design both extrinsic Raman label and capture surface and thereby to improve detection sensitivity. First, a highly uniform capture surface was designed by self-assembly of gold nanoflower (AuNF) onto a thiol-functionalized silicon wafer. Then, AuNF-based SERS immunoassay was carried out for the detection of pancreatic cancer marker MUC4 by tagging it with extrinsic Raman label constructed with AuNF, Raman reporter molecule and anti-MUC4 antibody. In addition to designing uniform SERS substrate, we employed Raman mapping over a large area to minimize the effect of spot-to-spot SERS signal variation. Furthermore, the use of a microwell plate for incubation of capture substrate minimized false positive error significantly. The designed method is sensitive enough to detect MUC4 down to 0.1 ng mL-1.
In the second approach, a SERS substrate with excellent uniformity and sensitivity has been designed by simple electrophoretic deposition of gold nanoflowers onto acid-etched Al-foil and into nanopores of anodized aluminum oxide (AAO). The SEM image and SERS mapping measurement over quite a large area confirmed the excellent uniformity of the substrates. Interestingly, the capping agent used to control the surface charge of nanoparticle can be wisely chosen so that it will also function as a linker molecule to capture a substance of interest onto the substrate. As aluminum has surface Plasmon property, LSPR coupling of gold nanoflowers with anodized aluminum nanoporous structure contributed about one order of magnitude to the overall enhancement compared to that of the silicon wafer.
In the last approach, a novel simplest ever method for fabrication of ultrasensitive and highly uniform SERS substrate and its application for label-free bacterial detection has been proposed. The idea of galvanic replacement reaction in combination with a seed-mediated particle-growth approach in the presence of hydroquinone has been employed, and Ag nanoparticle array was fabricated on Cu-foil in less than 3 min. Label-free detection of bacterial species has shown that the substrate is superior to highly acclaimed SERS substrates such as silver nanocubes. In contrast to most reported SERS detection of pathogens, the detection of bacteria was carried out by directly probing liquid samples. Interestingly, direct liquid bacteria sample analysis showed six-fold higher detection sensitivity than that of completely dried samples.
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